Patent Publication Number: US-2021161976-A1

Title: Bacteria engineered to treat diseases that benefit from reduced gut inflammation and/or tightened gut mucosal barrier

Description:
RELATED APPLICATIONS 
     This application is a 35 U.S.C. § 371 national stage filing of International Application No. PCT/US2017/016603, filed on Feb. 3, 2017, which in turn is a continuation-in-part of PCT Application No. PCT/US2016/020530, filed Mar. 2, 2016; a continuation-in-part of PCT Application No. PCT/US2016/050836, filed Sep. 8, 2016, a continuation-in-part of PCT/US2016/039444, filed Jun. 24, 2016; a continuation-in-part of PCT Application No. PCT/US2016/069052, filed Dec. 28, 2016; a continuation-in-part of PCT Application No. PCT/US2016/032565, filed May 13, 2016, and a continuation-in-part of U.S. application Ser. No. 15/260,319, filed Sep. 8, 2016; and claims the benefit of U.S. Provisional Application No. 62/291,461 filed Feb. 4, 2016; U.S. Provisional Application No. 62/291,468 filed Feb. 4, 2016; U.S. Provisional Application No. 62/291,470 filed Feb. 4, 2016; U.S. Provisional Application No. 62/347,508, filed Jun. 8, 2016; U.S. Provisional Application No. 62/354,682, filed Jun. 24, 2016; U.S. Provisional Application No. 62/362,954, filed Jul. 15, 2016; U.S. Provisional Application No. 62/385,235, filed Sep. 8, 2016; U.S. Provisional Application No. 62/423,170, filed Nov. 16, 2016; U.S. Provisional Application No. 62/439,871, filed Dec. 28, 2016; U.S. Provisional Application No. 62/347,576, filed Jun. 8, 2016 and U.S. Provisional Application No. 62/348,620, filed Jun. 10, 2016. The entire contents of each of the foregoing applications are expressly incorporated herein by reference in their entireties to provide continuity of disclosure. 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 1, 2019, is named 12671-2008-00-Corrected-Seq-Listing.txt and is 889,000 bytes in size. 
    
    
     BACKGROUND OF THE INVENTION 
     This disclosure relates to compositions and therapeutic methods for inhibiting inflammatory mechanisms in the gut, restoring and tightening gut mucosal barrier function, and/or treating and preventing autoimmune disorders. In certain aspects, the disclosure relates to genetically engineered bacteria that are capable of reducing inflammation in the gut and/or enhancing gut barrier function. In some embodiments, the genetically engineered bacteria are capable of reducing gut inflammation and/or enhancing gut barrier function, thereby ameliorating or preventing an autoimmune disorder. In some aspects, the compositions and methods disclosed herein may be used for treating or preventing autoimmune disorders as well as diseases and conditions associated with gut inflammation and/or compromised gut barrier function, e.g., diarrheal diseases, inflammatory bowel diseases, and related diseases. 
     Inflammatory bowel diseases (IBDs) are a group of diseases characterized by significant local inflammation in the gastrointestinal tract typically driven by T cells and activated macrophages and by compromised function of the epithelial barrier that separates the luminal contents of the gut from the host circulatory system (Ghishan et al., 2014). IBD pathogenesis is linked to both genetic and environmental factors and may be caused by altered interactions between gut microbes and the intestinal immune system. Current approaches to treat IBD are focused on therapeutics that modulate the immune system and suppress inflammation. These therapies include steroids, such as prednisone, and tumor necrosis factor (TNF) inhibitors, such as Humira® (Cohen et al., 2014). Drawbacks from this approach are associated with systemic immunosuppression, which includes greater susceptibility to infectious disease and cancer. 
     Other approaches have focused on treating compromised barrier function by supplying the short-chain fatty acid butyrate via enemas. Recently, several groups have demonstrated the importance of short-chain fatty acid production by commensal bacteria in regulating the immune system in the gut (Smith et al., 2013), showing that butyrate plays a direct role in inducing the differentiation of regulatory T cells and suppressing immune responses associated with inflammation in IBD (Atarashi et al., 2011; Furusawa et al., 2013). Butyrate is normally produced by microbial fermentation of dietary fiber and plays a central role in maintaining colonic epithelial cell homeostasis and barrier function (Hamer et al., 2008). Studies with butyrate enemas have shown some benefit to patients, but this treatment is not practical for long term therapy. More recently, patients with IBD have been treated with fecal transfer from healthy patients with some success (Ianiro et al., 2014). This success illustrates the central role that gut microbes play in disease pathology and suggests that certain microbial functions are associated with ameliorating the IBD disease process. However, this approach raises safety concerns over the transmission of infectious disease from the donor to the recipient. Moreover, the nature of this treatment has a negative stigma and thus is unlikely to be widely accepted. 
     Compromised gut barrier function also plays a central role in autoimmune diseases pathogenesis (Lerner et al., 2015a; Lerner et al., 2015b; Fasano et al., 2005; Fasano, 2012). A single layer of epithelial cells separates the gut lumen from the immune cells in the body. The epithelium is regulated by intercellular tight junctions and controls the equilibrium between tolerance and immunity to nonself-antigens (Fasano et al., 2005). Disrupting the epithelial layer can lead to pathological exposure of the highly immunoreactive subepithelium to the vast number of foreign antigens in the lumen (Lerner et al., 2015a) resulting in increased susceptibility to and both intestinal and extraintestinal autoimmune disorders can o ccur” (Fasano et al., 2005). Some foreign antigens are postulated to resemble self-antigens and can induce epitope-specific cross-reactivity that accelerates the progression of a pre-existing autoimmune disease or initiates an autoimmune disease (Fasano, 2012). Rheumatoid arthritis and celiac disease, for example, are autoimmune disorders that are thought to involve increased intestinal permeability (Lerner et al., 2015b). In individuals who are genetically susceptible to autoimmune disorders, dysregulation of intercellular tight junctions can lead to disease onset (Fasano, 2012). In fact, the loss of protective function of mucosal barriers that interact with the environment is necessary for autoimmunity to develop (Lerner et al., 2015a). 
     Changes in gut microbes can alter the host immune response (Paun et al., 2015; Sanz et al., 2014: Sanz et al., 2015; Wen et al., 2008). For example, in children with high genetic risk for type 1 diabetes, there are significant differences in the gut microbiome between children who develop autoimmunity for the disease and those who remain healthy (Richardson et al., 2015). Others have shown that gut bacteria are a potential therapeutic target in the prevention of asthma and exhibit strong immunomodulatory capacity . . . in lung inflammation (Arrieta et al., 2015). Thus, enhancing barrier function and reducing inflammation in the gastrointestinal tract are potential therapeutic mechanisms for the treatment or prevention of autoimmune disorders. 
     Recently there has been an effort to engineer microbes that produce anti-inflammatory molecules, such as IL-10, and administer them orally to a patient in order to deliver the therapeutic directly to the site of inflammation in the gut. The advantage of this approach is that it avoids systemic administration of immunosuppressive drugs and delivers the therapeutic directly to the gastrointestinal tract. However, while these engineered microbes have shown efficacy in some pre-clinical models, efficacy in patients has not been observed. One reason for the lack of success in treating patients is that the viability and stability of the microbes are compromised due to the constitutive production of large amounts of non-native proteins, e.g., human interleukin. Thus, there remains a great need for additional therapies to reduce gut inflammation, enhance gut barrier function, and/or treat autoimmune disorders, and that avoid undesirable side effects. 
     SUMMARY 
     The genetically engineered bacteria disclosed herein are capable of producing therapeutic anti-inflammation and/or gut barrier enhancer molecules. In some embodiments, the genetically engineered bacteria are functionally silent until they reach an inducing environment, e.g., a mammalian gut, wherein expression of the therapeutic molecule is induced. In certain embodiments, the genetically engineered bacteria are naturally non-pathogenic and may be introduced into the gut in order to reduce gut inflammation and/or enhance gut barrier function and may thereby further ameliorate or prevent an autoimmune disorder. In certain embodiments, the anti-inflammation and/or gut barrier enhancer molecule is stably produced by the genetically engineered bacteria, and/or the genetically engineered bacteria are stably maintained in vivo and/or in vitro. The invention also provides pharmaceutical compositions comprising the genetically engineered bacteria, and methods of treating diseases that benefit from reduced gut inflammation and/or tightened gut mucosal barrier function, e.g., an inflammatory bowel disease or an autoimmune disorder. 
     In some embodiments, the genetically engineered bacteria of the invention produce one or more therapeutic molecule(s) under the control of one or more promoters induced by an environmental condition, e.g., an environmental condition found in the mammalian gut, such as an inflammatory condition or a low oxygen condition. In on-limiting exemplary embodiments, the genetically engineered bacteria produce one or more therapeutic molecule(s) under the control of an oxygen level-dependent promoter, a reactive oxygen species (ROS)-dependent promoter, or a reactive nitrogen species (RNS)-dependent promoter, and a corresponding transcription factor. In some embodiments, the therapeutic molecule is butyrate; in an inducing environment, the butyrate biosynthetic gene cassette is activated, and butyrate is produced. Local production of butyrate induces the differentiation of regulatory T cells in the gut and/or promotes the barrier function of colonic epithelial cells. In some embodiments, the genetically engineered bacteria produce their therapeutic effect only in inducing environments such as the gut, thereby lowering the safety issues associated with systemic exposure. 
     Disclosed herein is a butyrate-producing bacterium comprising at least one gene or gene cassette encoding one or more non-native biosynthetic pathways for producing butyrate, wherein the bacteria produces acetyl CoA and wherein the bacterium has at least one mutation in or deletion of an endogenous pta gene. Such bacterium is capable of producing butyrate, but does not produce acetate. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous ldhA gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous frd gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene and an endogenous ldhA gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene and an endogenous frd gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous ldhA gene and an endogenous frd gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene, an endogenous frd gene, and an endogenous ldhA gene. In certain specific embodiments, the butyrate-producing bacterium comprises at least one gene or gene cassette encoding one or more non-native biosynthetic pathways for producing butyrate, wherein the bacteria produces acetyl CoA and wherein the bacterium has at least one mutation in or deletion of an endogenous pta gene and at least one mutation in or deletion of an endogenous gene selected from adhE gene and/or ldhA gene and/or frd gene. 
     In any of the above described embodiments of butyrate-producing bacteria, the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature. In any of the above described embodiments of butyrate-producing bacteria, the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature and is induced by exogenous environmental conditions found in a mammalian gut. 
     In some embodiments, the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous ldhA gene. In some embodiments, the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous adhE gene. In some embodiments, the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous frd gene. In some embodiments, the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous pta gene. In some embodiments, the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous gene selected from frd and/or ldhA and/or adhE and/or pta. In some embodiments, the butyrate-producing bacterium may produce an increased level of butyrate as compared to a bacterium which produces butyrate naturally or which comprises a gene or gene cassette for producing butyrate, but does not comprise at least one mutation in or deletion of an endogenous ldhA gene, frd gene, adhE gene, and pta gene. 
     In some embodiments, the bacterium described above comprises an endogenous pta gene and produces acetate. In these embodiments, the bacterium comprises at least one gene or gene cassette encoding one or more non-native biosynthetic pathways for producing butyrate, wherein the bacteria produces acetyl CoA and wherein the bacterium has an endogenous pta gene. Such bacterium is capable of producing butyrate and acetate. In some embodiments of this bacterium, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous ldhA gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous frd gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene and an endogenous ldhA gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene and an endogenous frd gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous ldhA gene and an endogenous frd gene. In some embodiments, the bacterium further has at least one mutation in or deletion of an endogenous adhE gene, an endogenous frd gene, and an endogenous ldhA gene. In certain specific embodiments, the butyrate-producing bacterium comprises at least one gene or gene cassette encoding one or more non-native biosynthetic pathways for producing butyrate, wherein the bacteria produces acetyl CoA and wherein the bacterium has an endogenous pta gene and at least one mutation in or deletion of an endogenous gene selected from adhE gene and/or ldhA gene and/or frd gene. 
     In any of the above-described embodiments of butyrate-producing bacterium, the at least one gene or gene cassette for producing butyrate may comprise ter, thiA1, hbd, crt2, pbt, and buk genes. In any of the above-described embodiments of butyrate-producing bacterium, the at least one gene or gene cassette for producing butyrate may comprise ter, thiA1, hbd, crt2, and tesB genes. 
     In any of the above described embodiments of butyrate- and acetate-producing bacteria, the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature. In any of the above described embodiments of butyrate- and acetate-producing bacteria, the at least one gene or gene cassette for producing butyrate is operably linked to a directly or indirectly inducible promoter that is not associated with the gene or gene cassette in nature and is induced by exogenous environmental conditions found in a mammalian gut. 
     In another aspect, disclosed herein is an acetate-producing bacterium that produces acetate but not butyrate. In any of these embodiments, the acetate-producing bacterium produces acetyl CoA and comprises a wild-type pta gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of a ldhA gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of an adhE gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of a frd gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of an ldhA gene and at least one mutation in or deletion of an adhE gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of a ldhA gene and at least one mutation in or deletion of an frd gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of an adhA gene and at least one mutation in or deletion of an frd gene. In some embodiments, the acetate-producing bacterium comprises at least one mutation in or deletion of an adhA gene, at least one mutation in or deletion of an frd gene, and at least one mutation in or deletion of an ldhA gene. 
     The bacterium may produce an increased level of acetate as compared to a bacterium which produces Acetyl CoA and comprises an endogenous pta gene, and has an endogenous frd gene and/or endogenous ldhA gene and/or endogenous adhA gene. The bacterium may produce an increased level of acetate as compared to a bacterium which produces Acetyl CoA and comprises an endogenous pta gene, and does not comprise at least one mutation in or deletion of an ldhA gene, an adhE gene, and/or a frd gene. 
     In any of the above-described embodiments comprising a gene or gene cassette for producing butyrate in which the gene or gene cassette is operably linked to a directly or indirectly inducible promoter, the promoter may be induced under low-oxygen or anaerobic conditions. In some embodiments, the promoter is selected from an FNR-responsive promoter, an ANR-responsive promoter, and a DNR-responsive promoter. In some embodiments, the promoter is an FNR-responsive promoter. In some embodiments, the promoter may be induced by the presence of reactive nitrogen species. In some embodiments, the promoter is selected from an NsrR-responsive promoter, NorR-responsive promoter, and a DNR-responsive promoter. In some embodiments, the promoter may be induced by the presence of reactive oxygen species. In some embodiments, the promoter is selected from an OxyR-responsive promoter, PerR-responsive promoter, OhrR-responsive promoter, SoxR-responsive promoter, or a RosR-responsive promoter. 
     In some embodiments, the gene and/or gene cassette is located on a chromosome in the bacterium. In some embodiments, the at least one gene and/or gene cassette is located on a plasmid in the bacterium. 
     In some embodiments, the bacterium is a probiotic bacterium. In some embodiments, the bacterium is selected from the group consisting of  Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus , and  Lactococcus . In some embodiments, the bacterium is  Escherichia coli  strain Nissle. 
     In some embodiments, the bacterium is an an auxotroph in a gene that is complemented when the bacterium is present in a mammalian gut. The bacterium may be an auxotroph in diaminopimelic acid or an enzyme in the thymine biosynthetic pathway. 
     Disclosed herein is a pharmaceutically acceptable composition comprising one or more of any of the bacterium disclosed herein; and a pharmaceutically acceptable carrier. In some embodiments, the composition is formulated for oral or rectal administration. 
     Disclosed herein is a method of treating or preventing an autoimmune disorder, comprising the step of administering to a patient in need thereof, a composition disclosed herein. 
     Disclosed herein is a method of treating a disease or condition associated with gut inflammation and/or compromised gut barrier function comprising the step of administering to a patient in need thereof, a composition. 
     The autoimmune disorder may be selected from the group consisting of acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison&#39;s disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticarial, Axonal &amp; neuronal neuropathies, Balo disease, Behcet&#39;s disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid/benign mucosal pemphigoid, Crohn&#39;s disease, Cogan syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST disease, Essential mixed cryoglobulinemia, Demyelinating neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic&#39;s disease (neuromyelitis optica), Discoid lupus, Dressier&#39;s syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture&#39;s syndrome, Granulomatosis with Polyangiitis (GPA), Graves&#39; disease, Guillain-Barre syndrome, Hashimoto&#39;s encephalitis, Hashimoto&#39;s thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, Immunoregulatory lipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenile arthritis, Juvenile idiopathic arthritis, Juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (Systemic Lupus Erythematosus), chronic Lyme disease, Meniere&#39;s disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren&#39;s ulcer, Mucha-Habermann disease, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic&#39;s), Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS (Pediatric autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, &amp; III autoimmune polyglandular syndromes, Polymyalgia rheumatic, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter&#39;s syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren&#39;s syndrome, sperm &amp; testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac&#39;s syndrome, sympathetic ophthalmia, Takayasu&#39;s arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener&#39;s granulomatosis. 
     The autoimmune disorder may be selected from the group consisting of type 1 diabetes, lupus, rheumatoid arthritis, ulcerative colitis, juvenile arthritis, psoriasis, psoriatic arthritis, celiac disease, and ankylosing spondylitis. 
     The disease or condition may be selected from an inflammatory bowel disease, including Crohn&#39;s disease and ulcerative colitis, and a diarrheal disease. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1A ,  FIG. 1B ,  FIG. 1C ,  FIG. 1D ,  FIG. 1E ,  FIG. 1F ,  FIG. 1G ,  FIG. 1H ,  FIG. 1I ,  FIG. 1J , and  FIG. 1K  depict schematics of  E. coli  that are genetically engineered to express a propionate biosynthesis cassette ( FIG. 1A ), a butyrate biosynthesis cassette ( FIG. 1B ), an acetate biosynthesis cassette ( FIG. 1C ), a cassette for the expression of GLP-2 ( FIG. 1D ), a cassette for the expression of human IL-10 ( FIG. 1E ) or v-IL-22 or hIL-22 ( FIG. 1F ) under the control of a FNR-responsive promoter. The genetically engineered  E. coli  depicted in  FIG. 1D ,  FIG. 1E , and  FIG. 1F  may further comprise a secretion system for secretion of the expressed polypeptide out of the cell.  FIG. 1G  depicts bacteria overexpressing butyrate (and not expressing acetate) by expressing a butyrate biosynthesis cassette in combination with deletions in adhE and pta ( FIG. 1G ),  FIG. 1H  depicts bacteria overexpressing butyrate by expressing a butyrate biosynthesis cassette in combination with deletions in ldhA,  FIG. 1I  depicts bacteria overexpressing butyrate by expressing a butyrate biosynthesis cassette in combination with deletions in adhE and frdA ( FIG. 1I ).  FIG. 1J  depicts bacteria overexpressing acetate by deletion in ldhA.  FIG. 1K  depicts bacteria overexpressing GLP-2 in combination with a deletion in adhE and pta. 
         FIG. 2A ,  FIG. 2B ,  FIG. 2C , and  FIG. 2D  depict schematics of a butyrate production pathway and schematics of different butyrate producing circuits.  FIG. 2A  depicts a metabolic pathway for butyrate production.  FIG. 2B  and  FIG. 2C  depict schematics of two different exemplary butyrate producing circuits, both under the control of a tetracycline inducible promoter.  FIG. 2B  depicts a bdc2 butyrate cassette under control of tet promoter on a plasmid. A “bdc2 cassette” or “bdc2 butyrate cassette” refres to a butyrate producing cassette that comprises at least the following genes: bcd2, etfB3, etfA3, hbd, crt2, pbt, and buk genes.  FIG. 2C  depicts a ter butyrate cassette (ter gene replaces the bcd2, etfB3, and etfA3 genes) under control of tet promoter on a plasmid. A “ter cassette” or “ter butyrate cassette” refers to a butyrate producing cassette that comprises at least the following genes: ter, thiA1, hbd, crt2, pbt, buk.  FIG. 2D  depicts a schematic of a third exemplary butyrate gene cassette under the control of a tetracycline inducible promoter, specifically, a tesB butyrate cassette (ter gene is present and tesB gene replaces the pbt gene and the buk gene) under control of tet promoter on a plasmid. A “tes or tesB cassette or “tes or tesB butyrate cassette” refers to a butyrate producing cassette that comprises at least ter, thiA1, hbd, crt2, and tesB genes. An alternative butyrate cassette of the disclosure comprises at least bcd2, etfB3, etfA3, thiA1, hbd, crt2, and tesB genes. In some embodiments, the tes or tesB cassette is under control of an inducible promoter other than tetracycline. Exemplary inducible promoters which may control the expression of the tesB cassette include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. 
         FIG. 3A ,  FIG. 3B ,  FIG. 3C ,  FIG. 3D ,  FIG. 3E , and  FIG. 3F  depict schematics of the gene organization of exemplary bacteria of the disclosure.  FIG. 3A  and  FIG. 3B  depict the gene organization of an exemplary engineered bacterium of the invention and its induction of butyrate production under low-oxygen conditions.  FIG. 3A  depicts relatively low butyrate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by “X”) FNR (boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the butyrate biosynthesis enzymes (hcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, and buk; white boxes) is expressed.  FIG. 3B  depicts increased butyrate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate.  FIG. 3C  and  FIG. 3D  depict the gene organization of an exemplary recombinant bacterium of the invention and its derepression in the presence of nitric oxide (NO). In  FIG. 3C , in the absence of NO, the NsrR transcription factor (circle, “NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, buk) is expressed. In  FIG. 3D , in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate. 
         FIG. 3E  and  FIG. 3F  depict the gene organization of an exemplary recombinant bacterium of the invention and its induction in the presence of H2O2. In  FIG. 3E , in the absence of H2O2, the OxyR transcription factor (circle, “OxyR”) binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, buk) is expressed. In  FIG. 3F , in the presence of H2O2, the OxyR transcription factor interacts with H2O2 and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate. 
         FIG. 4A ,  FIG. 4B ,  FIG. 4C ,  FIG. 4D ,  FIG. 4E , and  FIG. 4F  depict schematics of the gene organization of exemplary bacteria of the disclosure.  FIG. 4A  and  FIG. 4B  depict the gene organization of another exemplary engineered bacterium of the invention and its induction of butyrate production under low-oxygen conditions using a different butyrate circuit from that shown in  FIG. 3A .  FIG. 3B ,  FIG. 3C ,  FIG. 3D ,  FIG. 3E , and  FIG. 3F .  FIG. 4A  depicts relatively low butyrate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by “X”) FNR (boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, pbt, and buk; white boxes) is expressed.  FIG. 4B  depicts increased butyrate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate.  FIG. 4C  and  FIG. 4D  depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of NO. In  FIG. 4C , in the absence of NO, the NsrR transcription factor (circle, “NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, pbt, buk) is expressed. In  FIG. 4D , in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate.  FIG. 4E  and  FIG. 4F  depict the gene organization of another exemplary recombinant bacterium of the invention and its induction in the presence of H 2 O 2 . In  FIG. 4E , in the absence of H 2 O 2 , the OxyR transcription factor (circle, “OxyR”) binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, pbt, buk) is expressed. In  FIG. 4F , in the presence of H 2 O 2 , the OxyR transcription factor interacts with H 2 O 2  and is then capable of inducing the ox&#39;S promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate. 
         FIG. 5A ,  FIG. 5B ,  FIG. 5C ,  FIG. 5D ,  FIG. 5E , and  FIG. 5F  depict schematics of the gene organization of exemplary bacteria of the disclosure.  FIG. 5A  and  FIG. 5B  depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions.  FIG. 5A  depicts relatively low butyrate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by “X”) FNR (boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, and tesB) is expressed.  FIG. 5B  depicts increased butyrate production under low-oxygen conditions due to FNR dimerizing (two boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate.  FIG. 5C  and  FIG. 5D  depict the gene organization of another exemplary recombinant bacterium of the invention and its derepression in the presence of NO. In  FIG. 5C , in the absence of NO, the NsrR transcription factor (“NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, tesB) is expressed. In  FIG. 5D , in the presence of NO, the NsrR transcription factor interacts with NO, and no longer binds to or represses the regulatory sequence. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate.  FIG. 5E  and  FIG. 5F  depict the gene organization of another exemplary recombinant bacterium of the invention and its induction in the presence of H 2 O 2 . In  FIG. 5E , in the absence of H 2 O 2 , the OxyR transcription factor (circle, “OxyR”) binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (t/e; thiA1, hbd, crt2, tesB) is expressed. In  FIG. 6F , in the presence of H 2 O 2 , the OxyR transcription factor interacts with H 2 O 2  and is then capable of inducing the oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by black arrows and black squiggles) and ultimately to the production of butyrate. 
         FIG. 6A  and  FIG. 6B  depict schematics of the gene organization of exemplary bacteria of the disclosure for inducible propionate production.  FIG. 6A  depicts relatively low propionate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by “X”) FNR (boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the propionate biosynthesis enzymes (pt, lcdA, lcdB, lcdC, etfA, acrB, acrC) is expressed.  FIG. 6B  depicts increased propionate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the propionate biosynthesis enzymes, which leads to the production of propionate. In other embodiments, propionate production is induced by NO or H 2 O 2  as depicted and described for the butyrate cassette(s) in the preceding  FIG. 3C-3F ,  FIG. 4C-4F ,  FIG. 5C-5F . 
         FIG. 7  depicts an exemplary propionate biosynthesis gene cassette. 
         FIG. 8A ,  FIG. 8B , and  FIG. 8C  depict schematics of the gene organization of exemplary bacteria of the disclosure for inducible propionate production.  FIG. 8A  depicts relatively low propionate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by “X”) FNR (boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the propionate biosynthesis enzymes (thrA, thrB, thrC, ilvA, aceE, aceF, lpd) is expressed.  FIG. 8B  depicts increased propionate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the propionate biosynthesis enzymes, which leads to the production of propionate.  FIG. 8C  depicts an exemplary propionate biosynthesis gene cassette. In other embodiments, propionate production is induced by NO or H 2 O 2  as depicted and described for the butyrate cassette(s) in the preceding  FIG. 3C-3F ,  FIG. 4C-4F ,  FIG. 5C-5F . 
         FIG. 9A  and  FIG. 9B  depict schematics of the gene organization of exemplary bacteria of the disclosure for inducible propionate production.  FIG. 9A  depicts relatively low propionate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by “X”) FNR (boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the propionate biosynthesis enzymes (thrA, thrB, thrC, ilvA, aceE, aceF, lpd, tesB) is expressed.  FIG. 9B  depicts increased propionate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the propionate biosynthesis enzymes, which leads to the production of propionate. In other embodiments, propionate production is induced by NO or H 2 O 2  as depicted and described for the butyrate cassette(s) in the preceding  FIG. 3C-3F ,  FIG. 4C-4F ,  FIG. 5C-5F . 
         FIG. 10A ,  FIG. 10B , and  FIG. 10C  depict schematics of the sleeping beauty pathway and the gene organization of an exemplary bacterium of the disclosure.  FIG. 10A  depicts a schematic of a genetically engineered sleeping beauty metabolic pathway from  E. coli  for propionate production. The SBM pathway is cyclical and composed of a series of biochemical conversions forming propionate as a fermentative product while regenerating the starting molecule of succinyl-CoA.  FIG. 10B  and  FIG. 10C  depict schematics of the gene organization of another exemplary engineered bacterium of the invention and its induction of propionate production under low-oxygen conditions.  FIG. 10B  depicts relatively low propionate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by “X”) FNR (boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the propionate biosynthesis enzymes (sbm, ygfD, ygfG, ygfH) is expressed.  FIG. 10C  depicts increased propionate production under low-oxygen or anaerobic conditions due to FNR dimerizing (two boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the propionate biosynthesis enzymes, which leads to the production of propionate. In other embodiments, propionate production is induced by NO or H 2 O 2  as depicted and described for the butyrate cassette(s) in the preceding  FIG. 3C-3F ,  FIG. 4C-4F ,  FIG. 5C-5F . 
         FIG. 11  depicts a bar graph showing butyrate production of butyrate producing strains of the disclosure.  FIG. 11  shows butyrate production in strains pLOGIC031 and pLOGIC046 in the presence and absence of oxygen, in which there is no significant difference in butyrate production. Enhanced butyrate production was shown in Nissle in low copy plasmid expressing pLOGIC046 which contain a deletion of the final two genes (ptb-buk) and their replacement with the endogenous  E. coli  tesB gene (a thioesterase that cleaves off the butyrate portion from butyryl CoA). Overnight cultures of cells were diluted 1:100 in Lb and grown for 1.5 hours until early log phase was reached at which point anhydrous tet was added at a final concentration of 100 ng/ml to induce plasmid expression. After 2 hours induction, cells were washed and resuspended in M9 minimal media containing 0.5% glucose at OD600=0.5. Samples were removed at indicated times and cells spun down. The supernatant was tested for butyrate production using LC-MS. 
         FIG. 12  depicts a bar graph showing butyrate production of butyrate producing strains of the disclosure.  FIG. 12  shows butyrate production in strains comprising a tet-butyrate cassette having ter substitution (pLOGIC046) or the tesB substitution (ptb-buk deletion), demonstrating that the tesB substituted strain has greater butyrate production. 
         FIG. 13  depicts a graph of butyrate production using different butyrate-producing circuits comprising a nuoB gene deletion. Strains depicted are BW25113 comprising a bcd-butyrate cassette, with or without a nuoB deletion, and BW25113 comprising a ter-butyrate cassette, with or without a nuoB deletion. Strains with deletion are labeled with nuoB. The NuoB gene deletion results in greater levels of butyrate production as compared to a wild-type parent control in butyrate producing strains. NuoB is a main protein complex involved in the oxidation of NADH during respiratory growth. In some embodiments, preventing the coupling of NADH oxidation to electron transport increases the amount of NADH being used to support butyrate production. 
         FIG. 14A ,  FIG. 14B ,  FIG. 14C , and  FIG. 14D  depict schematics and graphs showing butyrate or biomarker production of a butyrate producing circuit under the control of an FNR promoter.  FIG. 14A  depicts a schematic showing a butyrate producing circuit under the control of an FNR promoter.  FIG. 14B  depicts a bar graph of anaerobic induction of butyrate production. FNR-responsive promoters were fused to butyrate cassettes containing either the bcd or ter circuits. Transformed cells were grown in LB to early log and placed in anaerobic chamber for 4 hours to induce expression of butyrate genes. Cells were washed and resuspended in minimal media w/l 0.5% glucose and incubated microaerobically to monitor butyrate production over time. SYN-501 led to significant butyrate production under anaerobic conditions.  FIG. 14C  depicts SYN-501 in the presence and absence of glucose and oxygen in vitro. SYN-501 comprises pSC101 PydfZ-ter butyrate plasmid; SYN-500 comprises pSC101 PydfZ-bcd butyrate plasmid; SYN-506 comprises pSC101 nirB-bcd butyrate plasmid.  FIG. 14D  depict levels of mouse lipocalin 2 (left) and calprotectin (right) quantified by ELISA using the fecal samples in an in vivo model. SYN-50 reduces inflammation and/or protects gut barrier function as compared to wild type Nissle control. 
         FIG. 15  depicts a graph measuring gut-barrier function in dextran sodium sulfate (DSS)-induced mouse models of IBD. The amount of FITC dextran found in the plasma of mice administered different concentrations of DSS was measured as an indicator of gut barrier function. 
         FIG. 16  depicts serum levels of FITC-dextran analyzed by spectrophotometry. FITC-dextran is a readout for gut barrier function in the DSS-induced mouse model of IBD. 
         FIG. 17  depicts a scatter graph of butyrate concentrations in the feces of mice gavaged with either H2O, 100 mM butyrate in H20, streptomycin resistant Nissle control or SYN501 comprising a PydfZ-ter→pbt-buk butyrate plasmid. Significantly greater levels of butyrate were detected in the feces of the mice gavaged with SYN501 as compared mice gavaged with the Nissle control or those given water only. Levels are close to 2 mM and higher than the levels seen in the mice fed with H20 (+) 200 mM butyrate. 
         FIG. 18  depicts a bar graph comparing butyrate concentrations produced in vitro by the butyrate cassette plasmid strain SYN501 as compared to Clostridia butyricum MIYARISAN (a Japanese probiotic strain),  Clostridium tyrobutyricum  VPI 5392 (Type Strain), and  Clostridium butyricum  NCTC 7423 (Type Strain) under aerobic and anaerobic conditions at the indicated timepoints. The Nissle strain comprising the butyrate cassette produces butyrate levels comparable to  Clostridium  spp. in RCM media. 
         FIG. 19A  depicts a bar graph showing butyrate concentrations produced in vitro by strains comprising chromsolmally integrated butyrate copies as compared to plasmid copies. Integrated butyrate strains, SYN1001 and SYN1002 (both integrated at the agaI/rsmI locus) gave comparable butyrate production to the plasmid strain SYN501. 
         FIG. 19B  and  FIG. 19C  depict bar graphs showing the effect of the supernatants from the engineered butyrate-producing strain, SYN1001, on alkaline phosphatase activity in HT-29 cells represented in bar ( FIG. 19B ) and nonlinear fit ( FIG. 19C ) graphical formats. 
         FIG. 20A  and  FIG. 20B  depicts the construction and gene organization of an exemplary plasmids.  FIG. 20A  depicts the construction and gene organization of an exemplary plasmids comprising a gene encoding NsrR, a regulatory sequence from norB, and a butyrogenic gene cassette (pLogic031-nsrR-norB-butyrate construct).  FIG. 20B  depicts the construction and gene organization of another exemplary plasmid comprising a gene encoding NsrR, a regulatory sequence from norB, and a butyrogenic gene cassette (pLogic046-nsrR-norB-butyrogenic gene cassette). 
         FIG. 21  depicts butyrate production using SYN001+tet (control wild-type Nissle comprising no plasmid), SYN067+tet (Nissle comprising the pLOGIC031 ATC-inducible butyrate plasmid), and SYN080+tet (Nissle comprising the pLOGIC046 ATC-inducible butyrate plasmid). 
         FIG. 22  depicts butyrate production by genetically engineered Nissle comprising the pLogic031-nsrR-norB-butyrate construct (SYN133) or the pLogic046-nsrR-norB-butyrate construct (SYN145), which produce more butyrate as compared to wild-type Nissle (SYN001). 
         FIG. 23  depicts the construction and gene organization of an exemplary plasmid comprising an oxyS promoter and butyrogenic gene cassette (pLogic031-oxyS-butyrogenic gene cassette). 
         FIG. 24  depicts the construction and gene organization of another exemplary plasmid comprising an oxyS promoter and butyrogenic gene cassette (pLogic046-oxyS-butyrogenic gene cassette). 
         FIG. 25  depicts a schematic illustrating a strategy for increasing butyrate and acetate production in engineered bacteria. Aerobic metabolism through the citric acid cycle (TCA cycle) (crossed out) is inactive in the anaerobic environment of the colon.  E. coli  makes high levels of acetate as an end production of fermentation. To improve acetate production, while still maintaining highlevels of butyrate production, targeted deletion can be introduced to prevent the production of unnecessary metabolic fermentative byproducts (thereby simultaneously increasing butyrate and acetate production). Non-limiting examples of competing routes (shown in in rounded boxes) are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions of interest therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
         FIG. 26A  and  FIG. 26B  depict line graphs showing acetate production over a 6 hour time course post-induction in 0.5% glucose MOPS (pH6.8) ( FIG. 26A ) and in 0.5% glucuronic acid MOPS (pH6.3) ( FIG. 26B ). Acetate production of an engineered  E. coli  Nissle strain comprising a deletion in the endenous ldh gene (SYN2001) was compared with streptomycin resistant Nissle (SYN94). 
         FIG. 26C  and  FIG. 26D  depict bar graphs showing acetate and butyrate production in 0.5% glucose MOPS (pH6.8) ( FIG. 26C ) and acetate and butyrate production in 0.5% glucuronic acid MOPS (pH6.3) ( FIG. 26D ). Deletions in endogenous adhE (Aldehyde-alcohol dehydrogenase) and ldh (lactate dehydrogenase) were introduced into Nissle strains with either integrated FNRS ter-tesB or FNRS-ter-pbt-buk butyrate cassettes. SYN2006 comprises a FNRS ter-tesB cassette integrated at the HA1/2 locus and a deletion in the endogenous adhE gene. SYN2007 comprises a FNRS ter-tesB cassette integrated at the HA1/2 locus and a deletion in the endogenous ldhA gene. SYN2008 comprises a FNRS-ter-pbt-buk butyrate cassette and a deletion in the endogenous adhE gene. SYN2003 comprises a FNRS-ter-pbt-buk butyrate cassette and a deletion in the endogenous ldhA gene. 
         FIG. 26E  depicts a bar graph showing acetate and butyrate production at the indicated time points post induction in 0.5% glucose MOPS (pH6.8). A strain comprising a FNRS-ter-tesB butyrate cassette integrated at the HA1/2 locus of the chromosome (SYN1004) was compared with a strain comprising the same integrated cassette and additionally a deletion in the endogenous frd gene (SYN2005). 
         FIG. 26F  depicts a bar graph showing acetate and butyrate production at 18 hours in 0.5% glucose MOPS (pH6.8), comparing three strains engineered to produce short chain fatty acids. SYN2001 comprises a deletion in the endenous ldh gene; SYN2002 comprises a FNRS-ter-tesB butyrate cassette integrated at the HA1/2 locus and deletions in the endogenous adhE and pta genes. SYN2003 comprises FNRS-ter-pbt-buk butyrate cassette integrated at the HA1/2 locus and a deletion in the endogenous ldhA gene. 
         FIG. 26G  and  FIG. 26H  depict line graphs showing the effect of supernatants from the engineered acetate-producing strain, SYN2001, on LPS-induced IFNγ secretion in primary human PBMC cells from donor 1 (D1) ( FIG. 26G ) and donor 2 (D2) ( FIG. 26H ). 
         FIG. 27  depicts a schematic of an exemplary propionate biosynthesis gene cassette. 
         FIG. 28  depicts a schematic of a construct comprising the sleeping beauty mutase operon from  E. coli  under the control of a heterologous FnrS promoter. 
         FIG. 29  depicts a bar graph of proprionate concentrations produced in vitro by the wild type  E. coli  BW25113 strain and a BW25113 strain which comprises the endogenous SBM operon under the control of the FnrS promoter, as depicted in the schematic in  FIG. 28 . 
         FIG. 30A ,  FIG. 30B , and  FIG. 30C  depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, which are secreted using components of the flagellar type III secretion system. A therapeutic polypeptide of interest, such as, GLP-2, IL-10, and IL-22, is assembled behind a fliC-5′UTR, and is driven by the native fliC and/or fliD promoter ( FIG. 30A  and  FIG. 30B ) or a tet-inducible promoter ( FIG. 30C ). In alternate embodiments, an inducible promoter such as oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by IBD specific molecules or promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose can be used. The therapeutic polypeptide of interest is either expressed from a plasmid (e.g., a medium copy plasmid) or integrated into fliC loci (thereby deleting all or a portion of fliC and/or fliD). Optionally, an N terminal part of FliC is included in the construct, as shown in  FIG. 30B  and  FIG. 30D . 
         FIG. 31A  and  FIG. 31B  depict schematics of the gene organization of exemplary circuits of the disclosure for the expression of therapeutic polypeptides, which are secreted via a diffusible outer membrane (DOM) system. The therapeutic polypeptide of interest is fused to a prototypical N-terminal Sec-dependent secretion signal or Tat-dependent secretion signal, which is is cleaved upon secretion into the periplasmic space. Exemplary secretion tags include sec-dependent PhoA, OmpF, OmpA, cvaC, and Tat-dependent tags (TorA, FdnG, DmsA). In certain embodiments, the genetically engineered bacteria comprise deletions in one or more of lpp, pal, tolA, and/or nlpI. Optionally, periplasmic proteases are also deleted, including, but not limited to, degP and ompT, e.g., to increase stability of the polypeptide in the periplasm. A FRT-KanR-FRT cassette is used for downstream integration. Expression is driven by a tet promoter ( FIG. 31A ) or an inducible promoter, such as oxygen level-dependent promoters (e.g., FNR-inducible promoter,  FIG. 31B ), promoters induced by IBD specific molecules or promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose. 
         FIG. 32A ,  FIG. 32B ,  FIG. 32C ,  FIG. 32D , and  FIG. 32E  depict schematics of non-limiting examples of constructs for the expression of GLP2 for bacterial secretion.  FIG. 32A  depicts a schematic of a human GLP2 construct inserted into the FliC locus, under the control of the native FliC promoter.  FIG. 32B  depicts a schematic of a human GLP2 construct, including the N terminal 20 amino acids of FliC, inserted into the FliC locus under the control of the native FliC promoter.  FIG. 32C  depicts a schematic of a human GLP2 construct, including the N-terminal 20 amino acids of FliC, inserted into the FliC locus under the control of a tet inducible promoter.  FIG. 32D  depicts a schematic of a human GLP2 construct with a N terminal OmpF secretion tag (sec-dependent secretion system) under the control of a tet inducible promoter.  FIG. 32E  depicts a schematic of a human GLP2 construct with a N terminal TorA secretion tag (tat secretion system) under the control of a tet inducible promoter. 
         FIG. 33A  and  FIG. 33B  depict line graphs of ELISA results.  FIG. 33A  depicts a line graph, showing an phopho-STAT3 (Tyr705) ELISA conducted on extracts from serum-starved Colo205 cells treated with supernatants from engineered bacteria comprising a PAL deletion and an integrated construct encoding hIL-22 with a phoA secretion tag. The data demonstrate that hIL-22 secreted from the engineered bacteria is functionally active.  FIG. 33B  depicts a line graph, showing an phopho-STAT3 (Tyr705) ELISA showing a antibody completion assay. Extracts from Colo205 cells were treated with the bacterial supernatants from the IL-22 overexpressing strain preincubated with increasing concentrations of neutralizing anti-IL-22 antibody. The data demonstrated that phospho-Stat3 signal induced by the secreted hIL-22 is competed away by the hIL-22 antibody MAB7821. 
         FIG. 33C  depicts a line graph showing SYN3001 (PhoA-IL-22 in pal mutant chassi), but not SYN3000 (pal mutant chassi) supernatant induces STAT3 activation. 
         FIG. 33D  depicts a line graph showing that anti IL-22 neutralizing antibody inhibits SYN3001-induced STAT3 activation (n=3). 
         FIG. 33E  depicts a Western blot analysis of bacterial supernatants from strain SYN2980 and SYN2982, using IL-10 antibody (IL-10 (D13A11) XP® Rabbit mAb #12163, Cell Signaling Technology). The secreted polypeptide has the same molecular weight as the standards, indicating that the signal sequence is cleaved from the native peptide. 
         FIG. 34  depicts a schematic of tryptophan metabolism along the kynurenine and the serotonin arms in humans. The abbreviations for the enzymes are as follows: 3-HAO: 3-hydroxyl-anthranilate 3,4-dioxidase: AAAD: aromatic-amino acid decarboxylase; ACMSD, alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase; HIOMT, hydroxyl-O-methyltransferase; IDO, indoleamine 2,3-dioxygenase; KAT, kynurenine amino transferases I-III; KMO: kynurenine 3-monooxygenase; KYNU, kynureninase; NAT, N-acetyltransferase; TDO, tryptophan 2,3-dioxygenase; TPH, tryptophan hydroxylase; QPRT, quinolinic acid phosphoribosyl transferase. 
         FIG. 35  depicts a schematic of bacterial tryptophan catabolism machinery, which is genetically and functionally homologous to IDO1 enzymatic activity, as described in Vujkovic-Cvijin et al., Dysbiosis of the gut microbiota is associated with HIV disease progression and tryptophan catabolism; Sci Transl Med. 2013 Jul. 10; 5(193): 193ra91, the contents of which is herein incorporated by reference in its entirety. In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes depicted in  FIG. 35 . In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in  FIG. 35 , including but not limited to, kynurenine, indole-3-aldehyde, indole-3-acetic acid, and/or indole-3 acetaldehyde. 
         FIG. 36A  and  FIG. 36B  depict schematics of indole metabolite mode of action ( FIG. 36A ) and indole biosynthesis ( FIG. 36B ).  FIG. 36A  depicts a schematic of molecular mechanisms of action of indole and its metabolites on host physiology and disease. Tryptophan catabolized by bacteria to yield indole and other indole metabolites, e.g., Indole-3-propionate (IPA) and Indole-3-aldehyde (13A), in the gut lumen. IPA acts on intestinal cells via pregnane X receptors (PXR) to maintain mucosal homeostasis and barrier function. 13A acts on the aryl hydrocarbon receptor (AhR) found on intestinal immune cells and promotes IL-22 production. Activation of AhR plays a crucial role in gut immunity, such as in maintaining the epithelial barrier function and promoting immune tolerance to promote microbial commensalism while protecting against pathogenic infections. Indole has a number of roles, such as a signaling molecule to intestinal L cells to produce glucagon-like protein 1 (GLP-1) or as a ligand for AhR (Zhang et al. Genome Med. 2016; 8: 46).  FIG. 36B  depicts a schematic of the trypophan catabolic pathway/indole biosynthesis pathways. Host and microbiota metabolites with AhR agonistic activity are in in diamond and circled, respectively (see, e.g., Lamas et al., CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands; Nature Medicine 22, 598-605 (2016). In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes which catalyze the reactions shown in  FIGS. 36A and 36B . In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in  FIGS. 36A and 36B , including but not limited to, kynurenine, indole-3-aldehyde, indole-3-acetic acid, and/or indole-3 acetaldehyde. 
         FIG. 37A  and  FIG. 37B  depict diagrams of bacterial tryptophan metabolism pathways.  FIG. 37A  depicts a schematic of the bacterial tryptophan metabolism, as described, e.g., in Enzymes are numbered as follows 1) Trp 2,3 dioxygenase (EC 1.13.11.11); 2) kynurenine formidase (EC 3.5.1.49); 3) kynureninase (EC 3.7.1.3); 4) tryptophanase (EC 4.1.99.1); 5) Trp aminotransferase (EC 2.6.1.27); 6) indole lactate dehydrogenase (EC1.1.1.110); 7) Trp decarboxylase (EC 4.1.1.28); 8) tryptamine oxidase (EC 1.4.3.4); 9) Trp side chain oxidase (EC 4.1.1.43); 10) indole acetaldehyde dehydrogenase (EC 1.2.1.3); 11) indole acetic acid oxidase; 13) Trp 2-monooxygenase (EC 1.13.12.3); and 14) indole acetamide hydrolase (EC 3.5.1.0). The dotted lines (-) indicate a spontaneous reaction.  FIG. 37B  Depicts a schematic of tryptophan derived pathways. Known AHR agonists are with asterisk. Abbreviations are as follows. Trp: Tryptophan; TrA: Tryptamine; IAAld: Indole-3-acetaldehyde: IAA: Indole-3-acetic acid; FICZ: 6-formylindolo(3,2-b)carbazole; IPyA: Indole-3-pyruvic acid; IAM: Indole-3-acetamine; IAOx: Indole-3-acetaldoxime; IAN: Indole-3-acetonitrile; N-formyl Kyn: N-formylkynurenine; Kyn:Kynurenine; KynA: Kynurenic acid; 13C: Indole-3-carbinol; IAld: Indole-3-aldehyde; DIM: 3,3′-Diindolylmethane; ICZ: Indolo(3,2-b)carbazole. Enzymes are numbered as follows: I. EC 1.13.11.11 (Tdo2, Bna2), EC 1.13.11.11 (ldo1); 2. EC 4.1.1.28 (Tdc); 3. EC 1.4.3.22, EC 1.4.3.4 (TynA); 4. EC 1.2.1.3 (lad1), EC 1.2.3.7 (Aao1); 5. EC 3.5.1.9 (Afmid Bna3); 6. EC 2.6.1.7 (Cclb1, Cclb2, Aadat, Got2); 7. EC 1.4.99.1 (TnaA); 8. EC 1.14.13.125 (CYP79B2, CYP79B3); 9. EC 1.4.3.2 (StaO), EC 2.6.1.27 (Aro9, aspC), EC 2.6.1.99 (Taa1), EC 1.4.1.19 (TrpDH); 10. EC 1.13.12.3 (laaM); 11. EC 4.1.1.74 (lpdC); 12. EC 1.14.13.168 (Yuc2); 13. EC 3.5.1.4 (laaH); 14. EC 3.5.5.1. (Nit1); 15. EC 4.2.1.84 (Nit1); 16. EC 4.99.1.6 (CYP71A13); 17. EC 3.2.1.147 (Pen2). In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes comprising one or more of the bacterial tryptophan metabolism enzymes depicted in  FIGS. 37A and 37B . In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in  FIGS. 37A and 37B . In certain embodiments, the one or more cassettes are on a plasmid; in other embodiments, the cassettes are integrated into the genome. In certain embodiments the one or more cassettes are under the control of inducible promoters which are induced under low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. 
         FIG. 38  depicts a schematic of the  E. coli  tryptophan synthesis pathway. In  Escherichia coli , tryptophan is biosynthesized from chorismate, the principal common precursor of the aromatic amino acids tryptophan, tyrosine and phenylalanine, as well as the essential compounds tetrahydrofolate, ubiquinone-8, menaquinone-8 and enterobactin (enterochelin), as shown in the superpathway of chorismate metabolism. Five genes encode five enzymes that catalyze tryptophan biosynthesis from chorismate. The five genes trpE trpD trpC trpB trpA form a single transcription unit, the trp operon. A weak internal promoter also exists within the trpD structural gene that provides low, constitutive levels of mRNA. 
         FIG. 39  depicts one embodiment of the disclosure in which the  E. coli  TRP synthesis enzymes are expressed from a construct under the control of a tetracycline inducible system. 
         FIG. 40A ,  FIG. 40B ,  FIG. 40C , and  FIG. 40D  depicts schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria comprise circuits for the production of tryptophan. Any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter. In certain embodiments the one or more cassettes are under the control of constitutive promoters. Exemplary inducible promoters which may control the expression of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. The bacteria may also include an auxotrophy, e.g., deletion of thyA (Δ thyA: thymidine dependence).  FIG. 40A  shows a schematic depicting an exemplary Tryptophan circuit. Tryptophan is produced from its precursor, chorismate, through expression of the trpE, trpG-D (also referred to as trpD), trpC-F (also referred to as trpC), trpB and trpA genes. Optional knockout of the tryptophan repressor trpR is also depicted. Optional production of chorismate through expression of aroG/F/H and aroB, aroD, aroE, aroK and aroC genes is also shown. The bacteria may optionally also include gene sequence(s) for the expression of YddG, which functions as a tryptophan exporter. The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in  FIG. 40B , and/or  FIG. 40C , and/or  FIG. 40D .  FIG. 40B  depicts a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production. Optionally, bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in  FIG. 40A  and/or described in the description of  FIG. 40A . The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in  FIG. 40C , and/or  FIG. 40D . Optionally, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced.  FIG. 40C  depicts a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production. The strain further comprises either a wild type or a feedback resistant SerA gene.  Escherichia coli  serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. This step is an oxidation of 3PG to 3-phosphohydroxypyruvate (3PHP) with the concomitant reduction of NADI to NADH.  E. coli  uses one serine for each tryptophan produced. As a result, by expressing serA, tryptophan production is improved. Optionally, bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in  FIG. 40A  and/or described in the description of  FIG. 40A . The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in  FIG. 40B , and/or  FIG. 40D . Optionally, Trp Repressor and/or the tnaA gene are deleted to further increase levels of tryptophan produced. The bacteria may optionally also include gene sequence(s) for the expression of YddG, which functions as a tryptophan exporter.  FIG. 40D  depicts a non-limiting example of a tryptophan producing strain, in which tryptophan is produced from the chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. AroG and TrpE are replaced with feedback resistant versions to improve tryptophan production. The strain further optionally comprises either a wild type or a feedback resistant SerA gene. Optionally, bacteria may comprise any of the transporters and/or additional tryptophan circuits depicted in  FIG. 40A  and/or described in the description of  FIG. 40A . The bacteria may optionally also comprise one or more gene sequence(s) depicted or described in  FIG. 40B , and/or  FIG. 40C . Optionally, Trp Repressor and/or the tnaA gene are deleted to further increase levels of tryptophan produced. The bacteria may optionally also include gene sequence(s) for the expression of YddG, which functions as a tryptophan exporter. Optionally, the bacteria may also comprise a deletion in PheA, which prevents conversion of chorismate into phenylalanine and thereby promotes the production of anthranilate and tryptophan. 
         FIG. 41A ,  FIG. 41B ,  FIG. 41D ,  FIG. 41D ,  FIG. 41E ,  FIG. 41F ,  FIG. 41G , and  FIG. 41H  depict schematics of non-limiting examples of embodiments of the disclosure. In all embodiments, optionally gene(s) which encode exporters may also be included.  FIG. 41A  depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce tryptamine from tryptophan. In certain embodiments the one or more cassettes are under the control of inducible promoters. In certain embodiments the one or more cassettes are under the control of constitutive promoters. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in  FIG. 40A  and/or and/or  FIG. 40B , and/or  FIG. 40C , and/or  FIG. 40D  for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit for Tryptophan decarboxylase, e.g., from  Catharanthus roseus , which converts tryptophan to tryptamine, e.g., under the control of an inducible promoter e.g., an FNR promoter.  FIG. 41B  depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-acetaldehyde and FICZ from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in  FIG. 40A  and/or  FIG. 40B , and/or  FIG. 40C , and/or  FIG. 40D  for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit for aro9 (L-tryptophan aminotransferase, e.g., from  S. cerevisae ) or aspC (aspartate aminotransferase, e.g., from  E. coli , or taa1 (L-tryptophan-pyruvate aminotransferase, e.g., from  Arabidopsis thaliana ) or staO (L-tryptophan oxidase, e.g., from  Streptomyces  sp. TP-A0274) or trpDH (Tryptophan dehydrogenase, e.g., from  Nostoc punctiforme  NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from  Enterobacter cloacae ) which together produce indole-3-acetaldehyde and FICZ from tryptophan, e.g., under the control of an inducible promoter e.g., an FNR promoter.  FIG. 41C  depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-acetaldehyde and FICZ from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in  FIG. 40A  and/or and/or  FIG. 40B , and/or  FIG. 40C , and/or  FIG. 40D  for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising tdc (Tryptophan decarboxylase, e.g., from  Catharanthus roseus  and/or  Clostridium sporogenes ), and tynA (Monoamine oxidase, e.g., from  E. coli ), which converts tryptophan to indole-3-acetaldehyde and FICZ, e.g., under the control of an inducible promoter e.g., an FNR promoter.  FIG. 41D  depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-acetonitrile from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in  FIG. 40A  and/or and/or  FIG. 40B , and/or  FIG. 40C , and/or  FIG. 40D  for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit for cyp79B2, (tryptophan N-monooxygenase, e.g., from  Arabidopsis thaliana ) or cyp79B3 (tryptophan N-monooxygenase, e.g., from  Arabidopsis thaliana ), which together convert tryptophan to indole-3-acetonitrile, e.g., under the control of an inducible promoter e.g., an FNR promoter.  FIG. 41E  depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce kynurenine from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in  FIG. 40A  and/or and/or  FIG. 40B , and/or  FIG. 40C , and/or  FIG. 40D  for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising IDO1 (indoleamine 2,3-dioxygenase, e.g., from  Homo sapiens  or TDO2 (tryptophan 2,3-dioxygenase, e.g., from  Homo sapiens ) or BNA2 (indoleamine 2,3-dioxygenase, e.g., from  S. cerevisiae ) and Afmid: Kynurenine formamidase, e.g., from mouse) or BNA3 (kynurenine-oxoglutarate transaminase, e.g., from  S. cerevisae ) which together convert tryptophan to kynurenine, e.g., under the control of an inducible promoter e.g., an FNR promoter.  FIG. 41F  depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce kynureninic acid from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in  FIG. 40A  and/or and/or  FIG. 40B , and/or  FIG. 40C , and/or  FIG. 40D  for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising IDO1 (indoleamine 2,3-dioxygenase, e.g., from  Homo sapiens  or TDO2 (tryptophan 2,3-dioxygenase, e.g., from  Homo sapiens ) or BNA2 (indoleamine 2,3-dioxygenase, e.g., from  S. cerevisiae ) and Afmid: Kynurenine formamidase, e.g., from mouse) or BNA3 (kynurenine-oxoglutarate transaminase, e.g., from  S. cerevisae ) and GOT2 (Aspartate aminotransferase, mitochondrial, e.g., from  Homo sapiens  or AADAT (Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial, e.g., from  Homo sapiens ), or CCLB1 (Kynurenine-oxoglutarate transaminase 1, e.g., from  Homo sapiens ) or CCLB2 (kynurenine-oxoglutarate transaminase 3, e.g., from  Homo sapiens , which together produce kynureninic acid from tryptophan, under the control of an inducible promoter, e.g., an FNR promoter.  FIG. 41G  depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole from tryptophan. The bacteria may comprise any of the transporters and/or tryptophan circuits depicted and described in  FIG. 40A  and/or and/or  FIG. 40B , and/or  FIG. 40C , and/or  FIG. 40D  for the production of tryptophan. Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit for tnaA (tryptophanase, e.g., from  E. coli ), which converts tryptophan to indole, e.g., under the control of an inducible promoter e.g., an FNR promoter.  FIG. 41H  depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-carbinol, indole-3-aldehyde, 3,3′ diindolylmethane (DIM), indolo(3,2-b) carbazole (ICZ) from indole glucosinolate taken up through the diet. The genetically engineered bacteria comprise a circuit comprising pne2 (myrosinase, e.g., from  Arabidopsis thaliana ) under the control of an inducible promoter, e.g. an FNR promoter. The engineered bacterium shown in any of  FIG. 41A ,  FIG. 41B ,  FIG. 41D ,  FIG. 41D ,  FIG. 41E ,  FIG. 41F ,  FIG. 41G  and  FIG. 41H  may also have an auxotrophy, e.g., in one example, the thyA gene can be been mutated in the  E. coli  Nissle genome, so thymidine must be supplied in the culture medium to support growth. 
         FIG. 42A ,  FIG. 42B ,  FIG. 42C ,  FIG. 42D , and  FIG. 42E  depict schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria convert tryptophan into indole-3-acetic acid. In certain embodiments, the one or more cassettes are under the control of inducible promoters. In certain embodiments, the one or more cassettes are under the control of constitutive promoters. In  FIG. 42A , the optional circuits for tryptophan production are as depicted and described in  FIG. 40A . The strain optionally comprises additional circuits as depicted and/or described in  FIG. 40B  and/or  FIG. 40C  and/or  FIG. 40D . Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising aro9 (L-tryptophan aminotransferase, e.g., from  S. cerevisae ) or aspC (aspartate aminotransferase, e.g., from  E. coli , or taa1 (L-tryptophan-pyruvate aminotransferase, e.g., from  Arabidopsis thaliana ) or staO (L-tryptophan oxidase, e.g., from  Streptomyces  sp. TP-A0274) or trpDH (Tryptophan dehydrogenase, e.g., from  Nostoc punctiforme  NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from  Enterobacter cloacae ) and iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from  Ustilago maydis ) or AAO1 (Indole-3-acetaldehyde oxidase, e.g., from  Arabidopsis thaliana ) which together produce indole-3-acetic acid from tryptophan, e.g., under the control of an inducible promoter e.g., an FNR promoter. In  FIG. 42B  the optional circuits for tryptophan production are as depicted and described in  FIG. 40A . The strain optionally comprises additional circuits as depicted and/or described in  FIG. 40B  and/or  FIG. 40C  and/or  FIG. 40D . Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising tdc (Tryptophan decarboxylase, e.g., from  Catharanthus roseus  and/or  Clostridium sporogenes ) ot tynA (Monoamine oxidase, e.g., from  E. coli ) and or iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from  Ustilago maydis ) or AAO1 (Indole-3-acetaldehyde oxidase, e.g., from  Arabidopsis thaliana ), e.g., under the control of an inducible promoter e.g., an FNR promoter. In  FIG. 42C  the optional circuits for tryptophan production are as depicted and described in  FIG. 40A . The strain optionally comprises additional circuits as depicted and/or described in  FIG. 40B  and/or  FIG. 40C  and/or  FIG. 40D . Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising aro9 (L-tryptophan aminotransferase, e.g., from  S. cerevisae ) or aspC (aspartate aminotransferase, e.g., from  E. coli , or taa1 (L-tryptophan-pyruvate aminotransferase, e.g., from  Arabidopsis thaliana ) or staO (L-tryptophan oxidase, e.g., from  Streptomyces  sp. TP-A0274) or trpDH (Tryptophan dehydrogenase, e.g., from  Nostoc punctiforme  NIES-2108) and yuc2 (indole-3-pyruvate monoxygenase, e.g., from  Arabidopsis thaliana ) e.g., under the control of an inducible promoter e.g., an FNR promoter. In  FIG. 42D  the optional circuits for tryptophan production are as depicted and described in  FIG. 40A . The strain optionally comprises additional circuits as depicted and/or described in  FIG. 40B  and/or  FIG. 40C  and/or  FIG. 40D . Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising laaM (Tryptophan 2-monooxygenase e.g., from  Pseudomonas savastanoi ) and iaaH (Indoleacetamide hydrolase, e.g., from  Pseudomonas savastanoi ), e.g., under the control of an inducible promoter e.g., an FNR promoter. In  FIG. 42E  the optional circuits for tryptophan production are as depicted and described in  FIG. 40A . The strain optionally comprises additional circuits as depicted and/or described in  FIG. 40B  and/or  FIG. 40C  and/or  FIG. 40D . Alternatively, optionally, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising cyp79B2 (tryptophan N-monooxygenase, e.g., from  Arabidopsis thaliana ) or cyp79B3 (tryptophan N-monooxygenase, e.g., from  Arabidopsis thaliana  and cyp71a13 (indoleacetaldoxime dehydratase, e.g., from  Arabidopsis thaliana ) and nit1 (Nitrilase, e.g., from  Arabidopsis thaliana ) and iaaH (Indoleacetamide hydrolase, e.g., from  Pseudomonas savastanoi ), e.g., under the control of an inducible promoter e.g., an FNR promoter. the engineered bacterium shown in any of  FIG. 42A ,  FIG. 42B ,  FIG. 42C ,  FIG. 42D , and  FIG. 42E  may also have an auxotrophy, e.g., in one example, the thyA gene can be been mutated in the  E. coli  Nissle genome, so thymidine must be supplied in the culture medium to support growth. 
       In  FIG. 42F  the optional circuits for tryptophan production are as depicted and described in  FIG. 40A . The strain optionally comprises additional circuits as depicted and/or described in  FIG. 40B  and/or  FIG. 40C  and/or  FIG. 40D . Alternatively, optionally, tryptophan can be imported through a transporter. Additionally, the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from  Nostoc punctiforme  NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from  Enterobacter cloacae ) which together produce indole-3-acetaldehyde and FICZ though an (indol-3yl)pyruvate intermediate, and iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from  Ustilago maydis ), which converts indole-3-acetaldehyde into indole-3-acetate. 
         FIG. 43A ,  FIG. 43B , and  FIG. 43C  depict schematics of exemplary embodiments of the disclosure, in which the genetically engineered bacteria comprise circuits for the production of tryptophan, tryptamine, indole acetic acid, and indole propionic acid. Any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter. In certain embodiments, the one or more cassettes are under the control of constitutive promoters. Exemplary inducible promoters which may control the expression of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. The bacteria may also include an auxotrophy, e.g., deletion of thyA (A thyA; thymidine dependence).  FIG. 43A  a depicts non-limiting example of a tryptamine producing strain. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in  FIG. 40A  and/or  FIG. 40B  and/or  FIG. 40C  and/or  FIG. 40D . Additionally, the strain comprises tdc (tryptophan decarboxylase, e.g., from  Catharanthus roseus  and/or  Clostridium sporogenes ), which converts tryptophan into tryptamine.  FIG. 43B  depicts a non-limiting example of an indole-3-acetate producing strain. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in  FIG. 40A  and/or  FIG. 40B  and/or  FIG. 40C  and/or  FIG. 40D . Additionally, the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from  Nostoc punctiforme  NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from  Enterobacter cloacae ) which together produce indole-3-acetaldehyde and FICZ though an (indol-3yl)pyruvate intermediate, and iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from  Ustilago maydis ), which converts indole-3-acetaldehyde into indole-3-acetate.  FIG. 43C  depicts a non-limiting example of an indole-3-propionate-producing strain. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in  FIG. 40A  and/or  FIG. 40B  and/or  FIG. 40C  and/or  FIG. 40D . Additionally, the strain comprises a circuit as described in  FIG. 48 , comprising trpDH (Tryptophan dehydrogenase, e.g., from  Nostoc punctiforme  NIES-2108, which produces (indol-3yl)pyruvate from tryptophan), fldA (indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from  Clostridium sporogenes , which converts converts indole-3-lactate and indol-3-propionyl-CoA to indole-3-propionic acid and indole-3-lactate-CoA), fldB and fldC (indole-3-lactate dehydratase e.g., from  Clostridium sporogenes , which converts indole-3-lactate-CoA to indole-3-acrylyl-CoA) fldD and/or AcuI: (indole-3-acrylyl-CoA reductase, e.g., from  Clostridium sporogenes  and/or acrylyl-CoA reductase, e.g., from  Rhodobacter sphaeroides , which convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA). The circuits further comprise fldH1 and/or fldH2 (indole-3-lactate dehydrogenase 1 and/or 2, e.g., from  Clostridium sporogenes ), which converts (indol-3-yl)pyruvate into indole-3-lactate). 
         FIG. 44A  and  FIG. 44B  depict schematics showing exemplary engineering strategies which can be employed for tryptophan production.  FIG. 44A  depicts a schematic showing intermediates in tryptophan biosynthesis and the gene products catalyzing the production of these intermediates. Phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) are used to generate 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP). DHAP is catabolized to chorismate and then anthranilate, which is converted to tryptophan (Trp) by the tryptophan operon. Alternatively, chorismate can be used in the synthesis of tyrosine (Tyr) and/or phenylalanine (Phe). In the serine biosynthesis pathway, D-3-phosphoglycerate is converted to serine, which can also be a source for tryptophan biosynthesis. AroG, AroF, AroH: DAHP synthase catalyzes an aldol reaction between phosphoenolpyruvate and D-erythrose 4-phosphate to generate 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP). There are three isozymes of DAHP synthase, each specifically feedback regulated by tyrosine (AroF), phenylalanine (AroG) or tryptophan (AroH). AroB: Dehydroquinate synthase (DHQ synthase) is involved in the second step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. DHQ synthase catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) to dehydroquinate (DHQ). AroD: 3-Dehydroquinate dehydratase (DHQ dehydratase) is involved in the 3rd step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. DHQ dehydratase catalyzes the conversion of DHQ to 3-dehydroshikimate and introduces the first double bond of the aromatic ring. AroE, YdiB:  E. coli  expresses two shikimate dehydrogenase paralogs, AroE and YdiB. Shikimate dehydrogenase is involved in the 4th step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. This enzyme converts 3-dehydroshikimate to shikimate by catalyzing the NADPH linked reduction of 3-dehydro-shikimate. AroL/AroK: Shikimate kinase is involved in the fifth step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. Shikimate kinase catalyzes the formation of shikimate 3-phosphate from shikimate and ATP. There are two shikimate kinase enzymes, 1 (AroK) and II (AroL). AroA: 3-Phosphoshikimate-1-carboxyvinyltransferase (EPSP synthase) is involved in the 6th step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. EPSP synthase catalyzes the transfer of the enolpyruvoyl moiety from phosphoenolpyruvate to the hydroxyl group of carbon 5 of shikimate 3-phosphate with the elimination of phosphate to produce 5-enolpyruvoyl shikimate 3-phosphate (EPSP). AroC: Chorismate synthase (AroC) is involved in the 7th and last step of the chorismate pathway, which leads to the biosynthesis of aromatic amino acids. This enzyme catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate into chorismate, which is the branch point compound that serves as the starting substrate for the three terminal pathways of aromatic amino acid biosynthesis. This reaction introduces a second double bond into the aromatic ring system. TrpEDCAB ( E. coli  trp operon): TrpE (anthranilate synthase) converts chorismate and L-glutamine into anthranilate, pyruvate and L-glutamate. Anthranilate phosphoribosyl transferase (TrpD) catalyzes the second step in the pathway of tryptophan biosynthesis. TrpD catalyzes a phosphoribosyltransferase reaction that generates N-(5′-phosphoribosyl)-anthranilate. The phosphoribosyl transferase and anthranilate synthase contributing portions of TrpD are present in different portions of the protein. Bifunctional phosphoribosylanthranilate isomerase/indole-3-glycerol phosphate synthase (TrpC) carries out the third and fourth steps in the tryptophan biosynthesis pathway. The phosphoribosylanthranilate isomerase activity of TrpC catalyzes the Amadori rearrangement of its substrate into carboxyphenylaminodeoxyribulose phosphate. The indole-glycerol phosphate synthase activity of TrpC catalyzes the ring closure of this product to yield indole-3-glycerol phosphate. The TrpA polypeptide (TSase α) functions as the a subunit of the tetrameric (α2-β2) tryptophan synthase complex. The TrpB polypeptide functions as the β subunit of the complex, which catalyzes the synthesis of L-tryptophan from indole and L-serine, also termed the β reaction. TnaA: Tryptophanase or tryptophan indole-lyase (TnaA) is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the cleavage of L-tryptophan to indole, pyruvate and NH4+. PheA: Bifunctional chorismate mutase/prephenate dehydratase (PheA) carries out the shared first step in the parallel biosynthetic pathways for the aromatic amino acids tyrosine and phenylalanine, as well as the second step in phenylalanine biosynthesis. TyrA: Bifunctional chorismate mutase/prephenate dehydrogenase (TyrA) carries out the shared first step in the parallel biosynthetic pathways for the aromatic amino acids tyrosine and phenylalanine, as well as the second step in tyrosine biosynthesis. TyrB, ilvE, AspC: Tyrosine aminotransferase (TyrB), also known as aromatic-amino acid aminotransferase, is a broad-specificity enzyme that catalyzes the final step in tyrosine, leucine, and phenylalanine biosynthesis. TyrB catalyzes the transamination of 2-ketoisocaproate, p-hydroxyphenylpyruvate, and phenylpyruvate to yield leucine, tyrosine, and phenylalanine, respectively. TyrB overlaps with the catalytic activities of branched-chain amino-acid aminotransferase (IlvE), which also produces leucine, and aspartate aminotransferase, PLP-dependent (AspC), which also produces phenylalanine. SerA: D-3-phosphoglycerate dehydrogenase catalyzes the first committed step in the biosynthesis of L-serine. SerC: The serC-encoded enzyme, phosphoserine/phosphohydroxythreonine aminotransferase, functions in the biosythesis of both serine and pyridoxine, by using different substrates. Pyridoxal 5′-phosphate is a cofactor for both enzyme activities. SerB: Phosphoserine phosphatase catalyzes the last step in serine biosynthesis. Steps which are negatively regulated by the Trp Repressor (2), Tyr Repressor (1), or tyrosine (3), phenylalanine (4), or tryptophan (4) or positively regulated by trptophan (6) are indicated.  FIG. 44B  depicts a schematic showing exemplary engineering strategies which can improve tryptophan production. Each of these exemplary strategies can be used alone or two or more strategies can be combined to increase tryptophan production. Intervention points are in bold, italics and underlined. In one embodiment of the disclosure, bacteria are engineered to express a feedback resistant from of AroG (AroGfbr). In one embodiment, bacteria are engineered to express AroL. In one embodiment, bacteria are engineered to comprise one or more copies of a feedback resistant form of TrpE (TrpEfbr). In one embodiment, bacteria are engineered to comprise one or more additional copies of the Trp operon, e.g., TrpE, e.g. TrpEfbr, and/or TrpD, and/or TrpC, and/or TrpA, and/or TrpB. In one embodiment, endogenous TnaA is knocked out through mutation(s) and/or deletion(s). In one embodiment, bacteria are engineered to comprise one or more additional copies of SerA. In one embodiment, bacteria are engineered to comprise one or more additional copies of YddG, a tryptophan exporter. In one embodiment, endogenous PheA is knocked out through mutation(s) and/or deletion(s). In one embodiment, two or more of the strategies depicted in the schematic of  FIG. 44B  are engineered into a bacterial strain. Alternatively, other gene products in this pathway may be mutated or overexpressed. 
         FIG. 45A  and  FIG. 45B  and  FIG. 45C  depict bar graphs showing tryptophan production by various engineered bacterial strains.  FIG. 45A  depicts a bar graph showing tryptophan production by various tryptophan producing strains. The data show expressing a feedback resistant form of AroG (AroG tbr ) is necessary to get tryptophan production. Additionally, using a feedback resistant trpE (trpE tbr ) has a positive effect on tryptophan production.  FIG. 45B  shows tryptophan production from a strain comprising a tet-trpE tbr DCBA, tet-aroG tbr  construct, comparing glucose and glucuronate as carbon sources in the presence and absence of oxygen. It takes  E. coli  two molecules of phosphoenolpyruvate (PEP) to produce one molecule of tryptophan. When glucose is used as the carbon source, 50% of all available PEP is used to import glucose into the cell through the PTS system (Phosphotransferase system). Tryptophan production is improved by using a non-PTS sugar (glucuronate) aerobically. The data also show the positive effect of deleting tnaA (only at early time point aerobically).  FIG. 45C  depicts a bar graph showing improved tryptophan production by engineered strain comprising ΔtrpRΔtnaA, tet-trpE fbr  DCBA, tet-aroG fbr  through the addition of serine. 
         FIG. 46  depicts a bar graph showing a comparison in tryptophan production in strains SYN2126, SYN2323, SYN2339, SYN2473, and SYN2476. SYN2126 ΔtrpRΔtnaA. ΔtrpRΔtnaA, tet-aroGfbr. SYN2339 comprises ΔtrpRΔtnaA, tet-aroGfbr, tet-trpEfbrDCBA. SYN2473 comprises ΔtrpRΔtnaA, tet-aroGfbr-serA, tet-trpEfbrDCBA. SYN2476 comprises ΔtrpRΔtnaA, tet-trpEfbrDCBA. Results indicate that expressing aroG is not sufficient nor necessary under these conditions to get Trp production and that expressing serA is beneficial for tryptophan production. 
         FIG. 47  depicts a schematic of an indole-3-propionic acid (IPA) synthesis circuit. IPA produced by the gut microbiota has a significant positive effect on barrier integrity. IPA does not signal through AhR, but rather through a different receptor (PXR) (Venkatesh et al., Symbiotic Bacterial Metabolites Regulate Gastrointestinal Bardrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296-310, Aug. 21, 2014). In some embodiments, IPA can be produced in a synthetic circuit by expressing two enzymes, a tryptophan ammonia lyase and an indole-3-acrylate reductase (e.g., Tryptophan ammonia lyase (WAL) (e.g., from  Rubrivivax benzoatilyticus ) and indole-3-acrylate reductase (e.g., from  Clostridium botulinum ). Tryptophan ammonia lyase converts tryptophan to indole-3-acrylic acid, and indole-3-acrylate reductase converts indole-3-acrylic acid into IPA. Without wishing to be bound by theory, no oxygen is needed for this reaction, allowing it to proceed under low or no oxygen conditions, e.g., as those found in the mammalian gut. In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in  FIG. 40  (A-D) and  FIG. 44  and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. 
         FIG. 48  depicts a schematic of indole-3-propionic acid (IPA), indole acetic acid (IAA), and tryptamine synthesis (TrA) circuits. Enzymes are as follows: 1. TrpDH: tryptophan dehydrogenase, e.g., from from  Nostoc punctiforme  NIES-2108; FldH1/FldH2: indole-3-lactate dehydrogenase, e.g., from  Clostridium sporogenes ; FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from  Clostridium sporogenes ; FldBC: indole-3-lactate dehydratase, e.g., from  Clostridium sporogenes ; FldD: indole-3-acrylyl-CoA reductase, e.g., from  Clostridium sporogenes ; AcuI: acrylyl-CoA reductase, e.g., from  Rhodobacter sphaeroides . lpdC: Indole-3-pyruvate decarboxylase, e.g., from  Enterobacter cloacae ; lad1: Indole-3-acetaldehyde dehydrogenase, e.g., from  Ustilago maydis ; Tdc: Tryptophan decarboxylase, e.g., from  Catharanthus roseus  or from  Clostridium sporogenes.    
       Tryptophan dehydrogenase (EC 1.4.1.19) is an enzyme that catalyzes the reversible chemical reaction converting L-tryptophan, NAD(P) and water to (indol-3-yl)pyruvate (IPyA), NH3, NAD(P)H and H + . Indole-3-lactate dehydrogenase ((EC 1.1.1.110, e.g.,  Clostridium sporogenes  or  Lactobacillus casei ) converts (indol-3yl)pyruvate (lpyA) and NADH and H+ to indole-3-lactate (ILA) and NAD+. Indole-3-propionyl-CoA:indole-3-lactate CoA transferase (FldA) converts indole-3-lactate (ILA) and indol-3-propionyl-CoA to indole-3-propionic acid (IPA) and indole-3-lactate-CoA. Indole-3-acrylyl-CoA reductase (FldD) and acrylyl-CoA reductase (AcuI) convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA. Indole-3-lactate dehydratase (FldBC) converts indole-3-lactate-CoA to indole-3-acrylyl-CoA. Indole-3-pyruvate decarboxylase (lpdC:) converts Indole-3-pyruvic acid (IPyA) into Indole-3-acetaldehyde (IAAld) lad1: Indole-3-acetaldehyde dehydrogenase coverts Indole-3-acetaldehyde (IAAld) into Indole-3-acetic acid (IAA) Tdc: Tryptophan decarboxylase converts tryptophan (Trp) into tryptamine (TrA). In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in  FIG. 40  (A-D) and  FIG. 44  and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. 
         FIG. 49  depicts a bar graph showing tryptophan and indole acetic acid production for strains SYN2126, SYN2339 and SYN2342. SYN2126: comprises ΔtrpR and ΔtnaA (ΔtrpRΔtnaA). SYN2339 comprises circuitry for the production of tryptophan (ΔtrpRΔtnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr (p15A)). SYN2342 comprises the same tryptophan production circuitry as the parental strain SYN2339, and additionally comprises ipdC-iad1 incorporated at the end of the second construct (ΔtrpRΔtnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr-trpDH-ipdC-iad1 (p15A)). SYN2126 produced no tryptophan, SYN2339 produces increasing tryptophan over the time points measured, and SYN2342 converts all trypophan it produces into IAA. 
         FIG. 50  depicts a bar graph showing tryptophan and tryptamine production for strains SYN2339, SYN2340, and SYN2794. SYN2339 is used as a control which can produce tryptophan but cannot convert it to tryptamine and comprises ΔtrpRΔtnaA, tetR-P tet -trpE fbr DCBA (pSC101), tetR-P tet -aroG tbr  (p15A). SYN2340 comprises ΔtrpRΔtnaA, tetR-P tet -trpE fbr DCBA (pSC101), tetR-P tet -aroG tbr -tdc Cs  (p15A). SYN2794 comprises ΔtrpRΔtnaA, tetR-P tet -trpE fbr DCBA (pSC101), tetR-P tet -aroG fbr -tdc Cs  (p15A). Results indicate that Tdc Cs  from  Clostridium sporogenes  is more efficient the Tdc Cr  from  Catharanthus roseus  in tryptamine production and converts all the tryptophan produced into tryptamine. 
         FIG. 51A ,  FIG. 51B ,  FIG. 51C ,  FIG. 51D ,  FIG. 51E  depict schematics of non-limiting examples of genetically engineered bacteria of the disclosure which comprises one or more gene sequence(s) and/or gene cassette(s) as described herein. 
         FIG. 52  depicts a map of integration sites within the  E. coli  Nissle chromosome. These sites indicate regions where circuit components may be inserted into the chromosome without interfering with essential gene expression. Backslashes (/) are used to show that the insertion will occur between divergently or convergently expressed genes. Insertions within biosynthetic genes, such as thyA, can be useful for creating nutrient auxotrophies. In some embodiments, an individual circuit component is inserted into more than one of the indicated sites. 
         FIG. 53  depicts an exemplary schematic of the  E. coli  1917 Nissle chromosome comprising multiple mechanisms of action (MoAs). 
         FIG. 54A  and  FIG. 54B  depict schematics of bacterial chromosomes, for example the  E. coli  Nissle 1917 Chromosome. For example,  FIG. 54A  depicts a schematic of an engineered bacterium comprising, a circuit for butyrate production, a circuit for propionate production, and a circuit for production of one or more interleukins relevant to IBD.  FIG. 54B  depicts a schematic of an engineered bacterium comprising three circuits, a circuit for butyrate production, a circuit for GLP-2 expression and and a circuit for production of one or more interleukins relevant to IBD. 
         FIG. 55  depicts a schematic of a secretion system based on the flagellar type III secretion in which an incomplete flagellum is used to secrete a therapeutic peptide of interest (star) by recombinantly fusing the peptide to an N-terminal flagellar secretion signal of a native flagellar component so that the intracellularly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment. 
         FIG. 56  depicts a schematic of a type V secretion system for the extracellular production of recombinant proteins in which a therapeutic peptide (star) can be fused to an N-terminal secretion signal, a linker and the beta-domain of an autotransporter. In this system, the N-terminal signal sequence directs the protein to the SecA-YEG machinery which moves the protein across the inner membrane into the periplasm, followed by subsequent cleavage of the signal sequence. The beta-domain is recruited to the Bam complex where the beta-domain is folded and inserted into the outer membrane as a beta-barrel structure. The therapeutic peptide is then thread through the hollow pore of the beta-barrel structure ahead of the linker sequence. The therapeutic peptide is freed from the linker system by an autocatalytic cleavage or by targeting of a membrane-associated peptidase (scissors) to a complementary protease cut site in the linker. 
         FIG. 57  depicts a schematic of a type 1 secretion system, which translocates a passenger peptide directly from the cytoplasm to the extracellular space using HlyB (an ATP-binding cassette transporter); HlyD (a membrane fusion protein); and TolC (an outer membrane protein) which form a channel through both the inner and outer membranes. The secretion signal-containing C-terminal portion of HlyA is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of this peptide. 
         FIG. 58  depicts a schematic of the outer and inner membranes of a gram-negative bacterium, and several deletion targets for generating a leaky or destabilized outer membrane, thereby facilitating the translocation of a therapeutic polypeptides to the extracellular space, e.g., therapeutic polypeptides of eukaryotic origin containing disulphide bonds. Deactivating mutations of one or more genes encoding a protein that tethers the outer membrane to the peptidoglycan skeleton, e.g., lpp, ompC, ompA, ompF, tolA, tolB, pal, and/or one or more genes encoding a periplasmic protease, e.g., degS, degP, nlpl, generates a leaky phenotype. Combinations of mutations may synergistically enhance the leaky phenotype. 
         FIG. 59  depicts a modified type 3 secretion system (T3SS) to allow the bacteria to inject secreted therapeutic proteins into the gut lumen. An inducible promoter (small arrow, top), e.g. a FNR-inducible promoter, drives expression of the T3 secretion system gene cassette (3 large arrows, top) that produces the apparatus that secretes tagged peptides out of the cell. An inducible promoter (small arrow, bottom), e.g. a FNR-inducible promoter, drives expression of a regulatory factor, e.g. T7 polymerase, that then activates the expression of the tagged therapeutic peptide (hexagons). 
         FIGS. 60A-60C  depict other non-limiting embodiments of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the ParaBAD promoter (P araBAD ), which induces expression of the Tet repressor (TetR) and an anti-toxin. The anti-toxin builds up in the recombinant bacterial cell, while TetR prevents expression of a toxin (which is under the control of a promoter having a TetR binding site). However, when arabinose is not present, both the anti-toxin and TetR are not expressed. Since TetR is not present to repress expression of the toxin, the toxin is expressed and kills the cell.  FIG. 60A  also depicts another non-limiting embodiment of the disclosure, wherein the expression of an essential gene not found in the recombinant bacteria is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of the essential gene under the control of the araBAD promoter and the bacterial cell cannot survive. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the essential gene and maintains viability of the bacterial cell.  FIG. 60B  depicts a non-limiting embodiment of the disclosure, where an anti-toxin is expressed from a constitutive promoter, and expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of TetR, thus preventing expression of a toxin. However, when arabinose is not present, TetR is not expressed, and the toxin is expressed, eventually overcoming the anti-toxin and killing the cell. The constitutive promoter regulating expression of the anti-toxin should be a weaker promoter than the promoter driving expression of the toxin. The araC gene is under the control of a constitutive promoter in this circuit.  FIG. 60C  depicts another non-limiting embodiment of the disclosure, wherein the expression of a heterologous gene is activated by an exogenous environmental signal. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the Tet repressor (TetR) and an anti-toxin. The anti-toxin builds up in the recombinant bacterial cell, while TetR prevents expression of a toxin (which is under the control of a promoter having a TetR binding site). However, when arabinose is not present, both the anti-toxin and TetR are not expressed. Since TetR is not present to repress expression of the toxin, the toxin is expressed and kills the cell. The araC gene is either under the control of a constitutive promoter or an inducible promoter (e.g., AraC promoter) in this circuit. 
         FIG. 61  depicts one non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips a toxin gene into an activated conformation, and the natural kinetics of the recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell. 
         FIG. 62  depicts another non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene, an anti-toxin, and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips a toxin gene into an activated conformation, but the presence of the accumulated anti-toxin suppresses the activity of the toxin. Once the exogenous environmental condition or cue(s) is no longer present, expression of the anti-toxin is turned off. The toxin is constitutively expressed, continues to accumulate, and kills the bacterial cell. 
         FIG. 63  depicts another non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and at least one recombinase from an inducible promoter or inducible promoters. The recombinase then flips at least one excision enzyme into an activated conformation. The at least one excision enzyme then excises one or more essential genes, leading to senescence, and eventual cell death. The natural kinetics of the recombinase and excision genes cause a time delay, the kinetics of which can be altered and optimized depending on the number and choice of essential genes to be excised, allowing cell death to occur within a matter of hours or days. The presence of multiple nested recombinases can be used to further control the timing of cell death. 
         FIG. 64  depicts one non-limiting embodiment of the disclosure, where an exogenous environmental condition or one or more environmental signals activates expression of a heterologous gene and a first recombinase from an inducible promoter or inducible promoters. The recombinase then flips a second recombinase from an inverted orientation to an active conformation. The activated second recombinase flips the toxin gene into an activated conformation, and the natural kinetics of the recombinase create a time delay in expression of the toxin, allowing the heterologous gene to be fully expressed. Once the toxin is expressed, it kills the cell. 
         FIG. 65  depicts the use of GeneGuards as an engineered safety component. All engineered DNA is present on a plasmid which can be conditionally destroyed. See, e.g., Wright et al., “GeneGuard: A Modular Plasmid System Designed for Biosafety,” ACS Synthetic Biology (2015) 4: 307-316. 
         FIG. 66  depicts β-galactosidase levels in samples comprising bacteria harboring a low-copy plasmid expressing lacZ from an FNR-responsive promoter selected from the exemplary FNR promoters shown in the tables (Pfnr1-5). Different FNR-responsive promoters were used to create a library of anaerobic-inducible reporters with a variety of expression levels and dynamic ranges. These promoters included strong ribosome binding sites. Bacterial cultures were grown in either aerobic (+O 2 ) or anaerobic conditions (−O 2 ). Samples were removed at 4 hrs and the promoter activity based on β-galactosidase levels was analyzed by performing standard β-galactosidase colorimetric assays. 
         FIGS. 67A-67C  depict a schematic representation of the lacZ gene under the control of an exemplary FNR promoter (P fnrS ) and corresponding graphical data.  FIG. 67A  depicts a schematic representation of the lacZ gene under the control of an exemplary FNR promoter (P fnrS ). LacZ encodes the β-galactosidase enzyme and is a common reporter gene in bacteria.  FIG. 67B  depicts FNR promoter activity as a function of β-galactosidase activity in SYN340. SYN340, an engineered bacterial strain harboring a low-copy fnrS-lacZ fusion gene, was grown in the presence or absence of oxygen. Values for standard β-galactosidase colorimetric assays are expressed in Miller units (Miller, 1972). These data suggest that the fnrS promoter begins to drive high-level gene expression within 1 hr under anaerobic conditions.  FIG. 67C  depicts the growth of bacterial cell cultures expressing lacZ over time, both in the presence and absence of oxygen. 
         FIGS. 68A-68D  depict bar graphs, schematic, and dot blot, respectively, showing the structure or activity of reporter constructs.  FIG. 68A  and  FIG. 68B  depict bar graphs of reporter constructs activity.  FIG. 68A  depicts a graph of an ATC-inducible reporter construct expression and  FIG. 68B  depicts a graph of a nitric oxide-inducible reporter construct expression. These constructs, when induced by their cognate inducer, lead to expression of GFP. Nissle cells harboring plasmids with either the control, ATC-inducible P tet -GFP reporter construct or the nitric oxide inducible P nsrR -GFP reporter construct induced across a range of concentrations. Promoter activity is expressed as relative florescence units.  FIG. 68C  depicts a schematic of the constructs.  FIG. 68D  depicts a dot blot of bacteria harboring a plasmid expressing NsrR under control of a constitutive promoter and the reporter gene gfp (green fluorescent protein) under control of an NsrR-inducible promoter. DSS-treated mice serve as exemplary models for HE. As in HE subjects, the guts of mice are damaged by supplementing drinking water with 2-3% dextran sodium sulfate (DSS). Chemiluminescent is shown for NsrR-regulated promoters induced in DSS-treated mice. 
         FIG. 69  depicts a graph of Nissle residence in vivo. Streptomycin-resistant Nissle was administered to mice via oral gavage without antibiotic pre-treatment. Fecal pellets from 6 total mice were monitored post-administration to determine the amount of administered Nissle still residing within the mouse gastrointestinal tract. The bars represent the number of bacteria administered to the mice. The line represents the number of Nissle recovered from the fecal samples each day for 10 consecutive days. 
         FIG. 70  depicts a bar graph of residence over time for streptomycin resistant Nissle in various compartments of the intestinal tract at 1, 4, 8, 12, 24, and 30 hours post gavage. Mice were treated with approximately 109 CFU, and at each timepoint, animals (n=4) were euthanized, and intestine, cecum, and colon were removed. The small intestine was cut into three sections, and the large intestine and colon each into two sections. Intestinal effluents gathered and CFUs in each compartment were determined by serial dilution plating. 
         FIG. 71A  and  FIG. 71B  depict a schematic diagrams of a wild-type clbA construct ( FIG. 71A ) and a schematic diagram of a clbA knockout construct ( FIG. 71B ). 
         FIG. 72  depicts a schematic of a design-build-test cycle. Steps are as follows: 1: Define the disease pathway; 2. Identify target metabolites; 3. Design genetic circuits; 4. Build synthetic biotic; 5. Activate circuit in vivo; 6. Characterize circuit activation kinetics; 7. Optimize in vitro productivity to disease threshold; 8. Test optimize circuit in animal disease model; 9. Assimilate into the microbiome; 10. Develop understanding of in vivo PK and dosing regimen. 
         FIG. 73  depicts a schematic of non-limiting manufacturing processes for upstream and downstream production of the genetically engineered bacteria of the present disclosure. Step 1 depicts the parameters for starter culture 1 (SC1): loop full—glycerol stock, duration overnight, temperature 37° C., shaking at 250 rpm. Step 2 depicts the parameters for starter culture 2 (SC2): 1/100 dilution from SC1, duration 1.5 hours, temperature 37° C., shaking at 250 rpm. Step 3 depicts the parameters for the production bioreactor: inoculum—SC2, temperature 37° C., pH set point 7.00, pH dead band 0.05, dissolved oxygen set point 50%, dissolved oxygen cascade agitation/gas FLO, agitation limits 300-1200 rpm, gas FLO limits 0.5-20 standard liters per minute, duration 24 hours. Step 4 depicts the parameters for harvest: centrifugation at speed 4000 rpm and duration 30 minutes, wash IX 10% glycerol/PBS, centrifugation, re-suspension 10% glycerol/PBS. Step 5 depicts the parameters for vial fill/storage: 1-2 mL aliquots, −80° C. 
         FIG. 74  depicts three bacterial strains which constitutively express red fluorescent protein (RFP). In strains 1-3, the rfp gene has been inserted into different sites within the bacterial chromosome, and results in varying degrees of brightness under fluorescent light. Unmodified  E. coli  Nissle (strain 4) is non-fluorescent. 
         FIG. 75A  depicts a graph showing bacterial cell growth of a Nissle thyA auxotroph strain (thyA knock-out) in various concentrations of thymidine. A chloramphenicol-resistant Nissle thyA auxotroph strain was grown overnight in LB+10 mM thymidine at 37 C. The next day, cells were diluted 1:100 in 1 mL LB+10 mM thymidine, and incubated at 37 C for 4 hours. The cells were then diluted 1:100 in 1 mL LB+varying concentrations of thymidine in triplicate in a 96-well plate. The plate is incubated at 37 C with shaking, and the OD600 is measured every 5 minutes for 720 minutes. This data shows that Nissle thyA auxotroph does not grow in environments lacking thymidine. 
         FIG. 75B  depicts a bar graph of Nissle residence in vivo of wildtype Nissle versus Nissle thyA auxotroph (thyA knock-out). Streptomycin-resistant Nissle (wildtype or thyA auxotroph) was administered to mice via oral gavage without antibiotic pre-treatment. Fecal pellets from 6 total mice were monitored post-administration to determine the amount of administered Nissle still residing within the mouse gastrointestinal tract. Each bar represents the number of Nissle recovered from the fecal samples each day for 7 consecutive days. There were no bacteria recovered in fecal samples from mice gavaged with Nissle thyA auxotroph bacteria after day 3. This data shows that the Nissle thyA auxotroph does not persist in vivo in mice. 
         FIG. 76  depicts a one non-limiting embodiment of the disclosure, which comprises a plasmid stability system with a plasmid that produces both a short-lived anti-toxin and a long-lived toxin. When the cell loses the plasmid, the anti-toxin is no longer produced, and the toxin kills the cell. In one embodiment, the genetically engineered bacteria produce an equal amount of a Hok toxin and a short-lived Sok antitoxin. In the upper panel, the cell produces equal amounts of toxin and anti-toxin and is stable. In the center panel, the cell loses the plasmid and anti-toxin begins to decay. In the lower panel, the anti-toxin decays completely, and the cell dies. 
         FIGS. 77A-77D  depict schematics of non-limiting examples of the gene organization of plasmids, which function as a component of a biosafety system ( FIG. 77A  and  FIG. 77B ), which also contains a chromosomal component (shown in  FIG. 77C  and  FIG. 77D ). The biosafety plasmid system vector comprises Kid Toxin and R6K minimal ori, dapA ( FIG. 77A ) and thyA ( FIG. 77B ) and promoter elements driving expression of these components. In some embodiments, bla is knocked out and replaced with one or more constructs described herein, in which a first protein of interest (POI1) and/or a second protein of interest, e.g., a transporter (POI2), and/or a third protein of interest (POI3) are expressed from an inducible or constitutive promoter.  FIG. 77C  and  FIG. 77D  depict schematics of the gene organization of the chromosomal component of a biosafety system.  FIG. 77C  depicts a construct comprising low copy Rep (Pi) and Kis antitoxin, in which transcription of Pi (Rep), which is required for the replication of the plasmid component of the system, is driven by a low copy RBS containing promoter.  FIG. 77D  depicts a construct comprising a medium-copy Rep (Pi) and Kis antitoxin, in which transcription of Pi (Rep), which is required for the replication of the plasmid component of the system, is driven by a medium copy RBS containing promoter. If the plasmid containing the functional DapA is used (as shown in  FIG. 77A ), then the chromosomal constructs shown in  FIG. 77C  and  FIG. 77D  are knocked into the DapA locus. If the plasmid containing the functional ThyA is used (as shown in  FIG. 77B ), then the chromosomal constructs shown in  FIG. 77C  and  FIG. 77D  are knocked into the ThyA locus. In this system, the bacteria comprising the chromosomal construct and a knocked out dapA or thyA gene can grow in the absence of dap or thymidine only in the presence of the plasmid. 
         FIG. 78  depicts a schematic of a polypeptide of interest displayed on the surface of the bacterium. A non-limiting example of such a therapeutic protein is a scFv. The polypeptide is expressed as a fusion protein, which comprises a outer membrane anchor from another protein, which was developed as part of a display system. Non-limiting examples of such anchors are described herein and include LppOmpA, NGIgAsig-NGIgAP, InaQ, Intimin, Invasin, pelB-PAL, and blcA/BAN. In a nonlimiting example a bacterial strain which has one or more diffusible outer membrane phenotype (“leaky membrane”) mutation, e.g., as described herein. 
         FIG. 79  depicts the gene organization of exemplary construct comprising FNRS24Y driven by the arabinose inducible promoter and araC in reverse direction. 
         FIG. 80A  depicts a “Oxygen bypass switch” useful for aerobic pre-induction of a strain comprising one or proteins of interest (POI), e.g., one or more anti-cancer molecules or immune modulatory effectors (POI1) and a second set of one or more proteins of interest (POI2), e.g., one or more transporter(s)/importer(s) and/or exporter(s), under the control of a low oxygen FNR promoter in vitro in a culture vessel (e.g., flask, fermenter or other vessel, e.g., used during with cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture). In some embodiments, it is desirable to pre-load a strain with active effector molecules prior to administration. This can be done by pre-inducing the expression of these effectors as the strains are propagated, (e.g., in flasks, fermenters or other appropriate vesicles) and are prepared for in vivo administration. In some embodiments, strains are induced under anaerobic and/or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more effectors or proteins of interest. In some embodiments, it is desirable to prepare, pre-load and pre-induce the strains under aerobic or microaerobic conditions with one or more effectors or proteins of interest. This allows more efficient growth and, in some cases, reduces the build-up of toxic metabolites. 
       FNRS24Y is a mutated form of FNR which is more resistant to inactivation by oxygen, and therefore can activate FNR promoters under aerobic conditions (see e.g., Jervis A J, The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci USA. 2009 Mar. 24; 106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). In this oxygen bypass system, FNRS24Y is induced by addition of arabinose and then drives the expression of one or more POIs by binding and activating the FNR promoter under aerobic conditions. Thus, strains can be grown, produced or manufactured efficiently under aerobic conditions, while being effectively pre-induced and pre-loaded, as the system takes advantage of the strong FNR promoter resulting in of high levels of expression of one or more POIs. This system does not interfere with or compromise in vivo activation, since the mutated FNRS24Y is no longer expressed in the absence of arabinose, and wild type FNR then binds to the FNR promoter and drives expression of the POIs in vivo. In some embodiments, a LacI promoter and IPTG induction are used in this system (in lieu of Para and arabinose induction). In some embodiments, a rhamnose inducible promoter is used in this system. In some embodiments, a temperature sensitive promoter is used to drive expression of FNRS24Y. 
         FIG. 80B  depicts a strategy to allow the expression of one or more POI(s) under aerobic conditions through the arabinose inducible expression of FNRS24Y. By using a ribosome binding site optimization strategy, the levels of Fnr S24Y  expression can be fine-tuned, e.g., under optimal inducing conditions (adequate amounts of arabinose for full induction). Fine-tuning is accomplished by selection of an appropriate RBS with the appropriate translation initiation rate. Bioinformatics tools for optimization of RBS are known in the art. 
         FIG. 80C  depicts a strategy to fine-tune the expression of a Para-POI construct by using a ribosome binding site optimization strategy. Bioinformatics tools for optimization of RBS are known in the art. In one strategy, arabinose controlled POI genes can be integrated into the chromosome to provide for efficient aerobic growth and pre-induction of the strain (e.g., in flasks, fermenters or other appropriate vesicles), while integrated versions of P fnrS -POI constructs are maintained to allow for strong in vivo induction. 
         FIG. 81  depicts the gene organization of an exemplary construct, e.g., comprised in SYN-PKU401, comprising a cloned POI gene under the control of a Tet promoter sequence and a Tet repressor gene. 
         FIG. 82  depicts the gene organization of an exemplary construct comprising LacI in reverse orientation, and a IPTG inducible promoter driving the expression of one or more POIs. In some embodiments, this construct is useful for pre-induction and pre-loading of a therapeutic strain prior to in vivo administration under aerobic conditions and in the presence of inducer, e.g., IPTG. In some embodiments, this construct is used alone. In some embodiments, the construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose or IPTG inducible constructs. In some embodiments, the construct is used in combination with a low-oxygen inducible construct which is active in an in vivo setting. 
       In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with construct expressing a second POI, e.g., a transporter, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. POI2 expression may be constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, or IPTG. In some embodiments, the construct is located on a plasmid, e.g., a low or high copy plasmid. In some embodiments, the construct is employed in a biosafety system, such as the system shown in  FIG. 77A ,  FIG. 77B ,  FIG. 77C , and  FIG. 77D . In some embodiments, the construct is integrated into the genome at one or more locations described herein. 
         FIG. 83A ,  FIG. 83B , and  FIG. 83C  depict schematics of non-limiting examples of constructs for the expression of proteins of interest POI(s).  FIG. 83A  depicts a schematic of a non-limiting example of the organization of a construct for POI expression under the control a lambda CI inducible promoter. The construct also provides the coding sequence of a mutant of CI, CI857, which is a temperature sensitive mutant of CI. The temperature sensitive CI repressor mutant, CI857, binds tightly at 30 degrees C. but is unable to bind (repress) at temperatures of 37 C and above. In some embodiments, this construct is used alone. In some embodiments, the temperature sensitive construct is used in combination with other constitutive or inducible POI constructs, e.g., low oxygen, arabinose, rhamnose, or IPTG inducible constructs. In some embodiments, the construct allows pre-induction and pre-loading of a POI10 and/or a POI2 prior to in vivo administration. In some embodiments, the construct provides in vivo activity. In some embodiments, the construct is located on a plasmid, e.g., a low copy or a high copy plasmid. In some embodiments, the construct is located on a plasmid component of a biosafety system. In some embodiments, the construct is integrated into the bacterial chromosome at one or more locations. In some embodiments, the construct is used in combination with a POI2 construct, which can either be provided on a plasmid or is integrated into the bacterial chromosome at one or more locations. POI2 expression may be constitutive or driven by an inducible promoter, e.g., low-oxygen, arabinose, rhamnose, or temperature sensitive. In some embodiments, the construct is used in combination with a POI3 expression construct. 
       In some embodiments, a temperature sensitive system can be used to set up a conditional auxotrophy. In a a strain comprising deltaThyA or deltaDapA, a dapA or thyA gene can be introduced into the strain under the control of a thermoregulated promoter system. The strain can grow in the absence of Thy and Dap only at the permissive temperature, e.g., 37 C (and not lower). 
         FIG. 84A  depicts a schematic of the gene organization of a PssB promoter. The ssB gene product protects ssDNA from degradation; SSB interacts directly with numerous enzymes of DNA metabolism and is believed to have a central role in organizing the nucleoprotein complexes and processes involved in DNA replication (and replication restart), recombination and repair. The PssB promoter was cloned in front of a LacZ reporter and beta-galactosidase activity was measured. 
         FIG. 84B  depicts a bar graph showing the reporter gene activity for the PssB promoter under aerobic and anaerobic conditions. Briefly, cells were grown aerobically overnight, then diluted 1:100 and split into two different tubes. One tube was placed in the anaerobic chamber, and the other was kept in aerobic conditions for the length of the experiment. At specific times, the cells were analyzed for promoter induction. The Pssb promoter is active under aerobic conditions, and shuts off under anaerobic conditions. This promoter can be used to express a gene of interest under aerobic conditions. This promoter can also be used to tightly control the expression of a gene product such that it is only expressed under anaerobic and/or low oxygen conditions. In this case, the oxygen induced PssB promoter induces the expression of a repressor, which represses the expression of a gene of interest. Thus, the gene of interest is only expressed in the absence of the repressor, i.e., under anaerobic and/or low oxygen conditions. This strategy has the advantage of an additional level of control for improved fine-tuning and tighter control. In one non-limiting example, this strategy can be used to control expression of thyA and/or dapA, e.g., to make a conditional auxotroph. The chromosomal copy of dapA or ThyA is knocked out. Under anaerobic and/or low oxygen conditions, dapA or thyA—as the case may be—are expressed, and the strain can grow in the absence of dap or thymidine. Under aerobic conditions, dapA or thyA expression is shut off, and the strain cannot grow in the absence of dap or thymidine. Such a strategy can, for example be employed to allow survival of bacteria under anaerobic and/or low oxygen conditions, e.g., the gut, but prevent survival under aerobic conditions (biosafety switch). 
         FIG. 85A  depicts a schematic diagram of a wild-type clbA construct. 
         FIG. 85B  depicts a schematic diagram of a clbA knockout construct. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     The present disclosure includes genetically engineered bacteria, pharmaceutical compositions thereof, and methods of reducing gut inflammation, enhancing gut barrier function, and/or treating or preventing autoimmune disorders. In some embodiments, the genetically engineered bacteria comprise at least one non-native gene and/or gene cassette for producing a non-native anti-inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, the at least one gene and/or gene cassette is further operably linked to a regulatory region that is controlled by a transcription factor that is capable of sensing an inducing condition, e.g., a low-oxygen environment, the presence of ROS, or the presence of RNS. The genetically engineered bacteria are capable of producing the anti-inflammation and/or gut barrier function enhancer molecule(s) in inducing environments, e.g., in the gut. Thus, the genetically engineered bacteria and pharmaceutical compositions comprising those bacteria may be used to treat or prevent autoimmune disorders and/or diseases or conditions associated with gut inflammation and/or compromised gut barrier function, including IBD. 
     In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description. 
     As used herein, “diseases and conditions associated with gut inflammation and/or compromised gut barrier function” include, but are not limited to, inflammatory bowel diseases, diarrheal diseases, and related diseases. “Inflammatory bowel diseases” and “IBD” are used interchangeably herein to refer to a group of diseases associated with gut inflammation, which include, but are not limited to, Crohn&#39;s disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, diversion colitis, Behcet&#39;s disease, and indeterminate colitis. As used herein, “diarrheal diseases” include, but are not limited to, acute watery diarrhea, e.g., cholera; acute bloody diarrhea, e.g., dysentery; and persistent diarrhea. As used herein, related diseases include, but are not limited to, short bowel syndrome, ulcerative proctitis, proctosigmoiditis, left-sided colitis, pancolitis, and fulminant colitis. 
     Symptoms associated with the aforementioned diseases and conditions include, but are not limited to, one or more of diarrhea, bloody stool, mouth sores, perianal disease, abdominal pain, abdominal cramping, fever, fatigue, weight loss, iron deficiency, anemia, appetite loss, weight loss, anorexia, delayed growth, delayed pubertal development, inflammation of the skin, inflammation of the eyes, inflammation of the joints, inflammation of the liver, and inflammation of the bile ducts. 
     A disease or condition associated with gut inflammation and/or compromised gut barrier function may be an autoimmune disorder. A disease or condition associated with gut inflammation and/or compromised gut barrier function may be co-morbid with an autoimmune disorder. As used herein, “autoimmune disorders” include, but are not limited to, acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison&#39;s disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticarial, axonal &amp; neuronal neuropathies, Balo disease, Behcet&#39;s disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn&#39;s disease, Cogan&#39;s syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devic&#39;s disease (neuromyelitis optica), discoid lupus, Dressler&#39;s syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture&#39;s syndrome, granulomatosis with polyangiitis (GPA), Graves&#39; disease, Guillain-Barre syndrome, Hashimoto&#39;s encephalitis, Hashimoto&#39;s thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile idiopathic arthritis, juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (systemic lupus erythematosus), chronic Lyme disease, Meniere&#39;s disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren&#39;s ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic&#39;s), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, &amp; III autoimmune polyglandular syndromes, polymyalgia rheumatic, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud&#39;s phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter&#39;s syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren&#39;s syndrome, sperm &amp; testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac&#39;s syndrome, sympathetic ophthalmia, Takayasu&#39;s arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener&#39;s granulomatosis. 
     As used herein, “anti-inflammation molecules” and/or “gut barrier function enhancer molecules” include, but are not limited to, short-chain fatty acids, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2 and analogs, GLP-1, IL-10, IL-27, TGF-β1, TGF-β2, N-acylphosphatidylethanolamines (NAPEs), elafin (also called peptidase inhibitor 3 and SKALP), trefoil factor, melatonin, tryptophan, PGD 2 , and kynurenic acid, indole metabolites, and other tryptophan metabolites, as well as other molecules disclosed herein. Such molecules may also include compounds that inhibit pro-inflammatory molecules, e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that neutralizes TNF-α, IFN-γ, IL-1β, IL-6, IL-8, IL-17, and/or chemokines, e.g., CXCL-8 and CCL2. Such molecules also include AHR agonists (e.g., which result in IL-22 production, e.g., indole acetic acid, indole-3-aldehyde, and indole) and and PXR agonists (e.g., IPA), as described herein. Such molecules also include HDAC inhibitors (e.g., butyrate), activators of GPR41 and/or GPR43 (e.g., butyrate and/or propionate and/or acetate), activators of GPR109A (e.g., butyrate), inhibitors of NF-kappaB signaling (e.g., butyrate), and modulators of PPARgamma (e.g., butyrate), activators of AMPK signaling (e.g., acetate), and modulators of GLP-1 secretion. Such molecules also include hydroxyl radical scavengers and antioxidants (e.g., IPA). A molecule may be primarily anti-inflammatory, e.g., IL-10, or primarily gut barrier function enhancing, e.g., GLP-2. A molecule may be both anti-inflammatory and gut barrier function enhancing. An anti-inflammation and/or gut barrier function enhancer molecule may be encoded by a single gene, e.g., elafin is encoded by the PI3 gene. Alternatively, an anti-inflammation and/or gut barrier function enhancer molecule may be synthesized by a biosynthetic pathway requiring multiple genes, e.g., butyrate. These molecules may also be referred to as therapeutic molecules. In some instances, the “anti-inflammation molecules” and/or “gut barrier function enhancer molecules” are referred to herein as “effector molecules” or “therapeutic molecules” or “therapeutic polypeptides”. 
     As used herein, the term “recombinant microorganism” refers to a microorganism, e.g., bacterial, yeast, or viral cell, or bacteria, yeast, or virus, that has been genetically modified from its native state. Thus, a “recombinant bacterial cell” or “recombinant bacteria” refers to a bacterial cell or bacteria that have been genetically modified from their native state. For instance, a recombinant bacterial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria or bacterial cell, or on a plasmid in the bacteria or bacterial cell. Recombinant bacterial cells disclosed herein may comprise exogenous nucleotide sequences on plasmids. Alternatively, recombinant bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome. 
     A “programmed or engineered microorganism” refers to a microorganism, e.g., bacterial or viral cell, or bacteria or virus, that has been genetically modified from its native state to perform a specific function. Thus, a “programmed or engineered bacterial cell” or “programmed or engineered bacteria” refers to a bacterial cell or bacteria that has been genetically modified from its native state to perform a specific function. In certain embodiments, the programmed or engineered bacterial cell has been modified to express one or more proteins, for example, one or more proteins that have a therapeutic activity or serve a therapeutic purpose. The programmed or engineered bacterial cell may additionally have the ability to stop growing or to destroy itself once the protein(s) of interest have been expressed. 
     As used herein, the term “gene” refers to a nucleic acid fragment that encodes a protein or fragment thereof, optionally including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. In one embodiment, a “gene” does not include regulatory sequences preceding and following the coding sequence. A “native gene” refers to a gene as found in nature, optionally with its own regulatory sequences preceding and following the coding sequence. A “chimeric gene” refers to any gene that is not a native gene, optionally comprising regulatory sequences preceding and following the coding sequence, wherein the coding sequences and/or the regulatory sequences, in whole or in part, are not found together in nature. Thus, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory and coding sequences that are derived from the same source, but arranged differently than is found in nature. 
     As used herein, the term “gene sequence” is meant to refer to a genetic sequence, e.g., a nucleic acid sequence. The gene sequence or genetic sequence is meant to include a complete gene sequence or a partial gene sequence. The gene sequence or genetic sequence is meant to include sequence that encodes a protein or polypeptide and is also meant to include genetic sequence that does not encode a protein or polypeptide, e.g., a regulatory sequence, leader sequence, signal sequence, or other non-protein coding sequence. 
     In some embodiments, the term “gene” or “gene sequence” is meant to refer to a nucleic acid sequence encoding any of the anti-inflammatory and gut barrier function enhancing molecules described herein, e.g., IL-2, IL-22, superoxide dismutase (SOD), kynurenine, GLP-2, GLP-1, IL-10, IL-27, TGF-β1, TGF-β2, N-acylphosphatidylethanolamines (NAPEs), elafin, and trefoil factor, as well as others. The nucleic acid sequence may comprise the entire gene sequence or a partial gene sequence encoding a functional molecule. The nucleic acid sequence may be a natural sequence or a synthetic sequence. The nucleic acid sequence may comprise a native or wild-type sequence or may comprise a modified sequence having one or more insertions, deletions, substitutions, or other modifications, for example, the nucleic acid sequence may be codon-optimized. 
     As used herein, a “heterologous” gene or “heterologous sequence” refers to a nucleotide sequence that is not normally found in a given cell in nature. As used herein, a heterologous sequence encompasses a nucleic acid sequence that is exogenously introduced into a given cell and can be a native sequence (naturally found or expressed in the cell) or non-native sequence (not naturally found or expressed in the cell) and can be a natural or wild-type sequence or a variant, non-natural, or synthetic sequence. “Heterologous gene” includes a native gene, or fragment thereof, that has been introduced into the host cell in a form that is different from the corresponding native gene. For example, a heterologous gene may include a native coding sequence that is a portion of a chimeric gene to include non-native regulatory regions that is reintroduced into the host cell. A heterologous gene may also include a native gene, or fragment thereof, introduced into a non-native host cell. Thus, a heterologous gene may be foreign or native to the recipient cell; a nucleic acid sequence that is naturally found in a given cell but expresses an unnatural amount of the nucleic acid and/or the polypeptide which it encodes; and/or two or more nucleic acid sequences that are not found in the same relationship to each other in nature. As used herein, the term “endogenous gene” refers to a native gene in its natural location in the genome of an organism. As used herein, the term “transgene” refers to a gene that has been introduced into the host organism, e.g., host bacterial cell, genome. 
     As used herein, a “non-native” nucleic acid sequence refers to a nucleic acid sequence not normally present in a microorganism, e.g., an extra copy of an endogenous sequence, or a heterologous sequence such as a sequence from a different species, strain, or substrain of bacteria or virus, or a sequence that is modified and/or mutated as compared to the unmodified sequence from bacteria or virus of the same subtype. In some embodiments, the non-native nucleic acid sequence is a synthetic, non-naturally occurring sequence (see, e.g., Purcell et al., 2013). The non-native nucleic acid sequence may be a regulatory region, a promoter, a gene, and/or one or more genes in gene cassette. In some embodiments, “non-native” refers to two or more nucleic acid sequences that are not found in the same relationship to each other in nature. The non-native nucleic acid sequence may be present on a plasmid or chromosome. In some embodiments, the genetically engineered microorganism of the disclosure comprises a gene that is operably linked to a promoter that is not associated with said gene in nature. For example, in some embodiments, the genetically engineered bacteria disclosed herein comprise a gene that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene in nature, e.g., an FNR responsive promoter (or other promoter disclosed herein) operably linked to an anti-inflammatory or gut barrier enhancer molecule. In some embodiments, the genetically engineered virus of the disclosure comprises a gene that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene in nature, e.g., a promoter operably linked to a gene encoding an anti-inflammatory or gut barrier enhancer molecule. 
     As used herein, the term “coding region” refers to a nucleotide sequence that codes for a specific amino acid sequence. The term “regulatory sequence” refers to a nucleotide sequence located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influences the transcription, RNA processing, RNA stability, or translation of the associated coding sequence. Examples of regulatory sequences include, but are not limited to, promoters, translation leader sequences, effector binding sites, signal sequences, and stem-loop structures. In one embodiment, the regulatory sequence comprises a promoter, e.g., an FNR responsive promoter or other promoter disclosed herein. 
     As used herein, a “gene cassette” or “operon” encoding a biosynthetic pathway refers to the two or more genes that are required to produce an anti-inflammatory or gut barrier enhancer molecule. In addition to encoding a set of genes capable of producing said molecule, the gene cassette or operon may also comprise additional transcription and translation elements, e.g., a ribosome binding site. 
     A “butyrogenic gene cassette,” “butyrate biosynthesis gene cassette,” and “butyrate operon” are used interchangeably to refer to a set of genes capable of producing butyrate in a biosynthetic pathway. Unmodified bacteria that are capable of producing butyrate via an endogenous butyrate biosynthesis pathway include, but are not limited to,  Clostridium, Peptoclostridium, Fusobacterium, Butyrivibrio, Eubacterium , and  Treponema . The genetically engineered bacteria of the invention may comprise butyrate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of butyrate biosynthesis genes from different species, strains, and/or substrains of bacteria. A butyrogenic gene cassette may comprise, for example, the eight genes of the butyrate production pathway from  Peptoclostridium difficile  (also called  Clostridium difficile ): bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, and buk, which encode butyryl-CoA dehydrogenase subunit, electron transfer flavoprotein subunit beta, electron transfer flavoprotein subunit alpha, acetyl-CoA C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase, respectively (Aboulnaga et al., 2013). One or more of the butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized.  Peptoclostridium difficile  strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiA1, hbd, crt2, pbt, and buk. A butyrogenic gene cassette may comprise bcd2, etfB3, etfA3, and thiA1 from  Peptoclostridium difficile  strain 630, and hbd, crt2, pbt, and buk from  Peptoclostridium difficile  strain 1296. Alternatively, a single gene from  Treponema denticola  (ter, encoding trans-2-enoynl-CoA reductase) is capable of functionally replacing all three of the bcd2, etfB3, and etfA3 genes from  Peptoclostridium difficile . Thus, a butyrogenic gene cassette may comprise thiA1, hbd, crt2, pbt, and buk from  Peptoclostridium difficile  and ter from  Treponema denticola . The butyrogenic gene cassette may comprise genes for the aerobic biosynthesis of butyrate and/or genes for the anaerobic or microaerobic biosynthesis of butyrate. In another example of a butyrate gene cassette, the pbt and buk genes are replaced with tesB (e.g., from  E. coli ). Thus a butyrogenic gene cassette may comprise ter, thiA1, hbd, crt2, and tesB. 
     Likewise, a “propionate gene cassette” or “propionate operon” refers to a set of genes capable of producing propionate in a biosynthetic pathway. Unmodified bacteria that are capable of producing propionate via an endogenous propionate biosynthesis pathway include, but are not limited to,  Clostridium propionicum, Megasphaera elsdenii , and  Prevotella ruminicola . The genetically engineered bacteria of the invention may comprise propionate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of propionate biosynthesis genes from different species, strains, and/or substrains of bacteria. In some embodiments, the propionate gene cassette comprises acrylate pathway propionate biosynthesis genes, e.g., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC, which encode propionate CoA-transferase, lactoyl-CoA dehydratase A, lactoyl-CoA dehydratase B, lactoyl-CoA dehydratase C, electron transfer flavoprotein subunit A, acryloyl-CoA reductase B, and acryloyl-CoA reductase C, respectively (Hetzel et al., 2003, Selmer et al., 2002, and Kandasamy 2012 Engineering  Escherichia coli  with acrylate pathway genes for propionic acid synthesis and its impact on mixed-acid fermentation). This operon catalyses the reduction of lactate to propionate. Dehydration of (R)-lactoyl-CoA leads to the production of the intermediate acryloyl-CoA by lactoyl-CoA dehydratase (LcdABC). Acrolyl-CoA is converted to propionyl-CoA by acrolyl-CoA reductase (EtfA, AcrBC). In some embodiments, the rate limiting step catalyzed by the enzymes encoded by etfA, acrB and acrC, are replaced by the acuI gene from  R. sphaeroides . This gene product catalyzes the NADPH-dependent acrylyl-CoA reduction to produce propionyl-CoA (Acrylyl-Coenzyme A Reductase, an Enzyme Involved in the Assimilation of 3-Hydroxypropionate by  Rhodobacter sphaeroides ; Asao 2013). Thus the propionate cassette comprises pct, lcdA, lcdB, lcdC, and acuI. In another embodiment, the homolog of AcuI in  E. coli , YhdH is used (see. e.g., Structure of  Escherichia coli  YhdH, a putative quinone oxidoreductase. Sulzenbacher 2004). This the propionate cassette comprises pct, lcdA, lcdB, lcdC, and yhdH. In alternate embodiments, the propionate gene cassette comprises pyruvate pathway propionate biosynthesis genes (see, e.g., Tseng et al., 2012), e.g., thrAfbr, thrB, thrC, ilvAfbr, aceE, aceF, and lpd, which encode homoserine dehydrogenase 1, homoserine kinase, L-threonine synthase, L-threonine dehydratase, pyruvate dehydrogenase, dihydrolipoamide acetyltrasferase, and dihydrolipoyl dehydrogenase, respectively. In some embodiments, the propionate gene cassette further comprises tesB, which encodes acyl-CoA thioesterase. 
     In another example of a propionate gene cassette comprises the genes of the Sleeping Beauty Mutase operon, e.g., from  E. coli  (sbm, ygfD, ygfG, ygfH). Recently, this pathway has been considered and utilized for the high yield industrial production of propionate from glycerol (Akawi et al., Engineering  Escherichia coli  for high-level production of propionate; J Ind Microbiol Biotechnol (2015) 42:1057-1072, the contents of which is herein incorporated by reference in its entirety). In addition, as described herein, it has been found that this pathway is also suitable for production of proprionate from glucose, e.g. by the genetically engineered bacteria of the disclosure. The SBM pathway is cyclical and composed of a series of biochemical conversions forming propionate as a fermentative product while regenerating the starting molecule of succinyl-CoA. Sbm (methylmalonyl-CoA mutase) converts succinyl CoA to L-methylmalonylCoA, YgfD is a Sbm-interacting protein kinase with GTPase activity, ygfG (methylmalonylCoA decarboxylase) converts L-methylmalonylCoA into PropionylCoA, and ygfH (propionyl-CoA/succinylCoA transferase) converts propionylCoA into propionate and succinate into succinylCoA (Sleeping beauty mutase (sbm) is expressed and interacts with ygfd in  Escherichia coli ; Froese 2009). This pathway is very similar to the oxidative propionate pathway of Propionibacteria, which also converts succinate to propionate. Succinyl-CoA is converted to R-methylmalonyl-CoA by methymalonyl-CoA mutase (mutAB). This is in turn converted to S-methylmalonyl-CoA via methymalonyl-CoA epimerase (GI:18042134). There are three genes which encode methylmalonyl-CoA carboxytransferase (mmdA, PFREUD_18870, bccp) which converts methylmalonyl-CoA to propionyl-CoA. 
     The propionate gene cassette may comprise genes for the aerobic biosynthesis of propionate and/or genes for the anaerobic or microaerobic biosynthesis of propionate. One or more of the propionate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized. 
     An “acetate gene cassette” or “acetate operon” refers to a set of genes capable of producing acetate in a biosynthetic pathway. Bacteria “synthesize acetate from a number of carbon and energy sources,” including a variety of substrates such as cellulose, lignin, and inorganic gases, and utilize different biosynthetic mechanisms and genes, which are known in the art (Ragsdale et al., 2008). The genetically engineered bacteria of the invention may comprise acetate biosynthesis genes from a different species, strain, or substrain of bacteria, or a combination of acetate biosynthesis genes from different species, strains, and/or substrains of bacteria.  Escherichia coli  are capable of consuming glucose and oxygen to produce acetate and carbon dioxide during aerobic growth (Kleman et al., 1994). Several bacteria, such as  Acetitomaculum, Acetoanaerobium, Acetohalobium, Acetonema, Balutia, Butyribacterium, Clostridium, Moorella, Oxobacter, Sporomusa , and  Thermoacetogenium , are acetogenic anaerobes that are capable of converting CO or CO 2 +H 2  into acetate, e.g., using the Wood-Ljungdahl pathway (Schiel-Bengelsdorf et al, 2012). Genes in the Wood-Ljungdahl pathway for various bacterial species are known in the art. The acetate gene cassette may comprise genes for the aerobic biosynthesis of acetate and/or genes for the anaerobic or microaerobic biosynthesis of acetate. One or more of the acetate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized. 
     Each gene or gene cassette may be present on a plasmid or bacterial chromosome. In addition, multiple copies of any gene, gene cassette, or regulatory region may be present in the bacterium, wherein one or more copies of the gene, gene cassette, or regulatory region may be mutated or otherwise altered as described herein. In some embodiments, the genetically engineered bacteria are engineered to comprise multiple copies of the same gene, gene cassette, or regulatory region in order to enhance copy number or to comprise multiple different components of a gene cassette performing multiple different functions. 
     Each gene or gene cassette may be operably linked to a promoter that is induced under low-oxygen conditions. “Operably linked” refers a nucleic acid sequence, e.g., a gene or gene cassette for producing an anti-inflammatory or gut barrier enhancer molecule, that is joined to a regulatory region sequence in a manner which allows expression of the nucleic acid sequence, e.g., acts in cis. A regulatory region “Operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. A regulatory element is operably linked with a coding sequence when it is capable of affecting the expression of the gene coding sequence, regardless of the distance between the regulatory element and the coding sequence. More specifically, operably linked refers to a nucleic acid sequence, e.g., a gene encoding an anti-inflammatory or gut barrier enhancer molecule, that is joined to a regulatory sequence in a manner which allows expression of the nucleic acid sequence, e.g., the gene encoding the anti-inflammatory or gut barrier enhancer molecule. In other words, the regulatory sequence acts in cis. In one embodiment, a gene may be “directly linked” to a regulatory sequence in a manner which allows expression of the gene. In another embodiment, a gene may be “indirectly linked” to a regulatory sequence in a manner which allows expression of the gene. In one embodiment, two or more genes may be directly or indirectly linked to a regulatory sequence in a manner which allows expression of the two or more genes. A regulatory region or sequence is a nucleic acid that can direct transcription of a gene of interest and may comprise promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5′ and 3′ untranslated regions, transcriptional start sites, termination sequences, polyadenylation sequences, and introns. 
     A “promoter” as used herein, refers to a nucleotide sequence that is capable of controlling the expression of a coding sequence or gene. Promoters are generally located 5′ of the sequence that they regulate. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from promoters found in nature, and/or comprise synthetic nucleotide segments. Those skilled in the art will readily ascertain that different promoters may regulate expression of a coding sequence or gene in response to a particular stimulus, e.g., in a cell- or tissue-specific manner, in response to different environmental or physiological conditions, or in response to specific compounds. Prokaryotic promoters are typically classified into two classes: inducible and constitutive. A “constitutive promoter” refers to a promoter that allows for continual transcription of the coding sequence or gene under its control. 
     “Constitutive promoter” refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and/or to which it is operably linked. Constitutive promoters and variants are well known in the art and include, but are not limited to, Ptac promoter, BBa_J23100, a constitutive  Escherichia coli  σS promoter (e.g., an osmY promoter (International Genetically Engineered Machine (iGEM) Registry of Standard Biological Parts Name BBa_J45992; BBa_J45993)), a constitutive  Escherichia coli  σ32 promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutive  Escherichia coli  σ70 promoter (e.g., lacq promoter (BBa_J54200; BBa_J56015),  E. coli  CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa_K119000; BBa_K119001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter (BBa_M13103). M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter (BBa_M13108), M13110 (BBa_M113110)), a constitutive  Bacillus subtilis  σA promoter (e.g., promoter veg (BBa_K143013), promoter 43 (BBa_K143013), PliaG (BBa_K823000), PlepA (BBa_K823002), Pveg (BBa_K823003)), a constitutive  Bacillus subtilis  σB promoter (e.g., promoter ctc (BBa K143010), promoter gsiB (BBa K143011)), a  Salmonella  promoter (e.g., Pspv2 from  Salmonella  (BBa_K112706), Pspv from  Salmonella  (BBa_K112707)), a bacteriophage T7 promoter (e.g., T7 promoter (BBa_I712074; BBa_I719005; BBa_J34814; BBa_J64997: BBa_K113010; BBa_K113011; BBa_K113012; BBa_R0085: BBa_R0180; BBa_R0181; BBa_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252; BBa_Z0253)), and a bacteriophage SP6 promoter (e.g., SP6 promoter (BBa_J64998)). 
     An “inducible promoter” refers to a regulatory region that is operably linked to one or more genes, wherein expression of the gene(s) is increased in the presence of an inducer of said regulatory region. An “inducible promoter” refers to a promoter that initiates increased levels of transcription of the coding sequence or gene under its control in response to a stimulus or an exogenous environmental condition. A “directly inducible promoter” refers to a regulatory region, wherein the regulatory region is operably linked to a gene encoding a protein or polypeptide, where, in the presence of an inducer of said regulatory region, the protein or polypeptide is expressed. An “indirectly inducible promoter” refers to a regulatory system comprising two or more regulatory regions, for example, a first regulatory region that is operably linked to a first gene encoding a first protein, polypeptide, or factor, e.g., a transcriptional regulator, which is capable of regulating a second regulatory region that is operably linked to a second gene, the second regulatory region may be activated or repressed, thereby activating or repressing expression of the second gene. Both a directly inducible promoter and an indirectly inducible promoter are encompassed by “inducible promoter.” Exemplary inducible promoters described herein include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. Examples of inducible promoters include, but are not limited to, an FNR responsive promoter, a ParaC promoter, a ParaBAD promoter, and a PTetR promoter, each of which are described in more detail herein. Examples of other inducible promoters are provided herein below. 
     As used herein, “stably maintained” or “stable” bacterium is used to refer to a bacterial host cell carrying non-native genetic material, e.g., a gene encoding one or more anti-inflammation and/or gut barrier enhancer molecule(s), which is incorporated into the host genome or propagated on a self-replicating extra-chromosomal plasmid, such that the non-native genetic material is retained, expressed, and propagated. The stable bacterium is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. For example, the stable bacterium may be a genetically engineered bacterium comprising a gene encoding a encoding a payload, e.g., one or more anti-inflammation and/or gut barrier enhancer molecule(s), in which the plasmid or chromosome carrying the gene is stably maintained in the bacterium, such that the payload can be expressed in the bacterium, and the bacterium is capable of survival and/or growth in vitro and/or in vivo. In some embodiments, copy number affects the stability of expression of the non-native genetic material. In some embodiments, copy number affects the level of expression of the non-native genetic material. 
     As used herein, the term “expression” refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA derived from a nucleic acid, and/or to translation of an mRNA into a polypeptide. 
     As used herein, the term “plasmid” or “vector” refers to an extrachromosomal nucleic acid, e.g., DNA, construct that is not integrated into a bacterial cell&#39;s genome. Plasmids are usually circular and capable of autonomous replication. Plasmids may be low-copy, medium-copy, or high-copy, as is well known in the art Plasmids may optionally comprise a selectable marker, such as an antibiotic resistance gene, which helps select for bacterial cells containing the plasmid and which ensures that the plasmid is retained in the bacterial cell. A plasmid disclosed herein may comprise a nucleic acid sequence encoding a heterologous gene, e.g., a gene encoding an anti-inflammatory or gut barrier enhancer molecule. 
     As used herein, the term “transform” or “transformation” refers to the transfer of a nucleic acid fragment into a host bacterial cell, resulting in genetically-stable inheritance. Host bacterial cells comprising the transformed nucleic acid fragment are referred to as “recombinant” or “transgenic” or “transformed” organisms. 
     The term “genetic modification,” as used herein, refers to any genetic change. Exemplary genetic modifications include those that increase, decrease, or abolish the expression of a gene, including, for example, modifications of native chromosomal or extrachromosomal genetic material. Exemplary genetic modifications also include the introduction of at least one plasmid, modification, mutation, base deletion, base addition, base substitution, and/or codon modification of chromosomal or extrachromosomal genetic sequence(s), gene over-expression, gene amplification, gene suppression, promoter modification or substitution, gene addition (either single or multi-copy), antisense expression or suppression, or any other change to the genetic elements of a host cell, whether the change produces a change in phenotype or not. Genetic modification can include the introduction of a plasmid, e.g., a plasmid comprising an anti-inflammatory or gut barrier enhancer molecule operably linked to a promoter, into a bacterial cell. Genetic modification can also involve a targeted replacement in the chromosome, e.g., to replace a native gene promoter with an inducible promoter, regulated promoter, strong promoter, or constitutive promoter. Genetic modification can also involve gene amplification, e.g., introduction of at least one additional copy of a native gene into the chromosome of the cell. Alternatively, chromosomal genetic modification can involve a genetic mutation. 
     As used herein, the term “genetic mutation” refers to a change or changes in a nucleotide sequence of a gene or related regulatory region that alters the nucleotide sequence as compared to its native or wild-type sequence. Mutations include, for example, substitutions, additions, and deletions, in whole or in part, within the wild-type sequence. Such substitutions, additions, or deletions can be single nucleotide changes (e.g., one or more point mutations), or can be two or more nucleotide changes, which may result in substantial changes to the sequence. Mutations can occur within the coding region of the gene as well as within the non-coding and regulatory sequence of the gene. The term “genetic mutation” is intended to include silent and conservative mutations within a coding region as well as changes which alter the amino acid sequence of the polypeptide encoded by the gene. A genetic mutation in a gene coding sequence may, for example, increase, decrease, or otherwise alter the activity (e.g., enzymatic activity) of the gene&#39;s polypeptide product. A genetic mutation in a regulatory sequence may increase, decrease, or otherwise alter the expression of sequences operably linked to the altered regulatory sequence. 
     As used herein, the term “transporter” is meant to refer to a mechanism, e.g., protein, proteins, or protein complex, for importing a molecule, e.g., amino acid, peptide (di-peptide, tri-peptide, polypeptide, etc), toxin, metabolite, substrate, as well as other biomolecules into the microorganism from the extracellular milieu. 
     As used herein, the phrase “exogenous environmental condition” or “exogenous environment signal” refers to settings, circumstances, stimuli, or biological molecules under which a promoter described herein is directly or indirectly induced. The phrase “exogenous environmental conditions” is meant to refer to the environmental conditions external to the engineered microorganism, but endogenous or native to the host subject environment. Thus, “exogenous” and “endogenous” may be used interchangeably to refer to environmental conditions in which the environmental conditions are endogenous to a mammalian body, but external or exogenous to an intact microorganism cell. In some embodiments, the exogenous environmental conditions are specific to the gut of a mammal. In some embodiments, the exogenous environmental conditions are specific to the upper gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the lower gastrointestinal tract of a mammal. In some embodiments, the exogenous environmental conditions are specific to the small intestine of a mammal. In some embodiments, the exogenous environmental conditions are low-oxygen, microaerobic, or anaerobic conditions, such as the environment of the mammalian gut. In some embodiments, exogenous environmental conditions are molecules or metabolites that are specific to the mammalian gut, e.g., propionate. In some embodiments, the exogenous environmental condition is a tissue-specific or disease-specific metabolite or molecule(s). In some embodiments, the exogenous environmental condition is specific to an inflammatory disease. In some embodiments, the exogenous environmental condition is a low-pH environment. In some embodiments, the genetically engineered microorganism of the disclosure comprises a pH-dependent promoter. In some embodiments, the genetically engineered microorganism of the disclosure comprise an oxygen level-dependent promoter. In some aspects, bacteria have evolved transcription factors that are capable of sensing oxygen levels. Different signaling pathways may be triggered by different oxygen levels and occur with different kinetics. An “oxygen level-dependent promoter” or “oxygen level-dependent regulatory region” refers to a nucleic acid sequence to which one or more oxygen level-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression. 
     Examples of oxygen level-dependent transcription factors include, but are not limited to, FNR (fumarate and nitrate reductase), ANR, and DNR. Corresponding FNR-responsive promoters, ANR (anaerobic nitrate respiration)-responsive promoters, and DNR (dissimilatory nitrate respiration regulator)-responsive promoters are known in the art (see, e.g., Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991; Hasegawa et al., 1998; Hoeren et al., 1993; Salmon et al., 2003), and non-limiting examples are shown in Table 1A. 
     In a non-limiting example, a promoter (PfnrS) was derived from the  E. coli  Nissle fumarate and nitrate reductase gene S (fnrS) that is known to be highly expressed under conditions of low or no environmental oxygen (Durand and Storz, 2010; Boysen et al, 2010). The PfnrS promoter is activated under anaerobic conditions by the global transcriptional regulator FNR that is naturally found in Nissle. Under anaerobic conditions, FNR forms a dimer and binds to specific sequences in the promoters of specific genes under its control, thereby activating their expression. However, under aerobic conditions, oxygen reacts with iron-sulfur clusters in FNR dimers and converts them to an inactive form. In this way, the PfnrS inducible promoter is adopted to modulate the expression of proteins or RNA. PfnrS is used interchangeably in this application as FNRS, fnrs, FNR, P-FNRS promoter and other such related designations to indicate the promoter PfnrS. 
     
       
         
           
               
             
               
                 TABLE 1A 
               
             
            
               
                   
               
               
                 Examples of transcription factors and 
               
               
                 responsive genes and regulatory regions 
               
            
           
           
               
               
               
            
               
                   
                 Transcription 
                 Examples of responsive genes, 
               
               
                   
                 Factor 
                 promoters, and/or regulatory regions: 
               
               
                   
                   
               
               
                   
                 FNR 
                 nirB, ydfZ, pdhR, focA, ndH, hlyE, narK, 
               
               
                   
                   
                 narX, narG, yfiD, tdcD 
               
               
                   
                 ANR 
                 arcDABC 
               
               
                   
                 DNR 
                 norb, norC 
               
               
                   
                   
               
            
           
         
       
     
     As used herein, a “tunable regulatory region” refers to a nucleic acid sequence under direct or indirect control of a transcription factor and which is capable of activating, repressing, derepressing, or otherwise controlling gene expression relative to levels of an inducer. In some embodiments, the tunable regulatory region comprises a promoter sequence. The inducer may be RNS, or other inducer described herein, and the tunable regulatory region may be a RNS-responsive regulatory region or other responsive regulatory region described herein. The tunable regulatory region may be operatively linked to a gene sequence(s) or gene cassette for the production of one or more payloads, e.g., a butyrogenic or other gene cassette or gene sequence(s). For example, in one specific embodiment, the tunable regulatory region is a RNS-derepressible regulatory region, and when RNS is present, a RNS-sensing transcription factor no longer binds to and/or represses the regulatory region, thereby permitting expression of the operatively linked gene or gene cassette. In this instance, the tunable regulatory region derepresses gene or gene cassette expression relative to RNS levels. Each gene or gene cassette may be operatively linked to a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one RNS. 
     In some embodiments, the exogenous environmental conditions are the presence or absence of reactive oxygen species (ROS). In other embodiments, the exogenous environmental conditions are the presence or absence of reactive nitrogen species (RNS). In some embodiments, exogenous environmental conditions are biological molecules that are involved in the inflammatory response, for example, molecules present in an inflammatory disorder of the gut. In some embodiments, the exogenous environmental conditions or signals exist naturally or are naturally absent in the environment in which the recombinant bacterial cell resides. In some embodiments, the exogenous environmental conditions or signals are artificially created, for example, by the creation or removal of biological conditions and/or the administration or removal of biological molecules. 
     In some embodiments, the exogenous environmental condition(s) and/or signal(s) stimulates the activity of an inducible promoter. In some embodiments, the exogenous environmental condition(s) and/or signal(s) that serves to activate the inducible promoter is not naturally present within the gut of a mammal. In some embodiments, the inducible promoter is stimulated by a molecule or metabolite that is administered in combination with the pharmaceutical composition of the disclosure, for example, tetracycline, arabinose, or any biological molecule that serves to activate an inducible promoter. In some embodiments, the exogenous environmental condition(s) and/or signal(s) is added to culture media comprising a recombinant bacterial cell of the disclosure. In some embodiments, the exogenous environmental condition that serves to activate the inducible promoter is naturally present within the gut of a mammal (for example, low oxygen or anaerobic conditions, or biological molecules involved in an inflammatory response). In some embodiments, the loss of exposure to an exogenous environmental condition (for example, in vivo) inhibits the activity of an inducible promoter, as the exogenous environmental condition is not present to induce the promoter (for example, an aerobic environment outside the gut). “Gut” refers to the organs, glands, tracts, and systems that are responsible for the transfer and digestion of food, absorption of nutrients, and excretion of waste. In humans, the gut comprises the gastrointestinal (GI) tract, which starts at the mouth and ends at the anus, and additionally comprises the esophagus, stomach, small intestine, and large intestine. The gut also comprises accessory organs and glands, such as the spleen, liver, gallbladder, and pancreas. The upper gastrointestinal tract comprises the esophagus, stomach, and duodenum of the small intestine. The lower gastrointestinal tract comprises the remainder of the small intestine, i.e., the jejunum and ileum, and all of the large intestine, i.e., the cecum, colon, rectum, and anal canal. Bacteria can be found throughout the gut, e.g., in the gastrointestinal tract, and particularly in the intestines. 
     As used herein, the term “low oxygen” is meant to refer to a level, amount, or concentration of oxygen (O 2 ) that is lower than the level, amount, or concentration of oxygen that is present in the atmosphere (e.g., &lt;21% O 2 :&lt;160 torr O 2 ). Thus, the term “low oxygen condition or conditions” or “low oxygen environment” refers to conditions or environments containing lower levels of oxygen than are present in the atmosphere. In some embodiments, the term “low oxygen” is meant to refer to the level, amount, or concentration of oxygen (O 2 ) found in a mammalian gut, e.g., lumen, stomach, small intestine, duodenum, jejunum, ileum, large intestine, cecum, colon, distal sigmoid colon, rectum, and anal canal. In some embodiments, the term “low oxygen” is meant to refer to a level, amount, or concentration of O 2  that is 0-60 mmHg O 2  (0-60 torr O 2 ) (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 mmHg O 2 ), including any and all incremental fraction(s) thereof (e.g., 0.2 mmHg, 0.5 mmHg O 2 , 0.75 mmHg O 2 , 1.25 mmHg O 2 , 2.175 mmHg O 2 , 3.45 mmHg O 2 , 3.75 mmHg O 2 , 4.5 mmHg O 2 , 6.8 mmHg O 2 , 11.35 mmHg O 2 , 46.3 mmHg O 2 , 58.75 mmHg, etc., which exemplary fractions are listed here for illustrative purposes and not meant to be limiting in any way). In some embodiments, “low oxygen” refers to about 60 mmHg O 2  or less (e.g., 0 to about 60 mmHg O 2 ). The term “low oxygen” may also refer to a range of O 2  levels, amounts, or concentrations between 0-60 mmHg O 2  (inclusive), e.g., 0-5 mmHg O 2 , &lt;1.5 mmHg O 2 , 6-10 mmHg, &lt;8 mmHg, 47-60 mmHg, etc. which listed exemplary ranges are listed here for illustrative purposes and not meant to be limiting in any way. See, for example, Albenberg et al., Gastroenterology, 147(5): 1055-1063 (2014); Bergofsky et al., J Clin. Invest., 41(11): 1971-1980 (1962); Crompton et al., J Exp. Biol., 43: 473-478 (1965); He et al., PNAS (USA), 96: 4586-4591 (1999); McKeown, Br. J. Radiol., 87:20130676 (2014) (doi: 10.1259/brj.20130676), each of which discusses the oxygen levels found in the mammalian gut of various species and each of which are incorporated by reference herewith in their entireties. In some embodiments, the term “low oxygen” is meant to refer to the level, amount, or concentration of oxygen (O 2 ) found in a mammalian organ or tissue other than the gut, e.g., urogenital tract, tumor tissue, etc. in which oxygen is present at a reduced level, e.g., at a hypoxic or anoxic level. In some embodiments, “low oxygen” is meant to refer to the level, amount, or concentration of oxygen (O 2 ) present in partially aerobic, semi aerobic, microaerobic, nanoaerobic, microoxic, hypoxic, anoxic, and/or anaerobic conditions. For example, Table 1B summarizes the amount of oxygen present in various organs and tissues. In some embodiments, the level, amount, or concentration of oxygen (O 2 ) is expressed as the amount of dissolved oxygen (“DO”) which refers to the level of free, non-compound oxygen (O 2 ) present in liquids and is typically reported in milligrams per liter (mg/L), parts per million (ppm; 1 mg/L=1 ppm), or in micromoles (umole) (1 umole O 2 =0.022391 mg/L O 2 ). Fondriest Environmental, Inc., “Dissolved Oxygen”, Fundamentals of Environmental Measurements, 19 Nov. 2013, www.fondriest.com/environmental-measurements/parameters/water-quality/dissolved-oxygen/&gt;. In some embodiments, the term “low oxygen” is meant to refer to a level, amount, or concentration of oxygen (O 2 ) that is about 6.0 mg/L DO or less, e.g., 6.0 mg/L, 5.0 mg/L, 4.0 mg/L, 3.0 mg/L, 2.0 mg/L, 1.0 mg/L, or 0 mg/L, and any fraction therein, e.g., 3.25 mg/L, 2.5 mg/L, 1.75 mg/L, 1.5 mg/L, 1.25 mg/L, 0.9 mg/L, 0.8 mg/L, 0.7 mg/L, 0.6 mg/L, 0.5 mg/L, 0.4 mg/L, 0.3 mg/L, 0.2 mg/L and 0.1 mg/L DO, which exemplary fractions are listed here for illustrative purposes and not meant to be limiting in any way. The level of oxygen in a liquid or solution may also be reported as a percentage of air saturation or as a percentage of oxygen saturation (the ratio of the concentration of dissolved oxygen (O 2 ) in the solution to the maximum amount of oxygen that will dissolve in the solution at a certain temperature, pressure, and salinity under stable equilibrium). Well-aerated solutions (e.g., solutions subjected to mixing and/or stirring) without oxygen producers or consumers are 100% air saturated. In some embodiments, the term “low oxygen” is meant to refer to 40% air saturation or less, e.g., 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0% air saturation, including any and all incremental fraction(s) thereof (e.g., 30.25%, 22.70%, 15.5%, 7.7%, 5.0%, 2.8%, 2.0%, 1.65%, 1.0%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%. 0.44%, 0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%. 0.032%, 0.025%, 0.01%, etc.) and any range of air saturation levels between 0-40%, inclusive (e.g., 0-5%, 0.05-0.1%, 0.1-0.2%, 0.1-0.5%, 0.5-2.0%, 0-10%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, etc.). The exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way. In some embodiments, the term “low oxygen” is meant to refer to 9% O 2  saturation or less, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0%, O 2  saturation, including any and all incremental fraction(s) thereof (e.g., 6.5%, 5.0%, 2.2%, 1.7%, 1.4%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%. 0.44%, 0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%. 0.032%, 0.025%, 0.01%, etc.) and any range of O 2  saturation levels between 0-9%, inclusive (e.g., 0-5%, 0.05-0.1%, 0.1-0.2%, 0.1-0.5%, 0.5-2.0%, 0-8%, 5-7%, 0.3-4.2% O 2 . etc.). The exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way. 
     
       
         
           
               
               
             
               
                 TABLE 1B 
               
               
                   
               
               
                 Compartment 
                 Oxygen Tension 
               
               
                   
               
             
            
               
                 stomach 
                 ~60 torr (e.g., 58 +/− 15 torr) 
               
               
                 duodenum and first part of 
                 ~30 torr (e.g., 32 +/− 8 torr); 
               
               
                 jejunum 
                 ~20% oxygen in ambient air 
               
               
                 Ileum (mid- small intestine) 
                 ~10 torr; ~6% oxygen in ambient air 
               
               
                   
                 (e.g., 11 +/− 3 torr) 
               
               
                 Distal sigmoid colon 
                 ~3 torr (e.g., 3 +/− 1 torr) 
               
               
                 colon 
                 &lt;2 torr 
               
               
                 Lumen of cecum 
                 &lt;1 torr 
               
               
                 tumor 
                 &lt;32 torr (most tumors are &lt;15 torr) 
               
               
                   
               
            
           
         
       
     
     “Microorganism” refers to an organism or microbe of microscopic, submicroscopic, or ultramicroscopic size that typically consists of a single cell. Examples of microrganisms include bacteria, viruses, parasites, fungi, certain algae, yeast, e.g.,  Saccharomyces , and protozoa. In some aspects, the microorganism is engineered (“engineered microorganism”) to produce one or more therapeutic molecules, e.g., an antiinflammatory or barrier enhancer molecule. In certain embodiments, the engineered microorganism is an engineered bacterium. In certain embodiments, the engineered microorganism is an engineered virus. 
     “Non-pathogenic bacteria” refer to bacteria that are not capable of causing disease or harmful responses in a host. In some embodiments, non-pathogenic bacteria are Gram-negative bacteria. In some embodiments, non-pathogenic bacteria are Gram-positive bacteria. In some embodiments, non-pathogenic bacteria do not contain lipopolysaccharides (LPS). In some embodiments, non-pathogenic bacteria are commensal bacteria. Examples of non-pathogenic bacteria include, but are not limited to certain strains belonging to the genus  Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Saccharomyces , and  Staphylococcus , e.g.,  Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Escherichia coli, Escherichia coli  Nissle,  Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis  and,  Saccharomyces boulardii  (Sonnenborn et al., 2009; Dinleyici et al., 2014; U.S. Pat. Nos. 6,835,376; 6,203,797; 5,589,168; 7,731,976). Non-pathogenic bacteria also include commensal bacteria, which are present in the indigenous microbiota of the gut. In one embodiment, the disclosure further includes non-pathogenic  Saccharomyces , such as  Saccharomyces boulardii . Naturally pathogenic bacteria may be genetically engineered to reduce or eliminate pathogenicity. 
     “Probiotic” is used to refer to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an appropriate amount of the microorganism. In some embodiments, the host organism is a mammal. In some embodiments, the host organism is a human. In some embodiments, the probiotic bacteria are Gram-negative bacteria. In some embodiments, the probiotic bacteria are Gram-positive bacteria. Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic bacteria. Examples of probiotic bacteria include, but are not limited to, certain strains belonging to the genus  Bifidobacteria, Escherichia coli, Lactobacillus , and  Saccharomyces  e.g.,  Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli  strain Nissle,  Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei , and  Lactobacillus plantarum , and  Saccharomyces boulardii  (Dinleyici et al., 2014; U.S. Pat. Nos. 5,589,168; 6,203,797; 6,835,376). The probiotic may be a variant or a mutant strain of bacterium (Arthur et al., 2012; Cuevas-Ramos et al., 2010; Olier et al., 2012; Nougayrede et al., 2006). Non-pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability. Non-pathogenic bacteria may be genetically engineered to provide probiotic properties. Probiotic bacteria may be genetically engineered to enhance or improve probiotic properties. 
     As used herein, the term “modulate” and its cognates means to alter, regulate, or adjust positively or negatively a molecular or physiological readout, outcome, or process, to effect a change in said readout, outcome, or process as compared to a normal, average, wild-type, or baseline measurement. Thus, for example, “modulate” or “modulation” includes up-regulation and down-regulation. A non-limiting example of modulating a readout, outcome, or process is effecting a change or alteration in the normal or baseline functioning, activity, expression, or secretion of a biomolecule (e.g. a protein, enzyme, cytokine, growth factor, hormone, metabolite, short chain fatty acid, or other compound). Another non-limiting example of modulating a readout, outcome, or process is effecting a change in the amount or level of a biomolecule of interest, e.g. in the serum and/or the gut lumen. In another non-limiting example, modulating a readout, outcome, or process relates to a phenotypic change or alteration in one or more disease symptoms. Thus, “modulate” is used to refer to an increase, decrease, masking, altering, overriding or restoring the normal functioning, activity, or levels of a readout, outcome or process (e.g, biomolecule of interest, and/or molecular or physiological process, and/or a phenotypic change in one or more disease symptoms). 
     As used herein, the term “auxotroph” or “auxotrophic” refers to an organism that requires a specific factor, e.g., an amino acid, a sugar, or other nutrient) to support its growth. An “auxotrophic modification” is a genetic modification that causes the organism to die in the absence of an exogenously added nutrient essential for survival or growth because it is unable to produce said nutrient. As used herein, the term “essential gene” refers to a gene which is necessary to for cell growth and/or survival. Essential genes are described in more detail infra and include, but are not limited to, DNA synthesis genes (such as thrA), cell wall synthesis genes (such as dapA), and amino acid genes (such as serA and metA). 
     As used herein, the terms “modulate” and “treat” a disease and their cognates refer to an amelioration of a disease, disorder, and/or condition, or at least one discernible symptom thereof. In another embodiment, “modulate” and “treat” refer to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient. In another embodiment, “modulate” and “treat” refer to inhibiting the progression of a disease, disorder, and/or condition, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. In another embodiment, “modulate” and “treat” refer to slowing the progression or reversing the progression of a disease, disorder, and/or condition. As used herein, “prevent” and its cognates refer to delaying the onset or reducing the risk of acquiring a given disease, disorder and/or condition or a symptom associated with such disease, disorder, and/or condition. 
     Those in need of treatment may include individuals already having a particular medical disorder, as well as those at risk of having, or who may ultimately acquire the disorder. The need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disorder, the presence or progression of a disorder, or likely receptiveness to treatment of a subject having the disorder. Treating autoimmune disorders and/or diseases and conditions associated with gut inflammation and/or compromised gut barrier function may encompass reducing or eliminating excess inflammation and/or associated symptoms, and does not necessarily encompass the elimination of the underlying disease. 
     Treating the diseases described herein may encompass increasing levels of butyrate, increasing levels of acetate, increasing levels of butyrate and increasing GLP-2, IL-22, and/o rIL-10, and/or modulating levels of tryptophan and/or its metabolites (e.g., kynurenine), and/or providing any other anti-inflammation and/or gut barrier enhancer molecule and does not necessarily encompass the elimination of the underlying disease. 
     As used herein a “pharmaceutical composition” refers to a preparation of genetically engineered microorganism of the disclosure, e.g., genetically engineered bacteria or virus, with other components such as a physiologically suitable carrier and/or excipient. 
     The phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be used interchangeably refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered bacterial or viral compound. An adjuvant is included under these phrases. 
     The term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples include, but are not limited to, calcium bicarbonate, sodium bicarbonate calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20. 
     The terms “therapeutically effective dose” and “therapeutically effective amount” are used to refer to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a condition, e.g., inflammation, diarrhea an autoimmune disorder. A therapeutically effective amount may, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms of an autoimmune a disorder and/or a disease or condition associated with gut inflammation and/or compromised gut barrier function. A therapeutically effective amount, as well as a therapeutically effective frequency of administration, can be determined by methods known in the art and discussed below. 
     As used herein, the term “bacteriostatic” or “cytostatic” refers to a molecule or protein which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of recombinant bacterial cell of the disclosure. 
     As used herein, the term “bactericidal” refers to a molecule or protein which is capable of killing the recombinant bacterial cell of the disclosure. 
     As used herein, the term “toxin” refers to a protein, enzyme, or polypeptide fragment thereof, or other molecule which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of the recombinant bacterial cell of the disclosure, or which is capable of killing the recombinant bacterial cell of the disclosure. The term “toxin” is intended to include bacteriostatic proteins and bactericidal proteins. The term “toxin” is intended to include, but not limited to, lytic proteins, bacteriocins (e.g., microcins and colicins), gyrase inhibitors, polymerase inhibitors, transcription inhibitors, translation inhibitors, DNases, and RNases. The term “anti-toxin” or “antitoxin,” as used herein, refers to a protein or enzyme which is capable of inhibiting the activity of a toxin. The term anti-toxin is intended to include, but not limited to, immunity modulators, and inhibitors of toxin expression. Examples of toxins and antitoxins are known in the art and described in more detail in/i a. 
     As used herein, “payload” refers to one or more molecules of interest to be produced by a genetically engineered microorganism, such as a bacteria or a virus. In some embodiments, the payload is a therapeutic payload, e.g. and antiinflammatory or gut barrier enhancer molecule, e.g. butyrate, acetate, propionate, GLP-2, IL-10, IL-22, IL-2, other interleukins, and/or tryptophan and/or one or more of its metabolites. In some embodiments, the payload is a regulatory molecule, e.g., a transcriptional regulator such as FNR. In some embodiments, the payload comprises a regulatory element, such as a promoter or a repressor. In some embodiments, the payload comprises an inducible promoter, such as from FNRS. In some embodiments the payload comprises a repressor element, such as a kill switch. In some embodiments the payload comprises an antibiotic resistance gene or genes. In some embodiments, the payload is encoded by a gene, multiple genes, gene cassette, or an operon. In alternate embodiments, the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway may optionally be endogenous to the microorganism. In alternate embodiments, the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway is not endogenous to the microorganism. In some embodiments, the genetically engineered microorganism comprises two or more payloads. 
     As used herein, the term “conventional treatment” or “conventional therapy” refers to treatment or therapy that is currently accepted, considered current standard of care, and/or used by most healthcare professionals for treating a disease or disorder associated with BCAA. It is different from alternative or complementary therapies, which are not as widely used. 
     As used herein, the term “polypeptide” includes “polypeptide” as well as “polypeptides,” and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (i.e., peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, “peptides,” “dipeptides,” “tripeptides, “oligopeptides,” “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of“polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology. In other embodiments, the polypeptide is produced by the genetically engineered bacteria or virus of the current invention. A polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides, which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, are referred to as unfolded. The term “peptide” or “polypeptide” may refer to an amino acid sequence that corresponds to a protein or a portion of a protein or may refer to an amino acid sequence that corresponds with non-protein sequence, e.g., a sequence selected from a regulatory peptide sequence, leader peptide sequence, signal peptide sequence, linker peptide sequence, and other peptide sequence. 
     An “isolated” polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required. Recombinantly produced polypeptides and proteins expressed in host cells, including but not limited to bacterial or mammalian cells, are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique. Recombinant peptides, polypeptides or proteins refer to peptides, polypeptides or proteins produced by recombinant DNA techniques, i.e. produced from cells, microbial or mammalian, transformed by an exogenous recombinant DNA expression construct encoding the polypeptide. Proteins or peptides expressed in most bacterial cultures will typically be free of glycan. Fragments, derivatives, analogs or variants of the foregoing polypeptides, and any combination thereof are also included as polypeptides. The terms “fragment,” “variant,” “derivative” and “analog” include polypeptides having an amino acid sequence sufficiently similar to the amino acid sequence of the original peptide and include any polypeptides, which retain at least one or more properties of the corresponding original polypeptide. Fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments. Fragments also include specific antibody or bioactive fragments or immunologically active fragments derived from any polypeptides described herein. Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants may be produced using mutagenesis methods known in the art. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. 
     Polypeptides also include fusion proteins. As used herein, the term “variant” includes a fusion protein, which comprises a sequence of the original peptide or sufficiently similar to the original peptide. As used herein, the term “fusion protein” refers to a chimeric protein comprising amino acid sequences of two or more different proteins. Typically, fusion proteins result from well known in vitro recombination techniques. Fusion proteins may have a similar structural function (but not necessarily to the same extent), and/or similar regulatory function (but not necessarily to the same extent), and/or similar biochemical function (but not necessarily to the same extent) and/or immunological activity (but not necessarily to the same extent) as the individual original proteins which are the components of the fusion proteins. “Derivatives” include but are not limited to peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. “Similarity” between two peptides is determined by comparing the amino acid sequence of one peptide to the sequence of a second peptide. An amino acid of one peptide is similar to the corresponding amino acid of a second peptide if it is identical or a conservative amino acid substitution. Conservative substitutions include those described in Dayhoff, M. O., ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, D.C. (1978), and in Argos, EMBO J. 8 (1989), 779-785. For example, amino acids belonging to one of the following groups represent conservative changes or substitutions: -Ala, Pro, Gly, Gln, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -Val, Ile, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and -Asp, Glu. 
     An antibody generally refers to a polypeptide of the immunoglobulin family or a polypeptide comprising fragments of an immunoglobulin that is capable of noncovalently, reversibly, and in a specific manner binding a corresponding antigen. An exemplary antibody structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD), connected through a disulfide bond. The recognized immunoglobulin genes include the κ, λ, α, γ, δ, ε, and μ constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these regions of light and heavy chains respectively. 
     As used herein, the term “antibody” or “antibodies” is meant to encompasses all variations of antibody and fragments thereof that possess one or more particular binding specificities. Thus, the term “antibody” or “antibodies” is meant to include full length antibodies, chimeric antibodies, humanized antibodies, single chain antibodies (ScFv, camelids), Fab, Fab′, multimeric versions of these fragments (e.g., F(ab′)2), single domain antibodies (sdAB, VHH framents), heavy chain antibodies (HCAb), nanobodies, diabodies, and minibodies. Antibodies can have more than one binding specificity, e.g., be bispecific. The term “antibody” is also meant to include so-called antibody mimetics. Antibody mimetics refers to small molecules, e.g., 3-30 kDa, which can be single amino acid chain molecules, which can specifically bind antigens but do not have an antibody-related structure. Antibody mimetics, include, but are not limited to, Affibody molecules (Z domain of Protein A), Affilins (Gamma-B crystalline), Ubiquitin, Affimers (Cystatin), Affitins (Sac7d (from  Sulfolobus acidocaldarius ), Alphabodies (Triple helix coiled coil), Anticalins (Lipocalins), Avimers (domains of various membrane receptors), DARPins (Ankyrin repeat motif), Fynomers (SH3 domain of Fyn), Kunitz domain peptides Kunitz domains of various protease inhibitors), Ecallantide (Kalbitor), and Monobodies. In certain aspects, the term “antibody” or “antibodies” is meant to refer to a single chain antibody(ies), single domain antibody(ies), and camelid antibody(ies). Utility of antibodies in the treatment of cancer and additional anti cancer antibodies can for example be found in Scott et al., Antibody Therapy for Cancer, Nature Reviews Cancer April 2012 Volume 12, incorporated by reference in its entirety. 
     A “single-chain antibody” or “single-chain antibodies” typically refers to a peptide comprising a heavy chain of an immunoglobulin, a light chain of an immunoglobulin, and optionally a linker or bond, such as a disulfide bond. The single-chain antibody lacks the constant Fc region found in traditional antibodies. In some embodiments, the single-chain antibody is a naturally occurring single-chain antibody, e.g., a camelid antibody. In some embodiments, the single-chain antibody is a synthetic, engineered, or modified single-chain antibody. In some embodiments, the single-chain antibody is capable of retaining substantially the same antigen specificity as compared to the original immunoglobulin despite the addition of a linker and the removal of the constant regions. In some aspects, the single chain antibody can be a “scFv antibody”, which refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins (without any constant regions), optionally connected with a short linker peptide of ten to about 25 amino acids, as described, for example, in U.S. Pat. No. 4,946,778, the contents of which is herein incorporated by reference in its entirety. The Fv fragment is the smallest fragment that holds a binding site of an antibody, which binding site may, in some aspects, maintain the specificity of the original antibody. Techniques for the production of single chain antibodies are described in U.S. Pat. No. 4,946,778. The Vh and VL sequences of the scFv can be connected via the N-terminus of the VH connecting to the C-terminus of the VL or via the C-terminus of the VH connecting to the N-terminus of the VL. ScFv fragments are independent folding entities that can be fused indistinctively on either end to other epitope tags or protein domains. Linkers of varying length can be used to link the Vh and VL sequences, which the linkers can be glycine rich (provides flexibility) and serine or threonine rich (increases solubility). Short linkers may prevent association of the two domains and can result in multimers (diabodies, tribodies, etc.). Long linkers may result in proteolysis or weak domain association (described in Voelkel et al el., 2011). Linkers of length between 15 and 20 amino acids or 18 and 20 amino acids are most often used. Additional non-limiting examples of linkers, including other flexible linkers are described in Chen et al., 2013 (Adv Drug Deliv Rev. 2013 Oct. 15; 65(10): 1357-1369. Fusion Protein Linkers: Property, Design and Functionality), the contents of which is herein incorporated by reference in its entirety. Flexible linkers are also rich in small or polar amino acids such as Glycine and Serine, but can contain additional amino acids such as Threonine and Alanine to maintain flexibility, as well as polar amino acids such as Lysine and Glutamate to improve solubility. Exemplary linkers include, but are not limited to, (Gly-Gly-Gly-Gly-Ser)n, KESGSVSSEQLAQFRSLD and EGKSSGSGSESKST, (Gly)8, and Gly and Ser rich flexible linker, GSAGSAAGSGEF. “Single chain antibodies” as used herein also include single-domain antibodies, which include camelid antibodies and other heavy chain antibodies, light chain antibodies, including nanobodies and single domains VH or VL domains derived from human, mouse or other species. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine. Single domain antibodies include domain antigen-binding units which have a camelid scaffold, derived from camels, llamas, or alpacas. Camelids produce functional antibodies devoid of light chains. The heavy chain variable (VH) domain folds autonomously and functions independently as an antigen-binding unit. Its binding surface involves only three CDRs as compared to the six CDRs in classical antigen-binding molecules (Fabs) or single chain variable fragments (scFvs). Camelid antibodies are capable of attaining binding affinities comparable to those of conventional antibodies. Camelid scaffold-based antibodies can be produced using methods well known in the art. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, ‘immunoglobulin new antigen receptor’), from which single-domain antibodies called VNAR fragments can be obtained. Alternatively, the dimeric variable domains from IgG from humans or mice can be split into monomers. Nanobodies are single chain antibodies derived from light chains. The term “single chain antibody” also refers to antibody mimetics. 
     In some embodiments, the antibodies expressed by the engineered microorganisms are bispecific. In certain embodiments, a bispecific antibody molecule comprises a scFv, or fragment thereof, have binding specificity for a first epitope and a scFv, or fragment thereof, have binding specificity for a second epitope. Antigen-binding fragments or antibody portions include bivalent scFv (diabody), bispecific scFv antibodies where the antibody molecule recognizes two different epitopes, single binding domains (dAbs), and minibodies. Monomeric single-chain diabodies (scDb) are readily assembled in bacterial and mammalian cells and show improved stability under physiological conditions (Voelkel et al., 2001 and references therein; Protein Eng. (2001) 14 (10): 815-823 (describes optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies). 
     As used herein, the term “sufficiently similar” means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common functional activity. For example, amino acid sequences that comprise a common structural domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, identical are defined herein as sufficiently similar. Preferably, variants will be sufficiently similar to the amino acid sequence of the peptides of the invention. Such variants generally retain the functional activity of the peptides of the present invention. Variants include peptides that differ in amino acid sequence from the native and wt peptide, respectively, by way of one or more amino acid deletion(s), addition(s), and/or substitution(s). These may be naturally occurring variants as well as artificially designed ones. 
     As used herein the term “linker”, “linker peptide” or “peptide linkers” or “linker” refers to synthetic or non-native or non-naturally-occurring amino acid sequences that connect or link two polypeptide sequences, e.g., that link two polypeptide domains. As used herein the term “synthetic” refers to amino acid sequences that are not naturally occurring. Exemplary linkers are described herein. Additional exemplary linkers are provided in US 20140079701, the contents of which are herein incorporated by reference in its entirety. 
     As used herein the term “codon-optimized” refers to the modification of codons in the gene or coding regions of a nucleic acid molecule to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the nucleic acid molecule. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of the host organism. A “codon-optimized sequence” refers to a sequence, which was modified from an existing coding sequence, or designed, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host organism. Many organisms display a bias or preference for use of particular codons to code for insertion of a particular amino acid in a growing polypeptide chain. Codon preference or codon bias, differences in codon usage between organisms, is allowed by the degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. 
     As used herein, the terms “secretion system” or “secretion protein” refers to a native or non-native secretion mechanism capable of secreting or exporting a biomolecule, e.g., polypeptide from the microbial, e.g., bacterial cytoplasm. The secretion system may comprise a single protein or may comprise two or more proteins assembled in a complex e.g., HlyBD. Non-limiting examples of secretion systems for gram negative bacteria include the modified type III flagellar, type I (e.g., hemolysin secretion system), type II, type IV, type V, type VI, and type VII secretion systems, resistance-nodulation-division (RND) multi-drug efflux pumps, various single membrane secretion systems. Non-limiting examples of secretion systems for gram positive bacteria include Sec and TAT secretion systems. In some embodiments, the polypeptide to be secreted include a “secretion tag” of either RNA or peptide origin to direct the polypeptide to specific secretion systems. In some embodiments, the secretion system is able to remove this tag before secreting the polypeptide from the engineered bacteria. For example, in Type V auto-secretion-mediated secretion the N-terminal peptide secretion tag is removed upon translocation of the “passenger” peptide from the cytoplasm into the periplasmic compartment by the native Sec system. Further, once the auto-secretor is translocated across the outer membrane the C-terminal secretion tag can be removed by either an autocatalytic or protease-catalyzed e.g., OmpT cleavage thereby releasing the antiinflammatory or barrier enhancer molecule(s) into the extracellular milieu. In some embodiments, the secretion system involves the generation of a “leaky” or de-stabilized outer membrane, which may be accomplished by deleting or mutagenizing genes responsible for tethering the outer membrane to the rigid peptidoglycan skeleton, including for example, lpp, ompC, ompA, ompF, tolA, tolB, pal, degS, degP, and nipl. Lpp functions as the primary ‘staple’ of the bacterial cell wall to the peptidoglycan. TolA-PAL and OmpA complexes function similarly to Lpp and are other deletion targets to generate a leaky phenotype. Additionally, leaky phenotypes have been observed when periplasmic proteases, such as degS, degP or nlpl, are deactivated. Thus, in some embodiments, the engineered bacteria have one or more deleted or mutated membrane genes, e.g., selected from lpp, ompA, ompA, ompF, tolA, tolB, and pal genes. In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes, e.g., selected from degS, degP, and nlpl. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from lpp, ompA, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl genes. 
     The articles “a” and “an,” as used herein, should be understood to mean “at least one,” unless clearly indicated to the contrary. 
     The phrase “and/or,” when used between elements in a list, is intended to mean either (1) that only a single listed element is present, or (2) that more than one element of the list is present. For example, “A, B, and/or C” indicates that the selection may be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C. The phrase “and/or” may be used interchangeably with “at least one of” or “one or more of” the elements in a list. 
     Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. 
     Bacteria 
     The genetically engineered microorganisms, or programmed microorganisms, such as genetically engineered bacteria of the disclosure are capable of producing one or more non-native anti-inflammation and/or gut barrier function enhancer molecules. In certain embodiments, the genetically engineered bacteria are obligate anaerobic bacteria. In certain embodiments, the genetically engineered bacteria are facultative anaerobic bacteria. In certain embodiments, the genetically engineered bacteria are aerobic bacteria. In some embodiments, the genetically engineered bacteria are Gram-positive bacteria. In some embodiments, the genetically engineered bacteria are Gram-positive bacteria and lack LPS. In some embodiments, the genetically engineered bacteria are Gram-negative bacteria. In some embodiments, the genetically engineered bacteria are Gram-positive and obligate anaerobic bacteria. In some embodiments, the genetically engineered bacteria are Gram-positive and facultative anaerobic bacteria. In some embodiments, the genetically engineered bacteria are non-pathogenic bacteria. In some embodiments, the genetically engineered bacteria are commensal bacteria. In some embodiments, the genetically engineered bacteria are probiotic bacteria. In some embodiments, the genetically engineered bacteria are naturally pathogenic bacteria that are modified or mutated to reduce or eliminate pathogenicity. Exemplary bacteria include, but are not limited to,  Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Caulobacter, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Listeria, Mycobacterium, Saccharomyces, Salmonella, Staphylococcus, Streptococcus, Vibrio, Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve  UCC2003,  Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium acetobutylicum, Clostridium butyricum, Clostridium butyricum  M-55,  Clostridium cochlearum, Clostridium felsineum, Clostridium histolyticum, Clostridium multifermentans, Clostridium novyi -NT,  Clostridium paraputrificum, Clostridium pasteureanum, Clostridium pectinovorum, Clostridium perfringens, Clostridium roseum, Clostridium sporogenes, Clostridium tertium, Clostridium tetani, Clostridium tyrobutyricum, Corynebacterium parvum, Escherichia coli  MG 1655,  Escherichia coli  Nissle 1917,  Listeria monocytogenes, Mycobacterium bovis, Salmonella choleraesuis, Salmonella typhimurium , and  Vibrio cholera . In certain embodiments, the genetically engineered bacteria are selected from the group consisting of  Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis , and  Saccharomyces boulardii, Clostridium  clusters IV and XIVa of Firmicutes (including species of  Eubacterium ),  Roseburia, Faecalibacterium, Enterobacter, Faecalibacterium prausnitzii, Clostridium difficile, Subdoligranulum, Clostridium sporogenes, Campylobacter jejuni, Clostridium saccharolyticum, Klebsiella, Citrobacter, Pseudobutyrivibrio , and  Ruminococcus . In certain embodiments, the the genetically engineered bacteria are selected from  Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Escherichia coli, Escherichia coli  Nissle,  Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri , and  Lactococcus lactis    
     In some embodiments, the genetically engineered bacterium is a Gram-positive bacterium, e.g.,  Clostridium , that is naturally capable of producing high levels of butyrate. In some embodiments, the genetically engineered bacterium is selected from the group consisting of  C. butyricum  ZJUCB,  C. butyricum  S21,  C. thermobutyricum  ATCC 49875,  C. beijerinckii, C. populeti  ATCC 35295,  C. tyrobutyricum  JM1,  C. tyrobutyricum  CIP 1-776,  C. tyrobutyricum  ATCC 25755,  C. tyrobutyricum  CNRZ 596, and  C. tyrobutyricum  ZJU 8235. In some embodiments, the genetically engineered bacterium is  C. butyricum  CBM588, a probiotic bacterium that is highly amenable to protein secretion and has demonstrated efficacy in treating IBD (Kanai et al., 2015). In some embodiments, the genetically engineered bacterium is  Bacillus , a probiotic bacterium that is highly genetically tractable and has been a popular chassis for industrial protein production; in some embodiments, the bacterium has highly active secretion and/or no toxic byproducts (Cutting, 2011). 
     In one embodiment, the bacterial cell is a  Bacteroides fragilis  bacterial cell. In one embodiment, the bacterial cell is a  Bacteroides thetaiotaomicron  bacterial cell. In one embodiment, the bacterial cell is a  Bacteroides subtilis  bacterial cell. In one embodiment, the bacterial cell is a  Bifidobacterium bifidum  bacterial cell. In one embodiment, the bacterial cell is a  Bifidobacterium infantis  bacterial cell. In one embodiment, the bacterial cell is a  Bifidobacterium lactis  bacterial cell. In one embodiment, the bacterial cell is a  Clostridium butyricum  bacterial cell. In one embodiment, the bacterial cell is an  Escherichia coli  bacterial cell. In one embodiment, the bacterial cell is a  Lactobacillus acidophilus  bacterial cell. In one embodiment, the bacterial cell is a  Lactobacillus plantarum  bacterial cell. In one embodiment, the bacterial cell is a  Lactobacillus reuteri  bacterial cell. In one embodiment, the bacterial cell is a  Lactococcus lactis  bacterial cell. 
     In some embodiments, the genetically engineered bacteria are  Escherichia coli  strain Nissle 1917 ( E. coli  Nissle), a Gram-negative bacterium of the Enterobacteriaceae family that has evolved into one of the best characterized probiotics (Ukena et al., 2007). The strain is characterized by its complete harmlessness (Schultz, 2008), and has GRAS (generally recognized as safe) status (Reister et al., 2014, emphasis added). Genomic sequencing confirmed that  E. coli  Nissle lacks prominent virulence factors (e.g.,  E. coli  α-hemolysin, P-fimbrial adhesins) (Schultz, 2008). In addition, it has been shown that  E. coli  Nissle does not carry pathogenic adhesion factors, does not produce any enterotoxins or cytotoxins, is not invasive, and not uropathogenic (Sonnenborn et al., 2009). As early as in 1917,  E. coli  Nissle was packaged into medicinal capsules, called Mutaflor, for therapeutic use.  E. coli  Nissle has since been used to treat ulcerative colitis in humans in vivo (Rembacken et al., 1999), to treat inflammatory bowel disease, Crohn&#39;s disease, and pouchitis in humans in vivo (Schultz, 2008), and to inhibit enteroinvasive  Salmonella, Legionella, Yersinia , and  Shigella  in vitro (Altenhoefer et al., 2004). It is commonly accepted that  E. coli  Nissle&#39;s therapeutic efficacy and safety have convincingly been proven (Ukena et al., 2007). In some embodiments, the genetically engineered bacteria are  E. coli  Nissle and are naturally capable of promoting tight junctions and gut barrier function. In some embodiments, the genetically engineered bacteria are  E. coli  and are highly amenable to recombinant protein technologies. 
     One of ordinary skill in the art would appreciate that the genetic modifications disclosed herein may be adapted for other species, strains, and subtypes of bacteria. It is known, for example, that the clostridial butyrogenic pathway genes are widespread in the genome-sequenced clostridia and related species (Aboulnaga et al., 2013). Furthermore, genes from one or more different species of bacteria can be introduced into one another, e.g., the butyrogenic genes from  Peptoclostridium difficile  have been expressed in  Escherichia coli  (Aboulnaga et al., 2013). 
     In one embodiment, the recombinant bacterial cell does not colonize the subject having the disorder. Unmodified  E. coli  Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al., 2009) or by activation of a kill switch, several hours or days after administration. Thus, the genetically engineered bacteria may require continued administration. Residence time in vivo may be calculated for the genetically engineered bacteria. In some embodiments, the residence time is calculated for a human subject. In some embodiments, residence time in vivo is calculated for the genetically engineered bacteria of the invention, e.g. as described herein. 
     In some embodiments, the bacterial cell is a genetically engineered bacterial cell. In another embodiment, the bacterial cell is a recombinant bacterial cell. In some embodiments, the disclosure comprises a colony of bacterial cells disclosed herein. 
     In another aspect, the disclosure provides a recombinant bacterial culture which comprises bacterial cells disclosed herein. 
     In some embodiments, the genetically engineered bacteria comprising an anti-inflammatory or gut barrier enhancer molecule further comprise a kill-switch circuit, such as any of the kill-switch circuits provided herein. For example, in some embodiments, the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter, and an inverted toxin sequence. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding a toxin under the control of a promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as ParaBAD. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an antitoxin. 
     In some embodiments, the genetically engineered bacteria is an auxotroph comprising gene sequence encoding an anti-inflammatory or gut barrier enhancer molecule and further comprises a kill-switch circuit, such as any of the kill-switch circuits described herein. 
     In some embodiments of the above described genetically engineered bacteria, the gene encoding an anti-inflammatory or gut barrier enhancer molecule is present on a plasmid in the bacterium. In some embodiments, the gene sequence(s) encoding an anti-inflammatory or gut barrier enhancer molecule is present in the bacterial chromosome. In some embodiments, a gene sequence encoding a secretion protein or protein complex, such as any of the secretion systems disclosed herein, for secreting a biomolecule (e.g. an anti-inflammatory or gut barrier enhancer molecule), is present on a plasmid in the bacterium. In some embodiments, the gene sequence encoding a secretion protein or protein complex for secreting a biomolecule, such as any of the secretion systems disclosed herein, is present in the bacterial chromosome. In some embodiments, the gene sequence(s) encoding an antibiotic resistance gene is present on a plasmid in the bacterium. In some embodiments, the gene sequence(s) encoding an antibiotic resistance gene is present in the bacterial chromosome. 
     Anti-Inflammation and/or Gut Barrier Function Enhancer Molecules 
     The genetically engineered bacteria comprise one or more gene sequence(s) and/or gene cassette(s) for producing a non-native anti-inflammation and/or gut barrier function enhancer molecule. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) for producing a non-native anti-inflammation and/or gut barrier function enhancer molecule. For example, the genetically engineered bacteria may comprise two or more gene sequence(s) for producing a non-native anti-inflammation and/or gut barrier function enhancer molecule. In some embodiments, the two or more gene sequences are multiple copies of the same gene. In some embodiments, the two or more gene sequences are sequences encoding different genes. In some embodiments, the two or more gene sequences are sequences encoding multiple copies of one or more different genes. In some embodiments, the genetically engineered bacteria comprise one or more gene cassette(s) for producing a non-native anti-inflammation and/or gut barrier function enhancer molecule. For example, the genetically engineered bacteria may comprise two or more gene cassette(s) for producing a non-native anti-inflammation and/or gut barrier function enhancer molecule. In some embodiments, the two or more gene cassettes are multiple copies of the same gene cassette. In some embodiments, the two or more gene cassettes are different gene cassettes for producing either the same or different anti-inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, the two or more gene cassettes are gene cassettes for producing multiple copies of one or more different anti-inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, the anti-inflammation and/or gut barrier function enhancer molecule is selected from the group consisting of a short-chain fatty acid, butyrate, propionate, acetate, IL-2, IL-22, superoxide dismutase (SOD), GLP-2, GLP-1, IL-10 (human or viral), IL-27, TGF-β1, TGF-β2, N-acylphosphatidylethanolamines (NAPEs), elafin (also known as peptidase inhibitor 3 or SKALP), trefoil factor, melatonin, PGD2, kynurenic acid, kynurenine, typtophan metabolite, indole, indole metabolite, a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that neutralizes TNF-α, IFN-γ, IL-1β, IL-6, IL-8, IL-17, and/or chemokines, e.g., CXCL-8 and CCL2, AHR agonist (e.g., indole acetic acid, indole-3-aldehyde, and indole), PXR agonist (e.g., IPA), HDAC inhibitor (e.g., butyrate), GPR41 and/or GPR43 activator (e.g., butyrate and/or propionate and/or acetate), GPR109A activator (e.g., butyrate), inhibitor of NF-kappaB signaling (e.g., butyrate), modulator of PPARgamma (e.g., butyrate), activator of AMPK signaling (e.g., acetate), modulator of GLP-1 secretion, and hydroxyl radical scavengers and antioxidants (e.g., IPA). A molecule may be primarily anti-inflammatory, e.g., IL-10, or primarily gut barrier function enhancing, e.g., GLP-2. Alternatively, a molecule may be both anti-inflammatory and gut barrier function enhancing. 
     In some embodiments, the genetically engineered bacteria of the invention express one or more anti-inflammation and/or gut barrier function enhancer molecule(s) that is encoded by a single gene, e.g., the molecule is elafin and encoded by the PI3 gene, or the molecule is interleukin-10 and encoded by the IL10 gene. In alternate embodiments, the genetically engineered bacteria of the invention encode one or more an anti-inflammation and/or gut barrier function enhancer molecule(s), e.g., butyrate, that is synthesized by a biosynthetic pathway requiring multiple genes. 
     The one or more gene sequence(s) and/or gene cassette(s) may be expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. In some embodiments, expression from the plasmid may be useful for increasing expression of the anti-inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, expression from the chromosome may be useful for increasing stability of expression of the anti-inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, the gene sequence(s) or gene cassette(s) for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is integrated into the bacterial chromosome at one or more integration sites in the genetically engineered bacteria. For example, one or more copies of the butyrate biosynthesis gene cassette may be integrated into the bacterial chromosome. In some embodiments, the gene sequence(s) or gene cassette(s) for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is expressed from a plasmid in the genetically engineered bacteria. In some embodiments, the gene sequence(s) or gene cassette(s) for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is inserted into the bacterial genome at one or more of the following insertion sites in  E. coli  Nissle: malE/K, araC/BAD, lacZ, thyA, malP/T. Any suitable insertion site may be used (see, e.g.,  FIG. 52  for exemplary insertion sites). The insertion site may be anywhere in the genome, e.g., in a gene required for survival and/or growth, such as thyA (to create an auxotroph); in an active area of the genome, such as near the site of genome replication; and/or in between divergent promoters in order to reduce the risk of unintended transcription, such as between AraB and AraC of the arabinose operon. 
     Short Chain Fatty Acids and Tryptophan Metabolites 
     One strategy in the treatment, prevention, and/or management of inflammatory bowel disorders may include approaches to help maintain and/or reestablish gut barrier function, e.g. through the prevention, treatment and/or management of inflammatory events at the root of increased permeability, e.g. through the administration of anti-inflammatory effectors. 
     For example, leading metabolites that play gut-protective roles are short chain fatty acids, e.g. acetate, butyrate and propionate, and those derived from tryptophan metabolism. These metabolites have been shown to play a major role in the prevention of inflammatory disease. As such one approach in the treatment, prevention, and/or management of gut barrier health may be to provide a treatment which contains one or more of such metabolites. 
     For example, butyrate and other SCFA, e.g., derived from the microbiota, are known to promote maintaining intestinal integrity (e.g., as reviewed in Thorburn et al., Diet, Metabolites, and “Western-Lifestyle” Inflammatory Diseases; Immunity Volume 40, Issue 6, 19 Jun. 2014, Pages 833-842). (A) SCFA-induced promotion of mucus by gut epithelial cells, possibly through signaling through metabolite sensing GPCRs; (B) SCFA-induced secretion of IgA by B cells; (C) SCFA-induced promotion of tissue repair and wound healing; (D) SCFA-induced promotion of Treg cell development in the gut in a process that presumably facilitates immunological tolerance; (E) SCFA-mediated enhancement of epithelial integrity in a process dependent on inflammasome activation (e.g., via NALP3) and IL-18 production; and (F) anti-inflammatory effects, inhibition of inflammatory cytokine production (e.g., TNF, 11-6, and IFN-gamma), and inhibition of NF-κB. Many of these actions of SCFAs in gut homeostatis can be ascribed to GPR43 and GPR109A, which are expressed by the colonic epithelium, by inflammatory leukocytes (e.g. neutrophils and macrophages) and by Treg cells. These receptors signal through G proteins, coupled to MAPK, PI3K and mTOR, as well as a separate arrestin-pathway, leading to NFkappa B inhibition. Other effects can be ascribed to SCFA-mediated HDAC inhibition, e.g. butyrate, which may regulate macrophage function and promote TReg cells. 
     In addition, a number of tryptophan metabolites, including kynurenine and kynurenic acid, as well as several indoles, such as indole-3 aldehyde, indole-3 propionic acid, and several other indole metabolites (which can be derived from microbiota or the diet) described infra, have been shown to be essential for gut homeostasis and promote gut-barrier health. These metabolites bind to aryl hydrocarbon receptor (Ahr). After agonist binding, AhR translocates to the nucleus, where it forms a heterodimer with AhR nuclear translocator (ARNT). AhR-dependent gene expression includes genes involved in the production of mediators important for gut homeostasis; these mediators include IL-22, antimicrobicidal factors, increased Th17 cell activity, and the maintenance of intraepithelial lymphocytes and RORγt+ innate lymphoid cells. 
     Tryptophan can also be transported across the epithelium by transport machinery comprising angiotensin I converting enzyme 2 (Ace2). Tryptophan is degraded to kynurenine, another AhR agonist, by the immune-regulatory enzyme indoleamine 2,3-dioxygenase (IDO), which is linked to suppression of T cell responses, promotion of Treg cells, and immune tolerance. Moreover, a number of tryptophan metabolites, including kynurenic acid and niacin, agonize metabolite-sensing GPCRs, such as GPR35 and GPR109A and thus multiple elements of tryptophan catabolism facilitate gut homeostasis. 
     In addition, some indole metabolites, e.g., indole 3-propionic acid (IPA), may exert their effect an activating ligand of Pregnane X receptor (PXR), which is thought to play a key role as an essential regulator of intestinal barrier function, through downregulation of TLR4 signaling (Venkatesh et al., 2014 Symbiotic Bacterial Metabolites Regulate Gastrointestinal Barrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296-310, Aug. 21, 2014). As a result, indole levels may through the activation of PXR regulate and balance the levels of TLR4 expression to promote homeostasis and gut barrier health. 
     Thus, in some embodiments, the genetically engineered bacteria of the disclosure produce one or more short chain fatty acids and/or one or more tryptophan metabolites. 
     Acetate 
     In some embodiments, the genetically engineered bacteria of the invention comprise an acetate gene cassette and are capable of producing acetate. The genetically engineered bacteria may include any suitable set of acetate biosynthesis genes. In other embodiments, the bacteria comprise an endogenous acetate biosynthetic gene or gene cassette and naturally produce acetate. Unmodified bacteria comprising acetate biosynthesis genes are known in the art and are capable of consuming various substrates to produce acetate under aerobic and/or anaerobic conditions (see, e.g., Ragsdale, 2008), and these endogenous acetate biosynthesis pathways may be a source of genes for the genetically engineered bacteria of the invention. In some embodiments, the genetically engineered bacteria of the invention comprise acetate biosynthesis genes from a different species, strain, or substrain of bacteria. In some embodiments, the native acetate biosynthesis genes in the genetically engineered bacteria are enhanced. In some embodiments, the genetically engineered bacteria comprise aerobic acetate biosynthesis genes, e.g., from  Escherichia coli . In some embodiments, the genetically engineered bacteria comprise anaerobic acetate biosynthesis genes, e.g., from  Acetitomaculum, Acetoanaerobium, Acetohalobium, Acetonema, Balutia, Butyribacterium, Clostridium, Moorella, Oxobacter, Sporomusa , and/or  Thermoacetogenium . The genetically engineered bacteria may comprise genes for aerobic acetate biosynthesis or genes for anaerobic or microaerobic acetate biosynthesis. In some embodiments, the genetically engineered bacteria comprise both aerobic and anaerobic or microaerobic acetate biosynthesis genes. In some embodiments, the genetically engineered bacteria comprise a combination of acetate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing acetate. In some embodiments, one or more of the acetate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or acetate production. In some embodiments, the genetically engineered bacteria are capable of expressing the acetate biosynthesis cassette and producing acetate under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing an alternate short-chain fatty acid. 
     In  E. coli  Nissle, acetate is generated as an end product of fermentation. In  E. coli , glucose fermentation occurs in two steps, (1) the glycolysis reactions and (2) the NADH recycling reactions, i.e. these reactions re-oxidize the NAD+ generated during the fermentation process.  E. coli  employs the “mixed acid” fermentation pathway (see, e.g.,  FIG. 25 ). Through the “mixed acid” pathway,  E. coli  generates several alternative end products and in variable amounts (e.g., lactate, acetate, formate, succinate, ethanol, carbon dioxide, and hydrogen) though various arms of the fermentation pathway, e.g., as shown in  FIG. 25 . Without wishing to be bound by theory, prevention or reduction of flux through one or more metabolic arm(s) generating metabolites other than acetate, e.g. through mutation, deletion and/or inhibition of one or more gene(s) encoding key enzymes in these metabolic arms, results in an increase in production of acetate for NAD recycling. As disclosed herein, e.g., in Example 20, deletions in gene(s) encoding such enzymes increase acetate production. Such enzymes include fumarate reductase (encoded by the frd genes), lactate dehydrogenase (encoded by the ldh gene), and aldehyde-alcohol dehydrogenase (encoded by the adhE gene). 
     LdhA is a soluble NAD-linked lactate dehydrogenase (LDH) that is specific for the production of D-lactate and is a homotetramer and shows positive homotropic cooperativity under higher pH conditions.  E. coli  carrying ldhA mutations show no observable growth defect and can still ferment sugars to a variety of products other than lactate. 
     In some embodiments, the genetically engineered bacteria producing acetate comprise a mutation and/or deletion in the endogenous ldhA gene. 
     AdhE is a homopolymeric protein with three catalytic functions: alcohol dehydrogenase, coenzyme A-dependent acetaldehyde dehydrogenase, and pyruvate formate-lyase deactivase. During fermentation, AdhE has catalyzes two steps towards the generation of ethanol: (1) the reduction of acetyl-CoA to acetaldehyde and (2) the reduction of acetaldehyde to to ethanol. Deletion of adhE has been employed to enhance production of certain metabolites industrially, including succinate, D-lactate, and polyhydroxyalkanoates (Singh et al, Manipulating redox and ATP balancing for improved production of succinate in  E. coli .; Metab Eng. 2011 January; 13(1):76-81; Zhou et al., Evaluation of genetic manipulation strategies on D-lactate production by  Escherichia coli , Curr Microbiol. 2011 March; 62(3):981-9; Jian et al., Production of polyhydroxyalkanoates by  Escherichia coli  mutants with defected mixed acid fermentation pathways, Appl Microbiol Biotechnol. 2010 August; 87(6):2247-56). 
     In some embodiments, the genetically engineered bacteria producing acetate comprise a mutation and/or deletion in the endogenous adhE gene. 
     The fumarate reductase enzyme complex, encoded by the frdABCD operon, allows  Escherichia coli  to utilize fumarate as a terminal electron acceptor for anaerobic oxidative phosphorylation. FrdA is one of two catalytic subunits in the four subunit fumarate reductase complex. FrdB is the second catalytic subunit of the complex. FrdC and FrdD are two integral membrane protein components of the fumarate reductase complex. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous frdA gene. 
     In some embodiments, the genetically engineered bacteria producing acetate comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding one or more enzyme(s) which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. 
     Phosphate acetyltransferase (Pta) catalyzes the reversible conversion between acetyl-CoA and acetylphosphate, a step in the metabolism of acetate (Campos-Bermudez et al., Functional dissection of  Escherichia coli  phosphotransacetylase structural domains and analysis of key compounds involved in activity regulation; FEBS J. 2010 April; 277(8):1957-66). Both pyruvate and phosphoenolpyruvate activate the enzyme in the direction of acetylphosphate synthesis and inhibit the enzyme in the direction of acetyl-CoA synthesis. The acetate formation from acetyl-CoA I pathway has been the target of metabolic engineering to reduce the flux to acetate and increase the production of commercially desired end products (see, e.g., Singh, et al., Manipulating redox and ATP balancing for improved production of succinate in  E. coli ; Metab Eng. 2011 January; 13(1):76-81). A pta mutant does not grow on acetate as the sole source of carbon (Brown et al., The enzymic interconversion of acetate and acetyl-coenzyme A in  Escherichia coli ; J Gen Microbiol. 1977 October; 102(2):327-36). 
     In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria produce butyrate. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta gene and also in one or more endogenous genes selected from the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. In some embodiments, the genetically engineered bacterias produce butyrate. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     Butyrate 
     In some embodiments, the genetically engineered bacteria of the invention comprise a butyrogenic gene cassette and are capable of producing butyrate under particular exogenous environmental conditions. The genetically engineered bacteria may include any suitable set of butyrogenic genes (see, e.g., Table 2 and Table 3). Unmodified bacteria comprising butyrate biosynthesis genes are known and include, but are not limited to,  Peptoclostridium, Clostridium, Fusobacterium, Butyrivibrio, Eubacterium , and  Treponema . In some embodiments, the genetically engineered bacteria of the invention comprise butyrate biosynthesis genes from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise the eight genes of the butyrate biosynthesis pathway from  Peptoclostridium difficile , e.g.,  Peptoclostridium difficile  strain 630: hcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, and buk (Aboulnaga et al., 2013) and are capable of producing butyrate.  Peptoclostridium difficile  strain 630 and strain 1296 are both capable of producing butyrate, but comprise different nucleic acid sequences for etfA3, thiA1, hbd, crt2, pbt, and buk. In some embodiments, the genetically engineered bacteria comprise a combination of butyrogenic genes from different species, strains, and/or substrains of bacteria and are capable of producing butyrate. For example, in some embodiments, the genetically engineered bacteria comprise bcd2, etfB3, etfA3, and thiA1 from  Peptoclostridium difficile  strain 630, and hbd, crt2, pbt, and buk from  Peptoclostridium difficile  strain 1296. Alternatively, a single gene from  Treponema denticola  (ter, encoding trans-2-enoynl-CoA reductase) is capable of functionally replacing all three of the bcd2, etfB3, and etfA3 genes from  Peptoclostridium difficile . Thus, a butyrogenic gene cassette may comprise thiA1, hbd, crt2, pbt, and buk from  Peptoclostridium difficile  and ter from  Treponema denticola . In another example of a butyrate gene cassette, the pbt and buk genes are replaced with tesB (e.g., from  E. coli ). Thus a butyrogenic gene cassette may comprise ter, thiA1, hbd, crt2, and tesB.n some embodiments, the genetically engineered bacteria are capable of expressing the butyrate biosynthesis cassette and producing butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. One or more of the butyrate biosynthesis genes may be functionally replaced or modified. e.g., codon optimized. 
     In some embodiments, additional genes may be mutated or knocked out, to further increase the levels of butyrate production. Production under anaerobic conditions depends on endogenous NADH pools. Therefore, the flux through the butyrate pathway may be enhanced by eliminating competing routes for NADH utilization. Non-limiting examples of such competing routes are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Thus, in certain embodiments, the genetically engineered bacteria further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
     Table 2 depicts the nucleic acid sequences of exemplary genes in exemplary butyrate biosynthesis gene cassettes. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Exemplary Butyrate Cassette Sequences 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 bcd2 
                 ATGGATTTAAATTCTAAAAAATATCAGATGCTTAAAGAGCTATATGTAAG 
               
               
                 SEQ ID NO: 1 
                 CTTCGCTGAAAATGAAGTTAAACCTTTAGCAACAGAACTTGATGAAGAAG 
               
               
                   
                 AAAGATTTCCTTATGAAACAGTGGAAAAAATGGCAAAAGCAGGAATGATG 
               
               
                   
                 GGTATACCATATCCAAAAGAATATGGTGGAGAAGGTGGAGACACTGTAGG 
               
               
                   
                 ATATATAATGGCAGTTGAAGAATTGTCTAGAGTTTGTGGTACTACAGGAG 
               
               
                   
                 TTATATTATCAGCTCATACATCTCTTGGCTCATGGCCTATATATCAATAT 
               
               
                   
                 GGTAATGAAGAACAAAAACAAAAATTCTTAAGACCACTAGCAAGTGGAGA 
               
               
                   
                 AAAATTAGGAGCATTTGGTCTTACTGAGCCTAATGCTGGTACAGATGCGT 
               
               
                   
                 CTGGCCAACAAACAACTGCTGTTTTAGACGGGGATGAATACATACTTAAT 
               
               
                   
                 GGCTCAAAAATATTTATAACAAACGCAATAGCTGGTGACATATATGTAGT 
               
               
                   
                 AATGGCAATGACTGATAAATCTAAGGGGAACAAAGGAATATCAGCATTTA 
               
               
                   
                 TAGTTGAAAAAGGAACTCCTGGGTTTAGCTTTGGAGTTAAAGAAAAGAAA 
               
               
                   
                 ATGGGTATAAGAGGTTCAGCTACGAGTGAATTAATATTTGAGGATTGCAG 
               
               
                   
                 AATACCTAAAGAAAATTTACTTGGAAAAGAAGGTCAAGGATTTAAGATAG 
               
               
                   
                 CAATGTCTACTCTTGATGGTGGTAGAATTGGTATAGCTGCACAAGCTTTA 
               
               
                   
                 GGTTTAGCACAAGGTGCTCTTGATGAAACTGTTAAATATGTAAAAGAAAG 
               
               
                   
                 AGTACAATTTGGTAGACCATTATCAAAATTCCAAAATACACAATTCCAAT 
               
               
                   
                 TAGCTGATATGGAAGTTAAGGTACAAGCGGCTAGACACCTTGTATATCAA 
               
               
                   
                 GCAGCTATAAATAAAGACTTAGGAAAACCTTATGGAGTAGAAGCAGCAAT 
               
               
                   
                 GGCAAAATTATTTGCAGCTGAAACAGCTATGGAAGTTACTACAAAAGCTG 
               
               
                   
                 TACAACTTCATGGAGGATATGGATACACTCGTGACTATCCAGTAGAAAGA 
               
               
                   
                 ATGATGAGAGATGCTAAGATAACTGAAATATATGAAGGAACTAGTGAAGT 
               
               
                   
                 TCAAAGAATGGTTATTTCAGGAAAACTATTAAAATAG 
               
               
                   
               
               
                 etfB3 
                 ATGAATATAGTCGTTTGTATAAAACAAGTTCCAGATACAACAGAAGTTAA 
               
               
                 SEQ ID NO: 2 
                 ACTAGATCCTAATACAGGTACTTTAATTAGAGATGGAGTACCAAGTATAA 
               
               
                   
                 TAAACCCTGATGATAAAGCAGGTTTAGAAGAAGCTATAAAATTAAAAGAA 
               
               
                   
                 GAAATGGGTGCTCATGTAACTGTTATAACAATGGGACCTCCTCAAGCAGA 
               
               
                   
                 TATGGCTTTAAAAGAAGCTTTAGCAATGGGTGCAGATAGAGGTATATTAT 
               
               
                   
                 TAACAGATAGAGCATTTGCGGGTGCTGATACTTGGGCAACTTCATCAGCA 
               
               
                   
                 TTAGCAGGAGCATTAAAAAATATAGATTTTGATATTATAATAGCTGGAAG 
               
               
                   
                 ACAGGCGATAGATGGAGATACTGCACAAGTTGGACCTCAAATAGCTGAAC 
               
               
                   
                 ATTTAAATCTTCCATCAATAACATATGCTGAAGAAATAAAAACTGAAGGT 
               
               
                   
                 GAATATGTATTAGTAAAAAGACAATTTGAAGATTGTTGCCATGACTTAAA 
               
               
                   
                 AGTTAAAATGCCATGCCTTATAACAACTCTTAAAGATATGAACACACCAA 
               
               
                   
                 GATACATGAAAGTTGGAAGAATATATGATGCTTTCGAAAATGATGTAGTA 
               
               
                   
                 GAAACATGGACTGTAAAAGATATAGAAGTTGACCCTTCTAATTTAGGTCT 
               
               
                   
                 TAAAGGTTCTCCAACTAGTGTATTTAAATCATTTACAAAATCAGTTAAAC 
               
               
                   
                 CAGCTGGTACAATATACAATGAAGATGCGAAAACATCAGCTGGAATTATC 
               
               
                   
                 ATAGATAAATTAAAAGAGAAGTATATCATATAA 
               
               
                   
               
               
                 etfA3 
                 ATGGGTAACGTTTTAGTAGTAATAGAACAAAGAGAAAATGTAATTCAAAC 
               
               
                 SEQ ID NO: 3 
                 TGTTTCTTTAGAATTACTAGGAAAGGCTACAGAAATAGCAAAAGATTATG 
               
               
                   
                 ATACAAAAGTTTCTGCATTACTTTTAGGTAGTAAGGTAGAAGGTTTAATA 
               
               
                   
                 GATACATTAGCACACTATGGTGCAGATGAGGTAATAGTAGTAGATGATGA 
               
               
                   
                 AGCTTTAGCAGTGTATACAACTGAACCATATACAAAAGCAGCTTATGAAG 
               
               
                   
                 CAATAAAAGCAGCTGACCCTATAGTTGTATTATTTGGTGCAACTTCAATA 
               
               
                   
                 GGTAGAGATTTAGCGCCTAGAGTTTCTGCTAGAATACATACAGGTCTTAC 
               
               
                   
                 TGCTGACTGTACAGGTCTTGCAGTAGCTGAAGATACAAAATTATTATTAA 
               
               
                   
                 TGACAAGACCTGCCTTTGGTGGAAATATAATGGCAACAATAGTTTGTAAA 
               
               
                   
                 GATTTCAGACCTCAAATGTCTACAGTTAGACCAGGGGTTATGAAGAAAAA 
               
               
                   
                 TGAACCTGATGAAACTAAAGAAGCTGTAATTAACCGTTTCAAGGTAGAAT 
               
               
                   
                 TTAATGATGCTGATAAATTAGTTCAAGTTGTACAAGTAATAAAAGAAGCT 
               
               
                   
                 AAAAAACAAGTTAAAATAGAAGATGCTAAGATATTAGTTTCTGCTGGACG 
               
               
                   
                 TGGAATGGGTGGAAAAGAAAACTTAGACATACTTTATGAATTAGCTGAAA 
               
               
                   
                 TTATAGGTGGAGAAGTTTCTGGTTCTCGTGCCACTATAGATGCAGGTTGG 
               
               
                   
                 TTAGATAAAGCAAGACAAGTTGGTCAAACTGGTAAAACTGTAAGACCAGA 
               
               
                   
                 CCTTTATATAGCATGTGGTATATCTGGAGCAATACAACATATAGCTGGTA 
               
               
                   
                 TGGAAGATGCTGAGTTTATAGTTGCTATAAATAAAAATCCAGAAGCTCCA 
               
               
                   
                 ATATTTAAATATGCTGATGTTGGTATAGTTGGAGATGTTCATAAAGTGCT 
               
               
                   
                 TCCAGAACTTATCAGTCAGTTAAGTGTTGCAAAAGAAAAAGGTGAAGTTT 
               
               
                   
                 TAGCTAACTAA 
               
               
                   
               
               
                 thiA1 
                 ATGAGAGAAGTAGTAATTGCCAGTGCAGCTAGAACAGCAGTAGGAAGTTT 
               
               
                 SEQ ID NO: 4 
                 TGGAGGAGCATTTAAATCAGTTTCAGCGGTAGAGTTAGGGGTAACAGCAG 
               
               
                   
                 CTAAAGAAGCTATAAAAAGAGCTAACATAACTCCAGATATGATAGATGAA 
               
               
                   
                 TCTCTTTTAGGGGGAGTACTTACAGCAGGTCTTGGACAAAATATAGCAAG 
               
               
                   
                 ACAAATAGCATTAGGAGCAGGAATACCAGTAGAAAAACCAGCTATGACTA 
               
               
                   
                 TAAATATAGTTTGTGGTTCTGGATTAAGATCTGTTTCAATGGGATCTCAA 
               
               
                   
                 CTTATAGCATTAGGTGATGCTGATATAATGTTAGTTGGTGGAGCTGAAAA 
               
               
                   
                 CATGAGTATGTCTCCTTATTTAGTACCAAGTGCGAGATATGGTGCAAGAA 
               
               
                   
                 TGGGTGATGCTGCTTTTGTTGATTCAATGATAAAAGATGGATTATCAGAC 
               
               
                   
                 ATATTTAATAACTATCACATGGGTATTACTGCTGAAAACATAGCAGAGCA 
               
               
                   
                 ATGGAATATAACTAGAGAAGAACAAGATGAATTAGCTCTTGCAAGTCAAA 
               
               
                   
                 ATAAAGCTGAAAAAGCTCAAGCTGAAGGAAAATTTGATGAAGAAATAGTT 
               
               
                   
                 CCTGTTGTTATAAAAGGAAGAAAAGGTGACACTGTAGTAGATAAAGATGA 
               
               
                   
                 ATATATTAAGCCTGGCACTACAATGGAGAAACTTGCTAAGTTAAGACCTG 
               
               
                   
                 CATTTAAAAAAGATGGAACAGTTACTGCTGGTAATGCATCAGGAATAAAT 
               
               
                   
                 GATGGTGCTGCTATGTTAGTAGTAATGGCTAAAGAAAAAGCTGAAGAACT 
               
               
                   
                 AGGAATAGAGCCTCTTGCAACTATAGTTTCTTATGGAACAGCTGGTGTTG 
               
               
                   
                 ACCCTAAAATAATGGGATATGGACCAGTTCCAGCAACTAAAAAAGCTTTA 
               
               
                   
                 GAAGCTGCTAATATGACTATTGAAGATATAGATTTAGTTGAAGCTAATGA 
               
               
                   
                 GGCATTTGCTGCCCAATCTGTAGCTGTAATAAGAGACTTAAATATAGATA 
               
               
                   
                 TGAATAAAGTTAATGTTAATGGTGGAGCAATAGCTATAGGACATCCAATA 
               
               
                   
                 GGATGCTCAGGAGCAAGAATACTTACTACACTTTTATATGAAATGAAGAG 
               
               
                   
                 AAGAGATGCTAAAACTGGTCTTGCTACACTTTGTATAGGCGGTGGAATGG 
               
               
                   
                 GAACTACTTTAATAGTTAAGAGATAG 
               
               
                   
               
               
                 hbd 
                 ATGAAATTAGCTGTAATAGGTAGTGGAACTATGGGAAGTGGTATTGTACA 
               
               
                 SEQ ID NO: 5 
                 AACTTTTGCAAGTTGTGGACATGATGTATGTTTAAAGAGTAGAACTCAAG 
               
               
                   
                 GTGCTATAGATAAATGTTTAGCTTTATTAGATAAAAATTTAACTAAGTTA 
               
               
                   
                 GTTACTAAGGGAAAAATGGATGAAGCTACAAAAGCAGAAATATTAAGTCA 
               
               
                   
                 TGTTAGTTCAACTACTAATTATGAAGATTTAAAAGATATGGATTTAATAA 
               
               
                   
                 TAGAAGCATCTGTAGAAGACATGAATATAAAGAAAGATGTTTTCAAGTTA 
               
               
                   
                 CTAGATGAATTATGTAAAGAAGATACTATCTTGGCAACAAATACTTCATC 
               
               
                   
                 ATTATCTATAACAGAAATAGCTTCTTCTACTAAGCGCCCAGATAAAGTTA 
               
               
                   
                 TAGGAATGCATTTCTTTAATCCAGTTCCTATGATGAAATTAGTTGAAGTT 
               
               
                   
                 ATAAGTGGTCAGTTAACATCAAAAGTTACTTTTGATACAGTATTTGAATT 
               
               
                   
                 ATCTAAGAGTATCAATAAAGTACCAGTAGATGTATCTGAATCTCCTGGAT 
               
               
                   
                 TTGTAGTAAATAGAATACTTATACCTATGATAAATGAAGCTGTTGGTATA 
               
               
                   
                 TATGCAGATGGTGTTGCAAGTAAAGAAGAAATAGATGAAGCTATGAAATT 
               
               
                   
                 AGGAGCAAACCATCCAATGGGACCACTAGCATTAGGTGATTTAATCGGAT 
               
               
                   
                 TAGATGTTGTTTTAGCTATAATGAACGTTTTATATACTGAATTTGGAGAT 
               
               
                   
                 ACTAAATATAGACCTCATCCACTTTTAGCTAAAATGGTTAGAGCTAATCA 
               
               
                   
                 ATTAGGAAGAAAAACTAAGATAGGATTCTATGATTATAATAAATAA 
               
               
                   
               
               
                 crt2 
                 ATGAGTACAAGTGATGTTAAAGTTTATGAGAATGTAGCTGTTGAAGTAGA 
               
               
                 SEQ ID NO: 6 
                 TGGAAATATATGTACAGTGAAAATGAATAGACCTAAAGCCCTTAATGCAA 
               
               
                   
                 TAAATTCAAAGACTTTAGAAGAACTTTATGAAGTATTTGTAGATATTAAT 
               
               
                   
                 AATGATGAAACTATTGATGTTGTAATATTGACAGGGGAAGGAAAGGCATT 
               
               
                   
                 TGTAGCTGGAGCAGATATTGCATACATGAAAGATTTAGATGCTGTAGCTG 
               
               
                   
                 CTAAAGATTTTAGTATCTTAGGAGCAAAAGCTTTTGGAGAAATAGAAAAT 
               
               
                   
                 AGTAAAAAAGTAGTGATAGCTGCTGTAAACGGATTTGCTTTAGGTGGAGG 
               
               
                   
                 ATGTGAACTTGCAATGGCATGTGATATAAGAATTGCATCTGCTAAAGCTA 
               
               
                   
                 AATTTGGTCAGCCAGAAGTAACTCTTGGAATAACTCCAGGATATGGAGGA 
               
               
                   
                 ACTCAAAGGCTTACAAGATTGGTTGGAATGGCAAAAGCAAAAGAATTAAT 
               
               
                   
                 CTTTACAGGTCAAGTTATAAAAGCTGATGAAGCTGAAAAAATAGGGCTAG 
               
               
                   
                 TAAATAGAGTCGTTGAGCCAGACATTTTAATAGAAGAAGTTGAGAAATTA 
               
               
                   
                 GCTAAGATAATAGCTAAAAATGCTCAGCTTGCAGTTAGATACTCTAAAGA 
               
               
                   
                 AGCAATACAACTTGGTGCTCAAACTGATATAAATACTGGAATAGATATAG 
               
               
                   
                 AATCTAATTTATTTGGTCTTTGTTTTTCAACTAAAGACCAAAAAGAAGGA 
               
               
                   
                 ATGTCAGCTTTCGTTGAAAAGAGAGAAGCTAACTTTATAAAAGGGTAA 
               
               
                   
               
               
                 pbt 
                 ATGAGAAGTTTTGAAGAAGTAATTAAGTTTGCAAAAGAAAGAGGACCTAA 
               
               
                 SEQ ID NO: 7 
                 AACTATATCAGTAGCATGTTGCCAAGATAAAGAAGTTTTAATGGCAGTTG 
               
               
                   
                 AAATGGCTAGAAAAGAAAAAATAGCAAATGCCATTTTAGTAGGAGATATA 
               
               
                   
                 GAAAAGACTAAAGAAATTGCAAAAAGCATAGACATGGATATCGAAAATTA 
               
               
                   
                 TGAACTGATAGATATAAAAGATTTAGCAGAAGCATCTCTAAAATCTGTTG 
               
               
                   
                 AATTAGTTTCACAAGGAAAAGCCGACATGGTAATGAAAGGCTTAGTAGAC 
               
               
                   
                 ACATCAATAATACTAAAAGCAGTTTTAAATAAAGAAGTAGGTCTTAGAAC 
               
               
                   
                 TGGAAATGTATTAAGTCACGTAGCAGTATTTGATGTAGAGGGATATGATA 
               
               
                   
                 GATTATTTTTCGTAACTGACGCAGCTATGAACTTAGCTCCTGATACAAAT 
               
               
                   
                 ACTAAAAAGCAAATCATAGAAAATGCTTGCACAGTAGCACATTCATTAGA 
               
               
                   
                 TATAAGTGAACCAAAAGTTGCTGCAATATGCGCAAAAGAAAAAGTAAATC 
               
               
                   
                 CAAAAATGAAAGATACAGTTGAAGCTAAAGAACTAGAAGAAATGTATGAA 
               
               
                   
                 AGAGGAGAAATCAAAGGTTGTATGGTTGGTGGGCCTTTTGCAATTGATAA 
               
               
                   
                 TGCAGTATCTTTAGAAGCAGCTAAACATAAAGGTATAAATCATCCTGTAG 
               
               
                   
                 CAGGACGAGCTGATATATTATTAGCCCCAGATATTGAAGGTGGTAACATA 
               
               
                   
                 TTATATAAAGCTTTGGTATTCTTCTCAAAATCAAAAAATGCAGGAGTTAT 
               
               
                   
                 AGTTGGGGCTAAAGCACCAATAATATTAACTTCTAGAGCAGACAGTGAAG 
               
               
                   
                 AAACTAAACTAAACTCAATAGCTTTAGGTGTTTTAATGGCAGCAAAGGCA 
               
               
                   
                 TAA 
               
               
                   
               
               
                 buk 
                 ATGAGCAAAATATTTAAAATCTTAACAATAAATCCTGGTTCGACATCAAC 
               
               
                 SEQ ID NO: 8 
                 TAAAATAGCTGTATTTGATAATGAGGATTTAGTATTTGAAAAAACTTTAA 
               
               
                   
                 GACATTCTTCAGAAGAAATAGGAAAATATGAGAAGGTGTCTGACCAATTT 
               
               
                   
                 GAATTTCGTAAACAAGTAATAGAAGAAGCTCTAAAAGAAGGTGGAGTAAA 
               
               
                   
                 AACATCTGAATTAGATGCTGTAGTAGGTAGAGGAGGACTTCTTAAACCTA 
               
               
                   
                 TAAAAGGTGGTACTTATTCAGTAAGTGCTGCTATGATTGAAGATTTAAAA 
               
               
                   
                 GTGGGAGTTTTAGGAGAACACGCTTCAAACCTAGGTGGAATAATAGCAAA 
               
               
                   
                 ACAAATAGGTGAAGAAGTAAATGTTCCTTCATACATAGTAGACCCTGTTG 
               
               
                   
                 TTGTAGATGAATTAGAAGATGTTGCTAGAATTTCTGGTATGCCTGAAATA 
               
               
                   
                 AGTAGAGCAAGTGTAGTACATGCTTTAAATCAAAAGGCAATAGCAAGAAG 
               
               
                   
                 ATATGCTAGAGAAATAAACAAGAAATATGAAGATATAAATCTTATAGTTG 
               
               
                   
                 CACACATGGGTGGAGGAGTTTCTGTTGGAGCTCATAAAAATGGTAAAATA 
               
               
                   
                 GTAGATGTTGCAAACGCATTAGATGGAGAAGGACCTTTCTCTCCAGAAAG 
               
               
                   
                 AAGTGGTGGACTACCAGTAGGTGCATTAGTAAAAATGTGCTTTAGTGGAA 
               
               
                   
                 AATATACTCAAGATGAAATTAAAAAGAAAATAAAAGGTAATGGCGGACTA 
               
               
                   
                 GTTGCATACTTAAACACTAATGATGCTAGAGAAGTTGAAGAAAGAATTGA 
               
               
                   
                 AGCTGGTGATGAAAAAGCTAAATTAGTATATGAAGCTATGGCATATCAAA 
               
               
                   
                 TCTCTAAAGAAATAGGAGCTAGTGCTGCAGTTCTTAAGGGAGATGTAAAA 
               
               
                   
                 GCAATATTATTAACTGGTGGAATCGCATATTCAAAAATGTTTACAGAAAT 
               
               
                   
                 GATTGCAGATAGAGTTAAATTTATAGCAGATGTAAAAGTTTATCCAGGTG 
               
               
                   
                 AAGATGAAATGATTGCATTAGCTCAAGGTGGACTTAGAGTTTTAACTGGT 
               
               
                   
                 GAAGAAGAGGCTCAAGTTTATGATAACTAA 
               
               
                   
               
               
                 ter 
                 ATGATCGTAAAACCTATGGTACGCAACAATATCTGCCTGAACGCCCATCC 
               
               
                 SEQ ID NO: 9 
                 TCAGGGCTGCAAGAAGGGAGTGGAAGATCAGATTGAATATACCAAGAAAC 
               
               
                   
                 GCATTACCGCAGAAGTCAAAGCTGGCGCAAAAGCTCCAAAAAACGTTCTG 
               
               
                   
                 GTGCTTGGCTGCTCAAATGGTTACGGCCTGGCGAGCCGCATTACTGCTGC 
               
               
                   
                 GTTCGGATACGGGGCTGCGACCATCGGCGTGTCCTTTGAAAAAGCGGGTT 
               
               
                   
                 CAGAAACCAAATATGGTACACCGGGATGGTACAATAATTTGGCATTTGAT 
               
               
                   
                 GAAGCGGCAAAACGCGAGGGTCTTTATAGCGTGACGATCGACGGCGATGC 
               
               
                   
                 GTTTTCAGACGAGATCAAGGCCCAGGTAATTGAGGAAGCCAAAAAAAAAG 
               
               
                   
                 GTATCAAATTTGATCTGATCGTATACAGCTTGGCCAGCCCAGTACGTACT 
               
               
                   
                 GATCCTGATACAGGTATCATGCACAAAAGCGTTTTGAAACCCTTTGGAAA 
               
               
                   
                 AACGTTCACAGGCAAAACAGTAGATCCGTTTACTGGCGAGCTGAAGGAAA 
               
               
                   
                 TCTCCGCGGAACCAGCAAATGACGAGGAAGCAGCCGCCACTGTTAAAGTT 
               
               
                   
                 ATGGGGGGTGAAGATTGGGAACGTTGGATTAAGCAGCTGTCGAAGGAAGG 
               
               
                   
                 CCTCTTAGAAGAAGGCTGTATTACCTTGGCCTATAGTTATATTGGCCCTG 
               
               
                   
                 AAGCTACCCAAGCTTTGTACCGTAAAGGCACAATCGGCAAGGCCAAAGAA 
               
               
                   
                 CACCTGGAGGCCACAGCACACCGTCTCAACAAAGAGAACCCGTCAATCCG 
               
               
                   
                 TGCCTTCGTGAGCGTGAATAAAGGCCTGGTAACCCGCGCAAGCGCCGTAA 
               
               
                   
                 TCCCGGTAATCCCTCTGTATCTCGCCAGCTTGTTCAAAGTAATGAAAGAG 
               
               
                   
                 AAGGGCAATCATGAAGGTTGTATTGAACAGATCACGCGTCTGTACGCCGA 
               
               
                   
                 GCGCCTGTACCGTAAAGATGGTACAATTCCAGTTGATGAGGAAAATCGCA 
               
               
                   
                 TTCGCATTGATGATTGGGAGTTAGAAGAAGACGTCCAGAAAGCGGTATCC 
               
               
                   
                 GCGTTGATGGAGAAAGTCACGGGTGAAAACGCAGAATCTCTCACTGACTT 
               
               
                   
                 AGCGGGGTACCGCCATGATTTCTTAGCTAGTAACGGCTTTGATGTAGAAG 
               
               
                   
                 GTATTAATTATGAAGCGGAAGTTGAACGCTTCGACCGTATCTGA 
               
               
                   
               
               
                 tesB 
                 ATGAGTCAGGCGCTAAAAAATTTACTGACATTGTTAAATCTGGAAAAAAT 
               
               
                 SEQ ID NO: 10 
                 TGAGGAAGGACTCTTTCGCGGCCAGAGTGAAGATTTAGGTTTACGCCAGG 
               
               
                   
                 TGTTTGGCGGCCAGGTCGTGGGTCAGGCCTTGTATGCTGCAAAAGAGACC 
               
               
                   
                 GTCCCTGAAGAGCGGCTGGTACATTCGTTTCACAGCTACTTTCTTCGCCC 
               
               
                   
                 TGGCGATAGTAAGAAGCCGATTATTTATGATGTCGAAACGCTGCGTGACG 
               
               
                   
                 GTAACAGCTTCAGCGCCCGCCGGGTTGCTGCTATTCAAAACGGCAAACCG 
               
               
                   
                 ATTTTTTATATGACTGCCTCTTTCCAGGCACCAGAAGCGGGTTTCGAACA 
               
               
                   
                 TCAAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCCTCCCTTCGGAAA 
               
               
                   
                 CGCAAATCGCCCAATCGCTGGCGCACCTGCTGCCGCCAGTGCTGAAAGAT 
               
               
                   
                 AAATTCATCTGCGATCGTCCGCTGGAAGTCCGTCCGGTGGAGTTTCATAA 
               
               
                   
                 CCCACTGAAAGGTCACGTCGCAGAACCACATCGTCAGGTGTGGATCCGCG 
               
               
                   
                 CAAATGGTAGCGTGCCGGATGACCTGCGCGTTCATCAGTATCTGCTCGGT 
               
               
                   
                 TACGCTTCTGATCTTAACTTCCTGCCGGTAGCTCTACAGCCGCACGGCAT 
               
               
                   
                 CGGTTTTCTCGAACCGGGGATTCAGATTGCCACCATTGACCATTCCATGT 
               
               
                   
                 GGTTCCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATAGCGTGGAG 
               
               
                   
                 AGCACCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGAGTTTTATAC 
               
               
                   
                 CCAAGACGGCGTACTGGTTGCCTCGACCGTTCAGGAAGGGGTGATGCGTA 
               
               
                   
                 ATCACAATTAA 
               
               
                   
               
            
           
         
       
     
     Exemplary polypeptide sequences for the production of butyrate by the genetically engineered bacteria are provided in Table 3. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Exemplary Polypeptide Sequences  
               
               
                 for Butyrate Production 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 Bcd2 
                 MDLNSKKYQMLKELYVSFAENEVKPLATELDEEER 
               
               
                 SEQ ID NO: 11 
                 FPYETVEKMAKAGMMGIPYPKEYGGEGGDTVGYIM 
               
               
                   
                 AVEELSRVCGTTGVILSAHTSLGSWPIYQYGNEEQK 
               
               
                   
                 QKFLRPLASGEKLGAFGLTEPNAGTDASGQQTTAVL 
               
               
                   
                 DGDEYILNGSKIFITNAIAGDIYVVMAMTDKSKGNK 
               
               
                   
                 GISAFIVEKGTPGFSFGVKEKKMGIRGSATSELIFEDC 
               
               
                   
                 RIPKENLLGKEGQGFKIAMSTLDGGRIGIAAQALGLA 
               
               
                   
                 QGALDETVKYVKERVQFGRPLSKFQNTQFQLADME 
               
               
                   
                 VKVQAARHLVYQAAINKDLGKPYGVEAAMAKLFA 
               
               
                   
                 AETAMEVTTKAVQLHGGYGYTRDYPVERMMRDAK 
               
               
                   
                 ITEIYEGTSEVQRMVISGKLLK 
               
               
                   
               
               
                 etfB3 
                 MNIVVCIKQVPDTTEVKLDPNTGTLIRDGVPSIINPDD 
               
               
                 SEQ ID NO: 12 
                 KAGLEEAIKLKEEMGAHVTVITMGPPQADMALKEA 
               
               
                   
                 LAMGADRGILLTDRAFAGADTWATSSALAGALKNI 
               
               
                   
                 DFDIIIAGRQAIDGDTAQVGPQIAEHLNLPSITYAEEIK 
               
               
                   
                 TEGEYVLVKRQFEDCCHDLKVKMPCLITTLKDMNT 
               
               
                   
                 PRYMKVGRIYDAFENDVVETWTVKDIEVDPSNLGL 
               
               
                   
                 KGSPTSVFKSFTKSVKPAGTIYNEDAKTSAGIIIDKLK 
               
               
                   
                 EKYII 
               
               
                   
               
               
                 etfA3 
                 MGNVLVVIEQRENVIQTVSLELLGKATEIAKDYDTK 
               
               
                 SEQ ID NO: 13 
                 VSALLLGSKVEGLIDTLAHYGADEVIVVDDEALAVY 
               
               
                   
                 TTEPYTKAAYEAIKAADPIVVLFGATSIGRDLAPRVS 
               
               
                   
                 ARIHTGLTADCTGLAVAEDTKLLLMTRPAFGGNIMA 
               
               
                   
                 TIVCKDFRPQMSTVRPGVMKKNEPDETKEAVINRFK 
               
               
                   
                 VEFNDADKLVQVVQVIKEAKKQVKIEDAKILVSAGR 
               
               
                   
                 GMGGKENLDILYELAEIIGGEVSGSRATIDAGWLDK 
               
               
                   
                 ARQVGQTGKTVRPDLYIACGISGAIQHIAGMEDAEFI 
               
               
                   
                 VAINKNPEAPIFKYADVGIVGDVHKVLPELISQLSVA 
               
               
                   
                 KEKGEVLAN 
               
               
                   
               
               
                 Ter 
                 MIVKPMVRNNICLNAHPQGCKKGVEDQIEYTKKRIT 
               
               
                 SEQ ID NO: 14 
                 AEVKAGAKAPKNVLVLGCSNGYGLASRITAAFGYG 
               
               
                   
                 AATIGVSFEKAGSETKYGTPGWYNNLAFDEAAKRE 
               
               
                   
                 GLYSVTIDGDAFSDEIKAQVIEEAKKKGIKFDLIVYSL 
               
               
                   
                 ASPVRTDPDTGIMHKSVLKPFGKTFTGKTVDPFTGEL 
               
               
                   
                 KEISAEPANDEEAAATVKVMGGEDWERWIKQLSKE 
               
               
                   
                 GLLEEGCITLAYSYIGPEATQALYRKGTIGKAKEHLE 
               
               
                   
                 ATAHRLNKENPSIRAFVSVNKGLVTRASAVIPVIPLY 
               
               
                   
                 LASLEKVMKEKGNHEGCIEQITRLYAERLYRKDGTIP 
               
               
                   
                 VDEENRIRIDDWELEEDVQKAVSALMEKVTGENAES 
               
               
                   
                 LTDLAGYRHDFLASNGFDVEGINYEAEVERFDRI 
               
               
                   
               
               
                 ThiA 
                 MREVVIASAARTAVGSFGGAFKSVSAVELGVTAAK 
               
               
                 SEQ ID NO: 15 
                 EAIKRANITPDMIDESLLGGVLTAGLGQNIARQIALG 
               
               
                   
                 AGIPVEKPAMTINIVCGSGLRSVSMASQLIALGDADI 
               
               
                   
                 MLVGGAENMSMSPYLVPSARYGARMGDAAFVDSM 
               
               
                   
                 IKDGLSDIFNNYHMGITAENIAEQWNITREEQDELAL 
               
               
                   
                 ASQNKAEKAQAEGKFDEEIVPVVIKGRKGDTVVDK 
               
               
                   
                 DEYIKPGTTMEKLAKLRPAFKKDGTVTAGNASGIND 
               
               
                   
                 GAAMLVVMAKEKAEELGIEPLATIVSYGTAGVDPKI 
               
               
                   
                 MGYGPVPATKKALEAANMTIEDIDLVEANEAFAAQ 
               
               
                   
                 SVAVIRDLNIDMNKVNVNGGAIAIGHPIGCSGARILT 
               
               
                   
                 TLLYEMKRRDAKTGLATLCIGGGMGTTLIVKR 
               
               
                   
               
               
                 Hbd 
                 MKLAVIGSGTMGSGIVQTFASCGHDVCLKSRTQGAI 
               
               
                 SEQ ID NO: 16 
                 DKCLALLDKNLTKLVTKGKMDEATKAEILSHVSSTT 
               
               
                   
                 NYEDLKDMDLIIEASVEDMNIKKDVFKLLDELCKED 
               
               
                   
                 TILATNTSSLSITEIASSTKRPDKVIGMHFFNPVPMMK 
               
               
                   
                 LVEVISGQLTSKVTFDTVFELSKSINKVPVDVSESPGF 
               
               
                   
                 VVNRILIPMINEAVGIYADGVASKEEIDEAMKLGAN 
               
               
                   
                 HPMGPLALGDLIGLDVVLAIMNVLYTEFGDTKYRPH 
               
               
                   
                 PLLAKMVRANQLGRKTKIGFYDYNK 
               
               
                   
               
               
                 Crt2 
                 MSTSDVKVYENVAVEVDGNICTVKMNRPKALNAIN 
               
               
                 SEQ ID NO: 17 
                 SKTLEELYEVFVDINNDETIDVVILTGEGKAFVAGAD 
               
               
                   
                 IAYMKDLDAVAAKDFSILGAKAFGEIENSKKVVIAA 
               
               
                   
                 VNGFALGGGCELAMACDIRIASAKAKFGQPEVTLGI 
               
               
                   
                 TPGYGGTQRLTRLVGMAKAKELIFTGQVIKADEAEK 
               
               
                   
                 IGLVNRVVEPDILIEEVEKLAKIIAKNAQLAVRYSKE 
               
               
                   
                 AIQLGAQTDINTGIDIESNLFGLCFSTKDQKEGMSAF 
               
               
                   
                 VEKREANFIKG 
               
               
                   
               
               
                 Pbt 
                 MRSFEEVIKFAKERGPKTISVACCQDKEVLMAVEMA 
               
               
                 SEQ ID NO: 18 
                 RKEKIANAILVGDIEKTKEIAKSIDMDIENYELIDIKD 
               
               
                   
                 LAEASLKSVELVSQGKADMVMKGLVDTSIILKAVLN 
               
               
                   
                 KEVGLRTGNVLSHVAVFDVEGYDRLFFVTDAAMNL 
               
               
                   
                 APDTNTKKQIIENACTVAHSLDISEPKVAAICAKEKV 
               
               
                   
                 NPKMKDTVEAKELEEMYERGEIKGCMVGGPFAIDN 
               
               
                   
                 AVSLEAAKHKGINHPVAGRADILLAPDIEGGNILYKA 
               
               
                   
                 LVFFSKSKNAGVIVGAKAPIILTSRADSEETKLNSIAL 
               
               
                   
                 GVLMAAKA 
               
               
                   
               
               
                 Buk 
                 MSKIFKILTINPGSTSTKIAVFDNEDLVFEKTLRHSSE 
               
               
                 SEQ ID NO: 19 
                 EIGKYEKVSDQFEFRKQVIEEALKEGGVKTSELDAV 
               
               
                   
                 VGRGGLLKPIKGGTYSVSAAMIEDLKVGVLGEHASN 
               
               
                   
                 LGGIIAKQIGEEVNVPSYIVDPVVVDELEDVARISGM 
               
               
                   
                 PEISRASVVHALNQKAIARRYAREINKKYEDINLIVA 
               
               
                   
                 HMGGGVSVGAHKNGKIVDVANALDGEGPFSPERSG 
               
               
                   
                 GLPVGALVKMCFSGKYTQDEIKKKIKGNGGLVAYL 
               
               
                   
                 NTNDAREVEERIEAGDEKAKLVYEAMAYQISKEIGA 
               
               
                   
                 SAAVLKGDVKAILLTGGIAYSKMFTEMIADRVKFIA 
               
               
                   
                 DVKVYPGEDEMIALAQGGLRVLTGEEEAQVYDN 
               
               
                   
               
               
                 TesB 
                 MSQALKNLLTLLNLEKIEEGLFRGQSEDLGLRQVFG 
               
               
                 SEQ ID NO: 20 
                 GQVVGQALYAAKETVPEERLVHSFHSYFLRPGDSKK 
               
               
                   
                 PIIYDVETLRDGNSFSARRVAAIQNGKPIFYMTASFQ 
               
               
                   
                 APEAGFEHQKTMPSAPAPDGLPSETQIAQSLAHLLPP 
               
               
                   
                 VLKDKFICDRPLEVRPVEFHNPLKGHVAEPHRQVWI 
               
               
                   
                 RANGSVPDDLRVHQYLLGYASDLNFLPVALQPHGIG 
               
               
                   
                 FLEPGIQIATIDHSMWFHRPFNLNEWLLYSVESTSAS 
               
               
                   
                 SARGFVRGEFYTQDGVLVASTVQEGVMRNHN* 
               
               
                   
               
            
           
         
       
     
     The gene products of the bcd2, etfA13, and etfB3 genes in  Clostridium difficile  form a complex that converts crotonyl-CoA to butyryl-CoA, which may function as an oxygen-dependent co-oxidant. In some embodiments, because the genetically engineered bacteria of the invention are designed to produce butyrate in a microaerobic or oxygen-limited environment, e.g., the mammalian gut, oxygen dependence could have a negative effect on butyrate production in the gut. It has been shown that a single gene from  Treponema denticola  (ter, encoding trans-2-enoynl-CoA reductase) can functionally replace this three-gene complex in an oxygen-independent manner. In some embodiments, the genetically engineered bacteria comprise a ter gene, e.g., from  Treponema denticola , which can functionally replace all three of the bcd2, etfB3, and etfA3 genes, e.g., from  Peptoclostridium difficile . In this embodiment, the genetically engineered bacteria comprise thiA1, hbd, crt2, pbt, and buk, e.g., from  Peptoclostridium difficile , and ter, e.g., from  Treponema denticola , and produce butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. 
     In some embodiments, the genetically engineered bacteria of the invention comprise thiA1, hbd, crt2, pbt, and buk, e.g., from  Peptoclostridium difficile ; ter, e.g., from  Treponema denticola ; one or more of bcd2, etfB3, and etfA3, e.g., from  Peptoclostridium difficile ; and produce butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. 
     The gene products of pbt and buk convert butyrylCoA to Butyrate. In some embodiments, the pbt and buk genes can be replaced by a tesB gene. tesB can be used to cleave off the CoA from butyryl-coA. In one embodiment, the genetically engineered bacteria comprise bcd2, etfB3, etfA3, thiA1, hbd, and crt2, e.g., from  Peptoclostridium difficile , and tesB from  E. coli  and produce butyrate in low-oxygen conditions, in the presence of molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In one embodiment, the genetically engineered bacteria comprise ter gene (encoding trans-2-enoynl-CoA reductase) e.g., from  Treponema denticola , thiA1, hbd, crt2, pbt, and buk, e.g., from  Peptoclostridium difficile , and tesB from  E. coli , and produce butyrate in low-oxygen conditions, in the presence of specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production in low-oxygen conditions or in the presence of specific molecules or metabolites, or molecules or metabolites associated with condition(s) such as inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. 
     In some embodiments, the local production of butyrate induces the differentiation of regulatory T cells in the gut and/or promotes the barrier function of colonic epithelial cells. In some embodiments, the genetically engineered bacteria comprise genes for aerobic butyrate biosynthesis and/or genes for anaerobic or microaerobic butyrate biosynthesis. In some embodiments, local butyrate production reduces gut inflammation, a symptom of IBD and other gut related disorders. 
     In one embodiment, the bcd2 gene has at least about 80% identity with SEQ ID NO: 1. In another embodiment, the bcd2 gene has at least about 85% identity with SEQ ID NO: 1. In one embodiment, the bcd2 gene has at least about 90% identity with SEQ ID NO: 1. In one embodiment, the bcd2 gene has at least about 95% identity with SEQ ID NO: 1. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. Accordingly, in one embodiment, the bcd2 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. In another embodiment, the bcd2 gene comprises the sequence of SEQ ID NO: 1. In yet another embodiment the bcd2 gene consists of the sequence of SEQ ID NO: 1. 
     In one embodiment, the etfB3 gene has at least about 80% identity with SEQ ID NO: 2. In another embodiment, the etfB3 gene has at least about 85% identity with SEQ ID NO: 2. In one embodiment, the etfB3 gene has at least about 90% identity with SEQ ID NO: 2. In one embodiment, the etfB3 gene has at least about 95% identity with SEQ ID NO: 2. In another embodiment, the etfB3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2. Accordingly, in one embodiment, the etfB3 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2. In another embodiment, the etfB33 gene comprises the sequence of SEQ ID NO: 2. In yet another embodiment the etfB3 gene consists of the sequence of SEQ ID NO: 2. 
     In one embodiment, the etfA3 gene has at least about 80% identity with SEQ ID NO: 3. In another embodiment, the etfA3 gene has at least about 85% identity with SEQ ID NO: 3. In one embodiment, the etfA3 gene has at least about 90% identity with SEQ ID NO: 3. In one embodiment, the etfA3 gene has at least about 95% identity with SEQ ID NO: 3. In another embodiment, the etfA3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3. Accordingly, in one embodiment, the etfA3 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3. In another embodiment, the etfA3 gene comprises the sequence of SEQ ID NO: 3. In yet another embodiment the etfA3 gene consists of the sequence of SEQ ID NO: 3. 
     In one embodiment, the thiA1 gene has at least about 80% identity with SEQ ID NO: 4. In another embodiment, the thiA1 gene has at least about 85% identity with SEQ ID NO: 4. In one embodiment, the thiA1 gene has at least about 90% identity with SEQ ID NO: 4. In one embodiment, the thiA1 gene has at least about 95% identity with SEQ ID NO: 4. In another embodiment, the thiA1 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. Accordingly, in one embodiment, the thiA1 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. In another embodiment, the thiA1 gene comprises the sequence of SEQ ID NO: 4. In yet another embodiment the thiA1 gene consists of the sequence of SEQ ID NO: 4. 
     In one embodiment, the hbd gene has at least about 80% identity with SEQ ID NO: 5. In another embodiment, the hbd gene has at least about 85% identity with SEQ ID NO: 5. In one embodiment, the hbd gene has at least about 90% identity with SEQ ID NO: 5. In one embodiment, the hbd gene has at least about 95% identity with SEQ ID NO: 5. In another embodiment, the hbd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5. Accordingly, in one embodiment, the hbd gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5. In another embodiment, the hbd gene comprises the sequence of SEQ ID NO: 5. In yet another embodiment the hbd gene consists of the sequence of SEQ ID NO: 5. 
     In one embodiment, the crt2 gene has at least about 80% identity with SEQ ID NO: 6. In another embodiment, the crt2 gene has at least about 85% identity with SEQ ID NO: 6. In one embodiment, the crt2 gene has at least about 90% identity with SEQ ID NO: 6. In one embodiment, the crt2 gene has at least about 95% identity with SEQ ID NO: 6. In another embodiment, the crt2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6. Accordingly, in one embodiment, the crt2 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6. In another embodiment, the crt2 gene comprises the sequence of SEQ ID NO: 6. In yet another embodiment the crt2 gene consists of the sequence of SEQ ID NO: 6. 
     In one embodiment, the pbt gene has at least about 80% identity with SEQ ID NO: 7. In another embodiment, the pbt gene has at least about 85% identity with SEQ ID NO: 7. In one embodiment, the pbt gene has at least about 90% identity with SEQ ID NO: 7. In one embodiment, the pbt gene has at least about 95% identity with SEQ ID NO: 7. In another embodiment, the pbt gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 7. Accordingly, in one embodiment, the pbt gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 7. In another embodiment, the pbt gene comprises the sequence of SEQ ID NO: 7. In yet another embodiment the pbt gene consists of the sequence of SEQ ID NO: 7. 
     In one embodiment, the buk gene has at least about 80% identity with SEQ ID NO: 8. In another embodiment, the buk gene has at least about 85% identity with SEQ ID NO: 8. In one embodiment, the buk gene has at least about 90% identity with SEQ ID NO: 8. In one embodiment, the buk gene has at least about 95% identity with SEQ ID NO: 8. In another embodiment, the buk gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 8. Accordingly, in one embodiment, the buk gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 8. In another embodiment, the buk gene comprises the sequence of SEQ ID NO: 8. In yet another embodiment the buk gene consists of the sequence of SEQ ID NO: 8. 
     In one embodiment, the ter gene has at least about 80% identity with SEQ ID NO: 9. In another embodiment, the ter gene has at least about 85% identity with SEQ ID NO: 9. In one embodiment, the ter gene has at least about 90% identity with SEQ ID NO: 9. In one embodiment, the ter gene has at least about 95% identity with SEQ ID NO: 9. In another embodiment, the ter gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9. Accordingly, in one embodiment, the ter gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9. In another embodiment, the ter gene comprises the sequence of SEQ ID NO: 9. In yet another embodiment the ter gene consists of the sequence of SEQ ID NO: 9. 
     In one embodiment, the tesB gene has at least about 80% identity with SEQ ID NO: 10. In another embodiment, the tesB gene has at least about 85% identity with SEQ ID NO: 10. In one embodiment, the tesB gene has at least about 90% identity with SEQ ID NO: 10. In one embodiment, the tesB gene has at least about 95% identity with SEQ ID NO: 10. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. Accordingly, in one embodiment, the tesB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. In another embodiment, the tesB gene comprises the sequence of SEQ ID NO: 10. In yet another embodiment the tesB gene consists of the sequence of SEQ ID NO: 10. 
     In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 11 through SEQ ID NO: 20. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 11 through SEQ ID NO: 20. In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 11 through SEQ ID NO: 20. In one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 11 through SEQ ID NO: 20. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 11 through SEQ ID NO: 20. Accordingly, in one embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 11 through SEQ ID NO: 20. In another embodiment, one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria comprise the sequence of with one or more of SEQ ID NO: 11 through SEQ ID NO: 20. In yet another embodiment one or more polypeptides encoded by the butyrate circuits and expressed by the genetically engineered bacteria consist of the sequence of with one or more of SEQ ID NO: 11 through SEQ ID NO: 20. 
     In some embodiments, one or more of the butyrate biosynthesis genes is a synthetic butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a  Treponema denticola  butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a  C. glutamicum  butyrate biosynthesis gene. In some embodiments, one or more of the butyrate biosynthesis genes is a  Peptoclostridicum difficile  butyrate biosynthesis gene. The butyrate gene cassette may comprise genes for the aerobic biosynthesis of butyrate and/or genes for the anaerobic or microaerobic biosynthesis of butyrate. 
     To improve acetate production, while maintaining high levels of butyrate production, one or more targeted deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby simultaneously increasing butyrate and acetate production). Non-limiting examples of such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more butyrate-producing cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more butyrate producing cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle, while maintaining high levels of butyrate production. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for butyrate production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for butyrate synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of butyrate and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of butyrate and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta and ldhA genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, pbt, and/or buk and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-pbt-buk butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation in the endogenous pta and ldhA genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from ter, thiA1, hbd, crt2, tesB and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more ter-thiA1-hbd-crt2-tesB butyrate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to. 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more butyrate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria comprise a combination of butyrate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing butyrate. In some embodiments, one or more of the butyrate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase butyrate production. In some embodiments, the local production of butyrate reduces food intake and ameliorates improves gut barrier function and reduces inflammation. In some embodiments, the genetically engineered bacteria are capable of expressing the butyrate biosynthesis cassette and producing butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. 
     In one embodiment, the butyrate gene cassette is directly operably linked to a first promoter. In another embodiment, the butyrate gene cassette is indirectly operably linked to a first promoter. In one embodiment, the promoter is not operably linked with the butyrate gene cassette in nature. 
     In some embodiments, the butyrate gene cassette is expressed under the control of a constitutive promoter. In another embodiment, the butyrate gene cassette is expressed under the control of an inducible promoter. In some embodiments, the butyrate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the butyrate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the butyrate gene cassette is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. Inducible promoters are described in more detail infra. 
     The butyrate gene cassette may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the butyrate gene cassette is located on a plasmid in the bacterial cell. In another embodiment, the butyrate gene cassette is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the butyrate gene cassette is located in the chromosome of the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the butyrate gene cassette is located on a plasmid in the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the butyrate gene cassette is located in the chromosome of the bacterial cell, and a butyrate gene cassette from a different species of bacteria is located in the chromosome of the bacterial cell. 
     In some embodiments, the butyrate gene cassette is expressed on a low-copy plasmid. In some embodiments, the butyrate gene cassette is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of butyrate. 
     Propionate 
     In alternate embodiments, the genetically engineered bacteria of the invention are capable of producing an anti-inflammatory or gut barrier enhancer molecule, e.g., propionate, that is synthesized by a biosynthetic pathway requiring multiple genes and/or enzymes. 
     In some embodiments, the genetically engineered bacteria of the invention comprise a propionate gene cassette and are capable of producing propionate under particular exogenous environmental conditions. The genetically engineered bacteria may express any suitable set of propionate biosynthesis genes (see, e.g., Table 4, Table 5, Table 6, Table 7). Unmodified bacteria that are capable of producing propionate via an endogenous propionate biosynthesis pathway include, but are not limited to,  Clostridium propionicum, Megasphaera elsdenii , and  Prevotella ruminicola . In some embodiments, the genetically engineered bacteria of the invention comprise propionate biosynthesis genes from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise the genes pct, lcd, and acr from  Clostridium propionicum . In some embodiments, the genetically engineered bacteria comprise acrylate pathway genes for propionate biosynthesis, e.g., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC. In some embodiments, the rate limiting step catalyzed by the Acr enzyme, is replaced by the AcuI from  R. sphaeroides , which catalyzes the NADPH-dependent acrylyl-CoA reduction to produce propionyl-CoA. Thus the propionate cassette comprises pct, lcdA, lcdB, lcdC, and acuI. In another embodiment, the homolog of AcuI in  E. coli , yhdH is used. This the propionate cassette comprises pct, lcdA, lcdB, lcdC, and yhdH. In alternate embodiments, the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, e.g., thrA fbr , thrB, thrC, ilvA fbr , aceE, aceF, and lpd, and optionally further comprise tesB. In another embodiment, the propionate gene cassette comprises the genes of the Sleepting Beauty Mutase operon, e.g., from  E. coli  (sbm, ygfD, ygfG, ygfH. The SBM pathway is cyclical and composed of a series of biochemical conversions forming propionate as a fermentative product while regenerating the starting molecule of succinyl-CoA. Sbm converts succinyl CoA to L-methylmalonylCoA, ygfG converts L-methylmalonylCoA into PropionylCoA, and ygfH converts propionylCoA into propionate and succinate into succinylCoA. 
     This pathway is very similar to the oxidative propionate pathway of Propionibacteria, which also converts succinate to propionate. Succinyl-CoA is converted to R-methylmalonyl-CoA by methymalonyl-CoA mutase (mutAB). This is in turn converted to S-methylmalonyl-CoA via methymalonyl-CoA epimerase (GI: 18042134). There are three genes which encode methylmalonyl-CoA carboxytransferase (mmdA, PFREUD_18870, bccp) which converts methylmalonyl-CoA to propionyl-CoA. 
     The genes may be codon-optimized, and translational and transcriptional elements may be added. Table 4-6 lists the nucleic acid sequences of exemplary genes in the propionate biosynthesis gene cassette. Table 7 lists the polypeptide sequences expressed by exemplary propionate biosynthesis genes. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Propionate Cassette Sequences (Acrylate Pathway) 
               
            
           
           
               
               
            
               
                 Gene sequence 
                 Description 
               
               
                   
               
               
                 pct SEQ ID NO: 21 
                 ATGCGCAAAGTGCCGATTATCACGGCTGACGAGGCCGCAAAAC 
               
               
                   
                 TGATCAAGGACGGCGACACCGTGACAACTAGCGGCTTTGTGGGT 
               
               
                   
                 AACGCGATCCCTGAGGCCCTTGACCGTGCAGTCGAAAAGCGTTT 
               
               
                   
                 CCTGGAAACGGGCGAACCGAAGAACATTACTTATGTATATTGCG 
               
               
                   
                 GCAGTCAGGGCAATCGCGACGGTCGTGGCGCAGAACATTTCGC 
               
               
                   
                 GCATGAAGGCCTGCTGAAACGTTATATCGCTGGCCATTGGGCGA 
               
               
                   
                 CCGTCCCGGCGTTAGGGAAAATGGCCATGGAGAATAAAATGGA 
               
               
                   
                 GGCCTACAATGTCTCTCAGGGCGCCTTGTGTCATCTCTTTCGCGA 
               
               
                   
                 TATTGCGAGCCATAAACCGGGTGTGTTCACGAAAGTAGGAATCG 
               
               
                   
                 GCACCTTCATTGATCCACGTAACGGTGGTGGGAAGGTCAACGAT 
               
               
                   
                 ATTACCAAGGAAGATATCGTAGAACTGGTGGAAATTAAAGGGC 
               
               
                   
                 AGGAATACCTGTTTTATCCGGCGTTCCCGATCCATGTCGCGCTG 
               
               
                   
                 ATTCGTGGCACCTATGCGGACGAGAGTGGTAACATCACCTTTGA 
               
               
                   
                 AAAAGAGGTAGCGCCTTTGGAAGGGACTTCTGTCTGTCAAGCGG 
               
               
                   
                 TGAAGAACTCGGGTGGCATTGTCGTGGTTCAGGTTGAGCGTGTC 
               
               
                   
                 GTCAAAGCAGGCACGCTGGATCCGCGCCATGTGAAAGTTCCGG 
               
               
                   
                 GTATCTATGTAGATTACGTAGTCGTCGCGGATCCGGAGGACCAT 
               
               
                   
                 CAACAGTCCCTTGACTGCGAATATGATCCTGCCCTTAGTGGAGA 
               
               
                   
                 GCACCGTCGTCCGGAGGTGGTGGGTGAACCACTGCCTTTATCCG 
               
               
                   
                 CGAAGAAAGTCATCGGCCGCCGTGGCGCGATTGAGCTCGAGAA 
               
               
                   
                 AGACGTTGCAGTGAACCTTGGGGTAGGTGCACCTGAGTATGTGG 
               
               
                   
                 CCTCCGTGGCCGATGAAGAAGGCATTGTGGATTTTATGACTCTC 
               
               
                   
                 ACAGCGGAGTCCGGCGCTATCGGTGGCGTTCCAGCCGGCGGTGT 
               
               
                   
                 TCGCTTTGGGGCGAGCTACAATGCTGACGCCTTGATCGACCAGG 
               
               
                   
                 GCTACCAATTTGATTATTACGACGGTGGGGGTCTGGATCTTTGTT 
               
               
                   
                 ACCTGGGTTTAGCTGAATGCGACGAAAAGGGTAATATCAATGTT 
               
               
                   
                 AGCCGCTTCGGTCCTCGTATCGCTGGGTGCGGCGGATTCATTAA 
               
               
                   
                 CATTACCCAAAACACGCCGAAAGTCTTCTTTTGTGGGACCTTTA 
               
               
                   
                 CAGCCGGGGGGCTGAAAGTGAAAATTGAAGATGGTAAGGTGAT 
               
               
                   
                 TATCGTTCAGGAAGGGAAACAGAAGAAATTCCTTAAGGCAGTG 
               
               
                   
                 GAGCAAATCACCTTTAATGGAGACGTGGCCTTAGCGAACAAGC 
               
               
                   
                 AACAAGTTACCTACATCACGGAGCGTTGCGTCTTCCTCCTCAAA 
               
               
                   
                 GAAGACGGTTTACACCTTTCGGAAATCGCGCCAGGCATCGATCT 
               
               
                   
                 GCAGACCCAGATTTTGGATGTTATGGACTTTGCCCCGATCATTG 
               
               
                   
                 ATCGTGACGCAAACGGGCAGATTAAACTGATGGACGCGGCGTT 
               
               
                   
                 ATTCGCAGAAGGGCTGATGGGCTTGAAAGAAATGAAGTCTTAA 
               
               
                   
               
               
                 lcdA SEQ ID NO: 22 
                 ATGAGCTTAACCCAAGGCATGAAAGCTAAACAACTGTTAGCAT 
               
               
                   
                 ACTTTCAGGGTAAAGCCGATCAGGATGCACGTGAAGCGAAAGC 
               
               
                   
                 CCGCGGTGAGCTGGTCTGCTGGTCGGCGTCAGTCGCGCCGCCGG 
               
               
                   
                 AATTTTGCGTAACAATGGGCATTGCCATGATCTACCCGGAGACT 
               
               
                   
                 CATGCAGCGGGCATCGGTGCCCGCAAAGGTGCGATGGACATGC 
               
               
                   
                 TGGAAGTTGCGGACCGCAAAGGCTACAACGTGGATTGTTGTTCC 
               
               
                   
                 TACGGCCGTGTAAATATGGGTTACATGGAATGTTTAAAAGAAGC 
               
               
                   
                 CGCCATCACGGGCGTCAAGCCGGAAGTTTTGGTTAATTCCCCTG 
               
               
                   
                 CTGCTGACGTTCCGCTTCCCGATTTGGTGATTACGTGTAATAATA 
               
               
                   
                 TCTGTAACACGCTGCTGAAATGGTACGAAAACTTAGCAGCAGA 
               
               
                   
                 ACTCGATATTCCTTGCATCGTGATCGACGTACCGTTTAATCATAC 
               
               
                   
                 CATGCCGATTCCGGAATATGCCAAGGCCTACATCGCGGACCAGT 
               
               
                   
                 TCCGCAATGCAATTTCTCAGCTGGAAGTTATTTGTGGCCGTCCGT 
               
               
                   
                 TCGATTGGAAGAAATTTAAGGAGGTCAAAGATCAGACCCAGCG 
               
               
                   
                 TAGCGTATACCACTGGAACCGCATTGCCGAGATGGCGAAATAC 
               
               
                   
                 AAGCCTAGCCCGCTGAACGGCTTCGATCTGTTCAATTACATGGC 
               
               
                   
                 GTTAATCGTGGCGTGCCGCAGCCTGGATTATGCAGAAATTACCT 
               
               
                   
                 TTAAAGCGTTCGCGGACGAATTAGAAGAGAATTTGAAGGCGGG 
               
               
                   
                 TATCTACGCCTTTAAAGGTGCGGAAAAAACGCGCTTTCAATGGG 
               
               
                   
                 AAGGTATCGCGGTGTGGCCACATTTAGGTCACACGTTTAAATCT 
               
               
                   
                 ATGAAGAATCTGAATTCGATTATGACCGGTACGGCATACCCCGC 
               
               
                   
                 CCTTTGGGACCTGCACTATGACGCTAACGACGAATCTATGCACT 
               
               
                   
                 CTATGGCTGAAGCGTACACCCGTATTTATATTAATACTTGTCTGC 
               
               
                   
                 AGAACAAAGTAGAGGTCCTGCTTGGGATCATGGAAAAAGGCCA 
               
               
                   
                 GGTGGATGGTACCGTATATCATCTGAATCGCAGCTGCAAACTGA 
               
               
                   
                 TGAGTTTCCTGAACGTGGAAACGGCTGAAATTATTAAAGAGAA 
               
               
                   
                 GAACGGTCTTCCTTACGTCTCCATTGATGGCGATCAGACCGATC 
               
               
                   
                 CTCGCGTTTTTTCTCCGGCCCAGTTTGATACCCGTGTTCAGGCCC 
               
               
                   
                 TGGTTGAGATGATGGAGGCCAATATGGCGGCAGCGGAATAA 
               
               
                   
               
               
                 lcdB SEQ ID NO: 23 
                 ATGTCACGCGTGGAGGCAATCCTGTCGCAGCTGAAAGATGTCGC 
               
               
                   
                 CGCGAATCCGAAAAAAGCCATGGATGACTATAAAGCTGAAACA 
               
               
                   
                 GGTAAGGGCGCGGTTGGTATCATGCCGATCTACAGCCCCGAAG 
               
               
                   
                 AAATGGTACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGC 
               
               
                   
                 GCCCAGGGCAAAACGATTAGTAAAGCGCGCACCTATCTGCCTGC 
               
               
                   
                 TTTTGCCTGCAGCGTAATGCAGCAGGTTATGGAATTACAGTGCG 
               
               
                   
                 AGGGCGCGTATGATGACCTGTCCGCAGTTATTTTTAGCGTACCG 
               
               
                   
                 TGCGACACTCTCAAATGTCTTAGCCAGAAATGGAAAGGTACGTC 
               
               
                   
                 CCCAGTGATTGTATTTACGCATCCGCAGAACCGCGGATTAGAAG 
               
               
                   
                 CGGCGAACCAATTCTTGGTTACCGAGTATGAACTGGTAAAAGCA 
               
               
                   
                 CAACTGGAATCAGTTCTGGGTGTGAAAATTTCAAACGCCGCCCT 
               
               
                   
                 GGAAAATTCGATTGCAATTTATAACGAGAATCGTGCCGTGATGC 
               
               
                   
                 GTGAGTTCGTGAAAGTGGCAGCGGACTATCCTCAAGTCATTGAC 
               
               
                   
                 GCAGTGAGCCGCCACGCGGTTTTTAAAGCGCGCCAGTTTATGCT 
               
               
                   
                 TAAGGAAAAACATACCGCACTTGTGAAAGAACTGATCGCTGAG 
               
               
                   
                 ATTAAAGCAACGCCAGTCCAGCCGTGGGACGGAAAAAAGGTTG 
               
               
                   
                 TAGTGACGGGCATTCTGTTGGAACCGAATGAGTTATTAGATATC 
               
               
                   
                 TTTAATGAGTTTAAGATCGCGATTGTTGATGATGATTTAGCGCA 
               
               
                   
                 GGAAAGCCGTCAGATCCGTGTTGACGTTCTGGACGGAGAAGGC 
               
               
                   
                 GGACCGCTCTACCGTATGGCTAAAGCGTGGCAGCAAATGTATGG 
               
               
                   
                 CTGCTCGCTGGCAACCGACACCAAGAAGGGTCGCGGCCGTATGT 
               
               
                   
                 TAATTAACAAAACGATTCAGACCGGTGCGGACGCTATCGTAGTT 
               
               
                   
                 GCAATGATGAAGTTTTGCGACCCAGAAGAATGGGATTATCCGGT 
               
               
                   
                 AATGTACCGTGAATTTGAAGAAAAAGGGGTCAAATCACTTATG 
               
               
                   
                 ATTGAGGTGGATCAGGAAGTATCGTCTTTCGAACAGATTAAAAC 
               
               
                   
                 CCGTCTGCAGTCATTCGTCGAAATGCTTTAA 
               
               
                   
               
               
                 lcdC SEQ ID NO: 24 
                 ATGTATACCTTGGGGATTGATGTCGGTTCTGCCTCTAGTAAAGC 
               
               
                   
                 GGTGATTCTGAAAGATGGAAAAGATATTGTCGCTGCCGAGGTTG 
               
               
                   
                 TCCAAGTCGGTACCGGCTCCTCGGGTCCCCAACGCGCACTGGAC 
               
               
                   
                 AAAGCCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACATCAGCTA 
               
               
                   
                 CACAGTAGCTACGGGCTATGGGCGCTTCAATTTTAGCGACGCGG 
               
               
                   
                 ATAAACAGATTTCGGAAATTAGCTGTCATGCCAAAGGCATTTAT 
               
               
                   
                 TTCTTAGTACCAACTGCGCGCACTATTATTGACATTGGCGGCCA 
               
               
                   
                 AGATGCGAAAGCCATCCGCCTGGACGACAAGGGGGGTATTAAG 
               
               
                   
                 CAATTCTTCATGAATGATAAATGCGCGGCGGGCACGGGGCGTTT 
               
               
                   
                 CCTGGAAGTCATGGCTCGCGTACTTGAAACCACCCTGGATGAAA 
               
               
                   
                 TGGCTGAACTGGATGAACAGGCGACTGACACCGCTCCCATTTCA 
               
               
                   
                 AGCACCTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAATT 
               
               
                   
                 GAGCAATGGTGTCTCACGCAACAACATCATTAAAGGTGTCCATC 
               
               
                   
                 TGAGCGTTGCGTCACGTGCGTGTGGTCTGGCGTATCGCGGCGGT 
               
               
                   
                 TTGGAGAAAGATGTTGTTATGACAGGTGGCGTGGCAAAAAATG 
               
               
                   
                 CAGGGGTGGTGCGCGCGGTGGCGGGCGTTCTGAAGACCGATGT 
               
               
                   
                 TATCGTTGCTCCGAATCCTCAGACGACCGGTGCACTGGGGGCAG 
               
               
                   
                 CGCTGTATGCTTATGAGGCCGCCCAGAAGAAGTA 
               
               
                   
               
               
                 etfA SEQ ID NO: 25 
                 ATGGCCTTCAATAGCGCAGATATTAATTCTTTCCGCGATATTTGG 
               
               
                   
                 GTGTTTTGTGAACAGCGTGAGGGCAAACTGATTAACACCGATTT 
               
               
                   
                 CGAATTAATTAGCGAAGGTCGTAAACTGGCTGACGAACGCGGA 
               
               
                   
                 AGCAAACTGGTTGGAATTTTGCTGGGGCACGAAGTTGAAGAAA 
               
               
                   
                 TCGCAAAAGAATTAGGCGGCTATGGTGCGGACAAGGTAATTGT 
               
               
                   
                 GTGCGATCATCCGGAACTTAAATTTTACACTACGGATGCTTATG 
               
               
                   
                 CCAAAGTTTTATGTGACGTCGTGATGGAAGAGAAACCGGAGGT 
               
               
                   
                 AATTTTGATCGGTGCCACCAACATTGGCCGTGATCTCGGACCGC 
               
               
                   
                 GTTGTGCTGCACGCTTGCACACGGGGCTGACGGCTGATTGCACG 
               
               
                   
                 CACCTGGATATTGATATGAATAAATATGTGGACTTTCTTAGCAC 
               
               
                   
                 CAGTAGCACCTTGGATATCTCGTCGATGACTTTCCCTATGGAAG 
               
               
                   
                 ATACAAACCTTAAAATGACGCGCCCTGCATTTGGCGGACATCTG 
               
               
                   
                 ATGGCAACGATCATTTGTCCACGCTTCCGTCCCTGTATGAGCAC 
               
               
                   
                 AGTGCGCCCCGGAGTGATGAAGAAAGCGGAGTTCTCGCAGGAG 
               
               
                   
                 ATGGCGCAAGCATGTCAAGTAGTGACCCGTCACGTAAATTTGTC 
               
               
                   
                 GGATGAAGACCTTAAAACTAAAGTAATTAATATCGTGAAGGAA 
               
               
                   
                 ACGAAAAAGATTGTGGATCTGATCGGCGCAGAAATTATTGTGTC 
               
               
                   
                 AGTTGGTCGTGGTATCTCGAAAGATGTCCAAGGTGGAATTGCAC 
               
               
                   
                 TGGCTGAAAAACTTGCGGACGCATTTGGTAACGGTGTCGTGGGC 
               
               
                   
                 GGCTCGCGCGCAGTGATTGATTCCGGCTGGTTACCTGCGGATCA 
               
               
                   
                 TCAGGTTGGACAAACCGGTAAGACCGTGCACCCGAAAGTCTAC 
               
               
                   
                 GTGGCGCTGGGTATTAGTGGGGCTATCCAGCATAAGGCTGGGAT 
               
               
                   
                 GCAAGACTCTGAACTGATCATTGCCGTCAACAAAGACGAAACG 
               
               
                   
                 GCGCCTATCTTCGACTGCGCCGATTATGGCATCACCGGTGATTT 
               
               
                   
                 ATTTAAAATCGTACCGATGATGATCGACGCGATCAAAGAGGGT 
               
               
                   
                 AAAAACGCATGA 
               
               
                   
               
               
                 acrB SEQ ID NO: 26 
                 ATGCGCATCTATGTGTGTGTGAAACAAGTCCCAGATACGAGCGG 
               
               
                   
                 CAAGGTGGCCGTTAACCCTGATGGGACCCTTAACCGTGCCTCAA 
               
               
                   
                 TGGCAGCGATTATTAACCCGGACGATATGTCCGCGATCGAACAG 
               
               
                   
                 GCATTAAAACTGAAAGATGAAACCGGATGCCAGGTTACGGCGC 
               
               
                   
                 TTACGATGGGTCCTCCTCCTGCCGAGGGCATGTTGCGCGAAATT 
               
               
                   
                 ATTGCAATGGGGGCCGACGATGGTGTGCTGATTTCGGCCCGTGA 
               
               
                   
                 ATTTGGGGGGTCCGATACCTTCGCAACCAGTCAAATTATTAGCG 
               
               
                   
                 CGGCAATCCATAAATTAGGCTTAAGCAATGAAGACATGATCTTT 
               
               
                   
                 TGCGGTCGTCAGGCCATTGACGGTGATACGGCCCAAGTCGGCCC 
               
               
                   
                 TCAAATTGCCGAAAAACTGAGCATCCCACAGGTAACCTATGGCG 
               
               
                   
                 CAGGAATCAAAAAATCTGGTGATTTAGTGCTGGTGAAGCGTATG 
               
               
                   
                 TTGGAGGATGGTTATATGATGATCGAAGTCGAAACTCCATGTCT 
               
               
                   
                 GATTACCTGCATTCAGGATAAAGCGGTAAAACCACGTTACATGA 
               
               
                   
                 CTCTCAACGGTATTATGGAATGCTACTCCAAGCCGCTCCTCGTTC 
               
               
                   
                 TCGATTACGAAGCACTGAAAGATGAACCGCTGATCGAACTTGAT 
               
               
                   
                 ACCATTGGGCTTAAAGGCTCCCCGACGAATATCTTTAAATCGTT 
               
               
                   
                 TACGCCGCCTCAGAAAGGCGTTGGTGTCATGCTCCAAGGCACCG 
               
               
                   
                 ATAAGGAAAAAGTCGAGGATCTGGTGGATAAGCTGATGCAGAA 
               
               
                   
                 ACATGTCATCTAA 
               
               
                   
               
               
                 acrC SEQ ID NO: 27 
                 ATGTTCTTACTGAAGATTAAAAAAGAACGTATGAAACGCATGG 
               
               
                   
                 ACTTTAGTTTAACGCGTGAACAGGAGATGTTAAAAAAACTGGCG 
               
               
                   
                 CGTCAGTTTGCTGAGATCGAGCTGGAACCGGTGGCCGAAGAGA 
               
               
                   
                 TTGATCGTGAGCACGTTTTTCCTGCAGAAAACTTTAAGAAGATG 
               
               
                   
                 GCGGAAATTGGCTTAACCGGCATTGGTATCCCGAAAGAATTTGG 
               
               
                   
                 TGGCTCCGGTGGAGGCACCCTGGAGAAGGTCATTGCCGTGTCAG 
               
               
                   
                 AATTCGGCAAAAAGTGTATGGCCTCAGCTTCCATTTTAAGCATT 
               
               
                   
                 CATCTTATCGCGCCGCAGGCAATCTACAAATATGGGACCAAAGA 
               
               
                   
                 ACAGAAAGAGACGTACCTGCCGCGTCTTACCAAAGGTGGTGAA 
               
               
                   
                 CTGGGCGCCTTTGCGCTGACAGAACCAAACGCCGGAAGCGATG 
               
               
                   
                 CCGGCGCGGTAAAAACGACCGCGATTCTGGACAGCCAGACAAA 
               
               
                   
                 CGAGTACGTGCTGAATGGCACCAAATGCTTTATCAGCGGGGGCG 
               
               
                   
                 GGCGCGCGGGTGTTCTTGTAATTTTTGCGCTTACTGAACCGAAA 
               
               
                   
                 AAAGGTCTGAAAGGGATGAGCGCGATTATCGTGGAGAAAGGGA 
               
               
                   
                 CCCCGGGCTTCAGCATCGGCAAGGTGGAGAGCAAGATGGGGAT 
               
               
                   
                 CGCAGGTTCGGAAACCGCGGAACTTATCTTCGAAGATTGTCGCG 
               
               
                   
                 TTCCGGCTGCCAACCTTTTAGGTAAAGAAGGCAAAGGCTTTAAA 
               
               
                   
                 ATTGCTATGGAAGCCCTGGATGGCGCCCGTATTGGCGTGGGCGC 
               
               
                   
                 TCAAGCAATCGGAATTGCCGAGGGGGCGATCGACCTGAGTGTG 
               
               
                   
                 AAGTACGTTCACGAGCGCATTCAATTTGGTAAACCGATCGCGAA 
               
               
                   
                 TCTGCAGGGAATTCAATGGTATATCGCGGATATGGCGACCAAAA 
               
               
                   
                 CCGCCGCGGCACGCGCACTTGTTGAGTTTGCAGCGTATCTTGAA 
               
               
                   
                 GACGCGGGTAAACCGTTCACAAAGGAATCTGCTATGTGCAAGCT 
               
               
                   
                 GAACGCCTCCGAAAACGCGCGTTTTGTGACAAATTTAGCTCTGC 
               
               
                   
                 AGATTCACGGGGGTTACGGTTATATGAAAGATTATCCGTTAGAG 
               
               
                   
                 CGTATGTATCGCGATGCTAAGATTACGGAAATTTACGAGGGGAC 
               
               
                   
                 ATCAGAAATCCATAAGGTGGTGATTGCGCGTGAAGTAATGAAA 
               
               
                   
                 CGCTAA 
               
               
                   
               
               
                 thrA fbr  SEQ ID NO: 28 
                 ATGCGAGTGTTGAAGTTCGGCGGTACATCAGTGGCAAATGCAG 
               
               
                   
                 AACGTTTTCTGCGTGTTGCCGATATTCTGGAAAGCAATGCCAGG 
               
               
                   
                 CAGGGGCAGGTGGCCACCGTCCTCTCTGCCCCCGCCAAAATCAC 
               
               
                   
                 CAACCACCTGGTGGCGATGATTGAAAAAACCATTAGCGGCCAG 
               
               
                   
                 GATGCTTTACCCAATATCAGCGATGCCGAACGTATTTGCCGA 
               
               
                   
                 ACTTTTGACGGGACTCGCCGCCGCCCAGCCGGGGTTCCCGCTGG 
               
               
                   
                 CGCAATTGAAAACTTTCGTCGATCAGGAATTTGCCCAAATAAAA 
               
               
                   
                 CATGTCCTGCATGGCATTAGTTTGTTGGGGCAGTGCCCGGATAG 
               
               
                   
                 CATCAACGCTGCGCTGATTTGCCGTGGCGAGAAAATGTCGATCG 
               
               
                   
                 CCATTATGGCCGGCGTATTAGAAGCGCGCGGTCACAACGTTACT 
               
               
                   
                 GTTATCGATCCGGTCGAAAAACTGCTGGCAGTGGGGCATTACCT 
               
               
                   
                 CGAATCTACCGTCGATATTGCTGAGTCCACCCGCCGTATTGCGG 
               
               
                   
                 CAAGCCGCATTCCGGCTGATCACATGGTGCTGATGGCAGGTTTC 
               
               
                   
                 ACCGCCGGTAATGAAAAAGGCGAACTGGTGGTGCTTGGACGCA 
               
               
                   
                 ACGGTTCCGACTACTCTGCTGCGGTGCTGGCTGCCTGTTTACGC 
               
               
                   
                 GCCGATTGTTGCGAGATTTGGACGGACGTTGACGGGGTCTATAC 
               
               
                   
                 CTGCGACCCGCGTCAGGTGCCCGATGCGAGGTTGTTGAAGTCGA 
               
               
                   
                 TGTCCTACCAGGAAGCGATGGAGCTTTCCTACTTCGGCGCTAAA 
               
               
                   
                 GTTCTTCACCCCCGCACCATTACCCCCATCGCCCAGTTCCAGATC 
               
               
                   
                 CCTTGCCTGATTAAAAATACCGGAAATCCTCAAGCACCAGGTAC 
               
               
                   
                 GCTCATTGGTGCCAGCCGTGATGAAGACGAATTACCGGTCAAGG 
               
               
                   
                 GCATTTCCAATCTGAATAACATGGCAATGTTCAGCGTTTCTGGT 
               
               
                   
                 CCGGGGATGAAAGGGATGGTCGGCATGGCGGCGCGCGTCTTTG 
               
               
                   
                 CAGCGATGTCACGCGCCCGTATTTCCGTGGTGCTGATTACGCAA 
               
               
                   
                 TCATCTTCCGAATACAGCATCAGTTTCTGCGTTCCACAAAGCGA 
               
               
                   
                 CTGTGTGCGAGCTGAACGGGCAATGCAGGAAGAGTTCTACCTG 
               
               
                   
                 GAACTGAAAGAAGGCTTACTGGAGCCGCTGGCAGTGACGGAAC 
               
               
                   
                 GGCTGGCCATTATCTCGGTGGTAGGTGATGGTATGCGCACCTTG 
               
               
                   
                 CGTGGGATCTCGGCGAAATTCTTTGCCGCACTGGCCCGCGCCAA 
               
               
                   
                 TATCAACATTGTCGCCATTGCTCAGAGATCTTCTGAACGCTCAA 
               
               
                   
                 TCTCTGTCGTGGTAAATAACGATGATGCGACCACTGGCGTGCGC 
               
               
                   
                 GTTACTCATCAGATGCTGTTCAATACCGATCAGGTTATCGAAGT 
               
               
                   
                 GTTTGTGATTGGCGTCGGTGGCGTTGGCGGTGCGCTGCTGGAGC 
               
               
                   
                 AACTGAAGCGTCAGCAAAGCTGGCTGAAGAATAAACATATCGA 
               
               
                   
                 CTTACGTGTCTGCGGTGTTGCCAACTCGAAGGCTCTGCTCACCA 
               
               
                   
                 ATGTACATGGCCTTAATCTGGAAAACTGGCAGGAAGAACTGGC 
               
               
                   
                 GCAAGCCAAAGAGCCGTTTAATCTCGGGCGCTTAATTCGCCTCG 
               
               
                   
                 TGAAAGAATATCATCTGCTGAACCCGGTCATTGTTGACTGCACT 
               
               
                   
                 TCCAGCCAGGCAGTGGCGGATCAATATGCCGACTTCCTGCGCGA 
               
               
                   
                 AGGTTTCCACGTTGTCACGCCGAACAAAAAGGCCAACACCTCGT 
               
               
                   
                 CGATGGATTACTACCATCAGTTGCGTTATGCGGCGGAAAAATCG 
               
               
                   
                 CGGCGTAAATTCCTCTATGACACCAACGTTGGGGCTGGATTACC 
               
               
                   
                 GGTTATTGAGAACCTGCAAAATCTGCTCAATGCAGGTGATGAAT 
               
               
                   
                 TGATGAAGTTCTCCGGCATTCTTTCTGGTTCGCTTTCTTATATCTT 
               
               
                   
                 CGGCAAGTTAGACGAAGGCATGAGTTTCTCCGAGGCGACCACG 
               
               
                   
                 CTGGCGCGGGAAATGGGTTATACCGAACCGGACCCGCGAGATG 
               
               
                   
                 ATCTTTCTGGTATGGATGTGGCGCGTAAACTATTGATTCTCGCTC 
               
               
                   
                 GTGAAACGGGACGTGAACTGGAGCTGGCGGATATTGAAATTGA 
               
               
                   
                 ACCTGTGCTGCCCGCAGAGTTTAACGCCGAGGGTGATGTTGCCG 
               
               
                   
                 CTTTTATGGCGAATCTGTCACAACTCGACGATCTCTTTGCCGCGC 
               
               
                   
                 GCGTGGCGAAGGCCCGTGATGAAGGAAAAGTTTTGCGCTATGTT 
               
               
                   
                 GGCAATATTGATGAAGATGGCGTCTGCCGCGTGAAGATTGCCGA 
               
               
                   
                 AGTGGATGGTAATGATCCGCTGTTCAAAGTGAAAAATGGCGAA 
               
               
                   
                 AACGCCCTGGCCTTCTATAGCCACTATTATCAGCCGCTGCCGTT 
               
               
                   
                 GGTACTGCGCGGATATGGTGCGGGCAATGACGTTACAGCTGCCG 
               
               
                   
                 GTGTCTTTGCTGATCTGCTACGTACCCTCTCATGGAAGTTAGGA 
               
               
                   
                 GTCTGA 
               
               
                   
               
               
                 thrB SEQ ID NO: 29 
                 ATGGTTAAAGTTTATGCCCCGGCTTCCAGTGCCAATATGAGCGT 
               
               
                   
                 CGGGTTTGATGTGCTCGGGGCGGCGGTGACACCTGTTGATGGTG 
               
               
                   
                 CATTGCTCGGAGATGTAGTCACGGTTGAGGCGGCAGAGACATTC 
               
               
                   
                 AGTCTCAACAACCTCGGACGCTTTGCCGATAAGCTGCCGTCAGA 
               
               
                   
                 ACCACGGGAAAATATCGTTTATCAGTGCTGGGAGCGTTTTTGCC 
               
               
                   
                 AGGAACTGGGTAAGCAAATTCCAGTGGCGATGACCCTGGAAAA 
               
               
                   
                 GAATATGCCGATCGGTTCGGGCTTAGGCTCCAGTGCCTGTTCGG 
               
               
                   
                 TGGTCGCGGCGCTGATGGCGATGAATGAACACTGCGGCAAGCC 
               
               
                   
                 GCTTAATGACACTCGTTTGCTGGCTTTGATGGGCGAGCTGGAAG 
               
               
                   
                 GCCGTATCTCCGGCAGCATTCATTACGACAACGTGGCACCGTGT 
               
               
                   
                 TTTCTCGGTGGTATGCAGTTGATGATCGAAGAAAACGACATCAT 
               
               
                   
                 CAGCCAGCAAGTGCCAGGGTTTGATGAGTGGCTGTGGGTGCTGG 
               
               
                   
                 CGTATCCGGGGATTAAAGTCTCGACGGCAGAAGCCAGGGCTATT 
               
               
                   
                 TTACCGGCGCAGTATCGCCGCCAGGATTGCATTGCGCACGGGCG 
               
               
                   
                 ACATCTGGCAGGCTTCATTCACGCCTGCTATTCCCGTCAGCCTG 
               
               
                   
                 AGCTTGCCGCGAAGCTGATGAAAGATGTTATCGCTGAACCCTAC 
               
               
                   
                 CGTGAACGGTTACTGCCAGGCTTCCGGCAGGCGCGGCAGGCGG 
               
               
                   
                 TCGCGGAAATCGGCGCGGTAGCGAGCGGTATCTCCGGCTCCGGC 
               
               
                   
                 CCGACCTTGTTCGCTCTGTGTGACAAGCCGGAAACCGCCCAGCG 
               
               
                   
                 CGTTGCCGACTGGTTGGGTAAGAACTACCTGCAAAATCAGGAA 
               
               
                   
                 GGTTTTGTTCATATTTGCCGGCTGGATACGGCGGGCGCACGAGT 
               
               
                   
                 ACTGGAAAACTAA 
               
               
                   
               
               
                 thrC SEQ ID NO: 30 
                 ATGAAACTCTACAATCTGAAAGATCACAACGAGCAGGTCAGCTT 
               
               
                   
                 TGCGCAAGCCGTAACCCAGGGGTTGGGCAAAAATCAGGGGCTG 
               
               
                   
                 TTTTTTCCGCACGACCTGCCGGAATTCAGCCTGACTGAAATTGA 
               
               
                   
                 TGAGATGCTGAAGCTGGATTTTGTCACCCGCAGTGCGAAGATCC 
               
               
                   
                 TCTCGGCGTTTATTGGTGATGAAATCCCACAGGAAATCCTGGAA 
               
               
                   
                 GAGCGCGTGCGCGCGGCGTTTGCCTTCCCGGCTCCGGTCGCCAA 
               
               
                   
                 TGTTGAAAGCGATGTCGGTTGTCTGGAATTGTTCCACGGGCCAA 
               
               
                   
                 CGCTGGCATTTAAAGATTTCGGCGGTCGCTTTATGGCACAAATG 
               
               
                   
                 CTGACCCATATTGCGGGTGATAAGCCAGTGACCATTCTGACCGC 
               
               
                   
                 GACCTCCGGTGATACCGGAGCGGCAGTGGCTCATGCTTTCTACG 
               
               
                   
                 GTTTACCGAATGTGAAAGTGGTTATCCTCTATCCACGAGGCAAA 
               
               
                   
                 ATCAGTCCACTGCAAGAAAAACTGTTCTGTACATTGGGCGGCAA 
               
               
                   
                 TATCGAAACTGTTGCCATCGACGGCGATTTCGATGCCTGTCAGG 
               
               
                   
                 CGCTGGTGAAGCAGGCGTTTGATGATGAAGAACTGAAAGTGGC 
               
               
                   
                 GCTAGGGTTAAACTCGGCTAACTCGATTAACATCAGCCGTTTGC 
               
               
                   
                 TGGCGCAGATTTGCTACTACTTTGAAGCTGTTGCGCAGCTGCCG 
               
               
                   
                 CAGGAGACGCGCAACCAGCTGGTTGTCTCGGTGCCAAGCGGAA 
               
               
                   
                 ACTTCGGCGATTTGACGGCGGGTCTGCTGGCGAAGTCACTCGGT 
               
               
                   
                 CTGCCGGTGAAACGTTTTATTGCTGCGACCAACGTGAACGATAC 
               
               
                   
                 CGTGCCACGTTTCCTGCACGACGGTCAGTGGTCACCCAAAGCGA 
               
               
                   
                 CTCAGGCGACGTTATCCAACGCGATGGACGTGAGTCAGCCGAA 
               
               
                   
                 CAACTGGCCGCGTGTGGAAGAGTTGTTCCGCCGCAAAATCTGGC 
               
               
                   
                 AACTGAAAGAGCTGGGTTATGCAGCCGTGGATGATGAAACCAC 
               
               
                   
                 GCAACAGACAATGCGTGAGTTAAAAGAACTGGGCTACACTTCG 
               
               
                   
                 GAGCCGCACGCTGCCGTAGCTTATCGTGCGCTGCGTGATCAGTT 
               
               
                   
                 GAATCCAGGCGAATATGGCTTGTTCCTCGGCACCGCGCATCCGG 
               
               
                   
                 CGAAATTTAAAGAGAGCGTGGAAGCGATTCTCGGTGAAACGTT 
               
               
                   
                 GGATCTGCCAAAAGAGCTGGCAGAACGTGCTGATTTACCCTTGC 
               
               
                   
                 TTTCACATAATCTGCCCGCCGATTTTGCTGCGTTGCGTAAATTGA 
               
               
                   
                 TGATGAATCATCAGTAA 
               
               
                   
               
               
                 ilvA fbr  SEQ ID NO: 31 
                 ATGAGTGAAACATACGTGTCTGAGAAAAGTCCAGGAGTGATGG 
               
               
                   
                 CTAGCGGAGCGGAGCTGATTCGTGCCGCCGACATTCAAACGGC 
               
               
                   
                 GCAGGCACGAATTTCCTCCGTCATTGCACCAACTCCATTGCAGT 
               
               
                   
                 ATTGCCCTCGTCTTTCTGAGGAAACCGGAGCGGAAATCTACCTT 
               
               
                   
                 AAGCGTGAGGATCTGCAGGATGTTCGTTCCTACAAGATCCGCGG 
               
               
                   
                 TGCGCTGAACTCTGGAGCGCAGCTCACCCAAGAGCAGCGCGAT 
               
               
                   
                 GCAGGTATCGTTGCCGCATCTGCAGGTAACCATGCCCAGGGCGT 
               
               
                   
                 GGCCTATGTGTGCAAGTCCTTGGGCGTTCAGGGACGCATCTATG 
               
               
                   
                 TTCCTGTGCAGACTCCAAAGCAAAAGCGTGACCGCATCATGGTT 
               
               
                   
                 CACGGCGGAGAGTTTGTCTCCTTGGTGGTCACTGGCAATAACTT 
               
               
                   
                 CGACGAAGCATCGGCTGCAGCGCATGAAGATGCAGAGCGCACC 
               
               
                   
                 GGCGCAACGCTGATCGAGCCTTTCGATGCTCGCAACACCGTCAT 
               
               
                   
                 CGGTCAGGGCACCGTGGCTGCTGAGATCTTGTCGCAGCTGACTT 
               
               
                   
                 CCATGGGCAAGAGTGCAGATCACGTGATGGTTCCAGTCGGCGGT 
               
               
                   
                 GGCGGACTTCTTGCAGGTGTGGTCAGCTACATGGCTGATATGGC 
               
               
                   
                 ACCTCGCACTGCGATCGTTGGTATCGAACCAGCGGGAGCAGCAT 
               
               
                   
                 CCATGCAGGCTGCATTGCACAATGGTGGACCAATCACTTTGGAG 
               
               
                   
                 ACTGTTGATCCCTTTGTGGACGGCGCAGCAGTCAAACGTGTCGG 
               
               
                   
                 AGATCTCAACTACACCATCGTGGAGAAGAACCAGGGTCGCGTG 
               
               
                   
                 CACATGATGAGCGCGACCGAGGGCGCTGTGTGTACTGAGATGCT 
               
               
                   
                 CGATCTTTACCAAAACGAAGGCATCATCGCGGAGCCTGCTGGCG 
               
               
                   
                 CGCTGTCTATCGCTGGGTTGAAGGAAATGTCCTTTGCACCTGGT 
               
               
                   
                 TCTGCAGTGGTGTGCATCATCTCTGGTGGCAACAACGATGTGCT 
               
               
                   
                 GCGTTATGCGGAAATCGCTGAGCGCTCCTTGGTGCACCGCGGTT 
               
               
                   
                 TGAAGCACTACTTCTTGGTGAACTTCCCGCAAAAGCCTGGTCAG 
               
               
                   
                 TTGCGTCACTTCCTGGAAGATATCCTGGGACCGGATGATGACAT 
               
               
                   
                 CACGCTGTTTGAGTACCTCAAGCGCAACAACCGTGAGACCGGTA 
               
               
                   
                 CTGCGTTGGTGGGTATTCACTTGAGTGAAGCATCAGGATTGGAT 
               
               
                   
                 TCTTTGCTGGAACGTATGGAGGAATCGGCAATTGATTCCCGTCG 
               
               
                   
                 CCTCGAGCCGGGCACCCCTGAGTACGAATACTTGACCTAA 
               
               
                   
               
               
                 aceE SEQ ID NO: 32 
                 ATGTCAGAACGTTTCCCAAATGACGTGGATCCGATCGAAACTCG 
               
               
                   
                 CGACTGGCTCCAGGCGATCGAATCGGTCATCCGTGAAGAAGGT 
               
               
                   
                 GTTGAGCGTGCTCAGTATCTGATCGACCAACTGCTTGCTGAAGC 
               
               
                   
                 CCGCAAAGGCGGTGTAAACGTAGCCGCAGGCACAGGTATCAGC 
               
               
                   
                 AACTACATCAACACCATCCCCGTTGAAGAACAACCGGAGTATCC 
               
               
                   
                 GGGTAATCTGGAACTGGAACGCCGTATTCGTTCAGCTATCCGCT 
               
               
                   
                 GGAACGCCATCATGACGGTGCTGCGTGCGTCGAAAAAAGACCT 
               
               
                   
                 CGAACTGGGCGGCCATATGGCGTCCTTCCAGTCTTCCGCAACCA 
               
               
                   
                 TTTATGATGTGTGCTTTAACCACTTCTTCCGTGCACGCAACGAGC 
               
               
                   
                 AGGATGGCGGCGACCTGGTTTACTTCCAGGGCCACATCTCCCCG 
               
               
                   
                 GGCGTGTACGCTCGTGCTTTCCTGGAAGGTCGTCTGACTCAGGA 
               
               
                   
                 GCAGCTGGATAACTTCCGTCAGGAAGTTCACGGCAATGGCCTCT 
               
               
                   
                 CTTCCTATCCGCACCCGAAACTGATGCCGGAATTCTGGCAGTTC 
               
               
                   
                 CCGACCGTATCTATGGGTCTGGGTCCGATTGGTGCTATTTACCA 
               
               
                   
                 GGCTAAATTCCTGAAATATCTGGAACACCGTGGCCTGAAAGATA 
               
               
                   
                 CCTCTAAACAAACCGTTTACGCGTTCCTCGGTGACGGTGAAATG 
               
               
                   
                 GACGAACCGGAATCCAAAGGTGCGATCACCATCGCTACCCGTG 
               
               
                   
                 AAAAACTGGATAACCTGGTCTTCGTTATCAACTGTAACCTGCAG 
               
               
                   
                 CGTCTTGACGGCCCGGTCACCGGTAACGGCAAGATCATCAACGA 
               
               
                   
                 ACTGGAAGGCATCTTCGAAGGTGCTGGCTGGAACGTGATCAAA 
               
               
                   
                 GTGATGTGGGGTAGCCGTTGGGATGAACTGCTGCGTAAGGATAC 
               
               
                   
                 CAGCGGTAAACTGATCCAGCTGATGAACGAAACCGTTGACGGC 
               
               
                   
                 GACTACCAGACCTTCAAATCGAAAGATGGTGCGTACGTTCGTGA 
               
               
                   
                 ACACTTCTTCGGTAAATATCCTGAAACCGCAGCACTGGTTGCAG 
               
               
                   
                 ACTGGACTGACGAGCAGATCTGGGCACTGAACCGTGGTGGTCA 
               
               
                   
                 CGATCCGAAGAAAATCTACGCTGCATTCAAGAAAGCGCAGGAA 
               
               
                   
                 ACCAAAGGCAAAGCGACAGTAATCCTTGCTCATACCATTAAAG 
               
               
                   
                 GTTACGGCATGGGCGACGCGGCTGAAGGTAAAAACATCGCGCA 
               
               
                   
                 CCAGGTTAAGAAAATGAACATGGACGGTGTGCGTCATATCCGC 
               
               
                   
                 GACCGTTTCAATGTGCCGGTGTCTGATGCAGATATCGAAAAACT 
               
               
                   
                 GCCGTACATCACCTTCCCGGAAGGTTCTGAAGAGCATACCTATC 
               
               
                   
                 TGCACGCTCAGCGTCAGAAACTGCACGGTTATCTGCCAAGCCGT 
               
               
                   
                 CAGCCGAACTTCACCGAGAAGCTTGAGCTGCCGAGCCTGCAAG 
               
               
                   
                 ACTTCGGCGCGCTGTTGGAAGAGCAGAGCAAAGAGATCTCTAC 
               
               
                   
                 CACTATCGCTTTCGTTCGTGCTCTGAACGTGATGCTGAAGAACA 
               
               
                   
                 AGTCGATCAAAGATCGTCTGGTACCGATCATCGCCGACGAAGCG 
               
               
                   
                 CGTACTTTCGGTATGGAAGGTCTGTTCCGTCAGATTGGTATTTAC 
               
               
                   
                 AGCCCGAACGGTCAGCAGTACACCCCGCAGGACCGCGAGCAGG 
               
               
                   
                 TTGCTTACTATAAAGAAGACGAGAAAGGTCAGATTCTGCAGGA 
               
               
                   
                 AGGGATCAACGAGCTGGGCGCAGGTTGTTCCTGGCTGGCAGCG 
               
               
                   
                 GCGACCTCTTACAGCACCAACAATCTGCCGATGATCCCGTTCTA 
               
               
                   
                 CATCTATTACTCGATGTTCGGCTTCCAGCGTATTGGCGATCTGTG 
               
               
                   
                 CTGGGCGGCTGGCGACCAGCAAGCGCGTGGCTTCCTGATCGGCG 
               
               
                   
                 GTACTTCCGGTCGTACCACCCTGAACGGCGAAGGTCTGCAGCAC 
               
               
                   
                 GAAGATGGTCACAGCCACATTCAGTCGCTGACTATCCCGAACTG 
               
               
                   
                 TATCTCTTACGACCCGGCTTACGCTTACGAAGTTGCTGTCATCAT 
               
               
                   
                 GCATGACGGTCTGGAGCGTATGTACGGTGAAAAACAAGAGAAC 
               
               
                   
                 GTTTACTACTACATCACTACGCTGAACGAAAACTACCACATGCC 
               
               
                   
                 GGCAATGCCGGAAGGTGCTGAGGAAGGTATCCGTAAAGGTATC 
               
               
                   
                 TACAAACTCGAAACTATTGAAGGTAGCAAAGGTAAAGTTCAGC 
               
               
                   
                 TGCTCGGCTCCGGTTCTATCCTGCGTCACGTCCGTGAAGCAGCT 
               
               
                   
                 GAGATCCTGGCGAAAGATTACGGCGTAGGTTCTGACGTTTATAG 
               
               
                   
                 CGTGACCTCCTTCACCGAGCTGGCGCGTGATGGTCAGGATTGTG 
               
               
                   
                 AACGCTGGAACATGCTGCACCCGCTGGAAACTCCGCGCGTTCCG 
               
               
                   
                 TATATCGCTCAGGTGATGAACGACGCTCCGGCAGTGGCATCTAC 
               
               
                   
                 CGACTATATGAAACTGTTCGCTGAGCAGGTCCGTACTTACGTAC 
               
               
                   
                 CGGCTGACGACTACCGCGTACTGGGTACTGATGGCTTCGGTCGT 
               
               
                   
                 TCCGACAGCCGTGAGAACCTGCGTCACCACTTCGAAGTTGATGC 
               
               
                   
                 TTCTTATGTCGTGGTTGCGGCGCTGGGCGAACTGGCTAAACGTG 
               
               
                   
                 GCGAAATCGATAAGAAAGTGGTTGCTGACGCAATCGCCAAATT 
               
               
                   
                 CAACATCGATGCAGATAAAGTTAACCCGCGTCTGGCGTAA 
               
               
                   
               
               
                 aceF SEQ ID NO: 33 
                 ATGGCTATCGAAATCAAAGTACCGGACATCGGGGCTGATGAAG 
               
               
                   
                 TTGAAATCACCGAGATCCTGGTCAAAGTGGGCGACAAAGTTGA 
               
               
                   
                 AGCCGAACAGTCGCTGATCACCGTAGAAGGCGACAAAGCCTCT 
               
               
                   
                 ATGGAAGTTCCGTCTCCGCAGGCGGGTATCGTTAAAGAGATCAA 
               
               
                   
                 AGTCTCTGTTGGCGATAAAACCCAGACCGGCGCACTGATTATGA 
               
               
                   
                 TTTTCGATTCCGCCGACGGTGCAGCAGACGCTGCACCTGCTCAG 
               
               
                   
                 GCAGAAGAGAAGAAAGAAGCAGCTCCGGCAGCAGCACCAGCG 
               
               
                   
                 GCTGCGGCGGCAAAAGACGTTAACGTTCCGGATATCGGCAGCG 
               
               
                   
                 ACGAAGTTGAAGTGACCGAAATCCTGGTGAAAGTTGGCGATAA 
               
               
                   
                 AGTTGAAGCTGAACAGTCGCTGATCACCGTAGAAGGCGACAAG 
               
               
                   
                 GCTTCTATGGAAGTTCCGGCTCCGTTTGCTGGCACCGTGAAAGA 
               
               
                   
                 GATCAAAGTGAACGTGGGTGACAAAGTGTCTACCGGCTCGCTG 
               
               
                   
                 ATTATGGTCTTCGAAGTCGCGGGTGAAGCAGGCGCGGCAGCTCC 
               
               
                   
                 GGCCGCTAAACAGGAAGCAGCTCCGGCAGCGGCCCCTGCACCA 
               
               
                   
                 GCGGCTGGCGTGAAAGAAGTTAACGTTCCGGATATCGGCGGTG 
               
               
                   
                 ACGAAGTTGAAGTGACTGAAGTGATGGTGAAAGTGGGCGACAA 
               
               
                   
                 AGTTGCCGCTGAACAGTCACTGATCACCGTAGAAGGCGACAAA 
               
               
                   
                 GCTTCTATGGAAGTTCCGGCGCCGTTTGCAGGCGTCGTGAAGGA 
               
               
                   
                 ACTGAAAGTCAACGTTGGCGATAAAGTGAAAACTGGCTCGCTG 
               
               
                   
                 ATTATGATCTTCGAAGTTGAAGGCGCAGCGCCTGCGGCAGCTCC 
               
               
                   
                 TGCGAAACAGGAAGCGGCAGCGCCGGCACCGGCAGCAAAAGCT 
               
               
                   
                 GAAGCCCCGGCAGCAGCACCAGCTGCGAAAGCGGAAGGCAAAT 
               
               
                   
                 CTGAATTTGCTGAAAACGACGCTTATGTTCACGCGACTCCGCTG 
               
               
                   
                 ATCCGCCGTCTGGCACGCGAGTTTGGTGTTAACCTTGCGAAAGT 
               
               
                   
                 GAAGGGCACTGGCCGTAAAGGTCGTATCCTGCGCGAAGACGTT 
               
               
                   
                 CAGGCTTACGTGAAAGAAGCTATCAAACGTGCAGAAGCAGCTC 
               
               
                   
                 CGGCAGCGACTGGCGGTGGTATCCCTGGCATGCTGCCGTGGCCG 
               
               
                   
                 AAGGTGGACTTCAGCAAGTTTGGTGAAATCGAAGAAGTGGAAC 
               
               
                   
                 TGGGCCGCATCCAGAAAATCTCTGGTGCGAACCTGAGCCGTAAC 
               
               
                   
                 TGGGTAATGATCCCGCATGTTACTCACTTCGACAAAACCGATAT 
               
               
                   
                 CACCGAGTTGGAAGCGTTCCGTAAACAGCAGAACGAAGAAGCG 
               
               
                   
                 GCGAAACGTAAGCTGGATGTGAAGATCACCCCGGTTGTCTTCAT 
               
               
                   
                 CATGAAAGCCGTTGCTGCAGCTCTTGAGCAGATGCCTCGCTTCA 
               
               
                   
                 ATAGTTCGCTGTCGGAAGACGGTCAGCGTCTGACCCTGAAGAAA 
               
               
                   
                 TACATCAACATCGGTGTGGCGGTGGATACCCCGAACGGTCTGGT 
               
               
                   
                 TGTTCCGGTATTCAAAGACGTCAACAAGAAAGGCATCATCGAGC 
               
               
                   
                 TGTCTCGCGAGCTGATGACTATTTCTAAGAAAGCGCGTGACGGT 
               
               
                   
                 AAGCTGACTGCGGGCGAAATGCAGGGCGGTTGCTTCACCATCTC 
               
               
                   
                 CAGCATCGGCGGCCTGGGTACTACCCACTTCGCGCCGATTGTGA 
               
               
                   
                 ACGCGCCGGAAGTGGCTATCCTCGGCGTTTCCAAGTCCGCGATG 
               
               
                   
                 GAGCCGGTGTGGAATGGTAAAGAGTTCGTGCCGCGTCTGATGCT 
               
               
                   
                 GCCGATTTCTCTCTCCTTCGACCACCGCGTGATCGACGGTGCTG 
               
               
                   
                 ATGGTGCCCGTTTCATTACCATCATTAACAACACGCTGTCTGAC 
               
               
                   
                 ATTCGCCGTCTGGTGATGTAA 
               
               
                   
               
               
                 lpd SEQ ID NO: 34 
                 ATGAGTACTGAAATCAAAACTCAGGTCGTGGTACTTGGGGCAG 
               
               
                   
                 GCCCCGCAGGTTACTCCGCTGCCTTCCGTTGCGCTGATTTAGGTC 
               
               
                   
                 TGGAAACCGTAATCGTAGAACGTTACAACACCCTTGGCGGTGTT 
               
               
                   
                 TGCCTGAACGTCGGCTGTATCCCTTCTAAAGCACTGCTGCACGT 
               
               
                   
                 AGCAAAAGTTATCGAAGAAGCCAAAGCGCTGGCTGAACACGGT 
               
               
                   
                 ATCGTCTTCGGCGAACCGAAAACCGATATCGACAAGATTCGTAC 
               
               
                   
                 CTGGAAAGAGAAAGTGATCAATCAGCTGACCGGTGGTCTGGCT 
               
               
                   
                 GGTATGGCGAAAGGCCGCAAAGTCAAAGTGGTCAACGGTCTGG 
               
               
                   
                 GTAAATTCACCGGGGCTAACACCCTGGAAGTTGAAGGTGAGAA 
               
               
                   
                 CGGCAAAACCGTGATCAACTTCGACAACGCGATCATTGCAGCG 
               
               
                   
                 GGTTCTCGCCCGATCCAACTGCCGTTTATTCCGCATGAAGATCC 
               
               
                   
                 GCGTATCTGGGACTCCACTGACGCGCTGGAACTGAAAGAAGTA 
               
               
                   
                 CCAGAACGCCTGCTGGTAATGGGTGGCGGTATCATCGGTCTGGA 
               
               
                   
                 AATGGGCACCGTTTACCACGCGCTGGGTTCACAGATTGACGTGG 
               
               
                   
                 TTGAAATGTTCGACCAGGTTATCCCGGCAGCTGACAAAGACATC 
               
               
                   
                 GTTAAAGTCTTCACCAAGCGTATCAGCAAGAAATTCAACCTGAT 
               
               
                   
                 GCTGGAAACCAAAGTTACCGCCGTTGAAGCGAAAGAAGACGGC 
               
               
                   
                 ATTTATGTGACGATGGAAGGCAAAAAAGCACCCGCTGAACCGC 
               
               
                   
                 AGCGTTACGACGCCGTGCTGGTAGCGATTGGTCGTGTGCCGAAC 
               
               
                   
                 GGTAAAAACCTCGACGCAGGCAAAGCAGGCGTGGAAGTTGACG 
               
               
                   
                 ACCGTGGTTTCATCCGCGTTGACAAACAGCTGCGTACCAACGTA 
               
               
                   
                 CCGCACATCTTTGCTATCGGCGATATCGTCGGTCAACCGATGCT 
               
               
                   
                 GGCACACAAAGGTGTTCACGAAGGTCACGTTGCCGCTGAAGTTA 
               
               
                   
                 TCGCCGGTAAGAAACACTACTTCGATCCGAAAGTTATCCCGTCC 
               
               
                   
                 ATCGCCTATACCAAACCAGAAGTTGCATGGGTGGGTCTGACTGA 
               
               
                   
                 GAAAGAAGCGAAAGAGAAAGGCATCAGCTATGAAACCGCCACC 
               
               
                   
                 TTCCCGTGGGCTGCTTCTGGTCGTGCTATCGCTTCCGACTGCGCA 
               
               
                   
                 GACGGTATGACCAAGCTGATTTTCGACAAAGAATCTCACCGTGT 
               
               
                   
                 GATCGGTGGTGCGATTGTCGGTACTAACGGCGGCGAGCTGCTGG 
               
               
                   
                 GTGAAATCGGCCTGGCAATCGAAATGGGTTGTGATGCTGAAGA 
               
               
                   
                 CATCGCACTGACCATCCACGCGCACCCGACTCTGCACGAGTCTG 
               
               
                   
                 TGGGCCTGGCGGCAGAAGTGTTCGAAGGTAGCATTACCGACCTG 
               
               
                   
                 CCGAACCCGAAAGCGAAGAAGAAGTAA 
               
               
                   
               
               
                 tesB SEQ ID NO: 10 
                 ATGAGTCAGGCGCTAAAAAATTTACTGACATTGTTAAATCTGGA 
               
               
                   
                 AAAAATTGAGGAAGGACTCTTTCGCGGCCAGAGTGAAGATTTA 
               
               
                   
                 GGTTTACGCCAGGTGTTTGGCGGCCAGGTCGTGGGTCAGGCCTT 
               
               
                   
                 GTATGCTGCAAAAGAGACCGTCCCTGAAGAGCGGCTGGTACATT 
               
               
                   
                 CGTTTCACAGCTACTTTCTTCGCCCTGGCGATAGTAAGAAGCCG 
               
               
                   
                 ATTATTTATGATGTCGAAACGCTGCGTGACGGTAACAGCTTCAG 
               
               
                   
                 CGCCCGCCGGGTTGCTGCTATTCAAAACGGCAAACCGATTTTTT 
               
               
                   
                 ATATGACTGCCTCTTTCCAGGCACCAGAAGCGGGTTTCGAACAT 
               
               
                   
                 CAAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCCTCCCTTC 
               
               
                   
                 GGAAACGCAAATCGCCCAATCGCTGGCGCACCTGCTGCCGCCA 
               
               
                   
                 GTGCTGAAAGATAAATTCATCTGCGATCGTCCGCTGGAAGTCCG 
               
               
                   
                 TCCGGTGGAGTTTCATAACCCACTGAAAGGTCACGTCGCAGAAC 
               
               
                   
                 CACATCGTCAGGTGTGGATCCGCGCAAATGGTAGCGTGCCGGAT 
               
               
                   
                 GACCTGCGCGTTCATCAGTATCTGCTCGGTTACGCTTCTGATCTT 
               
               
                   
                 AACTTCCTGCCGGTAGCTCTACAGCCGCACGGCATCGGTTTTCT 
               
               
                   
                 CGAACCGGGGATTCAGATTGCCACCATTGACCATTCCATGTGGT 
               
               
                   
                 TCCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATAGCGTG 
               
               
                   
                 GAGAGCACCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGA 
               
               
                   
                 GTTTTATACCCAAGACGGCGTACTGGTTGCCTCGACCGTTCAGG 
               
               
                   
                 AAGGGGTGATGCGTAATCACAATTAA 
               
               
                   
               
               
                 acuI SEQ ID NO: 35 
                 ATGCGTGCGGTACTGATCGAGAAGTCCGATGATACACAGTCCGT 
               
               
                   
                 CTCTGTCACCGAACTGGCTGAAGATCAACTGCCGGAAGGCGAC 
               
               
                   
                 GTTTTGGTAGATGTTGCTTATTCAACACTGAACTACAAAGACGC 
               
               
                   
                 CCTGGCAATTACCGGTAAAGCCCCCGTCGTTCGTCGTTTTCCGAT 
               
               
                   
                 GGTACCTGGAATCGACTTTACGGGTACCGTGGCCCAGTCTTCCC 
               
               
                   
                 ACGCCGACTTCAAGCCAGGTGATCGCGTAATCCTGAATGGTTGG 
               
               
                   
                 GGTGTGGGGGAAAAACATTGGGGCGGTTTAGCGGAGCGCGCTC 
               
               
                   
                 GCGTGCGCGGAGACTGGCTTGTTCCCTTGCCAGCCCCCCTGGAC 
               
               
                   
                 TTACGCCAAGCGGCCATGATCGGTACAGCAGGATACACGGCGA 
               
               
                   
                 TGTTGTGCGTTCTGGCGCTTGAACGTCACGGAGTGGTGCCGGGT 
               
               
                   
                 AATGGGGAAATCGTGGTGTCCGGTGCAGCAGGCGGCGTCGGCT 
               
               
                   
                 CCGTTGCGACGACCCTTCTTGCCGCTAAGGGCTATGAGGTAGCG 
               
               
                   
                 GCAGTGACTGGACGTGCGTCCGAAGCAGAATATCTGCGCGGTTT 
               
               
                   
                 GGGGGCGGCGAGCGTAATTGATCGTAACGAATTAACGGGGAAG 
               
               
                   
                 GTACGCCCGCTGGGTCAGGAGCGTTGGGCTOGCGGGATTGACGT 
               
               
                   
                 GGCGGGATCAACCGTGCTTGCGAACATGCTTTCTATGATGAAGT 
               
               
                   
                 ATCGCGGGGTAGTCGCTGCGTGTGGCCTGGCCGCGGGCATGGAT 
               
               
                   
                 CTGCCCGCGTCTGTCGCGCCCTTTATTCTTCGTGGGATGACGCTG 
               
               
                   
                 GCAGGGGTGGATAGCGTTATGTGCCCAAAGACAGATCGTTTAGC 
               
               
                   
                 AGCGTGGGCCCGTTTGGCGTCAGATCTTGACCCTGCCAAGCTGG 
               
               
                   
                 AGGAGATGACTACAGAGTTGCCGTTTAGTGAAGTAATCGAGAC 
               
               
                   
                 AGCACCCAAATTCTTGGACGGGACGGTTCGTGGCCGCATTGTTA 
               
               
                   
                 TCCCCGTAACGCCCTAA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 Propionate Cassette Sequences Sleeping Beauty Operon 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Sbm 
                 ATGTCTAACGTGCAGGAGTGGCAACAGCTTGCCAACAAGGAA 
               
               
                 SEQ ID  
                 TTGAGCCGTCGGGAGAAAACTGTCGACTCGCTGGTTCATCAAA 
               
               
                 NO: 36 
                 CCGCGGAAGGGATCGCCATCAAGCCGCTGTATACCGAAGCCG 
               
               
                   
                 ATCTCGATAATCTGGAGGTGACAGGTACCCTTCCTGGTTTGCC 
               
               
                   
                 GCCCTACGTTCGTGGCCCGCGTGCCACTATGTATACCGCCCAA 
               
               
                   
                 CCGTGGACCATCCGTCAGTATGCTGGTTTTTCAACAGCAAAAG 
               
               
                   
                 AGTCCAACGCTTTTTATCGCCGTAACCTGGCCGCCGGGCAAAA 
               
               
                   
                 AGGTCTTTCCGTTGCGTTTGACCTTGCCACCCACCGTGGCTAC 
               
               
                   
                 GACTCCGATAACCCGCGCGTGGCGGGCGACGTCGGCAAAGCG 
               
               
                   
                 GGCGTCGCTATCGACACCGTGGAAGATATGAAAGTCCTGTTCG 
               
               
                   
                 ACCAGATCCCGCTGGATAAAATGTCGGTTTCGATGACCATGAA 
               
               
                   
                 TGGCGCAGTGCTACCAGTACTGGCGTTTTATATCGTCGCCGCA 
               
               
                   
                 GAAGAGCAAGGTGTTACACCTGATAAACTGACCGGCACCATT 
               
               
                   
                 CAAAACGATATTCTCAAAGAGTACCTCTGCCGCAACACCTATA 
               
               
                   
                 TTTACCCACCAAAACCGTCAATGCGCATTATCGCCGACATCAT 
               
               
                   
                 CGCCTGGTGTTCCGGCAACATGCCGCGATTTAATACCATCAGT 
               
               
                   
                 ATCAGCGGTTACCACATGGGTGAAGCGGGTGCCAACTGCGTG 
               
               
                   
                 CAGCAGGTAGCATTTACGCTCGCTGATGGGATTGAGTACATCA 
               
               
                   
                 AAGCAGCAATCTCTGCCGGACTGAAAATTGATGACTTCGCTCC 
               
               
                   
                 TCGCCTGTCGTTCTTCTTCGGCATCGGCATGGATCTGTTTATGA 
               
               
                   
                 ACGTCGCCATGTTGCGTGCGGCACGTTATTTATGGAGCGAAGC 
               
               
                   
                 GGTCAGTGGATTTGGCGCACAGGACCCGAAATCACTGGCGCT 
               
               
                   
                 GCGTACCCACTGCCAGACCTCAGGCTGGAGCCTGACTGAACA 
               
               
                   
                 GGATCCGTATAACAACGTTATCCGCACCACCATTGAAGCGCTG 
               
               
                   
                 GCTGCGACGCTGGGCGGTACTCAGTCACTGCATACCAACGCCT 
               
               
                   
                 TTGACGAAGCGCTTGGTTTGCCTACCGATTTCTCAGCACGCAT 
               
               
                   
                 TGCCCGCAACACCCAGATCATCATCCAGGAAGAATCAGAACT 
               
               
                   
                 CTGCCGCACCGTCGATCCACTGGCCGGATCCTATTACATTGAG 
               
               
                   
                 TCGCTGACCGATCAAATCGTCAAACAAGCCAGAGCTATTATCC 
               
               
                   
                 AACAGATCGACGAAGCCGGTGGCATGGCGAAAGCGATCGAAG 
               
               
                   
                 CAGGTCTGCCAAAACGAATGATCGAAGAGGCCTCAGCGCGCG 
               
               
                   
                 AACAGTCGCTGATCGACCAGGGCAAGCGTGTCATCGTTGGTGT 
               
               
                   
                 CAACAAGTACAAACTGGATCACGAAGACGAAACCGATGTACT 
               
               
                   
                 TGAGATCGACAACGTGATGGTGCGTAACGAGCAAATTGCTTC 
               
               
                   
                 GCTGGAACGCATTCGCGCCACCCGTGATGATGCCGCCGTAACC 
               
               
                   
                 GCCGCGTTGAACGCCCTGACTCACGCCGCACAGCATAACGAA 
               
               
                   
                 AACCTGCTGGCTGCCGCTGTTAATGCCGCTCGCGTTCGCGCCA 
               
               
                   
                 CCCTGGGTGAAATTTCCGATGCGCTGGAAGTCGCTTTCGACCG 
               
               
                   
                 TTATCTGGTGCCAAGCCAGTGTGTTACCGGCGTGATTGCGCAA 
               
               
                   
                 AGCTATCATCAGTCTGAGAAATCGGCCTCCGAGTTCGATGCCA 
               
               
                   
                 TTGTTGCGCAAACGGAGCAGTTCCTTGCCGACAATGGTCGTCG 
               
               
                   
                 CCCGCGCATTCTGATCGCTAAGATGGGCCAGGATGGACACGA 
               
               
                   
                 TCGCGGCGCGAAAGTGATCGCCAGCGCCTATTCCGATCTCGGT 
               
               
                   
                 TTCGACGTAGATTTAAGCCCGATGTTCTCTACACCTGAAGAGA 
               
               
                   
                 TCGCCCGCCTGGCCGTAGAAAACGACGTTCACGTAGTGGGCG 
               
               
                   
                 CATCCTCACTGGCTGCCGGTCATAAAACGCTGATCCCGGAACT 
               
               
                   
                 GGTCGAAGCGCTGAAAAAATGGGGACGCGAAGATATCTGCGT 
               
               
                   
                 GGTCGCGGGTGGCGTCATTCCGCCGCAGGATTACGCCTTCCTG 
               
               
                   
                 CAAGAGCGCGGCGTGGCGGCGATTTATGGTCCAGGTACACCT 
               
               
                   
                 ATGCTCGACAGTGTGCGCGACGTACTGAATCTGATAAGCCAGC 
               
               
                   
                 ATCATGATTAA 
               
               
                   
               
               
                 ygfD 
                 ATGATTAATGAAGCCACGCTGGCAGAAAGTATTCGCCGCTTAC 
               
               
                 SEQ ID  
                 GTCAGGGTGAGCGTGCCACACTCGCCCAGGCCATGACGCTGG 
               
               
                 NO: 37 
                 TGGAAAGCCGTCACCCGCGTCATCAGGCACTAAGTACGCAGC 
               
               
                   
                 TGCTTGATGCCATTATGCCGTACTGCGGTAACACCCTGCGACT 
               
               
                   
                 GGGCGTTACCGGCACCCCCGGCGCGGGGAAAAGTACCTTTCTT 
               
               
                   
                 GAGGCCTTTGGCATGTTGTTGATTCGAGAGGGATTAAAGGTCG 
               
               
                   
                 CGGTTATTGCGGTCGATCCCAGCAGCCCGGTCACTGGCGGTAG 
               
               
                   
                 CATTCTCGGGGATAAAACCCGCATGAATGACCTGGCGCGTGCC 
               
               
                   
                 GAAGCGGCGTTTATTCGCCCGGTACCATCCTCCGGTCATCTGG 
               
               
                   
                 GCGGTGCCAGTCAGCGAGCGCGGGAATTAATGCTGTTATGCG 
               
               
                   
                 AAGCAGCGGGTTATGACGTAGTGATTGTCGAAACGGTTGGCG 
               
               
                   
                 TCGGGCAGTCGGAAACAGAAGTCGCCCGCATGGTGGACTGTT 
               
               
                   
                 TTATCTCGTTGCAAATTGCCGGTGGCGGCGATGATCTGCAGGG 
               
               
                   
                 CATTAAAAAAGGGCTGATGGAAGTGGCTGATCTGATCGTTATC 
               
               
                   
                 AACAAAGACGATGGCGATAACCATACCAATGTCGCCATTGCC 
               
               
                   
                 CGGCATATGTACGAGAGTGCCCTGCATATTCTGCGACGTAAAT 
               
               
                   
                 ACGACGAATGGCAGCCACGGGTTCTGACTTGTAGCGCACTGG 
               
               
                   
                 AAAAACGTGGAATCGATGAGATCTGGCACGCCATCATCGACT 
               
               
                   
                 TCAAAACCGCGCTAACTGCCAGTGGTCGTTTACAACAAGTGCG 
               
               
                   
                 GCAACAACAATCGGTGGAATGGCTGCGTAAGCAGACCGAAGA 
               
               
                   
                 AGAAGTACTGAATCACCTGTTCGCGAATGAAGATTTCGATCGC 
               
               
                   
                 TATTACCGCCAGACGCTTTTAGCGGTCAAAAACAATACGCTCT 
               
               
                   
                 CACCGCGCACCGGCCTGCGGCAGCTCAGTGAATTTATCCAGAC 
               
               
                   
                 GCAATATTTTGATTAA 
               
               
                   
               
               
                 ygfG 
                 ATGTCTTATCAGTATGTTAACGTTGTCACTATCAACAAAGTGG 
               
               
                 SEQ ID  
                 CGGTCATTGAGTTTAACTATGGCCGAAAACTTAATGCCTTAAG 
               
               
                 NO: 38 
                 TAAAGTCTTTATTGATGATCTTATGCAGGCGTTAAGCGATCTC 
               
               
                   
                 AACCGGCCGGAAATTCGCTGTATCATTTTGCGCGCACCGAGTG 
               
               
                   
                 GATCCAAAGTCTTCTCCGCAGGTCACGATATTCACGAACTGCC 
               
               
                   
                 GTCTGGCGGTCGCGATCCGCTCTCCTATGATGATCCATTGCGT 
               
               
                   
                 CAAATCACCCGCATGATCCAAAAATTCCCGAAACCGATCATTT 
               
               
                   
                 CGATGGTGGAAGGTAGTGTTTGGGGTGGCGCATTTGAAATGAT 
               
               
                   
                 CATGAGTTCCGATCTGATCATCGCCGCCAGTACCTCAACCTTC 
               
               
                   
                 TCAATGACGCCTGTAAACCTCGGCGTCCCGTATAACCTGGTCG 
               
               
                   
                 GCATTCACAACCTGACCCGCGACGCGGGCTTCCACATTGTCAA 
               
               
                   
                 AGAGCTGATTTTTACCGCTTCGCCAATCACCGCCCAGCGCGCG 
               
               
                   
                 CTGGCTGTCGGCATCCTCAACCATGTTGTGGAAGTGGAAGAAC 
               
               
                   
                 TGGAAGATTTCACCTTACAAATGGCGCACCACATCTCTGAGAA 
               
               
                   
                 AGCGCCGTTAGCCATTGCCGTTATCAAAGAAGAGCTGCGTGTA 
               
               
                   
                 CTGGGCGAAGCACACACCATGAACTCCGATGAATTTGAACGT 
               
               
                   
                 ATTCAGGGGATGCGCCGCGCGGTGTATGACAGCGAAGATTAC 
               
               
                   
                 CAGGAAGGGATGAACGCTTTCCTCGAAAAACGTAAACCTAAT 
               
               
                   
                 TTCGTTGGTCATTAA 
               
               
                   
               
               
                 ygfH 
                 ATGGAAACTCAGTGGACAAGGATGACCGCCAATGAAGCGGCA 
               
               
                 SEQ ID  
                 GAAATTATCCAGCATAACGACATGGTGGCATTTAGCGGCTTTA 
               
               
                 NO: 39 
                 CCCCGGCGGGTTCGCCGAAAGCCCTACCCACCGCGATTGCCCG 
               
               
                   
                 CAGAGCTAACGAACAGCATGAGGCCAAAAAGCCGTATCAAAT 
               
               
                   
                 TCGCCTTCTGACGGGTGCGTCAATCAGCGCCGCCGCTGACGAT 
               
               
                   
                 GTACTTTCTGACGCCGATGCTGTTTCCTGGCGTGCGCCATATC 
               
               
                   
                 AAACATCGTCCGGTTTACGTAAAAAGATCAATCAGGGCGCGG 
               
               
                   
                 TGAGTTTCGTTGACCTGCATTTGAGCGAAGTGGCGCAAATGGT 
               
               
                   
                 CAATTACGGTTTCTTCGGCGACATTGATGTTGCCGTCATTGAA 
               
               
                   
                 GCATCGGCACTGGCACCGGATGGTCGAGTCTGGTTAACCAGC 
               
               
                   
                 GGGATCGGTAATGCGCCGACCTGGCTGCTGCGGGCGAAGAAA 
               
               
                   
                 GTGATCATTGAACTCAATCACTATCACGATCCGCGCGTTGCAG 
               
               
                   
                 AACTGGCGGATATTGTGATTCCTGGCGCGCCACCGCGGCGCAA 
               
               
                   
                 TAGCGTGTCGATCTTCCATGCAATGGATCGCGTCGGTACCCGC 
               
               
                   
                 TATGTGCAAATCGATCCGAAAAAGATTGTCGCCGTCGTGGAA 
               
               
                   
                 ACCAACTTGCCCGACGCCGGTAATATGCTGGATAAGCAAAAT 
               
               
                   
                 CCCATGTGCCAGCAGATTGCCGATAACGTGGTCACGTTCTTAT 
               
               
                   
                 TGCAGGAAATGGCGCATGGGCGTATTCCGCCGGAATTTCTGCC 
               
               
                   
                 GCTGCAAAGTGGCGTGGGCAATATCAATAATGCGGTAATGGC 
               
               
                   
                 GCGTCTGGGGGAAAACCCGGTAATTCCTCCGTTTATGATGTAT 
               
               
                   
                 TCGGAAGTGCTACAGGAATCGGTGGTGCATTTACTGGAAACC 
               
               
                   
                 GGCAAAATCAGCGGGGCCAGCGCCTCCAGCCTGACAATCTCG 
               
               
                   
                 GCCGATTCCCTGCGCAAGATTTACGACAATATGGATTACTTTG 
               
               
                   
                 CCAGCCGCATTGTGTTGCGTCCGCAGGAGATTTCCAATAACCC 
               
               
                   
                 GGAAATCATCCGTCGTCTGGGCGTCATCGCTCTGAACGTCGGC 
               
               
                   
                 CTGGAGTTTGATATTTACGGGCATGCCAACTCAACACACGTAG 
               
               
                   
                 CCGGGGTCGATCTGATGAACGGCATCGGCGGCAGCGGTGATT 
               
               
                   
                 TTGAACGCAACGCGTATCTGTCGATCTTTATGGCCCCGTCGAT 
               
               
                   
                 TGCTAAAGAAGGCAAGATCTCAACCGTCGTGCCAATGTGCAG 
               
               
                   
                 CCATGTTGATCACAGCGAACACAGCGTCAAAGTGATCATCACC 
               
               
                   
                 GAACAAGGGATCGCCGATCTGCGCGGTCTTTCCCCGCTTCAAC 
               
               
                   
                 GCGCCCGCACTATCATTGATAATTGTGCACATCCTATGTATCG 
               
               
                   
                 GGATTATCTGCATCGCTATCTGGAAAATGCGCCTGGCGGACAT 
               
               
                   
                 ATTCACCACGATCTTAGCCACGTCTTCGACTTACACCGTAATTT 
               
               
                   
                 AATTGCAACCGGCTCGATGCTGGGTTAA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Sequences of Propionate Cassette from  
               
               
                 Propioni Bacteria 
               
            
           
           
               
               
            
               
                 Des- 
                   
               
               
                 cription 
                 Sequence 
               
               
                   
               
               
                 mutA 
                 ATGAGCAGCACGGATCAGGGGACCAACCCCGCCGACACTGAC 
               
               
                 SEQ ID  
                 GACCTCACTCCCACCACACTCAGTCTGGCCGGGGATTTCCCCA 
               
               
                 NO: 40 
                 AGGCCACTGAGGAGCAGTGGGAGCGCGAAGTTGAGAAGGTAT 
               
               
                   
                 TCAACCGTGGTCGTCCACCGGAGAAGCAGCTGACCTTCGCCGA 
               
               
                   
                 GTGTCTGAAGCGCCTGACGGTTCACACCGTCGATGGCATCGAC 
               
               
                   
                 ATCGTGCCGATGTACCGTCCGAAGGACGCGCCGAAGAAGCTG 
               
               
                   
                 GGTTACCCCGGCGTCACCCCCTTCACCCGCGGCACCACGGTGC 
               
               
                   
                 GCAACGGTGACATGGATGCCTGGGACGTGCGCGCCCTGCACG 
               
               
                   
                 AGGATCCCGACGAGAAGTTCACCCGCAAGGCGATCCTTGAAG 
               
               
                   
                 ACCTGGAGCGTGGCGTCACCTCCCTGTTGTTGCGCGTTGATCC 
               
               
                   
                 CGACGCGATCGCACCCGAGCACCTCGACGAGGTCCTCTCCGAC 
               
               
                   
                 GTCCTGCTGGAAATGACCAAGGTGGAGGTCTTCAGCCGCTACG 
               
               
                   
                 ACCAGGGTGCCGCCGCCGAGGCCTTGATGGGCGTCTACGAGC 
               
               
                   
                 GCTCCGACAAGCCGGCGAAGGACCTGGCCCTGAACCTGGGCC 
               
               
                   
                 TGGATCCCATCGGCTTCGCGGCCCTGCAGGGCACCGAGCCGG 
               
               
                   
                 ATCTGACCGTGCTCGGTGACTGGGTGCGCCGCCTGGCGAAGTT 
               
               
                   
                 CTCACCGGACTCGCGCGCCGTCACGATCGACGCGAACGTCTAC 
               
               
                   
                 CACAACGCCGGTGCCGGCGACGTGGCAGAGCTCGCTTGGGCA 
               
               
                   
                 CTGGCCACCGGCGCGGAGTACGTGCGCGCCCTGGTCGAACAG 
               
               
                   
                 GGCTTCAACGCCACAGAGGCCTTCGACACGATCAACTTCCGTG 
               
               
                   
                 TCACCGCCACCCACGACCAGTTCCTCACGATCGCCCGTCTTCG 
               
               
                   
                 CGCCCTGCGCGAGGCATGGGCCCGCATCGGCGAGGTCTTTGGC 
               
               
                   
                 GTGGACGAGGACAAGCGCGGCGCTCGCCAGAATGCGATCACC 
               
               
                   
                 AGTTGGCGTGAGCTCACCCGCGAAGACCCCTATGTCAACATCC 
               
               
                   
                 TTCGCGGTTCGATTGCCACCTTCTCCGCCTCCGTTGGCGGGGC 
               
               
                   
                 CGAGTCGATCACGACGCTGCCCTTCACCCAGGCCCTCGGCCTG 
               
               
                   
                 CCGGAGGACGACTTCCCGCTGCGCATCGCGCGCAACACGGGC 
               
               
                   
                 ATCGTGCTCGCCGAAGAGGTGAACATCGGCCGCGTCAACGAC 
               
               
                   
                 CCGGCCGGTGGCTCCTACTACGTCGAGTCGCTCACTCGCACCC 
               
               
                   
                 TGGCCGACGCTGCCTGGAAGGAATTCCAGGAGGTCGAGAAGC 
               
               
                   
                 TCGGTGGCATGTCGAAGGCGGTCATGACCGAGCACGTCACCA 
               
               
                   
                 AGGTGCTCGACGCCTGCAATGCCGAGCGCGCCAAGCGCCTGG 
               
               
                   
                 CCAACCGCAAGCAGCCGATCACCGCGGTCAGCGAGTTCCCGA 
               
               
                   
                 TGATCGGGGCCCGCAGCATCGAGACCAAGCCGTTCCCAACCG 
               
               
                   
                 CTCCGGCGCGCAAGGGCCTGGCCTGGCATCGCGATTCCGAGGT 
               
               
                   
                 GTTCGAGCAGCTGATGGATCGCTCCACCAGCGTCTCCGAGCGC 
               
               
                   
                 CCCAAGGTGTTCCTTGCCTGCCTGGGCACCCGTCGCGACTTCG 
               
               
                   
                 GTGGCCGCGAGGGCTTCTCCAGCCCGGTATGGCACATCGCCGG 
               
               
                   
                 TATCGACACCCCGCAGGTCGAAGGCGGCACCACCGCCGAGAT 
               
               
                   
                 CGTCGAGGCGTTCAAGAAGTCGGGCGCCCAGGTGGCCGATCT 
               
               
                   
                 CTGCTCGTCCGCCAAGATCTACGCGCAGCAGGGACTTGAGGTT 
               
               
                   
                 GCCAAGGCGCTCAAGGCCGCCGGCGCGAAGGCCCTGTATCTG 
               
               
                   
                 TCGGGCGCCTTCAAGGAGTTCGGCGATGACGCCGCCGAGGCC 
               
               
                   
                 GAGAAGCTGATCGACGGACGCCTGTACATGGGCATGGATGTC 
               
               
                   
                 GTCGACACCCTGTCCTCCACCCTTGATATCTTGGGAGTCGCGA 
               
               
                   
                 AGTGA 
               
               
                   
               
               
                 mutB 
                 GTGAGCACTCTGCCCCGTTTTGATTCAGTTGACCTGGGCAATG 
               
               
                 SEQ ID  
                 CCCCGGTTCCTGCTGATGCCGCACAGCGCTTCGAGGAGTTGGC 
               
               
                 NO: 41 
                 CGCCAAGGCCGGCACCGAAGAGGCGTGGGAGACGGCTGAGCA 
               
               
                   
                 GATTCCGGTTGGCACCCTGTTCAACGAAGACGTCTACAAGGAC 
               
               
                   
                 ATGGACTGGCTGGACACCTACGCCGGTATCCCGCCGTTCGTCC 
               
               
                   
                 ACGGCCCATATGCAACCATGTACGCGTTCCGTCCCTGGACGAT 
               
               
                   
                 TCGCCAGTACGCCGGCTTCTCCACGGCCAAGGAGTCCAACGCC 
               
               
                   
                 TTCTACCGCCGCAACCTTGCGGCGGGCCAGAAGGGCCTGTCGG 
               
               
                   
                 TTGCCTTCGACCTGCCCACCCACCGCGGCTACGACTCGGACAA 
               
               
                   
                 TCCCCGCGTCGCCGGTGACGTCGGCATGGCCGGGGTGGCCATC 
               
               
                   
                 GACTCCATCTATGACATGCGCGAGCTGTTCGCCGGCATTCCGC 
               
               
                   
                 TGGACCAGATGAGCGTGTCGATGACCATGAACGGCGCCGTGC 
               
               
                   
                 TGCCGATCCTGGCCCTCTATGTGGTGACCGCCGAGGAGCAGGG 
               
               
                   
                 CGTCAAGCCCGAGCAGCTCGCCGGGACGATCCAGAACGACAT 
               
               
                   
                 CCTCAAGGAGTTCATGGTTCGTAACACCTATATCTACCCGCCG 
               
               
                   
                 CAGCCGAGTATGCGAATCATCTCCGAGATCTTCGCCTACACGA 
               
               
                   
                 GTGCCAATATGCCGAAGTGGAATTCGATTTCCATTTCCGGCTA 
               
               
                   
                 CCACATGCAGGAAGCCGGCGCCACGGCCGACATCGAGATGGC 
               
               
                   
                 CTACACCCTGGCCGACGGTGTCGACTACATCCGCGCCGGCGAG 
               
               
                   
                 TCGGTGGGCCTCAATGTCGACCAGTTCGCGCCGCGTCTGTCCT 
               
               
                   
                 TCTTCTGGGGCATCGGCATGAACTTCTTCATGGAGGTTGCCAA 
               
               
                   
                 GCTGCGTGCCGCACGTATGTTGTGGGCCAAGCTGGTGCATCAG 
               
               
                   
                 TTCGGGCCGAAGAATCCGAAGTCGATGAGCCTGCGCACCCAC 
               
               
                   
                 TCGCAGACCTCCGGTTGGTCGCTGACCGCCCAGGACGTCTACA 
               
               
                   
                 ACAACGTCGTGCGTACCTGCATCGAGGCCATGGCCGCCACCCA 
               
               
                   
                 GGGCCATACCCAGTCGCTGCACACGAACTCGCTCGACGAGGC 
               
               
                   
                 CATTGCCCTACCGACCGATTTCAGCGCCCGCATCGCCCGTAAC 
               
               
                   
                 ACCCAGCTGTTCCTGCAGCAGGAATCGGGCACGACGCGCGTG 
               
               
                   
                 ATCGACCCGTGGAGCGGCTCGGCATACGTCGAGGAGCTCACC 
               
               
                   
                 TGGGACCTGGCCCGCAAGGCATGGGGCCACATCCAGGAGGTC 
               
               
                   
                 GAGAAGGTCGGCGGCATGGCCAAGGCCATCGAAAAGGGCATC 
               
               
                   
                 CCCAAGATGCGCATTGAGGAAGCCGCCGCCCGCACCCAGGCA 
               
               
                   
                 CGCATCGACTCCGGCCGTCAGCCGCTGATCGGCGTGAACAAGT 
               
               
                   
                 ACCGCCTGGAGCACGAGCCGCCGCTCGATGTGCTCAAGGTTG 
               
               
                   
                 ACAACTCCACGGTGCTCGCCGAGCAGAAGGCCAAGCTGGTCA 
               
               
                   
                 AGCTGCGCGCCGAGCGCGATCCCGAGAAGGTCAAGGCCGCCC 
               
               
                   
                 TCGACAAGATCACCTGGGCTGCCGCCAACCCCGACGACAAGG 
               
               
                   
                 ATCCGGATCGCAACCTGCTGAAGCTGTGCATCGACGCTGGCCG 
               
               
                   
                 CGCCATGGCGACGGTCGGCGAGATGAGCGACGCGCTCGAGAA 
               
               
                   
                 GGTCTTCGGACGCTACACCGCCCAGATTCGCACCATCTCCGGT 
               
               
                   
                 GTGTACTCGAAGGAAGTGAAGAACACGCCTGAGGTTGAGGAA 
               
               
                   
                 GCACGCGAGCTCGTTGAGGAATTCGAGCAGGCCGAGGGCCGT 
               
               
                   
                 CGTCCTCGCATCCTGCTGGCCAAGATGGGCCAGGACGGTCACG 
               
               
                   
                 ACCGTGGCCAGAAGGTCATCGCCACCGCCTATGCCGACCTCGG 
               
               
                   
                 TTTCGACGTCGACGTGGGCCCGCTGTTCCAGACCCCGGAGGAG 
               
               
                   
                 ACCGCACGTCAGGCCGTCGAGGCCGATGTGCACGTGGTGGGC 
               
               
                   
                 GTTTCGTCGCTCGCCGGCGGGCATCTGACGCTGGTTCCGGCCC 
               
               
                   
                 TGCGCAAGGAGCTGGACAAGCTCGGACGTCCCGACATCCTCA 
               
               
                   
                 TCACCGTGGGCGGCGTGATCCCTGAGCAGGACTTCGACGAGCT 
               
               
                   
                 GCGTAAGGACGGCGCCGTGGAGATCTACACCCCCGGCACCGT 
               
               
                   
                 CATTCCGGAGTCGGCGATCTCGCTGGTCAAGAAACTGCGGGCT 
               
               
                   
                 TCGCTCGATGCCTAG 
               
               
                   
               
               
                 GI: 
                 ATGAGTAATGAGGATCTTTTCATCTGTATCGATCACGTGGCAT 
               
               
                 18042134 
                 ATGCGTGCCCCGACGCCGACGAGGCTTCCAAGTACTACCAGG 
               
               
                 SEQ ID  
                 AGACCTTCGGCTGGCATGAGCTCCACCGCGAGGAGAACCCGG 
               
               
                 NO: 42 
                 AGCAGGGAGTCGTCGAGATCATGATGGCCCCGGCTGCGAAGC 
               
               
                   
                 TGACCGAGCACATGACCCAGGTTCAGGTCATGGCCCCGCTCAA 
               
               
                   
                 CGACGAGTCGACCGTTGCCAAGTGGCTTGCCAAGCACAATGG 
               
               
                   
                 TCGCGCCGGACTGCACCACATGGCATGGCGTGTCGATGACATC 
               
               
                   
                 GACGCCGTCAGCGCCACCCTGCGCGAGCGCGGCGTGCAGCTG 
               
               
                   
                 CTGTATGACGAGCCCAAGCTCGGCACCGGCGGCAACCGCATC 
               
               
                   
                 AACTTCATGCATCCCAAGTCGGGCAAGGGCGTGCTCATCGAGC 
               
               
                   
                 TCACCCAGTACCCGAAGAACTGA 
               
               
                   
               
               
                 mmdA 
                 ATGGCTGAAAACAACAATTTGAAGCTCGCCAGCACCATGGAA 
               
               
                 SEQ ID  
                 GGTCGCGTGGAGCAGCTCGCAGAGCAGCGCCAGGTGATCGAA 
               
               
                 NO: 43 
                 GCCGGTGGCGGCGAACGTCGCGTCGAGAAGCAACATTCCCAG 
               
               
                   
                 GGTAAGCAGACCGCTCGTGAGCGCCTGAACAACCTGCTCGAT 
               
               
                   
                 CCCCATTCGTTCGACGAGGTCGGCGCTTTCCGCAAGCACCGCA 
               
               
                   
                 CCACGTTGTTCGGCATGGACAAGGCCGTCGTCCCGGCAGATGG 
               
               
                   
                 CGTGGTCACCGGCCGTGGCACCATCCTTGGTCGTCCCGTGCAC 
               
               
                   
                 GCCGCGTCCCAGGACTTCACGGTCATGGGTGGTTCGGCTGGCG 
               
               
                   
                 AGACGCAGTCCACGAAGGTCGTCGAGACGATGGAACAGGCGC 
               
               
                   
                 TGCTCACCGGCACGCCCTTCCTGTTCTTCTACGATTCGGGCGG 
               
               
                   
                 CGCCCGGATCCAGGAGGGCATCGACTCGCTGAGCGGTTACGG 
               
               
                   
                 CAAGATGTTCTTCGCCAACGTGAAGCTGTCGGGCGTCGTGCCG 
               
               
                   
                 CAGATCGCCATCATTGCCGGCCCCTGTGCCGGTGGCGCCTCGT 
               
               
                   
                 ATTCGCCGGCACTGACTGACTTCATCATCATGACCAAGAAGGC 
               
               
                   
                 CCATATGTTCATCACGGGCCCCCAGGTCATCAAGTCGGTCACC 
               
               
                   
                 GGCGAGGATGTCACCGCTGACGAACTCGGTGGCGCTGAGGCC 
               
               
                   
                 CATATGGCCATCTCGGGCAATATCCACTTCGTGGCCGAGGACG 
               
               
                   
                 ACGACGCCGCGGAGCTCATTGCCAAGAAGCTGCTGAGCTTCCT 
               
               
                   
                 TCCGCAGAACAACACTGAGGAAGCATCCTTCGTCAACCCGAA 
               
               
                   
                 CAATGACGTCAGCCCCAATACCGAGCTGCGCGACATCGTTCCG 
               
               
                   
                 ATTGACGGCAAGAAGGGCTATGACGTGCGCGATGTCATTGCC 
               
               
                   
                 AAGATCGTCGACTGGGGTGACTACCTCGAGGTCAAGGCCGGC 
               
               
                   
                 TATGCCACCAACCTCGTGACCGCCTTCGCCCGGGTCAATGGTC 
               
               
                   
                 GTTCGGTGGGCATCGTGGCCAATCAGCCGTCGGTGATGTCGGG 
               
               
                   
                 TTGCCTCGACATCAACGCCTCTGACAAGGCCGCCGAATTCGTG 
               
               
                   
                 AATTTCTGCGATTCGTTCAACATCCCGCTGGTGCAGCTGGTCG 
               
               
                   
                 ACGTGCCGGGCTTCCTGCCCGGCGTGCAGCAGGAGTACGGCG 
               
               
                   
                 GCATCATTCGCCATGGCGCGAAGATGCTGTACGCCTACTCCGA 
               
               
                   
                 GGCCACCGTGCCGAAGATCACCGTGGTGCTCCGCAAGGCCTA 
               
               
                   
                 CGGCGGCTCCTACCTGGCCATGTGCAACCGTGACCTTGGTGCC 
               
               
                   
                 GACGCCGTGTACGCCTGGCCCAGCGCCGAGATTGCGGTGATG 
               
               
                   
                 GGCGCCGAGGGTGCGGCAAATGTGATCTTCCGCAAGGAGATC 
               
               
                   
                 AAGGCTGCCGACGATCCCGACGCCATGCGCGCCGAGAAGATC 
               
               
                   
                 GAGGAGTACCAGAACGCGTTCAACACGCCGTACGTGGCCGCC 
               
               
                   
                 GCCCGCGGTCAGGTCGACGACGTGATTGACCCGGCTGATACCC 
               
               
                   
                 GTCGAAAGATTGCTTCCGCCCTGGAGATGTACGCCACCAAGCG 
               
               
                   
                 TCAGACCCGCCCGGCGAAGAAGCATGGAAACTTCCCCTGCTG 
               
               
                   
                 A 
               
               
                   
               
               
                 PFREUD_ 
                 ATGAGTCCGCGAGAAATTGAGGTTTCCGAGCCGCGCGAGGTT 
               
               
                 18870 
                 GGTATCACCGAGCTCGTGCTGCGCGATGCCCATCAGAGCCTGA 
               
               
                 SEQ ID  
                 TGGCCACACGAATGGCAATGGAAGACATGGTCGGCGCCTGTG 
               
               
                 NO: 44 
                 CAGACATTGATGCTGCCGGGTACTGGTCAGTGGAGTGTTGGGG 
               
               
                   
                 TGGTGCCACGTATGACTCGTGTATCCGCTTCCTCAACGAGGAT 
               
               
                   
                 CCTTGGGAGCGTCTGCGCACGTTCCGCAAGCTGATGCCCAACA 
               
               
                   
                 GCCGTCTCCAGATGCTGCTGCGTGGCCAGAACCTGCTGGGTTA 
               
               
                   
                 CCGCCACTACAACGACGAGGTCGTCGATCGCTTCGTCGACAAG 
               
               
                   
                 TCCGCTGAGAACGGCATGGACGTGTTCCGTGTCTTCGACGCCA 
               
               
                   
                 TGAATGATCCCCGCAACATGGCGCACGCCATGGCTGCCGTCAA 
               
               
                   
                 GAAGGCCGGCAAGCACGCGCAGGGCACCATTTGCTACACGAT 
               
               
                   
                 CAGCCCGGTCCACACCGTTGAGGGCTATGTCAAGCTTGCTGGT 
               
               
                   
                 CAGCTGCTCGACATGGGTGCTGATTCCATCGCCCTGAAGGACA 
               
               
                   
                 TGGCCGCCCTGCTCAAGCCGCAGCCGGCCTACGACATCATCAA 
               
               
                   
                 GGCCATCAAGGACACCTACGGCCAGAAGACGCAGATCAACCT 
               
               
                   
                 GCACTGCCACTCCACCACGGGTGTCACCGAGGTCTCCCTCATG 
               
               
                   
                 AAGGCCATCGAGGCCGGCGTCGACGTCGTCGACACCGCCATC 
               
               
                   
                 TCGTCCATGTCGCTCGGCCCGGGCCACAACCCCACCGAGTCGG 
               
               
                   
                 TTGCCGAGATGCTCGAGGGCACCGGGTACACCACCAACCTTG 
               
               
                   
                 ACTACGATCGCCTGCACAAGATCCGCGATCACTTCAAGGCCAT 
               
               
                   
                 CCGCCCGAAGTACAAGAAGTTCGAGTCGAAGACGCTTGTCGA 
               
               
                   
                 CACCTCGATCTTCAAGTCGCAGATCCCCGGCGGCATGCTCTCC 
               
               
                   
                 AACATGGAGTCGCAGCTGCGCGCCCAGGGCGCCGAGGACAAG 
               
               
                   
                 ATGGACGAGGTCATGGCAGAGGTGCCGCGCGTCCGCAAGGCC 
               
               
                   
                 GCCGGCTTCCCGCCCCTGGTCACCCCGTCCAGCCAGATCGTCG 
               
               
                   
                 GCACGCAGGCCGTGTTCAACGTGATGATGGGCGAGTACAAGA 
               
               
                   
                 GGATGACCGGCGAGTTCGCCGACATCATGCTCGGCTACTACGG 
               
               
                   
                 CGCCAGCCCGGCCGATCGCGATCCGAAGGTGGTCAAGTTGGC 
               
               
                   
                 CGAGGAGCAGTCCGGCAAGAAGCCGATCACCCAGCGCCCGGC 
               
               
                   
                 CGATCTGCTGCCCCCCGAGTGGGAGGAGCAGTCCAAGGAGGC 
               
               
                   
                 CGCGGCCCTCAAGGGCTTCAACGGCACCGACGAGGACGTGCT 
               
               
                   
                 CACCTATGCACTGTTCCCGCAGGTCGCTCCGGTCTTCTTCGAG 
               
               
                   
                 CATCGCGCCGAGGGCCCGCACAGCGTGGCTCTCACCGATGCCC 
               
               
                   
                 AGCTGAAGGCCGAGGCCGAGGGCGACGAGAAGTCGCTCGCCG 
               
               
                   
                 TGGCCGGTCCCGTCACCTACAACGTGAACGTGGGCGGAACCG 
               
               
                   
                 TCCGCGAAGTCACCGTTCAGCAGGCGTGA 
               
               
                   
               
               
                 Bccp 
                 ATGAAACTGAAGGTAACAGTCAACGGCACTGCGTATGACGTT 
               
               
                 SEQ ID  
                 GACGTTGACGTCGACAAGTCACACGAAAACCCGATGGGCACC 
               
               
                 NO: 45 
                 ATCCTGTTCGGCGGCGGCACCGGCGGCGCGCCGGCACCGCGC 
               
               
                   
                 GCAGCAGGTGGCGCAGGCGCCGGTAAGGCCGGAGAGGGCGA 
               
               
                   
                 GATTCCCGCTCCGCTGGCCGGCACCGTCTCCAAGATCCTCGTG 
               
               
                   
                 AAGGAGGGTGACACGGTCAAGGCTGGTCAGACCGTGCTCGTT 
               
               
                   
                 CTCGAGGCCATGAAGATGGAGACCGAGATCAACGCTCCCACC 
               
               
                   
                 GACGGCAAGGTCGAGAAGGTCCTTGTCAAGGAGCGTGACGCC 
               
               
                   
                 GTGCAGGGCGGTCAGGGTCTCATCAAGATCGGCTGA 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence(s) of Table 4 (SEQ ID NO: 21-SEQ ID NO: 35, and SEQ ID NO: 10) or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid s sequence(s) of Table 4 (SEQ ID NO: 21-SEQ ID NO: 35, and SEQ ID NO: 10) or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence(s) of Table 4 (SEQ ID NO: 21-SEQ ID NO: 35, and SEQ ID NO: 10) or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence(s) of Table 4 (SEQ ID NO: 21-SEQ ID NO: 35, and SEQ ID NO: 10) or a functional fragment thereof. 
     In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence(s) of Table 5 (SEQ ID NO: 36-SEQ ID NO: 39) or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid s sequence(s) of Table 5 (SEQ ID NO: 36-SEQ ID NO: 39) or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence(s) of Table 5 (SEQ ID NO: 36-SEQ ID NO: 39) or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence(s) of Table 5 (SEQ ID NO: 36-SEQ ID NO: 39) or a functional fragment thereof. 
     In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence(s) of Table 6 (SEQ ID NO: 40-SEQ ID NO: 45) or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid s sequence(s) of Table 6 (SEQ ID NO: 40-SEQ ID NO: 45) or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence(s) of Table 6 (SEQ ID NO: 40-SEQ ID NO: 45) or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence(s) of Table 6 (SEQ ID NO: 40-SEQ ID NO: 45) or a functional fragment thereof. 
     Table 7 lists exemplary polypeptide sequences, which may be encoded by the propionate production gene(s) or cattette(s) of the genetically engineered bacteria. 
     
       
         
           
               
             
               
                 TABLE 7 
               
               
                   
               
               
                 Polypeptide Sequences for Propionate Synthesis 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Pct 
                 MRKVPIITADEAAKLIKDGDTVTTSGFVGNAIPEALDRAVEKRFLETGE 
               
               
                 SEQ ID 
                 PKNITYVYCGSQGNRDGRGAEHFAHEGLLKRYIAGHWATVPALGKM 
               
               
                 NO: 46 
                 AMENKMEAYNVSQGALCHLFRDIASHKPGVFTKVGIGTFIDPRNGGG 
               
               
                   
                 KVNDITKEDIVELVEIKGQEYLFYPAFPIHVALIRGTYADESGNITFEKE 
               
               
                   
                 VAPLEGTSVCQAVKNSGGIVVVQVERVVKAGTLDPRHVKVPGIYVDY 
               
               
                   
                 VVVADPEDHQQSLDCEYDPALSGEHRRPEVVGEPLPLSAKKVIGRRGA 
               
               
                   
                 IELEKDVAVNLGVGAPEYVASVADEEGIVDFMTLTAESGAIGGVPAGG 
               
               
                   
                 VRFGASYNADALIDQGYQFDYYDGGGLDLCYLGLAECDEKGNINVSR 
               
               
                   
                 FGPRIAGCGGFINITQNTPKVFFCGTFTAGGLKVKIEDGKVIIVQEGKQK 
               
               
                   
                 KFLKAVEQITFNGDVALANKQQVTYITERCVFLLKEDGLHLSEIAPGID 
               
               
                   
                 LQTQILDVMDFAPIIDRDANGQIKLMDAALFAEGLMGLKEMKS* 
               
               
                   
               
               
                 lcdA 
                 MSLTQGMKAKQLLAYFQGKADQDAREAKARGELVCWSASVAPPEFC 
               
               
                 SEQ ID 
                 VTMGIAMIYPETHAAGIGARKGAMDMLEVADRKGYNVDCCSYGRVN 
               
               
                 NO: 47 
                 MGYMECLKEAAITGVKPEVLVNSPAADVPLPDLVITCNNICNTLLKWY 
               
               
                   
                 ENLAAELDIPCIVIDVPFNHTMPIPEYAKAYIADQFRNAISQLEVICGRPF 
               
               
                   
                 DWKKFKEVKDQTQRSVYHWNRIAEMAKYKPSPLNGFDLFNYMALIV 
               
               
                   
                 ACRSLDYAEITFKAFADELEENLKAGIYAFKGAEKTRFQWEGIAVWPH 
               
               
                   
                 LGHTFKSMKNLNSIMTGTAYPALWDLHYDANDESMHSMAEAYTRIYI 
               
               
                   
                 NTCLQNKVEVLLGIMEKGQVDGTVYHLNRSCKLMSFLNVETAEIIKEK 
               
               
                   
                 NGLPYVSIDGDQTDPRVFSPAQFDTRVQALVEMMEANMAAAE* 
               
               
                   
               
               
                 lcdB 
                 MSRVEAILSQLKDVAANPKKAMDDYKAETGKGAVGIMPIYSPEEMVH 
               
               
                 SEQ ID 
                 AAGYLPMGIWGAQGKTISKARTYLPAFACSVMQQVMELQCEGAYDD 
               
               
                 NO: 48 
                 LSAVIFSVPCDTLKCLSQKWKGTSPVIVFTHPQNRGLEAANQFLVTEYE 
               
               
                   
                 LVKAQLESVLGVKISNAALENSIAIYNENRAVMREFVKVAADYPQVID 
               
               
                   
                 AVSRHAVFKARQFMLKEKHTALVKELIAEIKATPVQPWDGKKVVVTG 
               
               
                   
                 ILLEPNELLDIFNEFKIAIVDDDLAQESRQIRVDVLDGEGGPLYRMAKA 
               
               
                   
                 WQQMYGCSLATDTKKGRGRMLINKTIQTGADAIVVAMMKFCDPEEW 
               
               
                   
                 DYPVMYREFEEKGVKSLMIEVDQEVSSFEQIKTRLQSFVEML* 
               
               
                   
               
               
                 lcdC 
                 MYTLGIDVGSASSKAVILKDGKDIVAAEVVQVGTGSSGPQRALDKAFEV 
               
               
                 SEQ ID 
                 SGLKKEDISYTVATGYGRFNFSDADKQISEISCHAKGIYFLVPTARTIIDIG 
               
               
                 NO: 49 
                 GQDAKAIRLDDKGGIKQFFMNDKCAAGTGRFLEVMARVLETTLDEMAE 
               
               
                   
                 LDEQATDTAPISSTCTVFAESEVISQLSNGVSRNNIIKGVHLSVASRACGL 
               
               
                   
                 AYRGGLEKDVVMTGGVAKNAGVVRAVAGVLKTDVIVAPNPQTTGALG 
               
               
                   
                 AALYAYEAAQKKX 
               
               
                   
               
               
                 etfA 
                 MAFNSADINSFRDIWVFCEQREGKLINTDFELISEGRKLADERGSKLVG 
               
               
                 SEQ ID 
                 ILLGHEVEEIAKELGGYGADKVIVCDHPELKFYTTDAYAKVLCDVVME 
               
               
                 NO: 50 
                 EKPEVILIGATNIGRDLGPRCAARLHTGLTADCTHLDIDMNKYVDFLST 
               
               
                   
                 SSTLDISSMTFPMEDTNLKMTRPAFGGHLMATIICPRFRPCMSTVRPGV 
               
               
                   
                 MKKAEFSQEMAQACQVVTRHVNLSDEDLKTKVINIVKETKKIVDLIGA 
               
               
                   
                 EIIVSVGRGISKDVQGGIALAEKLADAFGNGVVGGSRAVIDSGWLPAD 
               
               
                   
                 HQVGQTGKTVHPKVYVALGISGAIQHKAGMQDSELIIAVNKDETAPIF 
               
               
                   
                 DCADYGITGDLFKIVPMMIDAIKEGKNA* 
               
               
                   
               
               
                 acrB 
                 MRIYVCVKQVPDTSGKVAVNPDGTLNRASMAAIINPDDMSAIEQALKL 
               
               
                 SEQ ID 
                 KDETGCQVTALTMGPPPAEGMLREIIAMGADDGVLISAREFGGSDTFA 
               
               
                 NO: 51 
                 TSQIISAAIHKLGLSNEDMIFCGRQAIDGDTAQVGPQIAEKLSIPQVTYG 
               
               
                   
                 AGIKKSGDLVLVKRMLEDGYMMIEVETPCLITCIQDKAVKPRYMTLN 
               
               
                   
                 GIMECYSKPLLVLDYEALKDEPLIELDTIGLKGSPTNIFKSFTPPQKGVG 
               
               
                   
                 VMLQGTDKEKVEDLVDKLMQKHVI* 
               
               
                   
               
               
                 acrC 
                 MFLLKIKKERMKRMDFSLTREQEMLKKLARQFAEIELEPVAEEIDREH 
               
               
                 SEQ ID 
                 VFPAENFKKMAEIGLTGIGIPKEFGGSGGGTLEKVIAVSEFGKKCMASA 
               
               
                 NO: 52 
                 SILSIHLIAPQAIYKYGTKEQKETYLPRLTKGGELGAFALTEPNAGSDAG 
               
               
                   
                 AVKTTAILDSQTNEYVLNGTKCFISGGGRAGVLVIFALTEPKKGLKGM 
               
               
                   
                 SAIIVEKGTPGFSIGKVESKMGIAGSETAELIFEDCRVPAANLLGKEGKG 
               
               
                   
                 FKIAMEALDGARIGVGAQAIGIAEGAIDLSVKYVHERIQFGKPIANLQGI 
               
               
                   
                 QWYIADMATKTAAARALVEFAAYLEDAGKPFTKESAMCKLNASENA 
               
               
                   
                 RFVTNLALQIHGGYGYMKDYPLERMYRDAKITEIYEGTSEIHKVVIAR 
               
               
                   
                 EVMKR* 
               
               
                   
               
               
                 thrAfbr 
                 MRVLKFGGTSVANAERFLRVADILESNARQGQVATVLSAPAKITNHLV 
               
               
                 SEQ ID 
                 AMIEKTISGQDALPNISDAERIFAELLTGLAAAQPGFPLAQLKTFVDQEF 
               
               
                 NO: 53 
                 AQIKHVLHGISLLGQCPDSINAALICRGEKMSIAIMAGVLEARGHNVTV 
               
               
                   
                 IDPVEKLLAVGHYLESTVDIAESTRRIAASRIPADHMVLMAGFTAGNEK 
               
               
                   
                 GELVVLGRNGSDYSAAVLAACLRADCCEIWTDVDGVYTCDPRQVPD 
               
               
                   
                 ARLLKSMSYQEAMELSYFGAKVLHPRTITPIAQFQIPCLIKNTGNPQAP 
               
               
                   
                 GTLIGASRDEDELPVKGISNLNNMAMFSVSGPGMKGMVGMAARVFA 
               
               
                   
                 AMSRARISVVLITQSSSEYSISFCVPQSDCVRAERAMQEEFYLELKEGLL 
               
               
                   
                 EPLAVTERLAIISVVGDGMRTLRGISAKFFAALARANINIVAIAQRSSER 
               
               
                   
                 SISVVVNNDDATTGVRVTHQMLFNTDQVIEVFVIGVGGVGGALLEQL 
               
               
                   
                 KRQQSWLKNKHIDLRVCGVANSKALLTNVHGLNLENWQEELAQAKE 
               
               
                   
                 PFNLGRLIRLVKEYHLLNPVIVDCTSSQAVADQYADFLREGFHVVTPN 
               
               
                   
                 KKANTSSMDYYHQLRYAAEKSRRKFLYDTNVGAGLPVIENLQNLLNA 
               
               
                   
                 GDELMKFSGILSGSLSYIFGKLDEGMSFSEATTLAREMGYTEPDPRDDL 
               
               
                   
                 SGMDVARKLLILARETGRELELADIEIEPVLPAEFNAEGDVAAFMANLS 
               
               
                   
                 QLDDLFAARVAKARDEGKVLRYVGNIDEDGVCRVKIAEVDGNDPLFK 
               
               
                   
                 VKNGENALAFYSHYYQPLPLVLRGYGAGNDVTAAGVFADLLRILSW 
               
               
                   
                 KLGV* 
               
               
                   
               
               
                 thrB 
                 MVKVYAPASSANMSVGFDVLGAAVTPVDGALLGDVVTVEAAETFSL 
               
               
                 SEQ ID 
                 NNLGRFADKLPSEPRENIVYQCWERFCQELGKQIPVAMTLEKNMPIGS 
               
               
                 NO: 54 
                 GLGSSACSVVAALMAMNEHCGKPLNDTRLLALMGELEGRISGSIHYD 
               
               
                   
                 NVAPCFLGGMQLMIEENDIISQQVPGFDEWLWVLAYPGIKVSTAEARA 
               
               
                   
                 ILPAQYRRQDCIAHGRHLAGFIHACYSRQPELAAKLMKDVIAEPYRER 
               
               
                   
                 LLPGFRQARQAVAEIGAVASGISGSGPTLFALCDKPETAQRVADWLGK 
               
               
                   
                 NYLQNQEGFVHICRLDTAGARVLEN* 
               
               
                   
               
               
                 thrC 
                 MKLYNLKDHNEQVSFAQAVTQGLGKNQGLFFPHDLPEFSLTEIDEML 
               
               
                 SEQ ID 
                 KLDFVTRSAKILSAFIGDEIPQEILEERVRAAFAFPAPVANVESDVGCLE 
               
               
                 NO: 55 
                 LFHGPTLAFKDFGGRFMAQMLTHIAGDKPVTILTATSGDTGAAVAHAF 
               
               
                   
                 YGLPNVKVVILYPRGKISPLQEKLFCTLGGNIETVAIDGDFDACQALVK 
               
               
                   
                 QAFDDEELKVALGLNSANSINISRLLAQICYYFEAVAQLPQETRNQLVV 
               
               
                   
                 SVPSGNFGDLTAGLLAKSLGLPVKRFIAATNVNDTVPRFLHDGQWSPK 
               
               
                   
                 ATQATLSNAMDVSQPNNWPRVEELFRRKIWQLKELGYAAVDDETTQ 
               
               
                   
                 QTMRELKELGYTSEPHAAVAYRALRDQLNPGEYGLFLGTAHPAKFKE 
               
               
                   
                 SVEAILGETLDLPKELAERADLPLLSHNLPADFAALRKLMMNHQ* 
               
               
                   
               
               
                 ilvA fbr   
                 MSETYVSEKSPGVMASGAELIRAADIQTAQARISSVIAPTPLQYCPRLSE 
               
               
                 SEQ ID 
                 ETGAEIYLKREDLQDVRSYKIRGALNSGAQLTQEQRDAGIVAASAGNH 
               
               
                 NO: 56 
                 AQGVAYVCKSLGVQGRIYVPVQTPKQKRDRIMVHGGEFVSLVVTGNN 
               
               
                   
                 FDEASAAAHEDAERTGATLIEPFDARNTVIGQGTVAAEILSQLTSMGKS 
               
               
                   
                 ADHVMVPVGGGGLLAGVVSYMADMAPRTAIVGIEPAGAASMQAALH 
               
               
                   
                 NGGPITLETVDPFVDGAAVKRVGDLNYTIVEKNQGRVHMMSATEGAV 
               
               
                   
                 CTEMLDLYQNEGIIAEPAGALSIAGLKEMSFAPGSAVVCIISGGNNDVL 
               
               
                   
                 RYAEIAERSLVHRGLKHYFLVNFPQKPGQLRHFLEDILGPDDDITLFEY 
               
               
                   
                 LKRNNRETGTALVGIHLSEASGLDSLLERMEESAIDSRRLEPGTPEYEY 
               
               
                   
                 LT* 
               
               
                   
               
               
                 ace 
                 MSERFPNDVDPIETRDWLQAIESVIREEGVERAQYLIDQLLAEARKGGV 
               
               
                 SEQ ID 
                 NVAAGTGISNYINTIPVEEQPEYPGNLELERRIRSAIRWNAIMTVLRASK 
               
               
                 NO: 57 
                 KDLELGGHMASFQSSATIYDVCFNHFFRARNEQDGGDLVYFQGHISPG 
               
               
                   
                 VYARAFLEGRLTQEQLDNFRQEVHGNGLSSYPHPKLMPEFWQFPTVS 
               
               
                   
                 MGLGPIGAIYQAKFLKYLEHRGLKDTSKQTVYAFLGDGEMDEPESKG 
               
               
                   
                 AITIATREKLDNLVFVINCNLQRLDGPVTGNGKIINELEGIFEGAGWNVI 
               
               
                   
                 KVMWGSRWDELLRKDTSGKLIQLMNETVDGDYQTFKSKDGAYVREH 
               
               
                   
                 FFGKYPETAALVADWTDEQIWALNRGGHDPKKIYAAFKKAQETKGK 
               
               
                   
                 ATVILAHTIKGYGMGDAAEGKNIAHQVKKMNMDGVRHIRDRFNVPVS 
               
               
                   
                 DADIEKLPYITFPEGSEEHTYLHAQRQKLHGYLPSRQPNFTEKLELPSLQ 
               
               
                   
                 DFGALLEEQSKEISTTIAFVRALNVMLKNKSIKDRLVPIIADEARTFGME 
               
               
                   
                 GLFRQIGIYSPNGQQYTPQDREQVAYYKEDEKGQILQEGINELGAGCS 
               
               
                   
                 WLAAATSYSTNNLPMIPFYIYYSMFGFQRIGDLCWAAGDQQARGFLIG 
               
               
                   
                 GTSGRTTLNGEGLQHEDGHSHIQSLTIPNCISYDPAYAYEVAVIMHDGL 
               
               
                   
                 ERMYGEKQENVYYYITTLNENYHMPAMPEGAEEGIRKGIYKLETIEGS 
               
               
                   
                 KGKVQLLGSGSILRHVREAAEILAKDYGVGSDVYSVTSFTELARDGQD 
               
               
                   
                 CERWNMLHPLETPRVPYIAQVMNDAPAVASTDYMKLFAEQVRTYVP 
               
               
                   
                 ADDYRVLGTDGFGRSDSRENLRHHFEVDASYVVVAALGELAKRGEID 
               
               
                   
                 KKVVADAIAKFNIDADKVNPRLA* 
               
               
                   
               
               
                 aceF 
                 MAIEIKVPDIGADEVEITEILVKVGDKVEAEQSLITVEGDKASMEVPSPQ 
               
               
                 SEQ ID 
                 AGIVKEIKVSVGDKTQTGALIMIFDSADGAADAAPAQAEEKKEAAPAA 
               
               
                 NO: 58 
                 APAAAAAKDVNVPDIGSDEVEVTEILVKVGDKVEAEQSLITVEGDKAS 
               
               
                   
                 MEVPAPFAGTVKEIKVNVGDKVSTGSLIMVFEVAGEAGAAAPAAKQE 
               
               
                   
                 AAPAAAPAPAAGVKEVNVPDIGGDEVEVTEVMVKVGDKVAAEQSLIT 
               
               
                   
                 VEGDKASMEVPAPFAGVVKELKVNVGDKVKTGSLIMIFEVEGAAPAA 
               
               
                   
                 APAKQEAAAPAPAAKAEAPAAAPAAKAEGKSEFAENDAYVHATPLIR 
               
               
                   
                 RLAREFGVNLAKVKGTGRKGRILREDVQAYVKEAIKRAEAAPAATGG 
               
               
                   
                 GIPGMLPWPKVDFSKFGEIEEVELGRIQKISGANLSRNWVMIPHVTHFD 
               
               
                   
                 KTDITELEAFRKQQNEEAAKRKLDVKITPVVFIMKAVAAALEQMPRFN 
               
               
                   
                 SSLSEDGQRLTLKKYINIGVAVDTPNGLVVPVFKDVNKKGIIELSRELM 
               
               
                   
                 TISKKARDGKLTAGEMQGGCFTISSIGGLGTTHFAPIVNAPEVAILGVSK 
               
               
                   
                 SAMEPVWNGKEFVPRLMLPISLSFDHRVIDGADGARFITIINNTLSDIRR 
               
               
                   
                 LVM* 
               
               
                   
               
               
                 Lpd 
                 MSTEIKTQVVVLGAGPAGYSAAFRCADLGLETVIVERYNTLGGVCLN 
               
               
                 SEQ ID 
                 VGCIPSKALLHVAKVIEEAKALAEHGIVFGEPKTDIDKIRTWKEKVINQ 
               
               
                 NO: 59 
                 LTGGLAGMAKGRKVKVVNGLGKFTGANTLEVEGENGKTVINFDNAII 
               
               
                   
                 AAGSRPIQLPFIPHEDPRIWDSTDALELKEVPERLLVMGGGIIGLEMGTV 
               
               
                   
                 YHALGSQIDVVEMFDQVIPAADKDIVKVFTKRISKKFNLMLETKVTAV 
               
               
                   
                 EAKEDGIYVTMEGKKAPAEPQRYDAVLVAIGRVPNGKNLDAGKAGV 
               
               
                   
                 EVDDRGFIRVDKQLRTNVPHIFAIGDIVGQPMLAHKGVHEGHVAAEVI 
               
               
                   
                 AGKKHYFDPKVIPSIAYTKPEVAWVGLTEKEAKEKGISYETATFPWAA 
               
               
                   
                 SGRAIASDCADGMTKLIFDKESHRVIGGAIVGTNGGELLGEIGLAIEMG 
               
               
                   
                 CDAEDIALTIHAHPTLHESVGLAAEVFEGSITDLPNPKAKKK* 
               
               
                   
               
               
                 tesB 
                 MSQALKNLLTLLNLEKIEEGLFRGQSEDLGLRQVFGGQVVGQALYAA 
               
               
                 SEQ ID 
                 KETVPEERLVHSFHSYFLRPGDSKKPIIYDVETLRDGNSFSARRVAAIQ 
               
               
                 NO: 20 
                 NGKPIFYMTASFQAPEAGFEHQKTMPSAPAPDGLPSETQIAQSLAHLLP 
               
               
                   
                 PVLKDKFICDRPLEVRPVEFHNPLKGHVAEPHRQVWIRANGSVPDDLR 
               
               
                   
                 VHQYLLGYASDLNFLPVALQPHGIGFLEPGIQIATIDHSMWFHRPFNLN 
               
               
                   
                 EWLLYSVESTSASSARGFVRGEFYTQDGVLVASTVQEGVMRNHN* 
               
               
                   
               
               
                 acuI 
                 MRAVLIEKSDDTQSVSVTELAEDQLPEGDVLVDVAYSTLNYKDALAIT 
               
               
                 SEQ ID 
                 GKAPVVRRFPMVPGIDFTGTVAQSSHADFKPGDRVILNGWGVGEKHW 
               
               
                 NO: 60 
                 GGLAERARVRGDWLVPLPAPLDLRQAAMIGTAGYTAMLCVLALERH 
               
               
                   
                 GVVPGNGEIVVSGAAGGVGSVATTLLAAKGYEVAAVTGRASEAEYLR 
               
               
                   
                 GLGAASVIDRNELTGKVRPLGQERWAGGIDVAGSTVLANMLSMMKY 
               
               
                   
                 RGVVAACGLAAGMDLPASVAPFILRGMTLAGVDSVMCPKTDRLAAW 
               
               
                   
                 ARLASDLDPAKLEEMTTELPFSEVIETAPKFLDGTVRGRIVIPVTP* 
               
               
                   
               
               
                 Sbm 
                 MSNVQEWQQLANKELSRREKTVDSLVHQTAEGIAIKPLYTEADLDNL 
               
               
                 SEQ ID 
                 EVTGTLPGLPPYVRGPRATMYTAQPWTIRQYAGFSTAKESNAFYRRNL 
               
               
                 NO: 61 
                 AAGQKGLSVAFDLATHRGYDSDNPRVAGDVGKAGVAIDTVEDMKVL 
               
               
                   
                 FDQIPLDKMSVSMTMNGAVLPVLAFYIVAAEEQGVTPDKLTGTIQNDI 
               
               
                   
                 LKEYLCRNTYIYPPKPSMRIIADIIAWCSGNMPRFNTISISGYHMGEAGA 
               
               
                   
                 NCVQQVAFTLADGIEYIKAAISAGLKIDDFAPRLSFFFGIGMDLFMNVA 
               
               
                   
                 MLRAARYLWSEAVSGFGAQDPKSLALRTHCQTSGWSLTEQDPYNNVI 
               
               
                   
                 RTTIEALAATLGGTQSLHTNAFDEALGLPTDFSARIARNTQIIIQEESELC 
               
               
                   
                 RTVDPLAGSYYIESLTDQIVKQARAIIQQIDEAGGMAKAIEAGLPKRMI 
               
               
                   
                 EEASAREQSLIDQGKRVIVGVNKYKLDHEDETDVLEIDNVMVRNEQIA 
               
               
                   
                 SLERIRATRDDAAVTAALNALTHAAQHNENLLAAAVNAARVRATLGE 
               
               
                   
                 ISDALEVAFDRYLVPSQCVTGVIAQSYHQSEKSASEFDAIVAQTEQFLA 
               
               
                   
                 DNGRRPRILIAKMGQDGHDRGAKVIASAYSDLGFDVDLSPMFSTPEEIA 
               
               
                   
                 RLAVENDVHVVGASSLAAGHKTLIPELVEALKKWGREDICVVAGGVIP 
               
               
                   
                 PQDYAFLQERGVAAIYGPGTPMLDSVRDVLNLISQHHD* 
               
               
                   
               
               
                 ygfD 
                 MINEATLAESIRRLRQGERATLAQAMTLVESRHPRHQALSTQLLDAIM 
               
               
                 SEQ ID 
                 PYCGNTLRLGVTGTPGAGKSTFLEAFGMLLIREGLKVAVIAVDPSSPVT 
               
               
                 NO: 62 
                 GGSILGDKTRMNDLARAEAAFIRPVPSSGHLGGASQRARELMLLCEAA 
               
               
                   
                 GYDVVIVETVGVGQSETEVARMVDCFISLQIAGGGDDLQGIKKGLME 
               
               
                   
                 VADLIVINKDDGDNHTNVAIARHMYESALHILRRKYDEWQPRVLTCS 
               
               
                   
                 ALEKRGIDEIWHAIIDFKTALTASGRLQQVRQQQSVEWLRKQTEEEVL 
               
               
                   
                 NHLFANEDFDRYYRQTLLAVKNNTLSPRTGLRQLSEFIQTQYFD* 
               
               
                   
               
               
                 ygfG 
                 MSYQYVNVVTINKVAVIEFNYGRKLNALSKVFIDDLMQALSDLNRPEI 
               
               
                 SEQ ID 
                 RCIILRAPSGSKVFSAGHDIHELPSGGRDPLSYDDPLRQITRMIQKFPKPI 
               
               
                 NO: 63 
                 ISMVEGSVWGGAFEMIMSSDLIIAASTSTFSMTPVNLGVPYNLVGIHNL 
               
               
                   
                 TRDAGFHIVKELIFTASPITAQRALAVGILNHVVEVEELEDFTLQMAHH 
               
               
                   
                 ISEKAPLAIAVIKEELRVLGEAHTMNSDEFERIQGMRRAVYDSEDYQEG 
               
               
                   
                 MNAFLEKRKPNFVGH* 
               
               
                   
               
               
                 ygfH 
                 METQWTRMTANEAAEIIQHNDMVAFSGFTPAGSPKALPTAIARRANEQ 
               
               
                 SEQ ID 
                 HEAKKPYQIRLLTGASISAAADDVLSDADAVSWRAPYQTSSGLRKKIN 
               
               
                 NO: 64 
                 QGAVSFVDLHLSEVAQMVNYGFFGDIDVAVIEASALAPDGRVWLTSGI 
               
               
                   
                 GNAPTWLLRAKKVIIELNHYHDPRVAELADIVIPGAPPRRNSVSIFHAM 
               
               
                   
                 DRVGTRYVQIDPKKIVAVVETNLPDAGNMLDKQNPMCQQIADNVVTF 
               
               
                   
                 LLQEMAHGRIPPEFLPLQSGVGNINNAVMARLGENPVIPPFMMYSEVL 
               
               
                   
                 QESVVHLLETGKISGASASSLTISADSLRKIYDNMDYFASRIVLRPQEIS 
               
               
                   
                 NNPEIIRRLGVIALNVGLEFDIYGHANSTHVAGVDLMNGIGGSGDFERN 
               
               
                   
                 AYLSIFMAPSIAKEGKISTVVPMCSHVDHSEHSVKVIITEQGIADLRGLS 
               
               
                   
                 PLQRARTIIDNCAHPMYRDYLHRYLENAPGGHIHHDLSHVFDLHRNLI 
               
               
                   
                 ATGSMLG* 
               
               
                   
               
               
                 mutA 
                 MSSTDQGTNPADTDDLTPTTLSLAGDFPKATEEQWEREVEKVFNRGRPP 
               
               
                 SEQ ID 
                 EKQLTFAECLKRLTVHTVDGIDIVPMYRPKDAPKKLGYPGVTPFTRGTT 
               
               
                 NO: 65 
                 VRNGDMDAWDVRALHEDPDEKFTRKAILEDLERGVTSLLLRVDPDAIA 
               
               
                   
                 PEHLDEVLSDVLLEMTKVEVFSRYDQGAAAEALMGVYERSDKPAKDLA 
               
               
                   
                 LNLGLDPIGFAALQGTEPDLTVLGDWVRRLAKFSPDSRAVTIDANVYHN 
               
               
                   
                 AGAGDVAELAWALATGAEYVRALVEQGFNATEAFDTINFRVTATHDQF 
               
               
                   
                 LTIARLRALREAWARIGEVFGVDEDKRGARQNAITSWRELTREDPYVNI 
               
               
                   
                 LRGSIATFSASVGGAESITTLPFTQALGLPEDDFPLRIARNTGIVLAEEVNI 
               
               
                   
                 GRVNDPAGGSYYVESLTRTLADAAWKEFQEVEKLGGMSKAVMTEHVT 
               
               
                   
                 KVLDACNAERAKRLANRKQPITAVSEFPMIGARSIETKPFPTAPARKGLA 
               
               
                   
                 WHRDSEVFEQLMDRSTSVSERPKVFLACLGTRRDFGGREGFSSPVWHIA 
               
               
                   
                 GIDTPQVEGGTTAEIVEAFKKSGAQVADLCSSAKIYAQQGLEVAKALKA 
               
               
                   
                 AGAKALYLSGAFKEFGDDAAEAEKLIDGRLYMGMDVVDTLSSTLDILG 
               
               
                   
                 VAK 
               
               
                   
               
               
                 mutB 
                 VSTLPRFDSVDLGNAPVPADAAQRFEELAAKAGTEEAWETAEQIPVGTL 
               
               
                 SEQ ID 
                 FNEDVYKDMDWLDTYAGIPPFVHGPYATMYAFRPWTIRQYAGFSTAKE 
               
               
                 NO: 66 
                 SNAFYRRNLAAGQKGLSVAFDLPTHRGYDSDNPRVAGDVGMAGVAIDS 
               
               
                   
                 IYDMRELFAGIPLDQMSVSMTMNGAVLPILALYVVTAEEQGVKPEQLA 
               
               
                   
                 GTIQNDILKEFMVRNTYIYPPQPSMRIISEIFAYTSANMPKWNSISISGYH 
               
               
                   
                 MQEAGATADIEMAYTLADGVDYIRAGESVGLNVDQFAPRLSFFWGIGM 
               
               
                   
                 NFFMEVAKLRAARMLWAKLVHQFGPKNPKSMSLRTHSQTSGWSLTAQ 
               
               
                   
                 DVYNNVVRTCIEAMAATQGHTQSLHTNSLDEAIALPTDFSARIARNTQL 
               
               
                   
                 FLQQESGTTRVIDPWSGSAYVEELTWDLARKAWGHIQEVEKVGGMAK 
               
               
                   
                 AIEKGIPKMRIEEAAARTQARIDSGRQPLIGVNKYRLEHEPPLDVLKVDN 
               
               
                   
                 STVLAEQKAKLVKLRAERDPEKVKAALDKITWAAANPDDKDPDRNLLK 
               
               
                   
                 LCIDAGRAMATVGEMSDALEKVFGRYTAQIRTISGVYSKEVKNTPEVEE 
               
               
                   
                 ARELVEEFEQAEGRRPRILLAKMGQDGHDRGQKVIATAYADLGFDVDV 
               
               
                   
                 GPLFQTPEETARQAVEADVHVVGVSSLAGGHLTLVPALRKELDKLGRP 
               
               
                   
                 DILITVGGVIPEQDFDELRKDGAVEIYTPGTVIPESAISLVKKLRASLDA 
               
               
                   
               
               
                 GI:180421 
                 MSNEDLFICIDHVAYACPDADEASKYYQETFGWHELHREENPEQGVVEI 
               
               
                 34 
                 MMAPAAKLTEHMTQVQVMAPLNDESTVAKWLAKHNGRAGLHHMAW 
               
               
                 SEQ ID 
                 RVDDIDAVSATLRERGVQLLYDEPKLGTGGNRINFMHPKSGKGVLIELT 
               
               
                 NO: 67 
                 QYPKN 
               
               
                   
               
               
                 mmdA 
                 MAENNNLKLASTMEGRVEQLAEQRQVIEAGGGERRVEKQHSQGKQTA 
               
               
                 SEQ ID 
                 RERLNNLLDPHSFDEVGAFRKHRTTLFGMDKAVVPADGVVTGRGTILG 
               
               
                 NO: 68 
                 RPVHAASQDFTVMGGSAGETQSTKVVETMEQALLTGTPFLFFYDSGGA 
               
               
                   
                 RIQEGIDSLSGYGKMFFANVKLSGVVPQIAIIAGPCAGGASYSPALTDFII 
               
               
                   
                 MTKKAHMFITGPQVIKSVTGEDVTADELGGAEAHMAISGNIHFVAEDD 
               
               
                   
                 DAAELIAKKLLSFLPQNNTEEASFVNPNNDVSPNTELRDIVPIDGKKGYD 
               
               
                   
                 VRDVIAKIVDWGDYLEVKAGYATNLVTAFARVNGRSVGIVANQPSVMS 
               
               
                   
                 GCLDINASDKAAEFVNFCDSFNIPLVQLVDVPGFLPGVQQEYGGIIRHGA 
               
               
                   
                 KMLYAYSEATVPKITVVLRKAYGGSYLAMCNRDLGADAVYAWPSAEI 
               
               
                   
                 AVMGAEGAANVIFRKEIKAADDPDAMRAEKIEEYQNAFNTPYVAAARG 
               
               
                   
                 QVDDVIDPADTRRKIASALEMYATKRQTRPAKKHGNFPC 
               
               
                   
               
               
                 PFREUD_ 
                 MSPREIEVSEPREVGITELVLRDAHQSLMATRMAMEDMVGACADIDAA 
               
               
                 18870 
                 GYWSVECWGGATYDSCIRFLNEDPWERLRTFRKLMPNSRLQMLLRGQN 
               
               
                 SEQ ID 
                 LLGYRHYNDEVVDRFVDKSAENGMDVFRVFDAMNDPRNMAHAMAAV 
               
               
                 NO: 69 
                 KKAGKHAQGTICYTISPVHTVEGYVKLAGQLLDMGADSIALKDMAALL 
               
               
                   
                 KPQPAYDIIKAIKDTYGQKTQINLHCHSTTGVTEVSLMKAIEAGVDVVD 
               
               
                   
                 TAISSMSLGPGHNPTESVAEMLEGTGYTTNLDYDRLHKIRDHFKAIRPKY 
               
               
                   
                 KKFESKTLVDTSIFKSQIPGGMLSNMESQLRAQGAEDKMDEVMAEVPR 
               
               
                   
                 VRKAAGFPPLVTPSSQIVGTQAVFNVMMGEYKRMTGEFADIMLGYYGA 
               
               
                   
                 SPADRDPKVVKLAEEQSGKKPITQRPADLLPPEWEEQSKEAAALKGFNG 
               
               
                   
                 TDEDVLTYALFPQVAPVFFEHRAEGPHSVALTDAQLKAEAEGDEKSLAV 
               
               
                   
                 AGPVTYNVNVGGTVREVTVQQA 
               
               
                   
               
               
                 Bccp 
                 MKLKVTVNGTAYDVDVDVDKSHENPMGTILFGGGTGGAPAPRAAGGA 
               
               
                 SEQ ID 
                 GAGKAGEGEIPAPLAGTVSKILVKEGDTVKAGQTVLVLEAMKMETEIN 
               
               
                 NO: 70 
                 APTDGKVEKVLVKERDAVQGGQGLIKIG 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the genetically engineered bacteria encode one or more polypeptide sequences of Table 7 (SEQ ID NO: 46-SEQ ID NO: 70, and SEQ ID NO: 20) or a functional fragment or variant thereof. In some embodiments, genetically engineered bacteria comprise a polypeptide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the polypeptide sequence of one or more polypeptide sequence of Table 7 (SEQ ID NO: 46-SEQ ID NO: 70, and SEQ ID NO: 20) or a functional fragment thereof. 
     In one embodiment, the bacterial cell comprises a non-native or heterologous propionate gene cassette. In some embodiments, the disclosure provides a bacterial cell that comprises a non-native or heterologous propionate gene cassette operably linked to a first promoter. In one embodiment, the first promoter is an inducible promoter. In one embodiment, the bacterial cell comprises a propionate gene cassette from a different organism, e.g., a different species of bacteria. In another embodiment, the bacterial cell comprises more than one copy of a native gene encoding a propionate gene cassette. In yet another embodiment, the bacterial cell comprises at least one native gene encoding a propionate gene cassette, as well as at least one copy of a propionate gene cassette from a different organism, e.g., a different species of bacteria. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of a gene encoding a propionate gene cassette. In one embodiment, the bacterial cell comprises multiple copies of a gene or genes encoding a propionate gene cassette. 
     Multiple distinct propionate gene cassettes are known in the art. In some embodiments, a propionate gene cassette is encoded by a gene cassette derived from a bacterial species. In some embodiments, a propionate gene cassette is encoded by a gene cassette derived from a non-bacterial species. In some embodiments, a propionate gene cassette is encoded by a gene derived from a eukaryotic species, e.g., a fungi. In one embodiment, the gene encoding the propionate gene cassette is derived from an organism of the genus or species that includes, but is not limited to,  Clostridium propionicum, Megasphaera elsdenii , or  Prevotella ruminicola.    
     In one embodiment, the propionate gene cassette has been codon-optimized for use in the engineered bacterial cell. In one embodiment, the propionate gene cassette has been codon-optimized for use in  Escherichia coli . In another embodiment, the propionate gene cassette has been codon-optimized for use in  Lactococcus . When the propionate gene cassette is expressed in the engineered bacterial cells, the bacterial cells produce more propionate than unmodified bacteria of the same bacterial subtype under the same conditions (e.g., culture or environmental conditions). Thus, the genetically engineered bacteria comprising a heterologous propionate gene cassette may be used to generate propionate to treat autoimmune disease, such as IBD. 
     The present disclosure further comprises genes encoding functional fragments of propionate biosynthesis enzymes or functional variants of a propionate biosynthesis enzyme. As used herein, the term “functional fragment thereof” or “functional variant thereof” relates to an element having qualitative biological activity in common with the wild-type enzyme from which the fragment or variant was derived. For example, a functional fragment or a functional variant of a mutated propionate biosynthesis enzyme is one which retains essentially the same ability to synthesize propionate as the propionate biosynthesis enzyme from which the functional fragment or functional variant was derived. For example a polypeptide having propionate biosynthesis enzyme activity may be truncated at the N-terminus or C-terminus, and the retention of propionate biosynthesis enzyme activity assessed using assays known to those of skill in the art, including the exemplary assays provided herein. In one embodiment, the engineered bacterial cell comprises a heterologous gene encoding a propionate biosynthesis enzyme functional variant. In another embodiment, the engineered bacterial cell comprises a heterologous gene encoding a propionate biosynthesis enzyme functional fragment. 
     As used herein, the term “percent (%) sequence identity” or “percent (%) identity,” also including “homology,” is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). 
     The present disclosure encompasses propionate biosynthesis enzymes comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein. Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions. A conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g., charge, structure, polarity, hydrophobicity/hydrophilicity) that are similar to those of the first amino acid. Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T. Similarly contemplated is replacing a basic amino acid with another basic amino acid (e.g., replacement among Lys, Arg, His), replacing an acidic amino acid with another acidic amino acid (e.g., replacement among Asp and Glu), replacing a neutral amino acid with another neutral amino acid (e.g., replacement among Ala, Gly, Ser, Met, Thr, Leu, Ile, Asn, Gin, Phe, Cys, Pro, Trp, Tyr, Val). 
     In some embodiments, a propionate biosynthesis enzyme is mutagenized; mutants exhibiting increased activity are selected; and the mutagenized gene encoding the propionate biosynthesis enzyme is isolated and inserted into the bacterial cell of the disclosure. The gene comprising the modifications described herein may be present on a plasmid or chromosome. 
     In one embodiment, the propionate biosynthesis gene cassette is from  Clostridium  spp. In one embodiment, the  Clostridium  spp. is  Clostridium propionicum . In another embodiment, the propionate biosynthesis gene cassette is from a  Megasphaera  spp. In one embodiment, the  Megasphaera  spp. is  Megasphaera elsdenii . In another embodiment, the propionate biosynthesis gene cassette is from  Prevotella  spp. In one embodiment, the  Prevotella  spp. is  Prevotella ruminicola . Other propionate biosynthesis gene cassettes are well-known to one of ordinary skill in the art. 
     In some embodiments, the genetically engineered bacteria comprise the genes pct, lcd, and acr from  Clostridium propionicum . In some embodiments, the genetically engineered bacteria comprise acrylate pathway genes for propionate biosynthesis, e.g., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC. In alternate embodiments, the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, e.g., thrA fbr , thrB, thrC, thrC, ilvA fbr , aceE, aceF, and lpd, and optionally further comprise tesB. The genes may be codon-optimized, and translational and transcriptional elements may be added. 
     In one embodiment, the pct gene has at least about 80% identity with SEQ ID NO: 21. In another embodiment, the pct gene has at least about 85% identity with SEQ ID NO: 21. In one embodiment, the pct gene has at least about 90% identity with SEQ ID NO: 21. In one embodiment, the pct gene has at least about 95% identity with SEQ ID NO: 21. In another embodiment, the pct gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 21. Accordingly, in one embodiment, the pct gene has at least about 80%, 821%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 921%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 21. In another embodiment, the pct gene comprises the sequence of SEQ ID NO: 21. In yet another embodiment the pct gene consists of the sequence of SEQ ID NO: 21. 
     In one embodiment, the lcdA gene has at least about 80% identity with SEQ ID NO: 22. In another embodiment, the lcdA gene has at least about 85% identity with SEQ ID NO: 22. In one embodiment, the lcdA gene has at least about 90% identity with SEQ ID NO: 22. In one embodiment, the lcdA gene has at least about 95% identity with SEQ ID NO: 22. In another embodiment, the lcdA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 22. Accordingly, in one embodiment, the lcdA gene has at least about 80%, 81%, 822%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 922%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 22. In another embodiment, the lcdA gene comprises the sequence of SEQ ID NO: 22. In yet another embodiment the lcdA gene consists of the sequence of SEQ ID NO: 22. 
     In one embodiment, the lcdB gene has at least about 80% identity with SEQ ID NO: 23. In another embodiment, the lcdB gene has at least about 85% identity with SEQ ID NO: 23. In one embodiment, the lcdB gene has at least about 90% identity with SEQ ID NO: 23. In one embodiment, the lcdB gene has at least about 95% identity with SEQ ID NO: 23. In another embodiment, the lcdB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 23. Accordingly, in one embodiment, the lcdB gene has at least about 80%, 81%, 82%, 823%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 923%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 23. In another embodiment, the lcdB gene comprises the sequence of SEQ ID NO: 23. In yet another embodiment the lcdB gene consists of the sequence of SEQ ID NO: 23. 
     In one embodiment, the lcdC gene has at least about 80% identity with SEQ ID NO: 24. In another embodiment, the lcdC gene has at least about 85% identity with SEQ ID NO: 24. In one embodiment, the lcdC gene has at least about 90% identity with SEQ ID NO: 24. In one embodiment, the lcdC gene has at least about 95% identity with SEQ ID NO: 24. In another embodiment, the lcdC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 24. Accordingly, in one embodiment, the lcdA gene has at least about 80%, 81%, 82%, 83%, 824%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 924%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 24. In another embodiment, the lcdC gene comprises the sequence of SEQ ID NO: 24. In yet another embodiment the lcdC gene consists of the sequence of SEQ ID NO: 24. 
     In one embodiment, the etfA gene has at least about 80% identity with SEQ ID NO: 25. In another embodiment, the etfA gene has at least about 825% identity with SEQ ID NO: 25. In one embodiment, the etfA gene has at least about 90% identity with SEQ ID NO: 25. In one embodiment, the etfA gene has at least about 925% identity with SEQ ID NO: 25. In another embodiment, the etfA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 25. Accordingly, in one embodiment, the etfA gene has at least about 80%, 81%, 82%, 83%, 84%, 825%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 925%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 25. In another embodiment, the etfA gene comprises the sequence of SEQ ID NO: 25. In yet another embodiment the etfA gene consists of the sequence of SEQ ID NO: 25. 
     In one embodiment, the acrB gene has at least about 80% identity with SEQ ID NO: 26. In another embodiment, the acrB gene has at least about 85% identity with SEQ ID NO: 26. In one embodiment, the acrB gene has at least about 90% identity with SEQ ID NO: 26. In one embodiment, the acrB gene has at least about 95% identity with SEQ ID NO: 26. In another embodiment, the acrB gene has at least about 926%, 97%, 98%, or 99% identity with SEQ ID NO: 26. Accordingly, in one embodiment, the acrB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 826%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 926%, 97%, 98%, or 99% identity with SEQ ID NO: 26. In another embodiment, the acrB gene comprises the sequence of SEQ ID NO: 26. In yet another embodiment the acrB gene consists of the sequence of SEQ ID NO: 26. 
     In one embodiment, the acrC gene has at least about 80% identity with SEQ ID NO: 27. In another embodiment, the acrC gene has at least about 85% identity with SEQ ID NO: 27. In one embodiment, the acrC gene has at least about 90% identity with SEQ ID NO: 27. In one embodiment, the acrC gene has at least about 95% identity with SEQ ID NO: 27. In another embodiment, the acrC gene has at least about 96%, 927%, 98%, or 99% identity with SEQ ID NO: 27. Accordingly, in one embodiment, the acrC gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 827%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 927%, 98%, or 99% identity with SEQ ID NO: 27. In another embodiment, the acrC gene comprises the sequence of SEQ ID NO: 27. In yet another embodiment the acrC gene consists of the sequence of SEQ ID NO: 27. 
     In one embodiment, the thrA fbr  gene has at least about 280% identity with SEQ ID NO: 28. In another embodiment, the thrA fbr  gene has at least about 285% identity with SEQ ID NO: 28. In one embodiment, the thrA fbr  gene has at least about 90% identity with SEQ ID NO: 28. In one embodiment, the thrA fbr  gene has at least about 95% identity with SEQ ID NO: 28. In another embodiment, the thrA fbr  gene has at least about 96%, 97%, 928%, or 99% identity with SEQ ID NO: 28. Accordingly, in one embodiment, the thrA fbr  gene has at least about 280%, 281%, 282%, 283%, 284%, 285%, 286%, 287%, 2828%, 289%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 928%, or 99% identity with SEQ ID NO: 28. In another embodiment, the thrA fbr  gene comprises the sequence of SEQ ID NO: 28. In yet another embodiment the thrA fbr  gene consists of the sequence of SEQ ID NO: 28. 
     In one embodiment, the thrB gene has at least about 80% identity with SEQ ID NO: 29. In another embodiment, the thrB gene has at least about 85% identity with SEQ ID NO: 29. In one embodiment, the thrB gene has at least about 290% identity with SEQ ID NO: 29. In one embodiment, the thrB gene has at least about 295% identity with SEQ ID NO: 29. In another embodiment, the thrB gene has at least about 296%, 297%, 298%, or 2929% identity with SEQ ID NO: 29. Accordingly, in one embodiment, the thrB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 829%, 290%, 291%, 292%, 293%, 294%, 295%, 296%, 297%, 298%, or 2929% identity with SEQ ID NO: 29. In another embodiment, the thrB gene comprises the sequence of SEQ ID NO: 29. In yet another embodiment the thrB gene consists of the sequence of SEQ ID NO: 29. 
     In one embodiment, the thrC gene has at least about 80% identity with SEQ ID NO: 30. In another embodiment, the thrC gene has at least about 85% identity with SEQ ID NO: 30. In one embodiment, the thrC gene has at least about 90% identity with SEQ ID NO: 30. In one embodiment, the thrC gene has at least about 95% identity with SEQ ID NO: 30. In another embodiment, the thrC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 30. Accordingly, in one embodiment, the thrC gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 30. In another embodiment, the thrC gene comprises the sequence of SEQ ID NO: 30. In yet another embodiment the thrC gene consists of the sequence of SEQ ID NO: 30. 
     In one embodiment, the ilvA fbr  gene has at least about 80% identity with SEQ ID NO: 31. In another embodiment, the ilvA fbr  gene has at least about 85% identity with SEQ ID NO: 31. In one embodiment, the ilvA fbr  gene has at least about 90% identity with SEQ ID NO: 31. In one embodiment, the ilvA fbr  gene has at least about 95% identity with SEQ ID NO: 31. In another embodiment, the ilvA fbr  gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 31. Accordingly, in one embodiment, the ilvA fbr  gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 31. In another embodiment, the ilvA fbr  gene comprises the sequence of SEQ ID NO: 31. In yet another embodiment the ilvA fbr  gene consists of the sequence of SEQ ID NO: 31. 
     In one embodiment, the aceE gene has at least about 80% identity with SEQ ID NO: 32. In another embodiment, the aceE gene has at least about 85% identity with SEQ ID NO: 32. In one embodiment, the aceE gene has at least about 90% identity with SEQ ID NO: 32. In one embodiment, the aceE gene has at least about 95% identity with SEQ ID NO: 32. In another embodiment, the aceE gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 32. Accordingly, in one embodiment, the aceE gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 32. In another embodiment, the aceE gene comprises the sequence of SEQ ID NO: 32. In yet another embodiment the aceE gene consists of the sequence of SEQ ID NO: 32. 
     In one embodiment, the aceF gene has at least about 80% identity with SEQ ID NO: 33. In another embodiment, the aceF gene has at least about 85% identity with SEQ ID NO: 33. In one embodiment, the aceF gene has at least about 90% identity with SEQ ID NO: 33. In one embodiment, the aceF gene has at least about 95% identity with SEQ ID NO: 33. In another embodiment, the aceF gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 33. Accordingly, in one embodiment, the aceF gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 33. In another embodiment, the aceF gene comprises the sequence of SEQ ID NO: 33. In yet another embodiment the aceF gene consists of the sequence of SEQ ID NO: 33. 
     In one embodiment, the lpd gene has at least about 80% identity with SEQ ID NO: 34. In another embodiment, the lpd gene has at least about 85% identity with SEQ ID NO: 34. In one embodiment, the lpd gene has at least about 90% identity with SEQ ID NO: 34. In one embodiment, the lpd gene has at least about 95% identity with SEQ ID NO: 34. In another embodiment, the lpd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 34. Accordingly, in one embodiment, the lpd gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 34. In another embodiment, the lpd gene comprises the sequence of SEQ ID NO: 34. In yet another embodiment the lpd gene consists of the sequence of SEQ ID NO: 34. 
     In one embodiment, the tesB gene has at least about 80% identity with SEQ ID NO: 10. In another embodiment, the tesB gene has at least about 85% identity with SEQ ID NO: 10. In one embodiment, the tesB gene has at least about 90% identity with SEQ ID NO: 10. In one embodiment, the tesB gene has at least about 95% identity with SEQ ID NO: 10. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. Accordingly, in one embodiment, the tesB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. In another embodiment, the tesB gene comprises the sequence of SEQ ID NO: 10. In yet another embodiment the tesB gene consists of the sequence of SEQ ID NO: 10. 
     In one embodiment, the acuI gene has at least about 80% identity with SEQ ID NO: 35. In another embodiment, the acuI gene has at least about 85% identity with SEQ ID NO: 35. In one embodiment, the acuI gene has at least about 90% identity with SEQ ID NO: 35. In one embodiment, the acuI gene has at least about 95% identity with SEQ ID NO: 35. In another embodiment, the acid gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 35. Accordingly, in one embodiment, the acuI gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 35. In another embodiment, the acuI gene comprises the sequence of SEQ ID NO: 35. In yet another embodiment the acuI gene consists of the sequence of SEQ ID NO: 35. 
     In one embodiment, the sbm gene has at least about 80% identity with SEQ ID NO: 36. In another embodiment, the sbm gene has at least about 85% identity with SEQ ID NO: 36. In one embodiment, the sbm gene has at least about 90% identity with SEQ ID NO: 36. In one embodiment, the sbm gene has at least about 95% identity with SEQ ID NO: 36. In another embodiment, the sbm gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 36.0. Accordingly, in one embodiment, the sbm gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 36. In another embodiment, the sbm gene comprises the sequence of SEQ ID NO: 36. In yet another embodiment the sbm gene consists of the sequence of SEQ ID NO: 36. 
     In one embodiment, the ygfD gene has at least about 80% identity with SEQ ID NO: 37. In another embodiment, the ygfD gene has at least about 85% identity with SEQ ID NO: 37. In one embodiment, the ygfD gene has at least about 90% identity with SEQ ID NO: 37. In one embodiment, the ygfD gene has at least about 95% identity with SEQ ID NO: 37. In another embodiment, the ygfD gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 37. Accordingly, in one embodiment, the ygfD gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 37. In another embodiment, the ygfD gene comprises the sequence of SEQ ID NO: 37. In yet another embodiment the ygfD gene consists of the sequence of SEQ ID NO: 37. 
     In one embodiment, the ygfG gene has at least about 80% identity with SEQ ID NO: 38. In another embodiment, the ybfG gene has at least about 85% identity with SEQ ID NO: 38. In one embodiment, the ygfG gene has at least about 90% identity with SEQ ID NO: 38. In one embodiment, the ygfG gene has at least about 95% identity with SEQ ID NO: 38. In another embodiment, the ygfG gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 38. Accordingly, in one embodiment, the ygfG gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 38. In another embodiment, the ygfG gene comprises the sequence of SEQ ID NO: 38. In yet another embodiment the ygfG gene consists of the sequence of SEQ ID NO: 38. 
     In one embodiment, the ygfH gene has at least about 80% identity with SEQ ID NO: 39. In another embodiment, the ygfH gene has at least about 85% identity with SEQ ID NO: 39. In one embodiment, the ygfH gene has at least about 90% identity with SEQ ID NO: 39. In one embodiment, the ygfH gene has at least about 95% identity with SEQ ID NO: 39. In another embodiment, the ygfH gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 39. Accordingly, in one embodiment, the ygfH gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 39. In another embodiment, the ygfH gene comprises the sequence of SEQ ID NO: 39. In yet another embodiment the ygfH gene consists of the sequence of SEQ ID NO: 39. 
     In one embodiment, the mutA gene has at least about 80% identity with SEQ ID NO: 40. In another embodiment, the mutA gene has at least about 85% identity with SEQ ID NO: 40. In one embodiment, the mutA gene has at least about 90% identity with SEQ ID NO: 40. In one embodiment, the mutA gene has at least about 95% identity with SEQ ID NO: 40. In another embodiment, the mutA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 40. Accordingly, in one embodiment, the mutA gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 40. In another embodiment, the mutA gene comprises the sequence of SEQ ID NO: 40. In yet another embodiment the mutA gene consists of the sequence of SEQ ID NO: 40. 
     In one embodiment, the mutB gene has at least about 80% identity with SEQ ID NO: 41. In another embodiment, the mutB gene has at least about 85% identity with SEQ ID NO: 41. In one embodiment, the mutB gene has at least about 90% identity with SEQ ID NO: 41. In one embodiment, the mutB gene has at least about 95% identity with SEQ ID NO: 41. In another embodiment, the mutB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 41. Accordingly, in one embodiment, the mutB gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 41. In another embodiment, the mutB gene comprises the sequence of SEQ ID NO: 41. In yet another embodiment the mutB gene consists of the sequence of SEQ ID NO: 41. 
     In one embodiment, the GI 18042134 gene has at least about 80% identity with SEQ ID NO: 42. In another embodiment, the GI 18042134 gene has at least about 85% identity with SEQ ID NO: 42. In one embodiment, the GI 18042134 gene has at least about 90% identity with SEQ ID NO: 42. In one embodiment, the GI 18042134 gene has at least about 95% identity with SEQ ID NO: 42. In another embodiment, the GI 18042134 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 42. Accordingly, in one embodiment, the GI 18042134 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 42. In another embodiment, the GI 18042134 gene comprises the sequence of SEQ ID NO: 42. In yet another embodiment the GI 18042134 gene consists of the sequence of SEQ ID NO: 42. 
     In one embodiment, the mmdA gene has at least about 80% identity with SEQ ID NO: 43. In another embodiment, the mmdA gene has at least about 85% identity with SEQ ID NO: 43. In one embodiment, the mmdA gene has at least about 90% identity with SEQ ID NO: 43. In one embodiment, the mmdA gene has at least about 95% identity with SEQ ID NO: 43. In another embodiment, the mmdA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 43. Accordingly, in one embodiment, the mmdA gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 43. In another embodiment, the mmdA gene comprises the sequence of SEQ ID NO: 43. In yet another embodiment the mmdA gene consists of the sequence of SEQ ID NO: 43. 
     In one embodiment, the PFREUD_188870 gene has at least about 80% identity with SEQ ID NO: 44. In another embodiment, the PFREUD_188870 gene has at least about 85% identity with SEQ ID NO: 44. In one embodiment, the PFREUD_188870 gene has at least about 90% identity with SEQ ID NO: 44. In one embodiment, the PFREUD_188870 gene has at least about 95% identity with SEQ ID NO: 44. In another embodiment, the PFREUD_188870 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 44. Accordingly, in one embodiment, the PFREUD_188870 gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 44. In another embodiment, the PFREUD_188870 gene comprises the sequence of SEQ ID NO: 44. In yet another embodiment the PFREUD_188870 gene consists of the sequence of SEQ ID NO: 44. 
     In one embodiment, the Bccp gene has at least about 80% identity with SEQ ID NO: 45. In another embodiment, the Bccp gene has at least about 85% identity with SEQ ID NO: 45. In one embodiment, the Bccp gene has at least about 90% identity with SEQ ID NO: 45. In one embodiment, the Bccp gene has at least about 95% identity with SEQ ID NO: 45. In another embodiment, the Bccp gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 45. Accordingly, in one embodiment, the Bccp gene has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 45. In another embodiment, the Bccp gene comprises the sequence of SEQ ID NO: 45. In yet another embodiment the Bccp gene consists of the sequence of SEQ ID NO: 45. 
     In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 46 through SEQ ID NO: 70. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 46 through SEQ ID NO: 70. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 46 through SEQ ID NO: 70. In one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 46 through SEQ ID NO: 70. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 46 through SEQ ID NO: 70. Accordingly, in one embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 46 through SEQ ID NO: 70. In another embodiment, one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 46 through SEQ ID NO: 70. In yet another embodiment one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria consist of or or more of SEQ ID NO: 46 through SEQ ID NO: 70. 
     In some embodiments, one or more of the propionate biosynthesis genes is a synthetic propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is an  E. coli  propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is a  C. glutamicum  propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is a  C. propionicum  propionate biosynthesis gene. In some embodiments, one or more of the propionate biosynthesis genes is a  R. sphaeroides  propionate biosynthesis gene. The propionate gene cassette may comprise genes for the aerobic biosynthesis of propionate and/or genes for the anaerobic or microaerobic biosynthesis of propionate. 
     To improve acetate production, while maintaining high levels of propionate production, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more propionate cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
     In some embodiments, the genetically engineered bacteria comprise one or more propionate cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH gene cassette(s) and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH gene cassette(s) and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH gene cassette(s) and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more propionate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more propionate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more propionate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle, while maintaining high levels of propionate production. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for propionate production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for propionate synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of propionate and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of propionate and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation in the endogenous pta and ldhA genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from sbm, ygfD, ygfG, and/or ygfH and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) comprising one or more sbm-ygfD-ygfG-ygfH propionate cassette(s) and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more propionate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more propionate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more propionate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria comprise a combination of propionate biosynthesis genes from different species, strains, and/or substrains of bacteria, and are capable of producing propionate. In some embodiments, one or more of the propionate biosynthesis genes is functionally replaced, modified, and/or mutated in order to enhance stability and/or increase propionate production. In some embodiments, the local production of propionate reduces food intake and improves gut barrier function and reduces inflammation In some embodiments, the genetically engineered bacteria are capable of expressing the propionate biosynthesis cassette and producing propionate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. 
     In one embodiment, the propionate gene cassette is directly operably linked to a first promoter. In another embodiment, the propionate gene cassette is indirectly operably linked to a first promoter. In one embodiment, the promoter is not operably linked with the propionate gene cassette in nature. 
     In some embodiments, the propionate gene cassette is expressed under the control of a constitutive promoter. In another embodiment, the propionate gene cassette is expressed under the control of an inducible promoter. In some embodiments, the propionate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the propionate gene cassette is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the propionate gene cassette is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. Inducible promoters are described in more detail infra. 
     The propionate gene cassette may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the propionate gene cassette is located on a plasmid in the bacterial cell. In another embodiment, the propionate gene cassette is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the propionate gene cassette is located in the chromosome of the bacterial cell, and a propionate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the propionate gene cassette is located on a plasmid in the bacterial cell, and a propionate gene cassette from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the propionate gene cassette is located in the chromosome of the bacterial cell, and a propionate gene cassette from a different species of bacteria is located in the chromosome of the bacterial cell. 
     In some embodiments, the propionate gene cassette is expressed on a low-copy plasmid. In some embodiments, the propionate gene cassette is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of propionate. 
     Tryptophan and Tryptophan Metabolism 
     Kynurenine 
     In some embodiments, the genetically engineered bacteria are capable of producing kynurenine. Kynurenine is a metabolite produced in the first, rate-limiting step of tryptophan catabolism. This step involves the conversion of tryptophan to kynurenine, and may be catalyzed by the ubiquitously-expressed enzyme indoleamine 2,3-dioxygenase (IDO-1), or by tryptophan dioxygenase (TDO), an enzyme which is primarily localized to the liver (Alvarado et al., 2015). Biopsies from human patients with IBD show elevated levels of IDO-1 expression compared to biopsies from healthy individuals, particularly near sites of ulceration (Ferdinande et al., 2008; Wolf et al., 2004). IDO-1 enzyme expression is similarly upregulated in trinitrobenzene sulfonic acid- and dextran sodium sulfate-induced mouse models of IBD; inhibition of IDO-1 significantly augments the inflammatory response caused by each inducer (Ciorba et al., 2010; Gurtner et al., 2003; Matteoli et al., 2010). Kynurenine has also been shown to directly induce apoptosis in neutrophils (El-Zaatari et al., 2014). Together, these observations suggest that IDO-1 and kynurenine play a role in limiting inflammation. The genetically engineered bacteria may comprise any suitable gene for producing kynurenine. In some embodiments, the genetically engineered bacteria may comprise a gene or gene cassette for producing a tryptophan transporter, a gene or gene cassette for producing IDO-1, and a gene or gene cassette for producing TDO. In some embodiments, the gene for producing kynurenine is modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions. 
     In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid. Kynurenic acid is produced from the irreversible transamination of kynurenine in a reaction catalyzed by the enzyme kynurenine-oxoglutarate transaminase. Kynurenic acid acts as an antagonist of ionotropic glutamate receptors (Turski et al., 2013). While glutamate is known to be a major excitatory neurotransmitter in the central nervous system, there is now evidence to suggest an additional role for glutamate in the peripheral nervous system. For example, the activation of NMDA glutamate receptors in the major nerve supply to the GI tract (i.e., the myenteric plexus) leads to an increase in gut motility (Forrest et al., 2003), but rats treated with kynurenic acid exhibit decreased gut motility and inflammation in the early phase of acute colitis (Varga et al., 2010). Thus, the elevated levels of kynurenic acid reported in IBD patients may represent a compensatory response to the increased activation of enteric neurons (Forrest et al., 2003). The genetically engineered bacteria may comprise any suitable gene, genes, or gene cassettes for producing kynurenic acid. In some embodiments, the gene for producing kynurenic acid is modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions 
     Tryptophan, Tryptophan Metabolism, and Tryptophan Metabolites 
     Typtophan and the Kynurenine Pathway 
     Tryptophan (TRP) is an essential amino acid that, after consumption, is either incorporated into proteins via new protein synthesis, or converted a number of biologically active metabolites with a number of differing roles in health and disease (Perez-De La Cruz et al., 2007 Kynurenine Pathway and Disease: An Overview; CNS&amp;Neurological Disorders—Drug Targets 2007, 6, 398-410). Along one arm of tryptophan catabolism, trytophan is converted to the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) by tryptophan hydroxylase. Serotonin can further be converted into the hormone melatonin. A large share of tryptophan, however, is metabolized to a number of bioactive metabolites, collectively called kynurenines, along a second arm called the kynurenine pathway (KP). In the first step of catabolism, TRP is converted to Kynurenine, (KYN), which has well-documented immune suppressive functions in several types of immune cells, and has recently been shown to be an activating ligand for the arylcarbon receptor (AhR; also known as dioxin receptor). KYN was initially shown in the cancer setting as an endogenous AHR ligand in immune and tumor cells, acting both in an autocrine and paracrine manner, and promoting tumor cell survival. In the gut, kynurenine pathway metabolism is regulated by gut microbiota, which can regulate tryptophan availability for kynurenine pathway metabolism. 
     More recently, additional tryptophan metabolites, collectively termed “indoles”, herein, including for example, indole-3 aldehyde, indole-3 acetate, indole-3 propoinic acid, indole, indole-3 acetaladehyde, indole-3acetonitrile, FICZ, etc. which are generated by the microbiota, some by the human host, some from the diet, which are also able to function as AhR agonists, see e.g., Table 8 and elsewhere herein, and Lama et al., Nat Med. 2016 June; 22(6):598-605; CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands. 
     Ahr best known as a receptor for xenobiotics such as polycyclic aromatic hydrocarbons AhR is a ligand-dependent cytosolic transcription factor that is able to translocate to the cell nucleus after ligand binding. The in addition to kynurenine, tryptophan metabolites L-kynurenine, 6-formylindolcarbazole (FICZ, a photoproduct of TRP), and KYNA are have recently been identified as endogenous AhR ligands mediating immunosuppressive functions. To induce transcription of AhR target genes in the nucleus, AhR partners with proteins such as AhR nuclear translocator (ARNT) or NF-κB subunit RelB. Studies on human cancer cells have shown that KYN activates the AhR-ARNT associated transcription of IL-6, which induced autocrine activation of IDO1 via STAT3. This AhR-IL-6-STAT3 loop is associated with a poor prognosis in lung cancer, supporting the idea that IDO/kynurenine-mediated immunosuppression enables the immune escape of tumor cells. 
     In the gut, tryptophan may also be transported across the epithelium by transport machinery comprising angiotensin I converting enzyme 2 (ACE2), and converted to kynurenine, where it functions in the suppression of T cell response and promotion of Treg cells. 
     The rate-limiting conversion of TRP to KYN may be mediated by either of two forms of indoleamine 2, 3-dioxygenase (IDO) or by tryptophan 2,3-dioxygenase (TDO). One characteristic of TRP metabolism is that the rate-limiting step of the catalysis from TRP to KYN is generated by both the hepatic enzyme tryptophan 2,3-dioxygenase (TDO) and the ubiquitous expressed enzyme IDO1. TDO is essential for homeostasis of TRP concentrations in organisms and has a lower affinity to TRP than IDO. Its expression is activated mainly by increased plasma TRP concentrations but can also be activated by glucocorticoids and glucagon. The tryptophan kynurenine pathway is also expressed in a large number of microbiota, most prominently in Enterobacteriaceae, and kynurenine and metabolites may be synthesized in the gut (as shown in the figures and the examples, and Sci Transl Med. 2013 Jul. 10; 5(193): 193ra91). In some embodiments, the genetically engineered bacteria comprise one or more heterologous bacterially derived genes from Enterobacteriaceae, e.g. whose gene products catalyze the conversion of TRP:KYN. Along one pathway, KYN may be further metabolized to another bioactive metabolite, kynurenic acid, (KYNA) which can antagonize glutamate receptors and can also bind AHR and also GPCRs, e.g., GPR35, glutamate receptors, N-methyl D-aspartate (NMDA)-receptors, and others. Along a third pathway of the KP, KYN can be converted to anthranilic acid (AA) and further downstream quinolinic acid (QUIN), which is a glutamate receptor agonist and has a neurotoxic role. 
     Therefore, finding a means to upregulate and/or downregulate the levels of flux through the KP and to reset relative amounts and/or ratios of tryptophan and its various bioactive metabolites may be useful in the prevention, treatment and/or management of a number of diseases as described herein. The present disclosure describes compositions for modulating, regulating and fine tuning trypophan and tryptophan metabolite levels, e.g., in the serum or in the gastrointestinal system, through genetically engineered bacteria which comprise circuitry enabling the synthesis, bacterial uptake and catabolism of tryptophan and/or tryptophan metabolites. and provides methods for using these compositions in the treatment, management and/or prevention of a number of different diseases. 
     Other Indole Tryptophan Metabolites 
     In addition to kynurenine and KYNA, numerous compounds have been proposed as endogenous AHR ligands, many of which are generated through pathways involved in the metabolism of tryptophan and indole (Bittinger et al., 2003; Chung and Gadupudi, 2011) A large number of metabolites generated through the tryptophan indole pathway are generated by microbiota in the gut. For example, bacteria take up tryptophan, which can be converted to mono-substituted indole compounds, such as indole acetic acid (IAA) and tryptamine, and other compounds, which have been found to activate the AHR (Hubbard et al., 2015, Adaptation of the human aryl hydrocarbon receptor to sense microbiota-derived indoles; Nature Scientific Reports 5:12689). 
     In the gastrointestinal tract, diet derived and bacterially AhR ligands promote IL-22 production by innate lymphoid cells, referred to as group 3 ILCs (Spits et al., 2013, Zelante et al., Tryptophan Catabolites from Microbiota Engage Aryl Hydrocarbon Receptor and Balance Mucosal Reactivity via Interleukin-22; Immunity 39, 372-385, Aug. 22, 2013). AHR is essential for IL-22-production in the intestinal lamina propria (Lee et al., Nature Immunology 13, 144-151 (2012); AHR drives the development of gut ILC22 cells and postnatal lymphoid tissues via pathways dependent on and independent of Notch). 
     Through initiation of Jak-STAT signaling pathways, IL-22 expression can trigger expression of antimicrobial compounds as well as a range of cell growth related pathways, both of which enhance tissue repair mechanisms. IL-22 is critical in promoting intestinal barrier fidelity and healing, while modulating inflammatory states. Murine models have demonstrated improved intestinal inflammation states following administration of 11-22. Additionally, IL-22 activates STAT3 signaling to promote enhanced mucus production to preserve barrier function. 
     Table 8 lists exemplary tryptophan metabolites which have been shown to bind to AhR and which can be produced by the genetically engineered bacteria of the disclosure. Thus, in some embodiments, the engineered bacteria comprises gene sequence(s) encoding one or more enzymes for the production of one or more metabolites listed in Table 8. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Indole Tryptophan Metabolites 
               
            
           
           
               
               
            
               
                 Origin 
                 Compound 
               
               
                   
               
               
                 Exogenous 
                 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) 
               
               
                 Dietary 
                 Indole-3-carbinol (I3C) 
               
               
                 Dietary 
                 Indole-3-acetonitrile (I3ACN) 
               
               
                 Dietary 
                 3.3′-Diindolylmethane (DIM) 
               
               
                 Dietary 
                 2-(indol-3-ylmethyl)-3.3′-diindolylmethane (Ltr-1) 
               
               
                 Dietary 
                 Indolo(3,2-b)carbazole (ICZ) 
               
               
                 Dietary 
                 2-(1′H-indole-3′-carbony)-thiazole-4-carboxylic 
               
               
                   
                 acid methyl ester (ITE) 
               
               
                 Microbial 
                 Indole 
               
               
                 Microbial 
                 Indole-3-acetic acid (IAA) 
               
               
                 Microbial 
                 Indole-3-aldehyde (IAId) 
               
               
                 Microbial 
                 Tryptamine 
               
               
                 Microbial 
                 3-methyl-indole (Skatole) 
               
               
                 Yeast 
                 Tryptanthrin 
               
               
                 Microbial/Host 
                 Indigo 
               
               
                 Metabolism 
               
               
                 Microbial/Host 
                 Indirubin 
               
               
                 Metabolism 
               
               
                 Microbial/Host 
                 Indoxyl-3-sulfate (I3S) 
               
               
                 Metabolism 
               
               
                 Host 
                 Kynurenine (Kyn) 
               
               
                 Metabolism 
               
               
                 Host 
                 Kynurenic acid (KA) 
               
               
                 Metabolism 
               
               
                 Host 
                 Xanthurenic acid 
               
               
                 Metabolism 
               
               
                 Host 
                 Cinnabarinic acid (CA) 
               
               
                 Metabolism 
               
               
                 UV-Light 
                 6-formylindolo(3,2-b)carbazole (FICZ) 
               
               
                 Oxidation 
               
               
                 Microbial 
               
               
                 metabolism 
               
               
                   
               
            
           
         
       
     
     In addition, some indole metabolites may exert their effect through Pregnane X receptor (PXR), which is thought to play a key role as an essential regulator of intestinal barrier function. PXR-deficient (Nrli2−/−) mice showed a distinctly “leaky” gut physiology coupled with upregulation of the Toll-like receptor 4 (TLR4), a receptor well known for recognizing LPS and activating the innate immune system (Venkatesh et al., 2014 Symbiotic Bacterial Metabolites Regulate Gastrointestinal Barrier Function via the Xenobiotic Sensor PXR and Toll-like Receptor 4; Immunity 41, 296-310, Aug. 21, 2014). In particular, indole 3-propionic acid (IPA), produced by microbiota in the gut, has been shown to be a ligand for PXR in vivo. 
     As a result of PXR agonism, indole levels e.g., produced by commensal bacteria, or by genetically engineered bacteria, may through the activation of PXR regulate and balance the levels of TLR4 expression to promote homeostasis and gut barrier health. I.e., low levels of IPA and/or PXR and an excess of TLR4 may lead to intestinally barrier dysfunction, while increasing levels of IPA may promote PXR activation and TLR4 downregulation, and improved gut barrier health. 
     In other embodiments, IPA producing circuits comprise enzymes depicted and described in the figures and elsewhere herein. Thus, in some embodiments, the engineered bacteria comprise gene sequence(s) encoding one or more enzymes selected from TrpDH: tryptophan dehydrogenase (e.g., from from  Nostoc punctiforme  NIES-2108); FldH1/FldH2: indole-3-lactate dehydrogenase (e.g., from  Clostridium sporogenes ); FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase (e.g., from  Clostridium sporogenes ); FldBC: indole-3-lactate dehydratase, (e.g., from  Clostridium sporogenes ); FldD: indole-3-acrylyl-CoA reductase (e.g., from  Clostridium sporogenes ); AcuI: acrylyl-CoA reductase (e.g., from  Rhodobacter sphaeroides ); lpdC: Indole-3-pyruvate decarboxylase (e.g., from  Enterobacter cloacae ); lad1: Indole-3-acetaldehyde dehydrogenase (e.g., from  Ustilago maydis ); and Tdc: Tryptophan decarboxylase (e.g., from  Catharanthus roseus  or from ( Clostridium sporogenes ). In some embodiments, the engineered bacteria comprise gene sequence(s) and/or gene cassette(s) for the production of one or more of the following: indole-3-propionic acid (IPA), indole acetic acid (IAA), and tryptamine synthesis (TrA). 
     Tryptophan dehydrogenase (EC 1.4.1.19) is an enzyme that catalyzes the reversible chemical reaction converting L-tryptophan, NAD(P) and water to (indol-3-yl)pyruvate (IPyA), NH 3 , NAD(P)H and H + . Indole-3-lactate dehydrogenase ((EC 1.1.1.110, e.g.,  Clostridium sporogenes  or  Lactobacillus casei ) converts (indol-3yl)pyruvate (IpyA) and NADH and H+ to indole-3-lactate (ILA) and NAD+. Indole-3-propionyl-CoA:indole-3-lactate CoA transferase (FldA) converts indole-3-lactate (ILA) and indol-3-propionyl-CoA to indole-3-propionic acid (IPA) and indole-3-lactate-CoA. Indole-3-acrylyl-CoA reductase (FldD) and acrylyl-CoA reductase (AcuI) convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA. Indole-3-lactate dehydratase (FldBC) converts indole-3-lactate-CoA to indole-3-acrylyl-CoA. Indole-3-pyruvate decarboxylase (lpdC:) converts Indole-3-pyruvic acid (IPyA) into Indole-3-acetaldehyde (IAAld) lad1: Indole-3-acetaldehyde dehydrogenase coverts Indole-3-acetaldehyde (IAAld) into Indole-3-acetic acid (IAA) Tdc: Tryptophan decarboxylase converts tryptophan (Trp) into tryptamine (TrA). 
     Although microbial degradation of tryptophan to indole-3-propionate has been shown in a number of microorganisms (see, e.g., Elsden et al., The end products of the metabolism of aromatic amino acids by Clostridia, Arch Microbiol. 1976 Apr. 1; 107(3):283-8), to date, the bacterial entire biosynthetic pathway from tryptophan to IPA is unknown. In  Clostridium sporogenes , tryptophan is catabolized via indole-3-pyruvate, indole-3-lactate, and indole-3-acrylate to indole-3-propionate (O&#39;Neill and DeMoss, Tryptophan transaminase from  Clostridium sporogenes , Arch Biochem Biophys. 1968 Sep. 20; 127(1):361-9). Two enzymes that have been purified from  C. sporogenes  are tryptophan transaminase and indole-3-lactate dehydrogenase (Jean and DeMoss, Indolelactate dehydrogenase from  Clostridium sporogenes , Can J Microbiol. 1968 April; 14(4):429-35).  Lactococcus lactis , catabolizes tryptophan by an aminotransferase to indole-3-pyruvate. In  Lactobacillus casei  and  Lactobacillus helveticus  tryptophan is also catabolized to indole-3-lactate through successive transamination and dehydrogenation (see, e.g., Tryptophan catabolism by  Lactobacillus casei  and  Lactobacillus helveticus  cheese flavor adjuncts Gummalla, S., Broadbent, J. R. J. Dairy Sci 82:2070-2077, and references therein). 
     L-tryptophan transaminase (e.g., EC 2.6.1.27, e.g.,  Clostridium sporogenes  or  Lactobacillus casei ) converts L-tryptophan and 2-oxoglutarate to (indol-3yl)pyruvate and L-glutamate). Indole-3-lactate dehydrogenase (EC 1.1.1.110, e.g.,  Clostridium sporogenes  or  Lactobacillus casei ) converts (indol-3yl) pyruvate and NADH and H+ to indole-3 lactate and NAD+. 
     In some embodiments, the engineered bacteria comprises gene sequence(s) encoding one or more enzymes selected from tryptophan transaminase (e.g., from  C. sporogenes ) and/or indole-3-lactate dehydrogenase (e.g., from  C. sporogenes ), and/or indole-3-pyruvate aminotransferase (e.g., from  Lactococcus lactis ). In other embodiments, such enzymes encoded by the bacteria are from  Lactobacillus casei  and/or  Lactobacillus helveticus.    
     In other embodiments, IPA producing circuits comprise enzymes depicted and described in  FIG. 47  and  FIG. 48  and elsewhere herein. 
     In some embodiments, the bacteria comprise gene sequence for producing one or more tryptophan metabolites, e.g., “indoles”. In some embodiments, the bacteria comprise gene sequence for producing and indole selected from indole-3 aldehyde, indole-3 acetate, indole-3 propoinic acid, indole, indole-3 acetaladehyde, indole-3acetonitrile, FICZ. In some embodiments, the bacteria comprise gene sequence for producing an indole that functions as an AhR agonist, see e.g., Table 8. 
     In some embodiments, the bacteria comprise any one or more of the circuits described and depicted in the figures and examples. 
     Methoxyindole Pathway, Serotonin and Melatonin 
     The methoxyindole pathway leads to formation of serotonin (5-HT) and melatonin. Serotonin (5-hydroxytryptamine, 5-HT) is a biogenic amine synthesized in a two-step enzymatic reaction: First, enzymes encoded by one of two tryptophan hydroxylase genes (Tph1 or Tph2) catalyze the rate-limiting conversion of tryptophan to 5-hydroxytryptophan (5-HTP). Subsequently, 5-HTP undergoes decarboxylation to serotonin. 
     The majority (95%-98%) of total body serotonin is found in the gut (Berger et al., 2009). Peripheral serotonin acts autonomously on many cells, tissues, and organs, including the cardiovascular, gastrointestinal, hematopoietic, and immune systems as well as bone, liver, and placenta (Amireault et al., 2013). Serotonin functions as a ligand for any of 15 membrane-bound mostly G protein-coupled serotonin receptors (5-HTRs) that are involved in various signal transduction pathways in both CNS and periphery. Intestinal serotonin is released by enterochromaffin cells and neurons and is regulated via the serotonin re-uptake transporter (SERT). The SERT is located on epithelial cells and neurons in the intestine. Gut microbiota are interconnected with serotonin signaling and are for example capable of increasing serotonin levels through host serotonin production (Jano et al., Cell. 2015 Apr. 9; 161(2):264-76. doi: 10. 1016/j.cell.2015.02.047. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis). 
     Modulation of tryptophan metabolism, especially serotonin synthesis is considered a novel potential strategy the treatment of gastrointestinal (GI) disorders, including IBD. 
     In some embodiments, the engineered bacteria comprise gene sequence encoding one or more tryptophan hydroxylase genes (Tph1 or Tph2). In some embodiments, the engineered bacteria further comprise gene sequence for decarboxylating 5-HTP. In some embodiments, the engineered bacteria comprise gene sequence for the production of 5-hydroxytryptophan (5-HTP). In some embodiments, the engineered bacteria comprise gene sequence for the production of seratonin. 
     In certain embodiments, the genetically engineered bacteria described herein may modulate serotonin levels in the gut, e.g., decrease or increase serotonin levels, e.g, in the gut and in the circulation. In certain embodiments, the genetically engineered bacteria influence serotonin synthesis, release, and/or degradation. In some embodiments, the genetically engineered bacteria may modulate the serotonin levels in the gut to improve gut barrier function, modulate the inflammatory status, otherwise ameliorate symptoms of A gastrointestinal disorder or inflammatory disorder. In some embodiments, the genetically engineered bacteria take up serotonin from the environment, e.g., the gut. In some embodiments, the genetically engineered bacteria release serotonin into the environment, e.g., the gut. In some embodiments, the genetically engineered modulate or influence serotonin levels produced by the host. In some embodiments, the genetically engineered bacteria counteract microbiota which are responsible for altered serotonin function in many metabolic diseases. 
     In some embodiments, the genetically engineered bacteria comprise gene sequence encoding tryptophan hydroxylase (TpH (1 and/or 2)) and/or 1-amino acid decarboxylase, e.g. for the treatment of constipation-associated metabolic disorders. In some embodiments, the genetically engineered bacteria comprise genetic cassettes which allow trptophan uptake and catalysis, reducing trptophan availability for serotonin synthesis (serotonin depletion). In some embodiments, the genetically engineered bacteria comprise cassettes which promote serotonin uptake from the environment, e.g., the gut, and serotonin catalysis. 
     Additionally, serotonin also functions a substrate for melatonin biosynthesis. Melatonin acts as a neurohormone and is associated with the development of circadian rhythm and the sleep-wake cycle. 
     In bacteria, melatonin is synthesized indirectly with tryptophan as an intermediate product of the shikimic acid pathway. In these cells, synthesis starts with d-erythrose-4-phosphate and phosphoenolpyruvate. In some embodiments, the genetically engineered bacteria comprise an endogenous or exogenous cassette for the production of melatonin. As a non-limiting example, the cassette is described in Bochkov, Denis V.; Sysolyatin, Sergey V.; Kalashnikov, Alexander I.; Surmacheva, Irina A. (2011). “Shikimic acid: review of its analytical, isolation, and purification techniques from plant and microbial sources”. Journal of Chemical Biology 5 (1): 5-17. doi:10.1007/s12154-011-0064-8. 
     In a non-limiting example, genetically engineered bacteria convert tryptophan and/or serotonin to melatonin by, e.g., tryptophan hydroxylase (TPH), hydroxyl-O-methyltransferase (HIOMT), N-acetyltransferase (NAT), and aromatic-amino acid decarboxylase (AAAD), or equivalents thereof, e.g., bacterial equivalents. 
     Exemplary Tryptophan and Tryptophan Metabolite Circuits 
     Decreasing Exogenous Tryptophan 
     In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan and/or the level of a tryptophan metabolite. In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding one or more aromatic amino acid transporter(s). In one embodiment, the amino acid transporter is a tryptophan transporter. Tryptophan transporters may be expressed or modified in the recombinant bacteria described herein in order to enhance tryptophan transport into the cell. Specifically, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the genetically engineered bacteria comprising a heterologous gene encoding a tryptophan transporter which may be used to import tryptophan into the bacteria. 
     The uptake of tryptophan into bacterial cells is mediated by proteins well known to those of skill in the art. For example, three different tryptophan transporters, distinguishable on the basis of their affinity for tryptophan have been identified in  E. coli  (see, e.g., Yanofsky et al. (1991) J. Bacteriol. 173: 6009-17). The bacterial genes mtr, aroP, and tnaB encode tryptophan permeases responsible for tryptophan uptake in bacteria. High affinity permease, Mtr, is negatively regulated by the trp repressor and positively regulated by the TyR product (see, e.g., Yanofsky et al. (1991) J. Bacteriol. 173: 6009-17 and Heatwole, et al. (1991) J. Bacteriol. 173: 3601-04), while AroP is negatively regulated by the tyR product (Chye et al. (1987) J. Bacteriol. 169:386-93). 
     In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding one or more aromatic amino acid transporter(s). In one embodiment, the amino acid transporter is a tryptophan transporter. In one embodiment, the at least one gene encoding a tryptophan transporter is a gene selected from the group consisting of mtr, aroP and tnaB. In one embodiment, the bacterial cell described herein has been genetically engineered to comprise at least one heterologous gene selected from the group consisting of mtr, aroP and tnaB. In one embodiment, the at least one gene encoding a tryptophan transporter is the  Escherichia coli  mtr gene. In one embodiment, the at least one gene encoding a tryptophan transporter is the  Escherichia coli  aroP gene. In one embodiment, the at least one gene encoding a tryptophan transporter is the  Escherichia coli  tnaB gene. 
     In some embodiments, the tryptophan transporter is encoded by a tryptophan transporter gene derived from a bacterial genus or species, including but not limited to,  Escherichia, Corynebacterium, Escherichia coli, Saccharomyces cerevisiae  or  Corynebacterium glutamicum . In some embodiments, the bacterial species is  Escherichia coli . In some embodiments, the bacterial species is  Escherichia coli  strain Nissle. 
     Assays for testing the activity of a tryptophan transporter, a functional variant of a tryptophan transporter, or a functional fragment of transporter of tryptophan are well known to one of ordinary skill in the art. For example, import of tryptophan may be determined using the methods as described in Shang et al. (2013) J. Bacteriol. 195:5334-42, the entire contents of each of which are expressly incorporated by reference herein. 
     In one embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import 10% more tryptophan into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In another embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more tryptophan into the bacterial cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import two-fold more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, when the tryptophan transporter is expressed in the recombinant bacterial cells described herein, the bacterial cells import three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more tryptophan into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In addition to the tryptophan uptake transporters, in some embodiments, the genetically engineered bacteria further comprise a circuit for the production of tryptophan metabolites, as described herein, e.g., for the production of kynurenine, kynurenine metabolites, or indole tryptophan metabolites as shown in Table 8. 
     In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan. In some embodiments, the engineered bacteria comprises one or more gene sequences for converting tryptophan to kynurenine. In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1). In some embodiments, the engineered bacteria comprises gene sequence(s) for encoding the enzyme tryptophan dioxygenase (TDO). In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1) and the enzyme tryptophan dioxygenase (TDO). In some embodiments, the genetically engineered bacteria comprise a gene cassette encoding Indoleamine 2, 3 dioxygenase (EC 1.13.11.52; producing N-formyl kynurenine from tryptophan) and Kynurenine formamidase (EC3.5.1.9) producing kynurenine from n-formylkynurenine). In some embodiments, the enzymes are bacterially derived, e.g., as described in Vujkovi-Cvijin et al. 2013. 
     In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan, e.g., in combination with the production of indole metabolites, through expression of gene(s) and gene cassette(s) described herein. In some embodiments, expression of the gene sequences(s) is driven by an inducible promoter, described in more detail herein. In some embodiments, the expression of the gene sequences(s) is driven by a constitutive promoter. 
     Increasing Kynurenine 
     In some embodiments, the genetically engineered bacteria are capable of producing kynurenine. 
     In some embodiments, the genetically engineered bacteria are capable of decreasing the level of tryptophan. In some embodiments, the engineered bacteria comprise one or more gene sequences for converting tryptophan to kynurenine. In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1). In some embodiments, the engineered bacteria comprise gene sequence(s) for encoding the enzyme tryptophan dioxygenase (TDO). In some embodiments, the engineered bacteria comprise on or more gene sequence(s) for encoding the enzyme indoleamine 2,3-dioxygenase (IDO-1) and the enzyme tryptophan dioxygenase (TDO). In some embodiments, the genetically engineered bacteria comprise a gene cassette encoding Indoleamine 2, 3 dioxygenase (EC 1.13.11.52; producing N-formyl kynurenine from tryptophan) and Kynurenine formamidase (EC3.5.1.9) producing kynurenine from n-formylkynurenine). In some embodiments, the enzymes are bacterially derived, e.g., as described in Vujkovi-Cvijin et al. 2013. 
     In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce kynurenine from tryptophan. Non-limiting example of such gene sequence(s) are shown the figures and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDO1 (indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDO1 from  Homo sapiens . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TDO2 (tryptophan 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TDO2 from  Homo sapiens . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 (indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 from  S. cerevisiae ). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase from mouse. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with one or more of ido1 and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with ido1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 (kynurenine-oxoglutarate transaminase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 from  S. cerevisae . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with one or more of ido1 and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with ido1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of ido1 and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of afmid and/or bna3. 
     In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of ido1 and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3. 
     In any of these embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in the figures and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce kynurenine from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites. 
     The genetically engineered bacteria may comprise any suitable gene for producing kynurenine. In some embodiments, the gene for producing kynurenine is modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria also have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) for the consumption of tryptophan and production of kynurenine, which are bacterially derived. In some embodiments, the enzymes for TRP to KYN conversion are derived from one or more of  Pseudomonas, Xanthomonas, Burkholderia, Stenotrophomonas, Shewanella , and  Bacillus , and/or members of the families Rhodobacteraceae, Micrococcaceae, and Halomonadaceae, In some embodiments the enzymes are derived from the species listed in table S7 of Vujkovic-Cvijin et al. (Dysbiosis of the gut microbiota is associated with HIV disease progression and tryptophan catabolism Sci Transl Med. 2013 Jul. 10; 5(193): 193ra91), the contents of which is herein incorporated by reference in its entirety. 
     In some embodiments, the one or more genes for producing kynurenine are modified and/or mutated, e.g., to enhance stability, increase kynurenine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein. 
     In any of the embodiments described above and elsewhere herein, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression, liver damage, or metabolic disease, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits. 
     Increasing Tryptophan 
     In some embodiments, the genetically engineered microorganisms of the present disclosure are capable of producing tryptophan. Exemplary circuits for the production of tryptophan are shown in the figures. 
     In some embodiments, the genetically engineered bacteria that produce tryptophan comprise one or more gene sequences encoding one or more enzymes of the tryptophan biosynthetic pathway. In some embodiments, the genetically engineered bacteria comprise a tryptophan operon. In some embodiments, the genetically engineered bacteria comprise the tryptophan operon of  E. coli . (Yanofsky, RNA (2007), 13:1141-1154). In some embodiments, the genetically engineered bacteria comprise the tryptophan operon of  B. subtilis . (Yanofsky, RNA (2007), 13:1141-1154). In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes from  E. coli . In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes from  B. subtilis.    
     Also, in any of these embodiments, the genetically engineered bacteria optionally comprise gene sequence(s) to produce the tryptophan precursor, chorismate. Thus, in some embodiments, the genetically engineered bacteria optionally comprise sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding one or more enzymes of the tryptophan biosynthetic pathway and one or more gene sequences encoding one or more enzymes of the chorismate biosynthetic pathway. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes from  E. coli  and sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes from  B. subtilis  and sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes. 
     In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding either a wild type or a feedback resistant SerA gene (Table 10).  Escherichia coli  serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. This step is an oxidation of 3PG to 3-phosphohydroxypyruvate (3PHP) with the concomitant reduction of NAD+ to NADH. As part of Tryptophan biosynthesis,  E. coli  uses one serine for each tryptophan produced. As a result, by expressing serA, tryptophan production is improved. 
     In any of these embodiments, AroG and TrpE are optionally replaced with feedback resistant versions to improve tryptophan production (Table 10 
     In any of these embodiments, the tryptophan repressor (trpR) optionally may be deleted, mutated, or modified so as to diminish or obliterate its repressor function. 
     In any of these embodiments the tnaA gene (encoding a tryptophanase converting Trp into indole) optionally may be deleted to prevent tryptophan catabolism along this pathway and to further increase levels of tryptophan produced (Table 10. 
     The inner membrane protein YddG of  Escherichia coli , encoded by the yddG gene, is a homologue of the known amino acid exporters RhtA and YdeD. Studies have shown that YddG is capable of exporting aromatic amino acids, including tryptophan. Thus, YddG c an function as a tryptophan exporter or a tryptophan secretion system (or tryptophan secretion protein). Other aromatic amino acid exporters are described in Doroshenko et al., FEMS Microbial Lett., 275:312-318 (2007). Thus, in some embodiments, the engineered bacteria optionally further comprise gene sequence(s) encoding YddG. In some embodiments, the engineered bacteria can over-express YddG. In some embodiments, the engineered bacteria optionally comprise one or more copies of yddG gene. 
     In some embodiments, the genetically engineered bacterium or genetically engineered microorganism comprises one or more genes for producing tryptophan, under the control of a promoter that is activated by low-oxygen conditions, by inflammatory conditions, liver damage, and or metabolic disease, such as any of the promoters activated by said conditions and described herein. In some embodiments, the genetically engineered bacteria expresses one or more genes for producing tryptophan. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. 
     Table 9A and 9B lists exemplary tryptophan synthesis cassettes encoded by the genetically engineered bacteria of the disclosure. 
     
       
         
           
               
             
               
                 TABLE 9A 
               
             
            
               
                   
               
               
                 Tryptophan Synthesis Cassette Sequences 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 Tet-regulated 
                 taagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaaggccgaataagaaggctggctct 
               
               
                 Tryptophan 
                 gcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcatactatcagtagtaggtgtttccctttct 
               
               
                 operon 
                 tctttagcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgccccacagcgctgagtgcatata 
               
               
                 SEQ ID NO: 
                 atgcattctctagtgaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcatactgtttttctgtagg 
               
               
                 71 
                 ccgtgtacctaaatgtacttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaa 
               
               
                   
                 aaaatcttgccagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcg 
               
               
                   
                 tcgagcaaagcccgcttattttttacatgccaatacaatgtaggctgctctacacctagcttctgggcgagtttacg 
               
               
                   
                 ggttgttaaaccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctagaca 
               
               
                   
                 tcattaattcctaatttttgttgacactctatcattgatagagttattttaccactccctatcagtgatagagaaaagtg 
               
               
                   
                 aactctagaaataattttgtttaactttaagaaggagatatacatatgcaaacacaaaaaccgactctcgaactgct 
               
               
                   
                 aacctgcgaaggcgcttatcgcgacaacccgactgcgctttttcaccagttgtgtggggatcgtccggcaacg 
               
               
                   
                 ctgctgctggaatccgcagatatcgacagcaaagatgatttaaaaagcctgctgctggtagacagtgcgctgc 
               
               
                   
                 gcattacagcattaagtgacactgtcacaatccaggcgctttccggcaatggagaagccctgttgacactactg 
               
               
                   
                 gataacgccttgcctgcgggtgtggaaaatgaacaatcaccaaactgccgcgtactgcgcttcccgcctgtca 
               
               
                   
                 gtccactgctggatgaagacgcccgcttatgctccctttcggtttttgacgctttccgcttattacagaatctgttga 
               
               
                   
                 atgtaccgaaggaagaacgagaagcaatgttcttcggcggcctgttctcttatgaccttgtggcgggatttgaaa 
               
               
                   
                 atttaccgcaactgtcagcggaaaatagctgccctgatttctgtttttatctcgctgaaacgctgatggtgattgac 
               
               
                   
                 catcagaaaaaaagcactcgtattcaggccagcctgtttgctccgaatgaagaagaaaaacaacgtctcactgc 
               
               
                   
                 tcgcctgaacgaactacgtcagcaactgaccgaagccgcgccgccgctgccggtggtttccgtgccgcatat 
               
               
                   
                 gcgttgtgaatgtaaccagagcgatgaagagttcggtggtgtagtgcgtttgttgcaaaaagcgattcgcgccg 
               
               
                   
                 gagaaattttccaggtggtgccatctcgccgtttctctctgccctgcccgtcaccgctggcagcctattacgtgct 
               
               
                   
                 gaaaaagagtaatcccagcccgtacatgttttttatgcaggataatgatttcaccctgtttggcgcgtcgccggaa 
               
               
                   
                 agttcgctcaagtatgacgccaccagccgccagattgagatttacccgattgccggaacacgtccacgcggtc 
               
               
                   
                 gtcgtgccgatggttcgctggacagagacctcgacagccgcatcgaactggagatgcgtaccgatcataaag 
               
               
                   
                 agctttctgaacatctgatgctggtggatctcgcccgtaatgacctggcacgcatttgcacacccggcagccgc 
               
               
                   
                 tacgtcgccgatctcaccaaagttgaccgttactcttacgtgatgcacctagtctcccgcgttgttggtgagctgc 
               
               
                   
                 gccacgatctcgacgccctgcacgcttaccgcgcctgtatgaatatggggacgttaagcggtgcaccgaaagt 
               
               
                   
                 acgcgctatgcagttaattgccgaagcagaaggtcgtcgacgcggcagctacggcggcgcggtaggttatttt 
               
               
                   
                 accgcgcatggcgatctcgacacctgcattgtgatccgctcggcgctggtggaaaacggtatcgccaccgtgc 
               
               
                   
                 aagccggtgctggcgtagtccttgattctgttccgcagtcggaagccgacgaaactcgtaataaagcccgcgc 
               
               
                   
                 tgtactgcgcgctattgccaccgcgcatcatgcacaggagacgttctaatggctgacattctgctgctcgataat 
               
               
                   
                 atcgactcttttacgtacaacctggcagatcagttgcgcagcaatggtcataacgtggtgatttaccgcaaccata 
               
               
                   
                 ttccggcgcagaccttaattgaacgcctggcgacgatgagcaatccggtgctgatgctttctcctggccccggt 
               
               
                   
                 gtgccgagcgaagccggttgtatgccggaactcctcacccgcttgcgtggcaagctgccaattattggcatttg 
               
               
                   
                 cctcggacatcaggcgattgtcgaagcttacgggggctatgtcggtcaggcgggcgaaattcttcacggtaaa 
               
               
                   
                 gcgtcgagcattgaacatgacggtcaggcgatgtttgccggattaacaaacccgctgccagtggcgcgttatc 
               
               
                   
                 actcgctggttggcagtaacattccggccggtttaaccatcaacgcccattttaatggcatggtgatggcggtgc 
               
               
                   
                 gtcacgatgcagatcgcgtttgtggattccagttccatccggaatccattcttactacccagggcgctcgcctgct 
               
               
                   
                 ggaacaaacgctggcctgggcgcagcagaaactagagccaaccaacacgctgcaaccgattctggaaaaa 
               
               
                   
                 ctgtatcaggcacagacgcttagccaacaagaaagccaccagctgttttcagcggtggtacgtggcgagctga 
               
               
                   
                 agccggaacaactggcggcggcgctggtgagcatgaaaattcgcggtgaacacccgaacgagatcgccgg 
               
               
                   
                 ggcagcaaccgcgctactggaaaacgccgcgccattcccgcgcccggattatctgtttgccgatatcgtcggt 
               
               
                   
                 actggcggtgacggcagcaacagcatcaatatttctaccgccagtgcgtttgtcgccgcggcctgcgggctga 
               
               
                   
                 aagtggcgaaacacggcaaccgtagcgtctccagtaaatccggctcgtcggatctgctggcggcgttcggtat 
               
               
                   
                 taatcttgatatgaacgccgataaatcgcgccaggcgctggatgagttaggcgtctgtttcctctttgcgccgaa 
               
               
                   
                 gtatcacaccggattccgccatgcgatgccggttcgccagcaactgaaaacccgcactctgttcaacgtgctg 
               
               
                   
                 ggaccattgattaacccggcgcatccgccgctggcgctaattggtgtttatagtccggaactggtgctgccgatt 
               
               
                   
                 gccgaaaccttgcgcgtgctggggtatcaacgcgcggcagtggtgcacagcggcgggatggatgaagtttc 
               
               
                   
                 attacacgcgccgacaatcgttgccgaactacatgacggcgaaattaagagctatcaattgaccgctgaagatt 
               
               
                   
                 ttggcctgacaccctaccaccaggagcaattggcaggcggaacaccggaagaaaaccgtgacattttaacac 
               
               
                   
                 gcttgttacaaggtaaaggcgacgccgcccatgaagcagccgtcgcggcgaatgtcgccatgttaatgcgcct 
               
               
                   
                 gcatggccatgaagatctgcaagccaatgcgcaaaccgttcttgaggtactgcgcagtggttccgcttacgaca 
               
               
                   
                 gagtcaccgcactggcggcacgagggtaaatgatgcaaaccgttttagcgaaaatcgtcgcagacaaggcg 
               
               
                   
                 atttgggtagaaacccgcaaagagcagcaaccgctggccagttttcagaatgaggttcagccgagcacgcga 
               
               
                   
                 catttttatgatgcacttcagggcgcacgcacggcgtttattctggagtgtaaaaaagcgtcgccgtcaaaaggc 
               
               
                   
                 gtgatccgtgatgatttcgatccggcacgcattgccgccatttataaacattacgcttcggcaatttcagtgctgac 
               
               
                   
                 tgatgagaaatattttcaggggagctttgatttcctccccatcgtcagccaaatcgccccgcagccgattttatgta 
               
               
                   
                 aagacttcattatcgatccttaccagatctatctggcgcgctattaccaggccgatgcctgcttattaatgctttcag 
               
               
                   
                 tactggatgacgaacaatatcgccagcttgcagccgtcgcccacagtctggagatgggtgtgctgaccgaagt 
               
               
                   
                 cagtaatgaagaggaactggagcgcgccattgcattgggggcaaaggtcgttggcatcaacaaccgcgatct 
               
               
                   
                 gcgcgatttgtcgattgatctcaaccgtacccgcgagcttgcgccgaaactggggcacaacgtgacggtaatc 
               
               
                   
                 agcgaatccggcatcaatacttacgctcaggtgcgcgagttaagccacttcgctaacggctttctgattggttcg 
               
               
                   
                 gcgttgatggcccatgacgatttgaacgccgccgtgcgtcgggtgttgctgggtgagaataaagtatgtggcct 
               
               
                   
                 gacacgtgggcaagatgctaaagcagcttatgacgcgggcgcgatttacggtgggttgatttttgttgcgacat 
               
               
                   
                 caccgcgttgcgtcaacgttgaacaggcgcaggaagtgatggctgcagcaccgttgcagtatgttggcgtgtt 
               
               
                   
                 ccgcaatcacgatattgccgatgtggcggacaaagctaaggtgttatcgctggcggcagtgcaactgcatggt 
               
               
                   
                 aatgaagatcagctgtatatcgacaatctgcgtgaggctctgccagcacacgtcgccatctggaaggctttaag 
               
               
                   
                 tgtcggtgaaactcttcccgcgcgcgattttcagcacatcgataaatatgtattcgacaacggtcagggcggga 
               
               
                   
                 gcggacaacgtttcgactggtcactattaaatggtcaatcgcttggcaacgttctgctggcggggggcttaggc 
               
               
                   
                 gcagataactgcgtggaagcggcacaaaccggctgcgccgggcttgattttaattctgctgtagagtcgcaac 
               
               
                   
                 cgggtatcaaagacgcacgtcttttggcctcggttttccagacgctgcgcgcatattaaggaaaggaacaatga 
               
               
                   
                 caacattacttaacccctattttggtgagtttggcggcatgtacgtgccacaaatcctgatgcctgctctgcgcca 
               
               
                   
                 gctggaagaagcttttgtcagcgcgcaaaaagatcctgaatttcaggctcagttcaacgacctgctgaaaaact 
               
               
                   
                 atgccgggcgtccaaccgcgctgaccaaatgccagaacattacagccgggacgaacaccacgctgtatctga 
               
               
                   
                 agcgcgaagatttgctgcacggcggcgcgcataaaactaaccaggtgctcggtcaggctttactggcgaagc 
               
               
                   
                 ggatgggtaaaactgaaattattgccgaaaccggtgccggtcagcatggcgtggcgtcggcccttgccagcg 
               
               
                   
                 ccctgctcggcctgaaatgccgaatttatatgggtgccaaagacgttgaacgccagtcgcccaacgttttccgg 
               
               
                   
                 atgcgcttaatgggtcggaagtgatcccggtacatagcggttccgcgaccctgaaagatgcctgtaatgagg 
               
               
                   
                 cgctacgcgactggtccggcagttatgaaaccgcgcactatatgctgggtaccgcagctggcccgcatcctta 
               
               
                   
                 cccgaccattgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgcagattctggaaagagaagg 
               
               
                   
                 tcgcctgccggatgccgttatcgcctgtgttggcggtggttcgaatgccatcggtatgtttgcagatttcatcaac 
               
               
                   
                 gaaaccgacgtcggcctgattggtgtggagcctggcggccacggtatcgaaactggcgagcacggcgcacc 
               
               
                   
                 gttaaaacatggtcgcgtgggcatctatttcggtatgaaagcgccgatgatgcaaaccgaagacgggcaaatt 
               
               
                   
                 gaagagtcttactccatttctgccgggctggatttcccgtccgtcggcccgcaacatgcgtatctcaacagcact 
               
               
                   
                 ggacgcgctgattacgtgtctattaccgacgatgaagccctggaagcctttaaaacgctttgcctgcatgaagg 
               
               
                   
                 gatcatcccggcgctggaatcctcccacgccctggcccatgcgctgaaaatgatgcgcgaaaatccggaaaa 
               
               
                   
                 agagcagctactggtggttaacctttccggtcgcggcgataaagacatcttcaccgttcacgatattttgaaagc 
               
               
                   
                 acgaggggaaatctgatggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattc 
               
               
                   
                 gttcctttcgtcaccctcggtgatccgggcattgagcagtcgttgaaaattatcgatacgctaattgaagccggtg 
               
               
                   
                 ctgacgcgctggagttaggcatccccttctccgacccactggcggatggcccgacgattcaaaacgccacact 
               
               
                   
                 gcgtgcttttgcggcgggagtaaccccggcgcagtgctttgagatgctggcactcattcgccagaagcacccg 
               
               
                   
                 accattcccatcggccttttgatgtatgccaacctggtgtttaacaaaggcattgatgagttttatgccgagtgcga 
               
               
                   
                 gaaagtcggcgtcgattcggtgctggttgccgatgtgcccgtggaagagtccgcgcccttccgccaggccgc 
               
               
                   
                 gttgcgtcataatgtcgcacctatctttatttgcccgccgaatgccgacgatgatttgctgcgccagatagcctctt 
               
               
                   
                 acggtcgtggttacacctatttgctgtcgcgagcgggcgtgaccggcgcagaaaaccgcgccgcgttacccc 
               
               
                   
                 tcaatcatctggttgcgaagctgaaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccgg 
               
               
                   
                 atcaggtaaaagccgcgattgatgcaggagctgcgggcgcgatttctggttcggccatcgttaaaatcatcgag 
               
               
                   
                 caacatattaatgagccagagaaaatgctggcggcactgaaagcttttgtacaaccgatgaaagcggcgacgc 
               
               
                   
                 gcagttaatacgcatggcatggatgaCCGATGGTAGTGTGGGGTCTCCCCATGCG 
               
               
                   
                 AGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGT 
               
               
                   
                 CGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGC 
               
               
                   
                 TCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGC 
               
               
                   
                 GAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAA 
               
               
                   
                 CTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCC 
               
               
                   
                 TTTTTGCGTGGCCAGTGCCAAGCTTGCATGCGTGC 
               
               
                   
               
               
                 trpE 
                 atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaacccgactgcgctttt 
               
               
                 SEQ ID NO: 
                 tcaccagttgtgtggggatcgtccggcaacgctgctgctggaatccgcagatatcgacagcaaagatgatttaa 
               
               
                 74 
                 aaagcctgctgaggtagacagtgcgctgcgcattacagcattaagtgacactgtcacaatccaggcgctttcc 
               
               
                   
                 ggcaatggagaagccctgttgacactactggataacgccttgcctgcgggtgtggaaaatgaacaatcaccaa 
               
               
                   
                 actgccgcgtactgcgcttcccgcctgtcagtccactgctggatgaagacgcccgcttatgctccctttcggtttt 
               
               
                   
                 tgacgattccgcttattacagaatctgttgaatgtaccgaaggaagaacgagaagcaatgttcggcggcct 
               
               
                   
                 gttctcttatgaccttgtggcgggatttgaaaatttaccgcaactgtcagcggaaaatagctgccctgatttctgttt 
               
               
                   
                 ttatctcgctgaaacgctgatggtgattgaccatcagaaaaaaagcactcgtattcaggccagcctgtttgctcc 
               
               
                   
                 gaatgaagaagaaaaacaacgtctcactgctcgcctgaacgaactacgtcagcaactgaccgaagccgcgc 
               
               
                   
                 cgccgctgccggtggtttccgtgccgcatatgcgttgtgaatgtaaccagagcgatgaagagttcggtggtgta 
               
               
                   
                 gtgcgtttgttgcaaaaagcgattcgcgccggagaaattttccaggtggtgccatctcgccgtttctctctgccct 
               
               
                   
                 gcccgtcaccgctggcagcctattacgtgctgaaaaagagtaatcccagcccgtacatgttttttatgcaggata 
               
               
                   
                 atgatttcaccctgtttggcgcgtcgccggaaagttcgctcaagtatgacgccaccagccgccagattgagattt 
               
               
                   
                 acccgattgccggaacacgtccacgcggtcgtcgtgccgatggttcgctggacagagacctcgacagccgc 
               
               
                   
                 atcgaactggagatgcgtaccgatcataaagagctttctgaacatctgatgctggtggatctcgcccgtaatgac 
               
               
                   
                 ctggcacgcatttgcacacccggcagccgctacgtcgccgatctcaccaaagttgaccgttactcttacgtgat 
               
               
                   
                 gcacctagtctcccgcgttgttggtgagctgcgccacgatctcgacgccctgcacgcttaccgcgcctgtatga 
               
               
                   
                 atatggggacgttaagcggtgcaccgaaagtacgcgctatgcagttaattgccgaagcagaaggtcgtcgac 
               
               
                   
                 gcggcagctacggcggcgcggtaggttattttaccgcgcatggcgatctcgacacctgcattgtgatccgctc 
               
               
                   
                 ggcgctggtggaaaacggtatcgccaccgtgcaagccggtgctggcgtagtccttgattctgttccgcagtcg 
               
               
                   
                 gaagccgacgaaactcgtaataaagcccgcgctgtactgcgcgctattgccaccgcgcatcatgcacaggag 
               
               
                   
                 acgttcta 
               
               
                   
               
               
                 trpD 
                 atggctgacattctgctgctcgataatatcgactcttttacgtacaacctggcagatcagttgcgcagcaatggtc 
               
               
                 SEQ ID NO: 
                 ataacgtggtgatttaccgcaaccatattccggcgcagaccttaattgaacgcctggcgacgatgagcaatccg 
               
               
                 76 
                 gtgctgatgctttctcctggccccggtgtgccgagcgaagccggttgtatgccggaactcctcacccgcttgcg 
               
               
                   
                 tggcaagctgccaattattggcatttgcctcggacatcaggcgattgtcgaagcttacgggggctatgtcggtca 
               
               
                   
                 ggcgggcgaaattcttcacggtaaagcgtcgagcattgaacatgacggtcaggcgatgtttgccggattaaca 
               
               
                   
                 aacccgctgccagtggcgcgttatcactcgctggttggcagtaacattccggccggtttaaccatcaacgccca 
               
               
                   
                 ttttaatggcatggtgatggcggtgcgtcacgatgcagatcgcgtttgtggattccagttccatccggaatccatt 
               
               
                   
                 cttactacccagggcgctcgcctgctggaacaaacgctggcctgggcgcagcagaaactagagccaaccaa 
               
               
                   
                 cacgctgcaaccgattctggaaaaactgtatcaggcacagacgcttagccaacaagaaagccaccagctgttt 
               
               
                   
                 tcagcggtggtacgtggcgagctgaagccggaacaactggcggcggcgctggtgagcatgaaaattcgcgg 
               
               
                   
                 tgaacacccgaacgagatcgccggggcagcaaccgcgctactggaaaacgccgcgccattcccgcgcccg 
               
               
                   
                 gattatctgtttgccgatatcgtcggtactggcggtgacggcagcaacagcatcaatatttctaccgccagtgcg 
               
               
                   
                 tttgtcgccgcggcctgcgggctgaaagtggcgaaacacggcaaccgtagcgtctccagtaaatccggctcg 
               
               
                   
                 tcggatctgctggcggcgttcggtattaatcttgatatgaacgccgataaatcgcgccaggcgctggatgagtta 
               
               
                   
                 ggcgtctgtttcctctttgcgccgaagtatcacaccggattccgccatgcgatgccggttcgccagcaactgaa 
               
               
                   
                 aacccgcactctgttcaacgtgctgggaccattgattaacccggcgcatccgccgctggcgctaattggtgata 
               
               
                   
                 tagtccggaactggtgctgccgattgccgaaaccttgcgcgtgctggggtatcaacgcgcggcagtggtgca 
               
               
                   
                 cagcggcgggatggatgaagtttcattacacgcgccgacaatcgttgccgaactacatgacggcgaaattaag 
               
               
                   
                 agctatcaattgaccgctgaagattttggcctgacaccctaccaccaggagcaattggcaggcggaacaccgg 
               
               
                   
                 aagaaaaccgtgacattttaacacgcttgttacaaggtaaaggcgacgccgcccatgaagcagccgtcgcgg 
               
               
                   
                 cgaatgtcgccatgttaatgcgcctgcatggccatgaagatctgcaagccaatgcgcaaaccgttcttgaggta 
               
               
                   
                 ctgcgcagtggttccgcttacgacagagtcaccgcactggcggcacgagggtaa 
               
               
                   
               
               
                 trpC 
                 atgcaaaccgttttagcgaaaatcgtcgcagacaaggcgatttgggtagaaacccgcaaagagcagcaaccg 
               
               
                 SEQ ID NO: 
                 ctggccagttttcagaatgaggttcagccgagcacgcgacatttttatgatgcacttcagggcgcacgcacggc 
               
               
                 78 
                 gtttattctggagtgtaaaaaagcgtcgccgtcaaaaggcgtgatccgtgatgatttcgatccggcacgcattgc 
               
               
                   
                 cgccatttataaacattacgcttcggcaatttcagtgctgactgatgagaaatattttcaggggagctttgatttcct 
               
               
                   
                 ccccatcgtcagccaaatcgccccgcagccgattttatgtaaagacttcattatcgatccttaccagatctatctg 
               
               
                   
                 gcgcgctattaccaggccgatgcctgcttattaatgctttcagtactggatgacgaacaatatcgccagcttgca 
               
               
                   
                 gccgtcgcccacagtctggagatgggtgtgctgaccgaagtcagtaatgaagaggaactggagcgcgccatt 
               
               
                   
                 gcattgggggcaaaggtcgttggcatcaacaaccgcgatctgcgcgatttgtcgattgatctcaaccgtacccg 
               
               
                   
                 cgagcttgcgccgaaactggggcacaacgtgacggtaatcagcgaatccggcatcaatacttacgctcaggt 
               
               
                   
                 gcgcgagttaagccacttcgctaacggctttctgattggttcggcgttgatggcccatgacgatttgaacgccgc 
               
               
                   
                 cgtgcgtcgggtgttgctgggtgagaataaagtatgtggcctgacacgtgggcaagatgctaaagcagcttat 
               
               
                   
                 gacgcgggcgcgatttacggtgggttgatttttgttgcgacatcaccgcgttgcgtcaacgttgaacaggcgca 
               
               
                   
                 ggaagtgatggctgcagcaccgttgcagtatgttggcgtgttccgcaatcacgatattgccgatgtggcggaca 
               
               
                   
                 aagctaaggtgttatcgctggcggcagtgcaactgcatggtaatgaagatcagctgtatatcgacaatctgcgt 
               
               
                   
                 gaggctctgccagcacacgtcgccatctggaaggctttaagtgtcggtgaaactcttcccgcgcgcgattttca 
               
               
                   
                 gcacatcgataaatatgtattcgacaacggtcagggcgggagcggacaacgtttcgactggtcactattaaatg 
               
               
                   
                 gtcaatcgcttggcaacgttctgctggcggggggcttaggcgcagataactgcgtggaagcggcacaaaccg 
               
               
                   
                 gctgcgccgggcttgattttaattctgctgtagagtcgcaaccgggtatcaaagacgcacgtcttttggcctcggt 
               
               
                   
                 tttccagacgctgcgcgcatattaa 
               
               
                   
               
               
                 trpB 
                 atgacaacattacttaacccctattttggtgagtttggcggcatgtacgtgccacaaatcctgatgcctgctctgcg 
               
               
                 SEQ ID NO: 
                 ccagctggaagaagcttttgtcagcgcgcaaaaagatcctgaatttcaggctcagttcaacgacctgctgaaaa 
               
               
                 80 
                 actatgccgggcgtccaaccgcgctgaccaaatgccagaacattacagccgggacgaacaccacgctgtatc 
               
               
                   
                 tgaagcgcgaagatttgctgcacggcggcgcgcataaaactaaccaggtgctcggtcaggctttactggcga 
               
               
                   
                 agcggatgggtaaaactgaaattattgccgaaaccggtgccggtcagcatggcgtggcgtcggcccttgcca 
               
               
                   
                 gcgccctgctcggcctgaaatgccgaatttatatgggtgccaaagacgttgaacgccagtcgcccaacgttttc 
               
               
                   
                 cggatgcgcttaatgggtgcggaagtgatcccggtacatagcggttccgcgaccctgaaagatgcctgtaatg 
               
               
                   
                 aggcgctacgcgactggtccggcagttatgaaaccgcgcactatatgctgggtaccgcagctggcccgcatc 
               
               
                   
                 cttacccgaccattgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgcagattctggaaagaga 
               
               
                   
                 aggtcgcctgccggatgccgttatcgcctgtgttggcggtggttcgaatgccatcggtatgtttgcagatttcatc 
               
               
                   
                 aacgaaaccgacgtcggcctgattggtgtggagcctggcggccacggtatcgaaactggcgagcacggcgc 
               
               
                   
                 accgttaaaacatggtcgcgtgggcatctatttcggtatgaaagcgccgatgatgcaaaccgaagacgggcaa 
               
               
                   
                 attgaagagtcttactccatttctgccgggctggatttcccgtccgtcggcccgcaacatgcgtatctcaacagc 
               
               
                   
                 actggacgcgctgattacgtgtctattaccgacgatgaagccctggaagcctttaaaacgctttgcctgcatgaa 
               
               
                   
                 gggatcatcccggcgctggaatcctcccacgccctggcccatgcgctgaaaatgatgcgcgaaaatccggaa 
               
               
                   
                 aaagagcagctactggtggttaacctttccggtcgcggcgataaagacatcttcaccgttcacgatattttgaaa 
               
               
                   
                 gcacgaggggaaatctga 
               
               
                   
               
               
                 trpA 
                 atggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgttcctttcgtcaccctc 
               
               
                 SEQ ID NO: 
                 ggtgatccgggcattgagcagtcgttgaaaattatcgatacgctaattgaagccggtgctgacgcgctggagtt 
               
               
                 82 
                 aggcatccccttctccgacccactggcggatggcccgacgattcaaaacgccacactgcgtgcttttgcggcg 
               
               
                   
                 ggagtaaccccggcgcagtgctttgagatgctggcactcattcgccagaagcacccgaccattcccatcggcc 
               
               
                   
                 ttttgatgtatgccaacctggtgtttaacaaaggcattgatgagttttatgccgagtgcgagaaagtcggcgtcga 
               
               
                   
                 ttcggtgctggttgccgatgtgcccgtggaagagtccgcgcccttccgccaggccgcgttgcgtcataatgtcg 
               
               
                   
                 cacctatctttatttgcccgccgaatgccgacgatgatttgctgcgccagatagcctcttacggtcgtggttacac 
               
               
                   
                 ctatttgctgtcgcgagcgggcgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcatctggttgcg 
               
               
                   
                 aagctgaaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccggatcaggtaaaagccg 
               
               
                   
                 cgattgatgcaggagctgcgggcgcgatttctggttcggccatcgttaaaatcatcgagcaacatattaatgagc 
               
               
                   
                 cagagaaaatgctggcggcactgaaagcttttgtacaaccgatgaaagcggcgacgcgcagttaa 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9B 
               
             
            
               
                   
               
               
                 Tryptophan Synthesis Polypeptide Sequences 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 TrpE 
                 MQTQKPTLELLTCEGAYRDNPTALFHQLCGDRPATLL 
               
               
                 SEQ ID NO: 75 
                 LESADIDSKDDLKSLLLVDSALRITALSDTVTIQALSGN 
               
               
                   
                 GEALLTLLDNALPAGVENEQSPNCRVLRFPPVSPLLDE 
               
               
                   
                 DARLCSLSVFDAFRLLQNLLNVPKEEREAMFFGGLFS 
               
               
                   
                 YDLVAGFENLPQLSAENSCPDFCFYLAETLMVIDHQK 
               
               
                   
                 KSTRIQASLFAPNEEEKQRLTARLNELRQQLTEAAPPL 
               
               
                   
                 PVVSVPHMRCECNQSDEEFGGVVRLLQKAIRAGEIFQ 
               
               
                   
                 VVPSRRFSLPCPSPLAAYYVLKKSNPSPYMFFMQDND 
               
               
                   
                 FTLFGASPESSLKYDATSRQIEIYPIAGTRPRGRRADGS 
               
               
                   
                 LDRDLDSRIELEMRTDHKELSEHLMLVDLARNDLARI 
               
               
                   
                 CTPGSRYVADLTKVDRYSYVMHLVSRVVGELRHDLD 
               
               
                   
                 ALHAYRACMNMGTLSGAPKVRAMQLIAEAEGRRRGS 
               
               
                   
                 YGGAVGYFTAHGDLDTCIVIRSALVENGIATVQAGAG 
               
               
                   
                 VVLDSVPQSEADETRNKARAVLRAIATAHHAQETF 
               
               
                   
               
               
                 TrpD 
                 MADILLLDNIDSFTYNLADQLRSNGHNVVIYRNHIPAQ 
               
               
                 SEQ ID NO: 77 
                 TLIERLATMSNPVLMLSPGPGVPSEAGCMPELLTRLRG 
               
               
                   
                 KLPIIGICLGHQAIVEAYGGYVGQAGEILHGKASSIEHD 
               
               
                   
                 GQAMFAGLTNPLPVARYHSLVGSNIPAGLTINAHFNG 
               
               
                   
                 MVMAVRHDADRVCGFQFHPESILTTQGARLLEQTLA 
               
               
                   
                 WAQQKLEPTNTLQPILEKLYQAQTLSQQESHQLFSAV 
               
               
                   
                 VRGELKPEQLAAALVSMKIRGEHPNEIAGAATALLEN 
               
               
                   
                 AAPFPRPDYLFADIVGTGGDGSNSINISTASAFVAAAC 
               
               
                   
                 GLKVAKHGNRSVSSKSGSSDLLAAFGINLDMNADKSR 
               
               
                   
                 QALDELGVCFLFAPKYHTGFRHAMPVRQQLKTRTLF 
               
               
                   
                 NVLGPLINPAHPPLALIGVYSPELVLPIAETLRVLGYQR 
               
               
                   
                 AAVVHSGGMDEVSLHAPTIVAELHDGEIKSYQLTAED 
               
               
                   
                 FGLTPYHQEQLAGGTPEENRDILTRLLQGKGDAAHEA 
               
               
                   
                 AVAANVAMLMRLHGHEDLQANAQTVLEVLRSGSAY 
               
               
                   
                 DRVTALAARG 
               
               
                   
               
               
                 TrpC 
                 MQTVLAKIVADKAIWVETRKEQQPLASFQNEVQPSTR 
               
               
                 SEQ ID NO: 79 
                 HFYDALQGARTAFILECKKASPSKGVIRDDFDPARIAA 
               
               
                   
                 IYKHYASAISVLTDEKYFQGSFDFLPIVSQIAPQPILCK 
               
               
                   
                 DFIIDPYQIYLARYYQADACLLMLSVLDDEQYRQLAA 
               
               
                   
                 VAHSLEMGVLTEVSNEEELERAIALGAKVVGINNRDL 
               
               
                   
                 RDLSIDLNRTRELAPKLGHNVTVISESGINTYAQVREL 
               
               
                   
                 SHFANGFLIGSALMAHDDLNAAVRRVLLGENKVCGL 
               
               
                   
                 TRGQDAKAAYDAGAIYGGLIFVATSPRCVNVEQAQE 
               
               
                   
                 VMAAAPLQYVGVFRNHDIADVADKAKVLSLAAVQL 
               
               
                   
                 HGNEDQLYIDNLREALPAHVAIWKALSVGETLPARDF 
               
               
                   
                 QHIDKYVFDNGQGGSGQRFDWSLLNGQSLGNVLLAG 
               
               
                   
                 GLGADNCVEAAQTGCAGLDFNSAVESQPGIKDARLL 
               
               
                   
                 ASVFQTLRAY 
               
               
                   
               
               
                 TrpB 
                 MTTLLNPYFGEFGGMYVPQILMPALRQLEEAFVSAQK 
               
               
                 SEQ ID NO: 81 
                 DPEFQAQFNDLLKNYAGRPTALTKCQNITAGTNTTLY 
               
               
                   
                 LKREDLLHGGAHKTNQVLGQALLAKRMGKTEIIAET 
               
               
                   
                 GAGQHGVASALASALLGLKCRIYMGAKDVERQSPNV 
               
               
                   
                 FRMRLMGAEVIPVHSGSATLKDACNEALRDWSGSYE 
               
               
                   
                 TAHYMLGTAAGPHPYPTIVREFQRMIGEETKAQILERE 
               
               
                   
                 GRLPDAVIACVGGGSNAIGMFADFINETDVGLIGVEPG 
               
               
                   
                 GHGIETGEHGAPLKHGRVGIYFGMKAPMMQTEDGQI 
               
               
                   
                 EESYSISAGLDFPSVGPQHAYLNSTGRADYVSITDDEA 
               
               
                   
                 LEAFKTLCLHEGIIPALESSHALAHALKMMRENPEKEQ 
               
               
                   
                 LLVVNLSGRGDKDIFTVHDILKARGEI 
               
               
                   
               
               
                 TrpA 
                 MERYESLFAQLKERKEGAFVPFVTLGDPGIEQSLKIID 
               
               
                 SEQ ID NO: 83 
                 TLIEAGADALELGIPFSDPLADGPTIQNATLRAFAAGV 
               
               
                   
                 TPAQCFEMLALIRQKHPTIPIGLLMYANLVFNKGIDEF 
               
               
                   
                 YAECEKVGVDSVLVADVPVEESAPFRQAALRHNVAPI 
               
               
                   
                 FICPPNADDDLLRQIASYGRGYTYLLSRAGVTGAENR 
               
               
                   
                 AALPLNHLVAKLKEYNAAPPLQGFGISAPDQVKAAID 
               
               
                   
                 AGAAGAISGSAIVKIIEQHINEPEKMLAALKAFVQPMK 
               
               
                   
                 AATRS 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 9A or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 9B or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence of Table 9A or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as one or more nucleic acid sequence of Table 9B or a functional fragment thereof. 
     In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 71 through SEQ ID NO: 83. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 71 through SEQ ID NO: 83. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 71 through SEQ ID NO: 83. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 71 through SEQ ID NO: 83. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 71 through SEQ ID NO: 83. Accordingly, in one embodiment, one or more polypeptides and/or polynucleotides expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 71 through SEQ ID NO: 83. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 71 through SEQ ID NO: 83. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically engineered bacteria consist of the sequence of one or more of SEQ ID NO: 71 through SEQ ID NO: 83. 
     Table 10A depicts exemplary polypeptide sequences feedback resistant AroG and TrpE. Table 10A also depicts an exemplary TnaA (tryptophanase from  E. coli ) sequence. IN some embodiments, the sequence is encoded in circuits for tryptophan catabolism to indole; in other embodiments, the sequence is deleted from the  E. coli  chromosome to increase levels of tryptophan. 
     
       
         
           
               
             
               
                 TABLE 10A 
               
             
            
               
                   
               
               
                 Feedback resistant AroG and TrpE and tryptophanase sequences 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 AroGfbr: feedback 
                 MNYQNDDLRIKEIKELLPPVALLEKFPATENAANTVAHARKAI 
               
               
                 resistant 2-dehydro- 
                 HKILKGNDDRLLVVIGPCSIHDPVAAKEYATRLLTLREELQDE 
               
               
                 3- 
                 LEIVMRVYFEKPRTTVGWKGLINDPHMDNSFQINDGLRIARK 
               
               
                 deoxyphosphohept 
                 LLLDINDSGLPAAGEFLDMITLQYLADLMSWGAIGARTTESQ 
               
               
                 onate aldolase from 
                 VHRELASGLSCPVGFKNGTDGTIKVAIDAINAAGAPHCFLSVT 
               
               
                 E. coli 
                 KWGHSAIVNTSGNGDCHIILRGGKEPNYSAKHVAEVKEGLNK 
               
               
                 SEQ ID NO: 84 
                 AGLPAQVMIDFSHANSSKQFKKQMDVCTDVCQQIAGGEKAII 
               
               
                   
                 GVMVESHLVEGNQSLESGEPLAYGKSITDACIGWDDTDALLR 
               
               
                   
                 QLASAVKARRG 
               
               
                   
               
               
                 TrpEfbr: feedback 
                 MQTQKPTLELLTCEGAYRDNPTALFHQLCGDRPATLLLEFADI 
               
               
                 resistant 
                 DSKDDLKSLLLVDSALRITALSDTVTIQALSGNGEALLTLLDN 
               
               
                 anthranilate 
                 ALPAGVENEQSPNCRVLRFPPVSPLLDEDARLCSLSVFDAFRL 
               
               
                 synthase 
                 LQNLLNVPKEEREAMFFGGLFSYDLVAGFENLPQLSAENSCP 
               
               
                 component I from 
                 DFCFYLAETLMVIDHQKKSTRIQASLFAPNEEEKQRLTARLNE 
               
               
                 E. coli 
                 LRQQLTEAAPPLPVVSVPHMRCECNQSDEEFGGVVRLLQKAI 
               
               
                 SEQ ID NO: 85 
                 RAGEIFQVVPSRRFSLPCPSPLAAYYVLKKSNPSPYMFFMQDN 
               
               
                   
                 DFTLFGASPESSLKYDATSRQIEIYPIAGTRPRGRRADGSLDRD 
               
               
                   
                 LDSRIELEMRTDHKELSEHLMLVDLARNDLARICTPGSRYVA 
               
               
                   
                 DLTKVDRYSYVMHLVSRVVGELRHDLDALHAYRACMNMGT 
               
               
                   
                 LSGAPKVRAMQLIAEAEGRRRGSYGGAVGYFTAHGDLDTCIV 
               
               
                   
                 IRSALVENGIATVQAGAGVVLDSVPQSEADETRNKARAVLRA 
               
               
                   
                 IATAHHAQETF 
               
               
                   
               
               
                 SerA: 2- 
                 MAKVSLEKDKIKFLLVEGVHQKALESLRAAGYTNIEFHKGAL 
               
               
                 oxoglutarate 
                 DDEQLKESIRDAHFIGLRSRTHLTEDVINAAEKLVAIGCFCIGT 
               
               
                 reductase from E. 
                 NQVDLDAAAKRGIPVFNAPFSNTRSVAELVIGELLLLLRGVPE 
               
               
                 coli Nissle 
                 ANAKAHRGVWNKLAAGSFEARGKKLGIIGYGHIGTQLGILAE 
               
               
                 SEQ ID NO: 86 
                 SLGMYVYFYDIENKLPLGNATQVQHLSDLLNMSDVVSLHVPE 
               
               
                   
                 NPSTKNMMGAKEISLMKPGSLLINASRGTVVDIPALCDALASK 
               
               
                   
                 HLAGAAIDVFPTEPATNSDPFTSPLCEFDNVLLTPHIGGSTQEA 
               
               
                   
                 QENIGLEVAGKLIKYSDNGSTLSAVNFPEVSLPLHGGRRLMHI 
               
               
                   
                 HENRPGVLTALNKIFAEQGVNIAAQYLQTSAQMGYVVIDIEA 
               
               
                   
                 DEDVAEKALQAMKAIPGTIRARLLY 
               
               
                   
               
               
                 SerAfbr: feedback 
                 MAKVSLEKDKIKFLLVEGVHQKALESLRAAGYTNIEFHKGAL 
               
               
                 resistant 2- 
                 DDEQLKESIRDAHFIGLRSRTHLTEDVINAAEKLVAIGCFCIGT 
               
               
                 oxoglutarate 
                 NQVDLDAAAKRGIPVFNAPFSNTRSVAELVIGELLLLLRGVPE 
               
               
                 reductase from E. 
                 ANAKAHRGVWNKLAAGSFEARGKKLGIIGYGHIGTQLGILAE 
               
               
                 coli Nissle 
                 SLGMYVYFYDIENKLPLGNATQVQHLSDLLNMSDVVSLHVPE 
               
               
                   
                 NPSTKNMMGAKEISLMKPGSLLINASRGTVVDIPALCDALASK 
               
               
                 SEQ ID NO: 87 
                 HLAGAAIDVFPTEPATNSDPFTSPLCEFDNVLLTPHIGGSTQEA 
               
               
                   
                 QENIGLEVAGKLIKYSDNGSTLSAVNFPEVSLPLHGGRRLMHI 
               
               
                   
                 AEARPGVLTALNKIFAEQGVNIAAQYLQTSAQMGYVVIDIEA 
               
               
                   
                 DEDVAEKALQAMKAIPGTIRARLLY 
               
               
                   
               
               
                 TnaA: 
                 MENFKHLPEPFRIRVIEPVKRTTRAYREEAIIKSGMNPFLLDSE 
               
               
                 tryptophanase from 
                 DVFIDLLTDSGTGAVTQSMQAAMMRGDEAYSGSRSYYALAE 
               
               
                 E. coli 
                 SVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKREQEKGLDRSKM 
               
               
                 SEQ ID NO: 88 
                 VAFSNYFFDTTQGHSQINGCTVRNVYIKEAFDTGVRYDFKGN 
               
               
                   
                 FDLEGLERGIEEVGPNNVPYIVATITSNSAGGQPVSLANLKVM 
               
               
                   
                 YSIAKKYDIPVVMDSARFAENAYFIKQREAEYKDWTIEQITRE 
               
               
                   
                 TYKYADMLAMSAKKDAMVPMGGLLCMKDDSFFDVYTECRT 
               
               
                   
                 LCVVQEGFPTYGGLEGGAMERLAVGLYDGMNLDWLAYRIA 
               
               
                   
                 QVQYLVDGLEEIGVVCQQAGGHAAFVDAGKLLPHIPADQFPA 
               
               
                   
                 QALACELYKVAGIRAVEIGSFLLGRDPKTGKQLPCPAELLRLTI 
               
               
                   
                 PRATYTQTHMDFIIEAFKHVKENAANIKGLTFTYEPKVLRHFT 
               
               
                   
                 AKLKEV 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
             
               
                 TABLE 10B 
               
               
                   
               
             
            
               
                 fbrAroG 
                 atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcg 
               
               
                 SEQ ID NO: 256 
                 cattgctggaaaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccga 
               
               
                   
                 aaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtggtgattggccca 
               
               
                   
                 tgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgtg 
               
               
                   
                 aagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtacta 
               
               
                   
                 cggtgggctggaaagggctgattaacgatccgcatatggataacagcttccagatcaac 
               
               
                   
                 gacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagcg 
               
               
                   
                 gcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggc 
               
               
                   
                 gcaattggcgcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttc 
               
               
                   
                 ttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggctatcgatgccatta 
               
               
                   
                 atgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcgatt 
               
               
                   
                 gtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaa 
               
               
                   
                 ctacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgcc 
               
               
                   
                 agcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcagat 
               
               
                   
                 ggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgt 
               
               
                   
                 gatggtggaaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgct 
               
               
                   
                 ggcctacggtaagagcatcaccgatgcctgcattggctgggatgataccgatgctctgtt 
               
               
                   
                 acgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaa 
               
               
                   
               
               
                 fbrTrpE 
                 atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaa 
               
               
                 SEQ ID NO: 274 
                 cccgactgcgctttttcaccagttgtgtggggatcgtccggcaacgctgctgctggaattc 
               
               
                   
                 gcagatatcgacagcaaagatgatttaaaaagcctgctgctggtagacagtgcgctgcg 
               
               
                   
                 cattacagcattaagtgacactgtcacaatccaggcgctttccggcaatggagaagccct 
               
               
                   
                 gttgacactactggataacgccttgcctgcgggtgtggaaaatgaacaatcaccaaactg 
               
               
                   
                 ccgcgtactgcgcttcccgcctgtcagtccactgctggatgaagacgcccgcttatgctc 
               
               
                   
                 cctttcggtttttgacgctttccgcttattacagaatctgttgaatgtaccgaaggaagaacg 
               
               
                   
                 agaagcaatgttctcggcggcctgttctcttatgaccttgtggcgggatttgaaaatttac 
               
               
                   
                 cgcaactgtcagcggaaaatagctgccctgatttctgtttttatctcgctgaaacgctgatg 
               
               
                   
                 gtgattgaccatcagaaaaaaagcactcgtattcaggccagcctgtttgctccgaatgaa 
               
               
                   
                 gaagaaaaacaacgtctcactgctcgcctgaacgaactacgtcagcaactgaccgaag 
               
               
                   
                 ccgcgccgccgctgccggtggtttccgtgccgcatatgcgttgtgaatgtaaccagagc 
               
               
                   
                 gatgaagagttcggtggtgtagtgcgtttgttgcaaaaagcgattcgcgccggagaaatt 
               
               
                   
                 ttccaggtggtgccatctcgccgtttctctctgccctgcccgtcaccgctggcagcctatta 
               
               
                   
                 cgtgctgaaaaagagtaatcccagcccgtacatgttttttatgcaggataatgatttcaccc 
               
               
                   
                 tgtttggcgcgtcgccggaaagttcgctcaagtatgacgccaccagccgccagattgag 
               
               
                   
                 atttacccgattgccggaacacgtccacgcggtcgtcgtgccgatggttcgctggacag 
               
               
                   
                 agacctcgacagccgcatcgaactggagatgcgtaccgatcataaagagctttctgaac 
               
               
                   
                 atctgatgctggtggatctcgcccgtaatgacctggcacgcatttgcacacccggcagc 
               
               
                   
                 cgctacgtcgccgatctcaccaaagttgaccgttactcttacgtgatgcacctagtctccc 
               
               
                   
                 gcgttgttggtgagctgcgccacgatctcgacgccctgcacgcttaccgcgcctgtatga 
               
               
                   
                 atatggggacgttaagcggtgcaccgaaagtacgcgctatgcagttaattgccgaagca 
               
               
                   
                 gaaggtcgtcgacgcggcagctacggcggcgcggtaggttattttaccgcgcatggcg 
               
               
                   
                 atctcgacacctgcattgtgatccgctcggcgctggtggaaaacggtatcgccaccgtg 
               
               
                   
                 caagccggtgctggcgtagtccttgattctgttccgcagtcggaagccgacgaaactcgt 
               
               
                   
                 aataaagcccgcgctgtactgcgcgctattgccaccgcgcatcatgcacaggagacgtt 
               
               
                   
                 cta 
               
               
                   
               
               
                 SerA 
                 atggcaaaggtatcgctggagaaagacaagattaagtttctgctggtagaaggcgtgca 
               
               
                 SEQ ID NO: 258 
                 ccaaaaggcgctggaaagccttcgtgcagctggttacaccaacatcgaatttcacaaag 
               
               
                   
                 gcgcgctggatgatgaacaattaaaagaatccatccgcgatgcccacttcatcggcctg 
               
               
                   
                 cgatcccgtacccatctgactgaagacgtgatcaacgccgcagaaaaactggtcgctat 
               
               
                   
                 tggctgtttctgtatcggaacaaatcaggttgatctggatgcggcggcaaagcgcgggat 
               
               
                   
                 cccggtatttaacgcaccgttctcaaatacgcgctctgttgcggagctggtgattggcga 
               
               
                   
                 actgctgctgctattgcgcggcgtgccagaagccaatgctaaagcgcatcgtggcgtgt 
               
               
                   
                 ggaacaaactggcggcgggttcttttgaagcgcgcggcaaaaagctgggtatcatcgg 
               
               
                   
                 ctacggtcatattggtacgcaattgggcattctggctgaatcgctgggaatgtatgtttactt 
               
               
                   
                 ttatgatattgaaaacaaactgccgctgggcaacgccactcaggtacagcatctttctgac 
               
               
                   
                 ctgctgaatatgagcgatgtggtgagtctgcatgtaccagagaatccgtccaccaaaaat 
               
               
                   
                 atgatgggcgcgaaagagatttcgctaatgaagcccggctcgctgctgattaatgcttcg 
               
               
                   
                 cgcggtactgtggtggatattccagcgctgtgtgacgcgctggcgagcaaacatctggc 
               
               
                   
                 gggggcggcaatcgacgtattcccgacggaaccggcgaccaatagcgatccatttacc 
               
               
                   
                 tctccgctgtgtgaattcgacaatgtccttctgacgccacacattggcggttcgactcagg 
               
               
                   
                 aagcgcaggagaatatcggcttggaagttgcgggtaaattgatcaagtattctgacaatg 
               
               
                   
                 gctcaacgctctctgcggtgaacttcccggaagtctcgctgccactgcacggtgggcgt 
               
               
                   
                 cgtctgatgcacatccacgaaaaccgtccgggcgtgctaactgcgctcaacaaaattttt 
               
               
                   
                 gccgagcagggcgtcaacatcgccgcgcaatatctacaaacttccgcccagatgggtt 
               
               
                   
                 atgtagttattgatattgaagccgacgaagacgttgccgaaaaagcgctgcaggcaatg 
               
               
                   
                 aaagctattccgggtaccattcgcgcccgtctgctgtactaa 
               
               
                   
               
               
                 SerAfbr 
                 atggcaaaggtatcgctggagaaagacaagattaagtttctgctggtagaaggcgtgca 
               
               
                 SEQ ID NO: 1510 
                 ccaaaaggcgctggaaagccttcgtgcagctggttacaccaacatcgaatttcacaaag 
               
               
                   
                 gcgcgctggatgatgaacaattaaaagaatccatccgcgatgcccacttcatcggcctg 
               
               
                   
                 cgatcccgtacccatctgactgaagacgtgatcaacgccgcagaaaaactggtcgctat 
               
               
                   
                 tggctgtttctgtatcggaacaaatcaggttgatctggatgcggcggcaaagcgcgggat 
               
               
                   
                 cccggtatttaacgcaccgttctcaaatacgcgctctgttgcggagctggtgattggcgaa 
               
               
                   
                 ctgctgctgctattgcgcggcgtgccagaagccaatgctaaagcgcatcgtggcgtgtgg 
               
               
                   
                 aacaaactggcggcgggttcttttgaagcgcgcggcaaaaagctgggtatcatcggcta 
               
               
                   
                 cggtcatattggtacgcaattgggcattctggctgaatcgctgggaatgtatgtttacttttatg 
               
               
                   
                 atattgaaaacaaactgccgctgggcaacgccactcaggtacagcatctttctgacctgc 
               
               
                   
                 tgaatatgagcgatgtggtgagtctgcatgtaccagagaatccgtccaccaaaaatatga 
               
               
                   
                 tgggcgcgaaagagatttcgctaatgaagcccggctcgctgctgattaatgcttcgcgcg 
               
               
                   
                 gtactgtggtggatattccagcgctgtgtgacgcgctggcgagcaaacatctggcgggg 
               
               
                   
                 gcggcaatcgacgtattcccgacggaaccggcgaccaatagcgatccatttacctctcc 
               
               
                   
                 gctgtgtgaattcgacaatgtccttctgacgccacacattggcggttcgactcaggaagcg 
               
               
                   
                 caggagaatatcggcttggaagttgcgggtaaattgatcaagtattctgacaatggctcaa 
               
               
                   
                 cgctctctgcggtgaacttcccggaagtctcgctgccactgcacggtgggcgtcgtctgat 
               
               
                   
                 gcacatcGCTgaaGCTcgtccgggcgtgctaactgcgctcaacaaaatttttgccga 
               
               
                   
                 gcagggcgtcaacatcgccgcgcaatatctacaaacttccgcccagatgggttatgtagt 
               
               
                   
                 tattgatattgaagccgacgaagacgttgccgaaaaagcgctgcaggcaatgaaagct 
               
               
                   
                 attccgggtaccattcgcgcccgtctgctgtactaa 
               
               
                   
               
            
           
         
       
     
     In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with one or more sequences of Table 10B. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with one or more sequences of Table 10B. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with one or more sequences of Table 10B. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with one or more sequences of Table 10B. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 96%, 97%, 98%, or 99% identity with one or more sequences of Table 10B. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more sequences of Table 10B. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of with one or more sequences of Table 10B. 
     In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 256. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 256. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 256. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 256. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 256. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 256. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 256. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 256. 
     In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 84 through SEQ ID NO: 87. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 85% identity with one or more of SEQ ID NO: 84 through SEQ ID NO: 87. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 90% identity with one or more of SEQ ID NO: 84 through SEQ ID NO: 87. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 95% identity with one or more of SEQ ID NO: 84 through SEQ ID NO: 87. In one embodiment, one or more polypeptides and/or polynucleotides encoded and expressed by the genetically engineered bacteria have have at least about 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 84 through SEQ ID NO: 87. Accordingly, in one embodiment, one or more polypeptides and/or polynucleotides expressed by the genetically engineered bacteria have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more of SEQ ID NO: 84 through SEQ ID NO: 87. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 84 through SEQ ID NO: 87. In another embodiment, one or more polynucleotides and/or polypeptides encoded and expressed by the genetically engineered bacteria consist of the sequence of one or more of SEQ ID NO: 84 through SEQ ID NO: 87. 
     In some embodiments, the endogenous TnaA polypeptide comprising SEQ ID NO: 88 is mutated or deleted. 
     To improve acetate production, while maintaining high levels of tryptophan production, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more tryptophan production cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
     In some embodiments, the genetically engineered bacteria comprise one or more tryptophan production cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more tryptophan than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more tryptophan than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more tryptophan than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle, while maintaining high levels of tryptophan production. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes described herein which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for tryptophan production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for tryptophan synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptophan and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of tryptophan and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and ΔtrpR, ΔtnaA, and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and ΔtrpR, ΔtnaA, and further comprise a mutation in the endogenous pta and ldhA genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and ΔtrpR, ΔtnaA, and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and ΔtrpR, ΔtnaA, and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and ΔtrpR, ΔtnaA, and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbr, trpDCBA, aroGfbr, SerAfbr and ΔtrpR, ΔtnaA, and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more tryptophan than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more tryptophan than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more tryptophan than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. 
     n some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits. 
     Producing Kynurenic Acid 
     In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid. Kynurenic acid is produced from the irreversible transamination of kynurenine in a reaction catalyzed by the enzyme kynurenine-oxoglutarate transaminase. Kynurenic acid acts as an antagonist of ionotropic glutamate receptors (Turski et al., 2013). While glutamate is known to be a major excitatory neurotransmitter in the central nervous system, there is now evidence to suggest an additional role for glutamate in the peripheral nervous system. For example, the activation of NMDA glutamate receptors in the major nerve supply to the GI tract (i.e., the myenteric plexus) leads to an increase in gut motility (Forrest et al., 2003), but rats treated with kynurenic acid exhibit decreased gut motility and inflammation in the early phase of acute colitis (Varga et al., 2010). Thus, the elevated levels of kynurenic acid reported in IBD patients may represent a compensatory response to the increased activation of enteric neurons (Forrest et al., 2003). The genetically engineered bacteria may comprise any suitable gene or genes for producing kynurenic acid. In some embodiments, the engineered bacteria comprise gene sequence(s) encoding one or more kynurenine-oxoglutarate transaminases (also referred to as kynurenine aminotransferases (e.g., KAT I, II, III)). 
     In some embodiments, the gene or genes for producing kynurenic acid is modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. 
     In some embodiments, the genetically engineered bacteria comprising one or more gene(s) or gene cassette(s) can alter the TRP:KYNA ratio, e.g. in the circulation. In some embodiments the TRP:KYNA ratio is increased. In some embodiments, TRP:KYNA ratio is decreased. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) for the consumption of tryptophan and production of kynurenic acid, which are bacterially derived. In some embodiments, the enzymes for producing kynureic acid are derived from one or more of  Pseudomonas, Xanthomonas, Burkholderia, Stenotrophomonas, Shewanella , and  Bacillus , and/or members of the families Rhodobacteraceae, Micrococcaceae, and Halomonadaceae, In some embodiments the enzymes are derived from the species listed in table S7 of Vujkovic-Cvijin et al. (Dysbiosis of the gut microbiota is associated with HIV disease progression and tryptophan catabolism Sci Transl Med. 2013 Jul. 10; 5(193): 193ra91), the contents of which is herein incorporated by reference in its entirety. 
     In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters and gene sequence(s) encoding kynureninase. In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters and gene sequence(s) encoding one or more kynurenine-oxoglutarate transaminases (kynurenine aminotransferases). In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding one or more tryptophan transporters, gene sequence(s) encoding kynureninase, and gene sequence(s) encoding one or more kynurenine-oxoglutarate transaminases (kynurenine aminotransferases). In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding kynureninase and gene sequence(s) encoding one or more kynurenine aminotransferases. 
     In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce kynurenic acid from tryptophan. Non-limiting example of such gene sequence(s) are shown in the figures and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDO1 (indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IDO1 from  Homo sapiens . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TDO2 (tryptophan 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode TDO2 from  Homo sapiens . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 (indoleamine 2,3-dioxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 from  S. cerevisiae ). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid: Kynurenine formamidase from mouse. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with one or more of ido1 and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with IDO. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with TDO2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode Afmid in combination with bna2. In one embodiment, the genetically engineered bacteria further comprise one or more gene sequence(s) which encode cclb1 and/or cclb2 and/or aadat and/or got2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 (kynurenine-oxoglutarate transaminase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA3 from  S. cerevisae . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with one or more of ido1 and/or tdo2 and/or bna2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with ido1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with tdo2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode BNA2 in combination with bna2. In one embodiment, the genetically engineered bacteria further comprise one or more gene sequence(s) which encode cclb1 and/or cclb2 and/or aadat and/or got2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of ido1 and/or tdo2 and/or bna2. 
     In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of afmid and/or bna3. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of ido1 and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode GOT2 (Aspartate aminotransferase, mitochondrial). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode GOT2 from  Homo sapiens . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AADAT (Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AADAT from  Homo sapiens . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB1 (Kynurenine-oxoglutarate transaminase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB1 from  Homo sapiens ). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB2 (kynurenine-oxoglutarate transaminase 3) In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode CCLB2 from  Homo sapiens . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cclb1 and/or cclb2 and/or aadat and/or got2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of ido1 and/or tdo2 and/or bna2, in combination with one or more of afmid and/or bna3, and in combination with one or more of cclb1 and/or cclb2 and/or aadat and/or got2. 
     In any of these embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in the figures and the examples and described elsewhere herein. In some embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce kynurenic acid from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites. 
     In some embodiments, the one or more genes for producing kynurenic acid are modified and/or mutated, e.g., to enhance stability, increase kynurenic acid production under inducing conditions. In some embodiments, the engineered bacteria have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell. 
     To improve acetate production, while maintaining high levels of kynurenic acid production, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more kynurenic acid production cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
     In some embodiments, the genetically engineered bacteria comprise one or more kynurenic acid production cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more kynurenic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more kynurenic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more kynurenic acid than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle, while maintaining high levels of kynurenic acid production. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes described herein which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for kynurenic acid production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for kynurenic acid synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of kynurenic acid and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of kynurenic acid and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more kynurenic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more kynurenic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more kynurenic acid than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenic acid in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. 
     In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. 
     In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits. 
     Producing Indole Tryptophan Metabolites and Tryptamine 
     In some embodiments, the genetically engineered bacteria comprise genetic circuits for the production of indole metabolites and/or tryptamine. Exemplary circuits for the production of indole metabolites/derivatives are shown in the figures. 
     In some embodiments, the genetically engineered bacteria comprise genetic circuitry for converting tryptophan to tryptamine. In some embodiments, the engineered bacteria comprise gene sequence encoding Tryptophan decarboxylase, e.g., from  Catharanthus roseus . In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-acetaldehyde and FICZ from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: aro9 (L-tryptophan aminotransferase, e.g., from  S. cerevisae ), aspC (aspartate aminotransferase, e.g., from  E. coli , taa1 (L-tryptophan-pyruvate aminotransferase, e.g., from  Arabidopsis thaliana ), staO (L-tryptophan oxidase, e.g., from  Streptomyces  sp. TP-A0274), trpDH (Tryptophan dehydrogenase, e.g., from  Nostoc punctiforme  NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from  Enterobacter cloacae ). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: tdc (Tryptophan decarboxylase, e.g., from  Catharanthus roseus  and/or  Clostridium sporogenes ), and tynA (Monoamine oxidase, e.g., from  E. coli ). In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-acetonitrile from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: cyp79B2, (tryptophan N-monooxygenase, e.g., from  Arabidopsis thaliana ), cyp79B3 (tryptophan N-monooxygenase, e.g., from  Arabidopsis thaliana ). In some embodiments, the engineered bacteria comprise genetic circuitry for producing kynurenine from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: IDO1 (indoleamine 2,3-dioxygenase, e.g., from  Homo sapiens  or TDO2 (tryptophan 2,3-dioxygenase, e.g., from  Homo sapiens ), BNA2 (indoleamine 2,3-dioxygenase, e.g., from  S. cerevisiae ) and Afmid: Kynurenine formamidase, e.g., from mouse), BNA3 (kynurenine-oxoglutarate transaminase, e.g., from  S. cerevisae ). In some embodiments, the engineered bacteria comprise genetic circuitry for producing kynureninic acid from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: IDO1 (indoleamine 2,3-dioxygenase, e.g., from  Homo sapiens  or TDO2 (tryptophan 2,3-dioxygenase, e.g., from  Homo sapiens ), BNA2 (indoleamine 2,3-dioxygenase, e.g., from  S. cerevisiae ) and Afmid: Kynurenine formamidase, e.g., from mouse), BNA3 (kynurenine-oxoglutarate transaminase, e.g., from  S. cerevisae ) and GOT2 (Aspartate aminotransferase, mitochondrial, e.g., from  Homo sapiens  or AADAT (Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial, e.g., from  Homo sapiens ), or CCLB1 (Kynurenine-oxoglutarate transaminase 1, e.g., from  Homo sapiens ) or CCLB2 (kynurenine-oxoglutarate transaminase 3, e.g., from  Homo sapiens . In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: tnaA (tryptophanase, e.g., from  E. coli ). In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-carbinol, indole-3-aldehyde, 3,3′ diindolylmethane (DIM), indolo(3,2-b) carbazole (ICZ) from indole glucosinolate (taken up through the diet). The genetically engineered bacteria comprise a gene sequence encoding pne2 (myrosinase, e.g., from  Arabidopsis thaliana ). In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-acetic acid from tryptophan. In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: aro9 (L-tryptophan aminotransferase, e.g., from  S. cerevisae ), aspC (aspartate aminotransferase, e.g., from  E. coli , taa1 (L-tryptophan-pyruvate aminotransferase, e.g., from  Arabidopsis thaliana ), staO (L-tryptophan oxidase, e.g., from  Streptomyces  sp. TP-A0274), trpDH (Tryptophan dehydrogenase, e.g., from  Nostoc punctiforme  NIES-2108), ipdC (Indole-3-pyruvate decarboxylase, e.g., from  Enterobacter cloacae ), iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from  Ustilago maydis ), AAO1 (Indole-3-acetaldehyde oxidase, e.g., from  Arabidopsis thaliana ). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: tdc (Tryptophan decarboxylase, e.g., from  Catharanthus roseus  and/or  Clostridium sporogenes ), tynA (Monoamine oxidase, e.g., from  E. coli ), iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from  Ustilago maydis ), AAO1 (Indole-3-acetaldehyde oxidase, e.g., from  Arabidopsis thaliana ). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: aro9 (L-tryptophan aminotransferase, e.g., from  S. cerevisae ), aspC (aspartate aminotransferase, e.g., from  E. coli , taa1 (L-tryptophan-pyruvate aminotransferase, e.g., from  Arabidopsis thaliana ), staO (L-tryptophan oxidase, e.g., from  Streptomyces  sp. TP-A0274), trpDH (Tryptophan dehydrogenase, e.g., from  Nostoc punctiforme  NIES-2108) and yuc2 (indole-3-pyruvate monoxygenase, e.g., from  Arabidopsis thaliana ). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: IaaM (Tryptophan 2-monooxygenase e.g., from  Pseudomonas savastanoi ), iaaH (Indoleacetamide hydrolase, e.g., from  Pseudomonas savastanoi ). In some embodiments, the genetically engineered bacteria comprise gene sequence encoding one or more of the following: cyp79B2 (tryptophan N-monooxygenase, e.g., from  Arabidopsis thaliana ), cyp79B3 (tryptophan N-monooxygenase, e.g., from  Arabidopsis thaliana , cyp71a13 (indoleacetaldoxime dehydratase, e.g., from  Arabidopsis thaliana ), nit1 (Nitrilase, e.g., from  Arabidopsis thaliana ), iaaH (Indoleacetamide hydrolase, e.g., from  Pseudomonas savastanoi ). In some embodiments, the genetically engineered bacteria comprises trpDH (Tryptophan dehydrogenase, e.g., from  Nostoc punctiforme  NIES-2108), ipdC (Indole-3-pyruvate decarboxylase, e.g., from  Enterobacter cloacae ) which together produce indole-3-acetaldehyde and FICZ though an (indol-3yl)pyruvate intermediate, and iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from  Ustilago maydis ), which converts indole-3-acetaldehyde into indole-3-acetate 
     In some embodiments, the genetically engineered bacteria comprise genetic circuits for the production of tryptophan, tryptamine, indole acetic acid, and indole propionic acid. In some embodiments, the engineered bacteria produces tryptamine. Tryptophan is optionally produced from chorismate precursor, and the bacteria optionally comprises circuits as depicted and/or described in  FIG. 40A  and/or  FIG. 40B  and/or  FIG. 40C  and/or  FIG. 40D . Additionally, the bacteria comprises tdc (tryptophan decarboxylase, e.g., from  Catharanthus roseus  and/or  Clostridium sporogenes ), which converts tryptophan into tryptamine. 
     In some embodiments, the engineered bacteria comprise genetic circuits for the production of indole-3-acetate. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in  FIG. 40A  and/or  FIG. 40B  and/or  FIG. 40C  and/or  FIG. 40D . Additionally, the strain comprises trpDH (Tryptophan dehydrogenase, e.g., from  Nostoc punctiforme  NIES-2108) and ipdC (Indole-3-pyruvate decarboxylase, e.g., from  Enterobacter cloacae ) which together produce indole-3-acetaldehyde and FICZ though an (indol-3yl)pyruvate intermediate, and iad1 (Indole-3-acetaldehyde dehydrogenase, e.g., from  Ustilago maydis ), which converts indole-3-acetaldehyde into indole-3-acetate. 
     In some embodiments, the engineered bacteria comprise genetic circuits for the production of indole-3-propionate. Tryptophan is optionally produced from chorismate precursor, and the strain optionally comprises circuits as depicted and/or described in  FIG. 40A  and/or  FIG. 40B  and/or  FIG. 40C  and/or  FIG. 40D . Additionally, the strain comprises a circuit as described in  FIG. 48 , comprising trpDH (Tryptophan dehydrogenase, e.g., from  Nostoc punctiforme  NIES-2108, which produces (indol-3yl)pyruvate from tryptophan), fldA (indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from  Clostridium sporogenes , which converts converts indole-3-lactate and indol-3-propionyl-CoA to indole-3-propionic acid and indole-3-lactate-CoA), fldB and fldC (indole-3-lactate dehydratase e.g., from  Clostridium sporogenes , which converts indole-3-lactate-CoA to indole-3-acrylyl-CoA) fldD and/or AcuI: (indole-3-acrylyl-CoA reductase, e.g., from  Clostridium sporogenes  and/or acrylyl-CoA reductase, e.g., from  Rhodobacter sphaeroides , which convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA). The circuits further comprise fldH1 and/or fldH2 (indole-3-lactate dehydrogenase 1 and/or 2, e.g., from  Clostridium sporogenes ), which converts (indol-3-yl)pyruvate into indole-3-lactate). 
     In some embodiments, the engineered bacteria comprises genetic circuitry for the production of indole-3-propionic acid (IPA). In some embodiments, the engineered bacteria comprises gene sequence encoding tryptophan ammonia lyase and an indole-3-acrylate reductase (e.g., Tryptophan ammonia lyase (WAL) (e.g., from  Rubrivivax benzoatilyticus ) and indole-3-acrylate reductase (e.g., from  Clostridium botulinum ). Tryptophan ammonia lyase converts tryptophan to indole-3-acrylic acid, and indole-3-acrylate reductase converts indole-3-acrylic acid into IPA. Without wishing to be bound by theory, no oxygen is needed for this reaction, allowing it to proceed under low or no oxygen conditions, e.g., as those found in the mammalian gut. In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in  FIG. 40  (A-D) and  FIG. 44  and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. 
     In some embodiments, the engineered bacteria comprise genetic circuitry for producing indole-3-propionic acid (IPA), indole acetic acid (IAA), and/or tryptamine synthesis (TrA) circuits. In some embodiments, the engineered bacteria comprise gene sequence encoding one or more of the following: TrpDH: tryptophan dehydrogenase, e.g., from from  Nostoc punctiforme  NIES-2108; FldH1/FldH2: indole-3-lactate dehydrogenase, e.g., from  Clostridium sporogenes ; FldA: indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from  Clostridium sporogenes ; FldBC: indole-3-lactate dehydratase, e.g., from  Clostridium sporogenes ; FldD: indole-3-acrylyl-CoA reductase, e.g., from  Clostridium sporogenes ; AcuI: acrylyl-CoA reductase, e.g., from  Rhodobacter sphaeroides . lpdC: Indole-3-pyruvate decarboxylase, e.g., from  Enterobacter cloacae ; lad1: Indole-3-acetaldehyde dehydrogenase, e.g., from  Ustilago maydis ; Tdc: Tryptophan decarboxylase, e.g., from  Catharanthus roseus  or from  Clostridium sporogenes.    
     In some embodiments, the engineered bacteria comprise genetic circuitry for producing (indol-3-yl)pyruvate (IPyA). In some embodiments, the engineered bacteria comprise gene sequence encoing one or more of the following: tryptophan dehydrogenase (EC 1.4.1.19) (enzyme that catalyzes the reversible chemical reaction converting L-tryptophan, NAD(P) and water to (indol-3-yl)pyruvate (IPyA), NH 3 , NAD(P)H and H + )); Indole-3-lactate dehydrogenase ((EC 1.1.1.110, e.g.,  Clostridium sporogenes  or  Lactobacillus casei ) (converts (indol-3yl)pyruvate (IpyA) and NADH and H+ to indole-3-lactate (ILA) and NAD+); Indole-3-propionyl-CoA:indole-3-lactate CoA transferase (FldA) (converts indole-3-lactate (ILA) and indol-3-propionyl-CoA to indole-3-propionic acid (IPA) and indole-3-lactate-CoA); Indole-3-acrylyl-CoA reductase (FldD) and acrylyl-CoA reductase (AcuI) (convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA); Indole-3-lactate dehydratase (FldBC) (converts indole-3-lactate-CoA to indole-3-acrylyl-CoA); Indole-3-pyruvate decarboxylase (lpdC:) (converts Indole-3-pyruvic acid (IPyA) into Indole-3-acetaldehyde (IAAld)); lad1: Indole-3-acetaldehyde dehydrogenase (coverts Indole-3-acetaldehyde (IAAld) into Indole-3-acetic acid (IAA)); Tdc: Tryptophan decarboxylase (converts tryptophan (Trp) into tryptamine (TrA)). In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in  FIG. 40  (A-D) and  FIG. 44  and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. 
     In any of the described embodiments, any of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) are optionally expressed from an inducible promoter. In certain embodiments, the one or more cassettes are under the control of constitutive promoters. Exemplary inducible promoters which may control the expression of the gene(s), gene sequence(s) and/or gene circuit(s) or cassette(s) include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. The bacteria may also include an auxotrophy, e.g., deletion of thyA (Δ thyA; thymidine dependence). 
     In some embodiments, the genetically engineered bacteria further comprise one or more circuits for the production of tryptophan, e.g., as shown in  FIG. 40  (A-D) and  FIG. 44  and as described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. 
     In in any of these embodiments the expression of the gene sequences for the production of the indole and other tryptophan metabolites, including, but not limited to, tryptamine and/or indole-3 acetaladehyde, indole-3acetonitrile, indole, indole acetic acid FICZ, indole-3-propionic acid, is under the control of an inducible promoter. Exemplary inducible promoters which may control the expression of the biosynthetic cassettes include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite characteristic of a disorder described herein, or that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. 
     In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (6) combinations of one or more of such additional circuits. 
     Tryptamine 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce tryptamine from tryptophan. The monoamine alkaloid, tryptamine, is derived from the direct decarboxylation of tryptophan. Tryptophan is converted to indole-3-acetic acid (IAA) via the enzymes tryptophan monooxygenase (IaaM) and indole-3-acetamide hydrolase (IaaH), which constitute the indole-3-acetamide (IAM) pathway, as described in the figures and examples. 
     A non-limiting example of such as strain is shown in  FIG. 41A . Another non-limiting example of such as strain is shown in  FIG. 43A . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s), e.g., from  Catharanthus roseus . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s), e.g., from  Clostridium sporgenenes . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more Tryptophan decarboxylase(s) e.g., from Ruminococcus Gnavus. 
     Table 15, Table 11A, and Table 12 lists exemplary sequences for tryptamine production in genetically engineered bacteria. 
     In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in  FIG. 40 ,  FIG. 44A  and/or  FIG. 44B  and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, In some embodiments, the genetically engineered bacteria which produce tryptamine from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites. 
     To improve acetate production, while maintaining high levels of tryptamine production, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more tryptamine production cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
     In some embodiments, the genetically engineered bacteria comprise one or more tryptamine production cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and ΔtrpR and ΔtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and ΔtrpR and ΔtnaA, and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and ΔtrpR and ΔtnaA, and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and ΔtrpR and ΔtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and ΔtrpR and ΔtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and ΔtrpR and ΔtnaA, and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and ΔtrpR and ΔtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more tryptamine than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more tryptamine than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more tryptamine than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle, while maintaining high levels of tryptamine production. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes described herein which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for tryptamine production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for tryptamine synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of tryptamine and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of tryptamine and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and ΔtrpR and ΔtnaA, and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and ΔtrpR and ΔtnaA, and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and ΔtrpR and ΔtnaA, and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and ΔtrpR and ΔtnaA, and further comprise a mutation in the endogenous pta and ldhA genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and ΔtrpR and ΔtnaA, and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and ΔtrpR and ΔtnaA, and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and ΔtrpR and ΔtnaA, and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and ΔtrpR and ΔtnaA, and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, tdc, SerAfbr, and ΔtrpR and ΔtnaA, and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more tryptamine than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more tryptamine than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more tryptamine than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria are capable of producing Tryptamine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose and others described herein. 
     Indole-3-Acetaldehyde and FICZ 
     In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-acetaldehyde and FICZ from tryptophan. Exemplary gene cassettes for the production of produce indole-3-acetaldehyde and FICZ from tryptophan are shown in  FIG. 41B . 
     In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 (L-tryptophan aminotransferase). In one embodiment, the (L-tryptophan aminotransferase is from  S. cerevisiae . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from  Enterobacter cloacae ). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from  E. coli . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taa1 (L-tryptophan-pyruvate aminotransferase, In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taa1 from  Arabidopsis thaliana . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taa1 and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L-tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from  Streptomyces  sp. TP-A0274. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from  Nostoc punctiforme  NIES-2108. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH and ipdC. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taa1 or staO or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taa1 or staO or trpDH and ipdC. 
     Further exemplary gene cassettes for the production of produce indole-3-acetaldehyde and FICZ from tryptophan are shown in  FIG. 41C . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc (Tryptophan decarboxylase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from  Catharanthus roseus . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA (Monoamine oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA from  E. coli . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA. 
     In any of these embodiments, the genetically engineered bacteria which produce produce indole-3-acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in  FIG. 40 ,  FIG. 44A  and/or  FIG. 44B  and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce indole-3-acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole-3-acetaldehyde and FICZ also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites. 
     To improve acetate production, while maintaining high levels of Indole-3-acetaldehyde and/or FICZ production, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more Indole-3-acetaldehyde and/or FICZ production cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
     In some embodiments, the genetically engineered bacteria comprise one or more Indole-3-acetaldehyde and/or FICZ production cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more Indole-3-acetaldehyde and/or FICZ than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more Indole-3-acetaldehyde and/or FICZ than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more Indole-3-acetaldehyde and/or FICZ than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle, while maintaining high levels of Indole-3-acetaldehyde and/or FICZ production. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes described herein which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for Indole-3-acetaldehyde and/or FICZ production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for Indole-3-acetaldehyde and/or FICZ synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of Indole-3-acetaldehyde and/or FICZ and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more Indole-3-acetaldehyde and/or FICZ than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more Indole-3-acetaldehyde and/or FICZ than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more Indole-3-acetaldehyde and/or FICZ than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria are capable of producing Indole-3-aldehyde under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. 
     In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. 
     Indole-3-Acetic Acid 
     In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes which convert tryptophan to Indole-3-aldehyde and Indole Acetic Acid, e.g., via a tryptophan aminotransferase cassette. A non-limiting example of such a tryptophan aminotransferase expressed by the genetically engineered bacteria is in the tables. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter, and further produce Indole-3-aldehyde and Indole Acetic Acid from tryptophan. In some embodiments, the genetically engineered bacteria optionally comprise a tryptophan and/or indole metabolite exporter. 
     The genetically engineered bacteria may comprise any suitable gene for producing Indole-3-aldehyde and/or Indole Acetic Acid and/or Tryptamine. In some embodiments, the gene for producing kynurenine is modified and/or mutated, e.g., to enhance stability, increase Indole-3-aldehyde and/or Indole Acetic Acid and/or Tryptamine production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the engineered bacteria also have enhanced uptake or import of tryptophan, e.g., comprise a transporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the engineered bacteria also have enhanced export of a indole tryptophan metabolite, e.g., comprise an exporter or other mechanism for increasing the uptake of tryptophan into the bacterial cell, as discussed in detail above. In some embodiments, the genetically engineered bacteria are capable of producing Indole-3-aldehyde and/or Indole Acetic Acid and/or Tryptamine under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. 
     In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-acetic acid. 
     Non-limiting example of such gene sequence(s) are shown in  FIG. 42A ,  FIG. 42B ,  FIG. 42C ,  FIG. 42D , and  FIG. 42E , and  FIG. 43B  and  FIG. 43E . 
     In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 (L-tryptophan aminotransferase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 from  S. cerevisae ). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase), In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from  E. coli . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taa1 (L-tryptophan-pyruvate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taa1 from  Arabidopsis thaliana ). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L-tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from  Streptomyces  sp. TP-A0274). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from  Nostoc punctiforme  NIES-2108). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iad1 (Indole-3-acetaldehyde dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iad1 from  Ustilago maydis . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAO1 (Indole-3-acetaldehyde oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAO1 from  Arabidopsis thaliana . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from  Enterobacter cloacae ). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from  Enterobacter cloacae ) in combination with one or more sequences encoding enzymes selected from aro9 and/or aspC and/or taa1 and/or staO and/or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from  Enterobacter cloacae ) in combination with one or more sequences encoding enzymes selected from iad1 and/or aao1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from  Enterobacter cloacae ) in combination with one or more sequences encoding enzymes selected from aro9 and/or aspC and/or taa1 and/or staO and in combination with one or more sequences encoding enzymes selected from iad1 and/or aao1 (see, e.g.,  FIG. 42A ). 
     Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in  FIG. 42B . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc (Tryptophan decarboxylase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc from  Catharanthus roseus ). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA (Monoamine oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA from  E. coli ). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iad1 (Indole-3-acetaldehyde dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iad1 from  Ustilago maydis ). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAO1 (Indole-3-acetaldehyde oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode AAO1 from  Arabidopsis thaliana ). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and one or more sequence(s) selected from iad1 and/or aao1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tynA and one or more sequence(s) selected from iad1 and/or aao1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tdc and tynA and one or more sequence(s) selected from iad1 and/or aao1. 
     Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in  FIG. 42C . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode yuc2 (indole-3-pyruvate monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode yuc2 from  Enterobacter cloacae . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 (L-tryptophan aminotransferase). In one embodiment, the (L-tryptophan aminotransferase is from  S. cerevisiae . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aro9 and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC (aspartate aminotransferase. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC from  E. coli . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode aspC and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taa1 (L-tryptophan-pyruvate aminotransferase, In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taa1 from  Arabidopsis thaliana . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode taa1 and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO (L-tryptophan oxidase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO from  Streptomyces  sp. TP-A0274. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode staO and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from  Nostoc punctiforme  NIES-2108. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH and yuc2. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taa1 or staO or trpDH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of aro9 or aspC or taa1 or staO or trpDH and yuc2. 
     Another non-limiting example of gene sequence(s) for the production of acetic acid are shown in  FIG. 42D . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM (Tryptophan 2-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM from  Pseudomonas savastanoi ). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH (Indoleacetamide hydrolase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH from  Pseudomonas savastanoi ). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode IaaM and iaaH. 
     Another non-limiting example of gene sequence(s) for the production of acetic acid are shown in  FIG. 42E . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13 (indoleacetaldoxime dehydratase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13 from  Arabidopsis thaliana . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode nit1 (Nitrilase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode nit1 from  Arabidopsis thaliana . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH (Indoleacetamide hydrolase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iaaH from  Pseudomonas savastanoi ). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 (tryptophan N-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from  Arabidopsis thaliana . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from  Arabidopsis thaliana . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and nit1 and/or iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 (tryptophan N-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from  Arabidopsis thaliana . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13 and nit1 and/or iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13, and nit1 and/or iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from  Arabidopsis thaliana . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13 and nit1 and iaaH. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13 and nit1 and iaaH. 
     Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in  FIG. 42F . Another non-limiting example of gene sequence(s) for the production of indole-3-acetic acid are shown in  FIG. 343E . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH (Tryptophan dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode trpDH from  Nostoc punctiforme  NIES-2108. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode ipdC (Indole-3-pyruvate decarboxylase, e.g., from  Enterobacter cloacae ). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iad1 (Indole-3-acetaldehyde dehydrogenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode iad1 from  Ustilago maydis . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of trpDH and/or ipdC and/or iad1. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more of trpDH and ipdC and iad1. 
     In any of these embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in  FIG. 40 ,  FIG. 44A  and/or  FIG. 44B  and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole acetic acid also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites. 
     To improve acetate production, while maintaining high levels of indole-3-acetic acid production, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more indole-3-acetic acid production cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
     In some embodiments, the genetically engineered bacteria comprise one or more indole-3-acetic acid production cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of indole-3-acetic acid and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of indole-3-acetic acid and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of indole-3-acetic acid and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of indole-3-acetic acid and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of indole-3-acetic acid and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of indole-3-acetic acid and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of indole-3-acetic acid and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of indole-3-acetic acid and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of indole-3-acetic acid and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iad, and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iad, and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iad, and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iad, and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iad, and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iad, and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iad, and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more indole-3-acetic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more indole-3-acetic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more indole-3-acetic acid than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle, while maintaining high levels of indole-3-acetic acid production. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes described herein which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for indole-3-acetic acid production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for indole-3-acetic acid synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of indole-3-acetic acid and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of indole-3-acetic acid and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of indole-3-acetic acid and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of indole-3-acetic acid and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of indole-3-acetic acid and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of indole-3-acetic acid and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of indole-3-acetic acid and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of indole-3-acetic acid and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of indole-3-acetic acid and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iad, and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iad, and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iad, and ΔtrpR, ΔtnaA, and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iad, and ΔtrpR, ΔtnaA, and further comprise a mutation in the endogenous pta and ldhA genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iad, and ΔtrpR, ΔtnaA, and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iad, and ΔtrpR, ΔtnaA, and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iad, and ΔtrpR, ΔtnaA, and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iad, and ΔtrpR, ΔtnaA, and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from trpEfbrDCBA, aroGfbr, SerAfbr, trpDH, ipdC, iad, and ΔtrpR, ΔtnaA, and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more indole-3-acetic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more indole-3-acetic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more indole-3-acetic acid than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria are capable of producing Indole Acetic Acid and under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. 
     In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. 
     Indole-3-Acetonitrile 
     In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-acetonitrile from tryptophan. A non-limiting example of such gene sequence(s) which allow in which the genetically engineered bacteria to produce indole-3-acetonitrile from tryptophan is depicted in the figures and examples. 
     In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 (tryptophan N-monooxygenase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 from  Arabidopsis thaliana . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13 (indoleacetaldoxime dehydratase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp71a13 from  Arabidopsis thaliana . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B2 and cyp71a13. 
     In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 (tryptophan N-monooxygenase) In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 from  Arabidopsis thaliana . In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3 and cyp71a13. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode cyp79B3, cyp79B2 and cyp71a13. 
     In any of these embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in  FIG. 40 ,  FIG. 44A  and/or  FIG. 44B  and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. 
     In some embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole-3-acetonitrile from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites. 
     To improve acetate production, while maintaining high levels of Indole-3-acetonitrile production, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more Indole-3-acetonitrile production cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
     In some embodiments, the genetically engineered bacteria comprise one or more Indole-3-acetonitrile production cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetonitrile and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetonitrile and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetonitrile and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetonitrile and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetonitrile and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetonitrile and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetonitrile and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetonitrile and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetonitrile and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more Indole-3-acetonitrile than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more Indole-3-acetonitrile than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more Indole-3-acetonitrile than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle, while maintaining high levels of Indole-3-acetonitrile production. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes described herein which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for indole-3-acetonitrile production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for Indole-3-acetonitrile synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetonitrile and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetonitrile and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetonitrile and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetonitrile and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetonitrile and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetonitrile and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetonitrile and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-acetonitrile and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of Indole-3-acetonitrile and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more Indole-3-acetonitrile than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more Indole-3-acetonitrile than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more Indole-3-acetonitrile than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. 
     Indole-3-Propionic Acid (IPA) 
     In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-propionic acid from tryptophan.  FIG. 47  and  FIG. 48 , and  FIG. 43C  depict schematics of exemplary circuits for the production of indole-3-propionic acid. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding tryptophan ammonia lyase. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding tryptophan ammonia lyase from  Rubrivivax benzoatilyticus . In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding indole-3-acrylate reductase. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding indole-3-acrylate reductase from  Clostridium botulinum . In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding a tryptophan ammonia lyase and an indole-3-acrylate reductase. In some embodiments, the indole-3-propionate-producing strain optionally produces tryptophan from a chorismate precursor, and the strain optionally comprises additional circuits for tryptophan production and/or tryptophan uptake/transport s described herein. 
     The genetically engineered bacteria comprise a circuit, comprising trpDH (Tryptophan dehydrogenase, e.g., from  Nostoc punctiforme  NIES-2108, which produces (indol-3yl)pyruvate from tryptophan), fldA (indole-3-propionyl-CoA:indole-3-lactate CoA transferase, e.g., from  Clostridium sporogenes , which converts converts indole-3-lactate and indol-3-propionyl-CoA to indole-3-propionic acid and indole-3-lactate-CoA), fldB and fldC (indole-3-lactate dehydratase e.g., from  Clostridium sporogenes , which converts indole-3-lactate-CoA to indole-3-acrylyl-CoA) fldD and/or AcuI: (indole-3-acrylyl-CoA reductase, e.g., from  Clostridium sporogenes  and/or acrylyl-CoA reductase, e.g., from  Rhodobacter sphaeroides , which convert indole-3-acrylyl-CoA to indole-3-propionyl-CoA). The circuits further comprise fldH1 and/or fldH2 (indole-3-lactate dehydrogenase 1 and/or 2, e.g., from  Clostridium sporogenes ), which converts (indol-3-yl)pyruvate into indole-3-lactate) (see, e.g.,  FIG. 48 ). 
     Another embodiment of the IPA producing strain is shown in  FIG. 47 . 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH (Tryptophan dehydrogenase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH from  Nostoc punctiforme  NIES-2108. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldA (indole-3-propionyl-CoA:indole-3-lactate CoA transferase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldA from  Clostridium sporogenes . In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldB and fldC (indole-3-lactate dehydratase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldB and fldC  Clostridium sporogenes . In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldD (indole-3-acrylyl-CoA reductase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldD from  Clostridium sporogenes . In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding AcuI (acrylyl-CoA reductase). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding AcuI from  Rhodobacter sphaeroides . In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH1 (3-lactate dehydrogenase 1). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH1 from  Clostridium sporogenes . In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH2 (indole-3-lactate dehydrogenase 2). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding fldH2 from  Clostridium sporogenes ). In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or flD and/or fldH1. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or flD and/or fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or acuI and/or fldH1. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and/or fldA and/or fldB and/or acuI and/or fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and flD and fldH1. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and flD and fldH2. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and acuI and fldH1. In some embodiments, the genetically engineered bacteria comprise one or more gene sequences encoding trpDH and fldA and fldB and acuI and fldH2. 
     In any of these embodiments, the genetically engineered bacteria which produce indole-3-propionic acid also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in  FIG. 40 ,  FIG. 44A  and/or  FIG. 44B  and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce indole-3-propionic acid also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole-3-propionic acid also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites. 
     To improve acetate production, while maintaining high levels of Indole-3-propionic acid production, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more Indole-3-propionic acid production cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
     In some embodiments, the genetically engineered bacteria comprise one or more Indole-3-propionic acid production cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-propionic acid and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-propionic acid and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-propionic acid and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-propionic acid and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-propionic acid and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-propionic acid and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-propionic acid and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-propionic acid and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-propionic acid and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more Indole-3-propionic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more Indole-3-propionic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more Indole-3-propionic acid than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle, while maintaining high levels of Indole-3-propionic acid production. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes described herein which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for Indole-3-propionic acid production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for Indole-3-propionic acid synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-propionic acid and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-propionic acid and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-propionic acid and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-propionic acid and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-propionic acid and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-propionic acid and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-propionic acid and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole-3-propionic acid and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of Indole-3-propionic acid and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more Indole-3-propionic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more Indole-3-propionic acid than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more Indole-3-propionic acid than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of tryptophan metabolites. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 different tryptophan metabolites. In certain embodiments the bacteria comprise one or more gene sequence(s) encoding one or more enzymes for the production of tryptophan metabolites selected from tryptamine and/or indole-3 acetaladehyde, indole-3acetonitrile, kynurenine, kynurenic acid, indole, indole acetic acid FICZ, indole-3-propionic acid. 
     In some embodiments, the genetically engineered bacteria are capable of producing such tryptophan metabolites under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing such tryptophan metabolites in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. 
     In some embodiments, the gene sequences(s) are controlled by an inducible promoter. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constritutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. 
     Indole 
     In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole from tryptophan. Non-limiting example of such gene sequence(s) are shown  FIG. 41G  and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tnaA (tryptophanase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode tnaA from  E. coli.    
     In any of these embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in  FIG. 40 ,  FIG. 44A  and/or  FIG. 44B  and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria which produce indole from tryptophan also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites. 
     To improve acetate production, while maintaining high levels of Indole production, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more Indole production cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
     In some embodiments, the genetically engineered bacteria comprise one or more Indole production cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more Indole than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more Indole than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more Indole than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle, while maintaining high levels of Indole production. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes described herein which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for Indole production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for indole synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Indole and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of Indole and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more Indole than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more Indole than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more Indole than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria are capable of producing Indole-3-acetonitrile under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. 
     Other Indole Metabolites 
     In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more tryptophan catabolism enzymes, which produce indole-3-carbinol, indole-3-aldehyde, 3,3′ diindolylmethane (DIM), indolo(3,2-b) carbazole (ICZ) from indole glucosinolate taken up through the diet. Non-limiting example of such gene sequence(s) are shown  FIG. 41H  and described elsewhere herein. In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode pne2 (myrosinase). In one embodiment, the genetically engineered bacteria comprise one or more gene sequence(s) which encode pne2from  Arabidopsis thaliana.    
     In any of these embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) comprising one or more enzymes for tryptophan production, and gene deletions/or mutations as depicted and described in  FIG. 40 ,  FIG. 44A  and/or  FIG. 44B  and described elsewhere herein. In some embodiments, AroG and/or TrpE are replaced with feedback resistant versions to improve tryptophan production in the genetically engineered bacteria. In some embodiments, trpR and/or the tnaA gene (encoding a tryptophanase converting tryptophan into indole) are deleted to further increase levels of tryptophan produced. In some embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) which encode one or more transporter(s) as described herein, through which tryptophan can be imported. Optionally, in some embodiments, the genetically engineered bacteria also optionally comprise one or more gene sequence(s) which encode an exporter as described herein, which can export tryptophan or any of its metabolites. 
     To improve acetate production, while maintaining high levels of Other indoles production, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more Other indoles production cassette(s) and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
     In some embodiments, the genetically engineered bacteria comprise one or more Other indoles production cassette(s) described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Other indoles and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Other indoles and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Other indoles and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Other indoles and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Other indoles and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Other indoles and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Other indoles and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Other indoles and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Other indoles and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more Other indoles than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more Other indoles than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more Other indoles than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle, while maintaining high levels of Other indoles production. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes described herein which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for Other indoles production. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for Other indoles synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Other indoles and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Other indoles and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Other indoles and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Other indoles and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Other indoles and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Other indoles and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Other indoles and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzymes described herein for the production of Other indoles and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more enzyme(s) for the production of Other indoles and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more Other indoles than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more Other indoles than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more Other indoles than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria are capable of producing these metabolites under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. 
     Tryptophan Catabolic Pathway Enzymes 
     Table 11A and Table 11B comprise polypeptide and polynucleotide sequences of such enzymes which are encoded by the genetically engineered bacteria of the disclosure. 
     
       
         
           
               
             
               
                 TABLE 11A 
               
             
            
               
                   
               
               
                 Tryptophan Pathway Catabolic Enzymes 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 TDC: Tryptophan 
                 MGSIDSTNVAMSNSPVGEFKPLEAEEFRKQAHRMVDFIADYY 
               
               
                 decarboxylase from 
                 KNVETYPVLSEVEPGYLRKRIPETAPYLPEPLDDIMKDIQKDII 
               
               
                 Catharanthus roseus 
                 PGMTNWMSPNFYAFFPATVSSAAFLGEMLSTALNSVGFTWV 
               
               
                 SEQ ID NO: 141 
                 SSPAATELEMIVMDWLAQILKLPKSFMFSGTGGGVIQNTTSES 
               
               
                   
                 ILCTIIAARERALEKLGPDSIGKLVCYGSDQTHTMFPKTCKLA 
               
               
                   
                 GIYPNNIRLIPTTVETDFGISPQVLRKMVEDDVAAGYVPLFLC 
               
               
                   
                 ATLGTTSTTATDPVDSLSEIANEFGIWIHVDAAYAGSACICPEF 
               
               
                   
                 RHYLDGIERVDSLSLSPHKWLLAYLDCTCLWVKQPHLLLRAL 
               
               
                   
                 TTNPEYLKNKQSDLDKVVDFKNWQIATGRKFRSLKLWLILRS 
               
               
                   
                 YGVVNLQSHIRSDVAMGKMFEEWVRSDSRFEIVVPRNFSLVC 
               
               
                   
                 FRLKPDVSSLHVEEVNKKLLDMLNSTGRVYMTHTIVGGIYML 
               
               
                   
                 RLAVGSSLTEEHHVRRVWDLIQKLTDDLLKEA 
               
               
                   
               
               
                 TDC: Tryptophan 
                 MKFWRKYTQQEMDEKITESLEKTLNYDNTKTIGIPGTKLDDT 
               
               
                 decarboxylase from 
                 VFYDDHSFVKHSPYLRTFIQNPNHIGCHTYDKADILFGGTFDIE 
               
               
                 Clostridium 
                 RELIQLLAIDVLNGNDEEFDGYVTQGGTEANIQAMWVYRNY 
               
               
                 sporogenes 
                 FKKERKAKHEEIAIITSADTHYSAYKGSDLLNIDIIKVPVDFYS 
               
               
                 SEQ ID NO: 142 
                 RKIQENTLDSIVKEAKEIGKKYFIVISNMGTTMFGSVDDPDLY 
               
               
                   
                 ANIFDKYNLEYKIHVDGAFGGFIYPIDNKECKTDFSNKNVSSIT 
               
               
                   
                 LDGHKMLQAPYGTGIFVSRKNLIHNTLTKEATYIENLDVTLSG 
               
               
                   
                 SRSGSNAVAIWMVLASYGPYGWMEKINKLRNRTKWLCKQL 
               
               
                   
                 NDMRIKYYKEDSMNIVTIEEQYVNKEIAEKYFLVPEVHNPTN 
               
               
                   
                 NWYKIVVMEHVELDILNSLVYDLRKFNKEHLKAM 
               
               
                   
               
               
                 TYNA: Monoamine 
                 MGSPSLYSARKTTLALAVALSFAWQAPVFAHGGEAHMVPM 
               
               
                 oxidase from E. coli 
                 DKTLKEFGADVQWDDYAQLFTLIKDGAYVKVKPGAQTAIVN 
               
               
                 SEQ ID NO: 143 
                 GQPLALQVPVVMKDNKAWVSDTFINDVFQSGLDQTFQVEKR 
               
               
                   
                 PHPLNALTADEIKQAVEIVKASADFKPNTRFTEISLLPPDKEAV 
               
               
                   
                 WAFALENKPVDQPRKADVIMLDGKHIIEAVVDLQNNKLLSW 
               
               
                   
                 QPIKDAHGMVLLDDFASVQNIINNSEEFAAAVKKRGITDAKK 
               
               
                   
                 VITTPLTVGYFDGKDGLKQDARLLKVISYLDVGDGNYWAHPI 
               
               
                   
                 ENLVAVVDLEQKKIVKIEEGPVVPVPMTARPFDGRDRVAPAV 
               
               
                   
                 KPMQIIEPEGKNYTITGDMIHWRNWDFHLSMNSRVGPMISTV 
               
               
                   
                 TYNDNGTKRKVMYEGSLGGMIVPYGDPDIGWYFKAYLDSGD 
               
               
                   
                 YGMGTLTSPIARGKDAPSNAVLLNETIADYTGVPMEIPRAIAV 
               
               
                   
                 FERYAGPEYKHQEMGQPNVSTERRELVVRWISTVGNYDYIFD 
               
               
                   
                 WIFHENGTIGIDAGATGIEAVKGVKAKTMHDETAKDDTRYGT 
               
               
                   
                 LIDHNIVGTTHQHIYNFRLDLDVDGENNSLVAMDPVVKPNTA 
               
               
                   
                 GGPRTSTMQVNQYNIGNEQDAAQKFDPGTIRLLSNPNKENRM 
               
               
                   
                 GNPVSYQIIPYAGGTHPVAKGAQFAPDEWIYHRLSFMDKQLW 
               
               
                   
                 VTRYHPGERFPEGKYPNRSTHDTGLGQYSKDNESLDNTDAV 
               
               
                   
                 VWMTTGTTHVARAEEWPIMPTEWVHTLLKPWNFFDETPTLG 
               
               
                   
                 ALKKDK 
               
               
                   
               
               
                 AAO1: Indole-3- 
                 MGEKAIDEDKVEAMKSSKTSLVFAINGQRFELELSSIDPSTTL 
               
               
                 acetaldehyde oxidase 
                 VDFLRNKTPFKSVKLGCGEGGCGACVVLLSKYDPLLEKVDEF 
               
               
                 from Arabidopsis 
                 TISSCLTLLCSIDGCSITTSDGLGNSRVGFHAVHERIAGFHATQ 
               
               
                 thaliana 
                 CGFCTPGMSVSMFSALLNADKSHPPPRSGFSNLTAVEAEKAV 
               
               
                 SEQ ID NO: 144 
                 SGNLCRCTGYRPLVDACKSFAADVDIEDLGFNAFCKKGENRD 
               
               
                   
                 EVLRRLPCYDHTSSHVCTFPEFLKKEIKNDMSLHSRKYRWSSP 
               
               
                   
                 VSVSELQGLLEVENGLSVKLVAGNTSTGYYKEEKERKYERFI 
               
               
                   
                 DIRKIPEFTMVRSDEKGVELGACVTISKAIEVLREEKNVSVLA 
               
               
                   
                 KIATHMEKIANRFVRNTGTIGGNIMMAQRKQFPSDLATILVA 
               
               
                   
                 AQATVKIMTSSSSQEQFTLEEFLQQPPLDAKSLLLSLEIPSWHS 
               
               
                   
                 AKKNGSSEDSILLFETYRAAPRPLGNALAFLNAAFSAEVTEAL 
               
               
                   
                 DGIVVNDCQINFGAYGTKHAHRAKKVEEFLTGKVISDEVLM 
               
               
                   
                 EAISLLKDEIVPDKGTSNPGYRSSLAVTFLFEFFGSLTKKNAKT 
               
               
                   
                 TNGWLNGGCKEIGFDQNVESLKPEAMLSSAQQIVENQEHSPV 
               
               
                   
                 GKGITKAGACLQASGEAVYVDDIPAPENCLYGAFIYSTMPLA 
               
               
                   
                 RIKGIRFKQNRVPEGVLGIITYKDIPKGGQNIGTNGFFTSDLLF 
               
               
                   
                 AEEVTHCAGQIIAFLVADSQKHADIAANLVVIDYDTKDLKPPI 
               
               
                   
                 LSLEEAVENFSLFEVPPPLRGYPVGDITKGMDEAEHKILGSKIS 
               
               
                   
                 FGSQYFFYMETQTALAVPDEDNCMVVYSSTQTPEFVHQTIAG 
               
               
                   
                 CLGVPENNVRVITRRVGGGFGGKAVKSMPVAAACALAASK 
               
               
                   
                 MQRPVRTYVNRKTDMITTGGRHPMKVTYSVGFKSNGKITAL 
               
               
                   
                 DVEVLLDAGLTEDISPLMPKGIQGALMKYDWGALSFNVKVC 
               
               
                   
                 KTNTVSRTALRAPGDVQGSYIGEAIIEKVASYLSVDVDEIRKV 
               
               
                   
                 NLHTYESLRLFHSAKAGEFSEYTLPLLWDRIDEFSGFNKRRKV 
               
               
                   
                 VEEFNASNKWRKRGISRVPAVYAVNMRSTPGRVSVLGDGSIV 
               
               
                   
                 VEVQGIEIGQGLWTKVKQMAAYSLGLIQCGTTSDELLKKIRVI 
               
               
                   
                 QSDTLSMVQGSMTAGSTTSEASSEAVRICCDGLVERLLPVKT 
               
               
                   
                 ALVEQTGGFVTWDSLISQAYQQSINMSVSSKYMPDSTGEYLN 
               
               
                   
                 YGIAASEVEVNVLTGETTILRTDIIYDCGKSLNPAVDLGQIEGA 
               
               
                   
                 FVQGLGFFMLEEFLMNSDGLVVTDSTWTYKIPTVDTIPRQFN 
               
               
                   
                 VEILNSGQHKNRVLSSKASGEPPLLLAASVHCAVRAAVKEAR 
               
               
                   
                 KQILSWNSNKQGTDMYFELPVPATMPIVKEFCGLDVVEKYLE 
               
               
                   
                 WKIQQRKNV 
               
               
                   
               
               
                 ARO9: L-tryptophan 
                 MTAGSAPPVDYTSLKKNFQPFLSRRVENRSLKSFWDASDISD 
               
               
                 aminotransferase 
                 DVIELAGGMPNERFFPIESMDLKISKVPFNDNPKWHNSFTTAH 
               
               
                 from S. cerevisae 
                 LDLGSPSELPIARSFQYAETKGLPPLLHFVKDFVSRINRPAFSD 
               
               
                 SEQ ID NO: 145 
                 ETESNWDVILSGGSNDSMFKVFETICDESTTVMIEEFTFTPAM 
               
               
                   
                 SNVEATGAKVIPIKMNLTFDRESQGIDVEYLTQLLDNWSTGP 
               
               
                   
                 YKDLNKPRVLYTIATGQNPTGMSVPQWKREKIYQLAQRHDF 
               
               
                   
                 LIVEDDPYGYLYFPSYNPQEPLENPYHSSDLTTERYLNDFLMK 
               
               
                   
                 SFLTLDTDARVIRLETFSKIFAPGLRLSFIVANKFLLQKILDLAD 
               
               
                   
                 ITTRAPSGTSQAIVYSTIKAMAESNLSSSLSMKEAMFEGWIRW 
               
               
                   
                 IMQIASKYNHRKNLTLKALYETESYQAGQFTVMEPSAGMFIII 
               
               
                   
                 KINWGNFDRPDDLPQQMDILDKFLLKNGVKVVLGYKMAVCP 
               
               
                   
                 NYSKQNSDFLRLTIAYARDDDQLIEASKRIGSGIKEFFDNYKS 
               
               
                   
               
               
                 aspC: aspartate 
                 MFENITAAPADPILGLADLFRADERPGKINLGIGVYKDETGKT 
               
               
                 aminotransferase 
                 PVLTSVKKAEQYLLENETTKNYLGIDGIPEFGRCTQELLFGKG 
               
               
                 from E. coli 
                 SALINDKRARTAQTPGGTGALRVAADFLAKNTSVKRVWVSN 
               
               
                 SEQ ID NO: 146 
                 PSWPNHKSVFNSAGLEVREYAYYDAENHTLDFDALINSLNEA 
               
               
                   
                 QAGDVVLFHGCCHNPTGIDPTLEQWQTLAQLSVEKGWLPLF 
               
               
                   
                 DFAYQGFARGLEEDAEGLRAFAAMHKELIVASSYSKNFGLYN 
               
               
                   
                 ERVGACTLVAADSETVDRAFSQMKAAIRANYSNPPAHGASV 
               
               
                   
                 VATILSNDALRAIWEQELTDMRQRIQRMRQLFVNTLQEKGAN 
               
               
                   
                 RDFSFIIKQNGMFSFSGLTKEQVLRLREEFGVYAVASGRVNVA 
               
               
                   
                 GMTPDNMAPLCEAIVAVL 
               
               
                   
               
               
                 TAA1: L-tryptophan- 
                 MVKLENSRKPEKISNKNIPMSDFVVNLDHGDPTAYEEYWRK 
               
               
                 pyruvate 
                 MGDRCTVTIRGCDLMSYFSDMTNLCWFLEPELEDAIKDLHGV 
               
               
                 aminotransferase 
                 VGNAATEDRYIVVGTGSTQLCQAAVHALSSLARSQPVSVVA 
               
               
                 from Arabidopsis 
                 AAPFYSTYVEETTYVRSGMYKWEGDAWGFDKKGPYIELVTS 
               
               
                 thaliana 
                 PNNPDGTIRETVVNRPDDDEAKVIHDFAYYWPHYTPITRRQD 
               
               
                 SEQ ID NO: 147 
                 HDIMLFTFSKITGHAGSRIGWALVKDKEVAKKMVEYIIVNSIG 
               
               
                   
                 VSKESQVRTAKILNVLKETCKSESESENFFKYGREMMKNRWE 
               
               
                   
                 KLREVVKESDAFTLPKYPEAFCNYFGKSLESYPAFAWLGTKE 
               
               
                   
                 ETDLVSELRRHKVMSRAGERCGSDKKHVRVSMLSREDVFNV 
               
               
                   
                 FLERLANMKLIKSIDL 
               
               
                   
               
               
                 STAO: L-tryptophan 
                 MTAPLQDSDGPDDAIGGPKQVTVIGAGIAGLVTAYELERLGH 
               
               
                 oxidase from 
                 HVQIIEGSDDIGGRIHTHRFSGAGGPGPFAEMGAMRIPAGHRL 
               
               
                 streptomyces sp. TP- 
                 TMHYIAELGLQNQVREFRTLFSDDAAYLPSSAGYLRVREAHD 
               
               
                 A0274 
                 TLVDEFATGLPSAHYRQDTLLFGAWLDASIRAIAPRQFYDGL 
               
               
                 SEQ ID NO: 148 
                 HNDIGVELLNLVDDIDLTPYRCGTARNRIDLHALFADHPRVR 
               
               
                   
                 ASCPPRLERFLDDVLDETSSSIVRLKDGMDELPRRLASRIRGKI 
               
               
                   
                 SLGQEVTGIDVHDDTVTLTVRQGLRTVTRTCDYVVCTIPFTVL 
               
               
                   
                 RTLRLTGFDQDKLDIVHETKYWPATKIAFHCREPFWEKDGIS 
               
               
                   
                 GGASFTGGHVRQTYYPPAEGDPALGAVLLASYTIGPDAEALA 
               
               
                   
                 RMDEAERDALVAKELSVMHPELRRPGMVLAVAGRDWGARR 
               
               
                   
                 WSRGAATVRWGQEAALREAERRECARPQKGLFFAGEHCSSK 
               
               
                   
                 PAWIEGAIESAIDAAHEIEWYEPRASRVFAASRLSRSDRSA 
               
               
                   
               
               
                 ipdC: Indole-3- 
                 MRTPYCVADYLLDRLTDCGADHLFGVPGDYNLQFLDHVIDS 
               
               
                 pyruvate 
                 PDICWVGCANELNASYAADGYARCKGFAALLTTFGVGELSA 
               
               
                 decarboxylase from 
                 MNGIAGSYAEHVPVLHIVGAPGTAAQQRGELLHHTLGDGEFR 
               
               
                 Enterobacter cloacae 
                 HFYHMSEPITVAQAVLTEQNACYEIDRVLTTMLRERRPGYLM 
               
               
                 SEQ ID NO: 149 
                 LPADVAKKAATPPVNALTHKQAHADSACLKAFRDAAENKLA 
               
               
                   
                 MSKRTALLADFLVLRHGLKHALQKWVKEVPMAHATMLMG 
               
               
                   
                 KGIFDERQAGFYGTYSGSASTGAVKEAIEGADTVLCVGTRFT 
               
               
                   
                 DTLTAGFTHQLTPAQTIEVQPHAARVGDVWFTGIPMNQAIET 
               
               
                   
                 LVELCKQHVHAGLMSSSSGAIPFPQPDGSLTQENFWRTLQTFI 
               
               
                   
                 RPGDIILADQGTSAFGAIDLRLPADVNFIVQPLWGSIGYTLAA 
               
               
                   
                 AFGAQTACPNRRVIVLTGDGAAQLTIQELGSMLRDKQHPIILV 
               
               
                   
                 LNNEGYTVERAIHGAEQRYNDIALWNWTHIPQALSLDPQSEC 
               
               
                   
                 WRVSEAEQLADVLEKVAHHERLSLIEVMLPKADIPPLLGALT 
               
               
                   
                 KALEACNNA 
               
               
                   
               
               
                 IAD1 : Indole-3- 
                 MPTLNLDLPNGIKSTIQADLFINNKFVPALDGKTFATINPSTGK 
               
               
                 acetaldehyde 
                 EIGQVAEASAKDVDLAVKAAREAFETTWGENTPGDARGRLLI 
               
               
                 dehydrogenase from 
                 KLAELVEANIDELAAIESLDNGKAFSIAKSFDVAAVAANLRY 
               
               
                 Ustilago maydis 
                 YGGWADKNHGKVMEVDTKRLNYTRHEPIGVCGQIIPWNFPL 
               
               
                 SEQ ID NO: 150 
                 LMFAWKLGPALATGNTIVLKTAEQTPLSAIKMCELIVEAGFPP 
               
               
                   
                 GVVNVISGFGPVAGAAISQHMDIDKIAFTGSTLVGRNIMKAA 
               
               
                   
                 ASTNLKKVTLELGGKSPNIIFKDADLDQAVRWSAFGIMFNHG 
               
               
                   
                 QCCCAGSRVYVEESIYDAFMEKMTAHCKALQVGDPFSANTF 
               
               
                   
                 QGPQVSQLQYDRIMEYIESGKKDANLALGGVRKGNEGYFIEP 
               
               
                   
                 TIFTDVPHDAKIAKEEIFGPVVVVSKFKDEKDLIRIANDSIYGL 
               
               
                   
                 AAAVFSRDISRAIETAHKLKAGTVWVNCYNQLIPQVPFGGYK 
               
               
                   
                 ASGIGRELGEYALSNYTNIKAVHVNLSQPAPI 
               
               
                   
               
               
                 YUC2: indole-3- 
                 MEFVTETLGKRIHDPYVEETRCLMIPGPIIVGSGPSGLATAACL 
               
               
                 pyruvate 
                 KSRDIPSLILERSTCIASLWQHKTYDRLRLHLPKDFCELPLMPF 
               
               
                 monoxygenase from 
                 PSSYPTYPTKQQFVQYLESYAEHFDLKPVFNQTVEEAKFDRR 
               
               
                 Arabidopsis thaliana 
                 CGLWRVRTTGGKKDETMEYVSRWLVVATGENAEEVMPEID 
               
               
                 SEQ ID NO: 151 
                 GIPDFGGPILHTSSYKSGEIFSEKKILVVGCGNSGMEVCLDLCN 
               
               
                   
                 FNALPSLVVRDSVHVLPQEMLGISTFGISTSLLKWFPVHVVDR 
               
               
                   
                 FLLRMSRLVLGDTDRLGLVRPKLGPLERKIKCGKTPVLDVGT 
               
               
                   
                 LAKIRSGHIKVYPELKRVMHYSAEFVDGRVDNFDAIILATGY 
               
               
                   
                 KSNVPMWLKGVNMFSEKDGFPHKPFPNGWKGESGLYAVGF 
               
               
                   
                 TKLGLLGAAIDAKKIAEDIEVQRHFLPLARPQHC 
               
               
                   
               
               
                 IaaM: Tryptophan 2- 
                 MYDHFNSPSIDILYDYGPFLKKCEMTGGIGSYSAGTPTPRVAI 
               
               
                 monooxygenase from 
                 VGAGISGLVAATELLRAGVKDVVLYESRDRIGGRVWSQVFD 
               
               
                 Pseudomonas 
                 QTRPRYIAEMGAMRFPPSATGLFHYLKKFGISTSTTFPDPGVV 
               
               
                 savastanoi 
                 DTELHYRGKRYHWPAGKKPPELFRRVYEGWQSLLSEGYLLE 
               
               
                 SEQ ID NO: 152 
                 GGSLVAPLDITAMLKSGRLEEAAIAWQGWLNVFRDCSFYNAI 
               
               
                   
                 VCIFTGRHPPGGDRWARPEDFELFGSLGIGSGGFLPVFQAGFT 
               
               
                   
                 EILRMVINGYQSDQRLIPDGISSLAARLADQSFDGKALRDRVC 
               
               
                   
                 FSRVGRISREAEKIIIQTEAGEQRVFDRVIVTSSNRAMQMIHCL 
               
               
                   
                 TDSESFLSRDVARAVRETHLTGSSKLFILTRTKFWIKNKLPTTI 
               
               
                   
                 QSDGLVRGVYCLDYQPDEPEGHGVVLLSYTWEDDAQKMLA 
               
               
                   
                 MPDKKTRCQVLVDDLAAIHPTFASYLLPVDGDYERYVLHHD 
               
               
                   
                 WLTDPHSAGAFKLNYPGEDVYSQRLFFQPMTANSPNKDTGL 
               
               
                   
                 YLAGCSCSFAGGWIEGAVQTALNSACAVLRSTGGQLSKGNPL 
               
               
                   
                 DCINASYRY 
               
               
                   
               
               
                 iaaH: 
                 MHEIITLESLCQALADGEIAAAELRERALDTEARLARLNCFIRE 
               
               
                 Indoleacetamide 
                 GDAVSQFGEADHAMKGTPLWGMPVSFKDNICVRGLPLTAGT 
               
               
                 hydrolase from 
                 RGMSGFVSDQDAAIVSQLRALGAVVAGKNNMHELSFGVTSI 
               
               
                 Pseudomonas 
                 NPHWGTVGNPVAPGYCAGGSSGGSAAAVASGIVPLSVGTDT 
               
               
                 savastanoi 
                 GGSIRIPAAFCGITGFRPTTGRWSTAGIIPVSHTKDCVGLLTRT 
               
               
                 SEQ ID NO: 153 
                 AGDAGFLYGLLSGKQQSFPLSRTAPCRIGLPVSMWSDLDGEV 
               
               
                   
                 ERACVNALSLLRKTGFEFIEIDDADIVELNQTLTFTVPLYEFFA 
               
               
                   
                 DLAQSLLSLGWKHGIHHIFAQVDDANVKGIINHHLGEGAIKP 
               
               
                   
                 AHYLSSLQNGELLKRKMDELFARHNIELLGYPTVPCRVPHLD 
               
               
                   
                 HADRPEFFSQAIRNTDLASNAMLPSITIPVGPEGRLPVGLSFDA 
               
               
                   
                 LRGRDALLLSRVSAIEQVLGFVRKVLPHTT 
               
               
                   
               
               
                 TrpDH: Tryptophan 
                 MLLFETVREMGHEQVLFCHSKNPEIKAIIAIHDTTLGPAMGAT 
               
               
                 dehydrogenase from 
                 RILPYINEEAALKDALRLSRGMTYKAACANIPAGGGKAVIIAN 
               
               
                 Nostoc punctiforme 
                 PENKTDDLLRAYGRFVDSLNGRFITGQDVNITPDDVRTISQET 
               
               
                 NIES-2108 
                 KYVVGVSEKSGGPAPITSLGVFLGIKAAVESRWQSKRLDGMK 
               
               
                 SEQ ID NO: 154 
                 VAVQGLGNVGKNLCRHLHEHDVQLFVSDVDPIKAEEVKRLF 
               
               
                   
                 GATVVEPTEIYSLDVDIFAPCALGGILNSHTIPFLQASIIAGAAN 
               
               
                   
                 NQLENEQLHSQMLAKKGILYSPDYVINAGGLINVYNEMIGYD 
               
               
                   
                 EEKAFKQVHNIYDTLLAIFEIAKEQGVTTNDAARRLAEDRINN 
               
               
                   
                 SKRSKSKAIAA 
               
               
                   
               
               
                 CYP79B2: 
                 MNTFTSNSSDLTTTATETSSFSTLYLLSTLQAFVAITLVMLLKK 
               
               
                 tryptophan N- 
                 LMTDPNKKKPYLPPGPTGWPIIGMIPTMLKSRPVFRWLHSIMK 
               
               
                 monooxygenase from 
                 QLNTEIACVKLGNTHVITVTCPKIAREILKQQDALFASRPLTY 
               
               
                 Arabidopsis thaliana 
                 AQKILSNGYKTCVITPFGDQFKKMRKVVMTELVCPARHRWL 
               
               
                 SEQ ID NO: 155 
                 HQKRSEENDHLTAWVYNMVKNSGSVDFRFMTRHYCGNAIK 
               
               
                   
                 KLMFGTRTFSKNTAPDGGPTVEDVEHMEAMFEALGFTFAFCI 
               
               
                   
                 SDYLPMLTGLDLNGHEKIMRESSAIMDKYHDPIIDERIKMWR 
               
               
                   
                 EGKRTQIEDFLDIFISIKDEQGNPLLTADEIKPTIKELVMAAPDN 
               
               
                   
                 PSNAVEWAMAEMVNKPEILRKAMEEIDRVVGKERLVQESDIP 
               
               
                   
                 KLNYVKAILREAFRLHPVAAFNLPHVALSDTTVAGYHIPKGS 
               
               
                   
                 QVLLSRYGLGRNPKVWADPLCFKPERHLNECSEVTLTENDLR 
               
               
                   
                 FISFSTGKRGCAAPALGTALTTMMLARLLQGFTWKLPENETR 
               
               
                   
                 VELMESSHDMFLAKPLVMVGDLRLPEHLYPTVK 
               
               
                   
               
               
                 CYP79B3: 
                 MDTLASNSSDLTTKSSLGMSSFTNMYLLTTLQALAALCFLMI 
               
               
                 tryptophan N- 
                 LNKIKSSSRNKKLHPLPPGPTGFPIVGMIPAMLKNRPVFRWLH 
               
               
                 monooxygenase from 
                 SLMKELNTEIACVRLGNTHVIPVTCPKIAREIFKQQDALFASRP 
               
               
                 Arabidopsis thaliana 
                 LTYAQKILSNGYKTCVITPFGEQFKKMRKVIMTEIVCPARHR 
               
               
                 SEQ ID NO: 156 
                 WLHDNRAEETDHLTAWLYNMVKNSEPVDLRFVTRHYCGNA 
               
               
                   
                 IKRLMFGTRTFSEKTEADGGPTLEDIEHMDAMFEGLGFTFAFC 
               
               
                   
                 ISDYLPMLTGLDLNGHEKIMRESSAIMDKYHDPIIDERIKMWR 
               
               
                   
                 EGKRTQIEDFLDIFISIKDEAGQPLLTADEIKPTIKELVMAAPDN 
               
               
                   
                 PSNAVEWAIAEMINKPEILHKAMEEIDRVVGKERFVQESDIPK 
               
               
                   
                 LNYVKAIIREAFRLHPVAAFNLPHVALSDTTVAGYHIPKGSQV 
               
               
                   
                 LLSRYGLGRNPKVWSDPLSFKPERHLNECSEVTLTENDLRFIS 
               
               
                   
                 FSTGKRGCAAPALGTAITTMMLARLLQGFKWKLAGSETRVE 
               
               
                   
                 LMESSHDMFLSKPLVLVGELRLSEDLYPMVK 
               
               
                   
               
               
                 CYP71A13: 
                 MSNIQEMEMILSISLCLTTLITLLLLRRFLKRTATKVNLPPSPW 
               
               
                 indoleacetaldoxime 
                 RLPVIGNLHQLSLHPHRSLRSLSLRYGPLMLLHFGRVPILVVSS 
               
               
                 dehydratase from 
                 GEAAQEVLKTHDHKFANRPRSKAVHGLMNGGRDVVFAPYG 
               
               
                 Arabidopis thaliana 
                 EYWRQMKSVCILNLLTNKMVESFEKVREDEVNAMIEKLEKA 
               
               
                 SEQ ID NO: 157 
                 SSSSSSENLSELFITLPSDVTSRVALGRKHSEDETARDLKKRVR 
               
               
                   
                 QIMELLGEFPIGEYVPILAWIDGIRGFNNKIKEVSRGFSDLMDK 
               
               
                   
                 VVQEHLEASNDKADFVDILLSIEKDKNSGFQVQRNDIKFMILD 
               
               
                   
                 MFIGGTSTTSTLLEWTMTELIRSPKSMKKLQDEIRSTIRPHGSY 
               
               
                   
                 IKEKEVENMKYLKAVIKEVLRLHPSLPMILPRLLSEDVKVKGY 
               
               
                   
                 NIAAGTEVIINAWAIQRDTAIWGPDAEEFKPERHLDSGLDYHG 
               
               
                   
                 KNLNYIPFGSGRRICPGINLALGLAEVTVANLVGRFDWRVEA 
               
               
                   
                 GPNGDQPDLTEAIGIDVCRKFPLIAFPSSVV 
               
               
                   
               
               
                 PEN2: myrosinase 
                 MAHLQRTFPTEMSKGRASFPKGFLFGTASSSYQYEGAVNEGA 
               
               
                 from Arabidopsis 
                 RGQSVWDHFSNRFPHRISDSSDGNVAVDFYHRYKEDIKRMK 
               
               
                 thaliana 
                 DINMDSFRLSIAWPRVLPYGKRDRGVSEEGIKFYNDVIDELLA 
               
               
                 SEQ ID NO: 158 
                 NEITPLVTIFHWDIPQDLEDEYGGFLSEQIIDDFRDYASLCFERF 
               
               
                   
                 GDRVSLWCTMNEPWVYSVAGYDTGRKAPGRCSKYVNGASV 
               
               
                   
                 AGMSGYEAYIVSHNMLLAHAEAVEVFRKCDHIKNGQIGIAHN 
               
               
                   
                 PLWYEPYDPSDPDDVEGCNRAMDFMLGWHQHPTACGDYPE 
               
               
                   
                 TMKKSVGDRLPSFTPEQSKKLIGSCDYVGINYYSSLFVKSIKH 
               
               
                   
                 VDPTQPTWRTDQGVDWMKTNIDGKQIAKQGGSEWSFTYPTG 
               
               
                   
                 LRNILKYVKKTYGNPPILITENGYGEVAEQSQSLYMYNPSIDT 
               
               
                   
                 ERLEYIEGHIHAIHQAIHEDGVRVEGYYVWSLLDNFEWNSGY 
               
               
                   
                 GVRYGLYYIDYKDGLRRYPKMSALWLKEFLRFDQEDDSSTS 
               
               
                   
                 KKEEKKESYGKQLLHSVQDSQFVHSIKDSGALPAVLGSLFVV 
               
               
                   
                 SATVGTSLFFKGANN 
               
               
                   
               
               
                 Nit1: Nitrilase from 
                 MSSTKDMSTVQNATPFNGVAPSTTVRVTIVQSSTVYNDTPATI 
               
               
                 Arabidopsis thaliana 
                 DKAEKYIVEAASKGAELVLFPEGFIGGYPRGFRFGLAVGVHN 
               
               
                 SEQ ID NO: 159 
                 EEGRDEFRKYHASAIHVPGPEVARLADVARKNHVYLVMGAI 
               
               
                   
                 EKEGYTLYCTVLFFSPQGQFLGKHRKLMPTSLERCIWGQGDG 
               
               
                   
                 STIPVYDTPIGKLGAAICWENRMPLYRTALYAKGIELYCAPTA 
               
               
                   
                 DGSKEWQSSMLHIAIEGGCFVLSACQFCQRKHFPDHPDYLFT 
               
               
                   
                 DWYDDKEHDSIVSQGGSVIISPLGQVLAGPNFESEGLVTADID 
               
               
                   
                 LGDIARAKLYFDSVGHYSRPDVLHLTVNEHPRKSVTFVTKVE 
               
               
                   
                 KAEDDSNK 
               
               
                   
               
               
                 IDO1: indoleamine 
                 MAHAMENSWTISKEYHIDEEVGFALPNPQENLPDFYNDWMFI 
               
               
                 2,3-dioxygenase from 
                 AKHLPDLIESGQLRERVEKLNMLSIDHLTDHKSQRLARLVLG 
               
               
                 homo sapiens 
                 CITMAYVWGKGHGDVRKVLPRNIAVPYCQLSKKLELPPILVY 
               
               
                 SEQ ID NO: 160 
                 ADCVLANWKKKDPNKPLTYENMDVLFSFRDGDCSKGFFLVS 
               
               
                   
                 LLVEIAAASAIKVIPTVFKAMQMQERDTLLKALLEIASCLEKA 
               
               
                   
                 LQVFHQIHDHVNPKAFFSVLRIYLSGWKGNPQLSDGLVYEGF 
               
               
                   
                 WEDPKEFAGGSAGQSSVFQCFDVLLGIQQTAGGGHAAQFLQ 
               
               
                   
                 DMRRYMPPAHRNFLCSLESNPSVREFVLSKGDAGLREAYDA 
               
               
                   
                 CVKALVSLRSYHLQIVTKYILIPASQQPKENKTSEDPSKLEAK 
               
               
                   
                 GTGGTDLMNFLKTVRSTTEKSLLKEG 
               
               
                   
               
               
                 TDO2: tryptophan 
                 MSGCPFLGNNFGYTFKKLPVEGSEEDKSQTGVNRASKGGLIY 
               
               
                 2,3-dioxygenase from 
                 GNYLHLEKVLNAQELQSETKGNKIHDEHLFIITHQAYELWFK 
               
               
                 homo sapiens 
                 QILWELDSVREIFQNGHVRDERNMLKVVSRMHRVSVILKLLV 
               
               
                 SEQ ID NO: 161 
                 QQFSILETMTALDFNDFREYLSPASGFQSLQFRLLENKIGVLQ 
               
               
                   
                 NMRVPYNRRHYRDNFKGEENELLLKSEQEKTLLELVEAWLE 
               
               
                   
                 RTPGLEPHGFNFWGKLEKNITRGLEEEFIRIQAKEESEEKEEQV 
               
               
                   
                 AEFQKQKEVLLSLFDEKRHEHLLSKGERRLSYRALQGALMIY 
               
               
                   
                 FYREEPRFQVPFQLLTSLMDIDSLMTKWRYNHVCMVHRMLG 
               
               
                   
                 SKAGTGGSSGYHYLRSTVSDRYKVFVDLFNLSTYLIPRHWIPK 
               
               
                   
                 MNPTIHKFLYTAEYCDSSYFSSDESD 
               
               
                   
               
               
                 BNA2: indoleamine 
                 MNNTSITGPQVLHRTKMRPLPVLEKYCISPHHGFLDDRLPLTR 
               
               
                 2,3-dioxygenase from 
                 LSSKKYMKWEEIVADLPSLLQEDNKVRSVIDGLDVLDLDETIL 
               
               
                 S. cerevisiae 
                 GDVRELRRAYSILGFMAHAYIWASGTPRDVLPECIARPLLETA 
               
               
                 SEQ ID NO: 162 
                 HILGVPPLATYSSLVLWNFKVTDECKKTETGCLDLENITTINTF 
               
               
                   
                 TGTVDESWFYLVSVRFEKIGSACLNHGLQILRAIRSGDKGDA 
               
               
                   
                 NVIDGLEGLAATIERLSKALMEMELKCEPNVFYFKIRPFLAGW 
               
               
                   
                 TNMSHMGLPQGVRYGAEGQYRIFSGGSNAQSSLIQTLDILLG 
               
               
                   
                 VKHTANAAHSSQGDSKINYLDEMKKYMPREHREFLYHLESV 
               
               
                   
                 CNIREYVSRNASNRALQEAYGRCISMLKIFRDNHIQIVTKYIIL 
               
               
                   
                 PSNSKQHGSNKPNVLSPIEPNTKASGCLGHKVASSKTIGTGGT 
               
               
                   
                 RLMPFLKQCRDETVATADIKNEDKN 
               
               
                   
               
               
                 Afmid: Kynurenine 
                 MAFPSLSAGQNPWRNLSSEELEKQYSPSRWVIHTKPEEVVGN 
               
               
                 formamidase from 
                 FVQIGSQATQKARATRRNQLDVPYGDGEGEKLDIYFPDEDSK 
               
               
                 mouse 
                 AFPLFLFLHGGYWQSGSKDDSAFMVNPLTAQGIVVVIVAYDI 
               
               
                 SEQ ID NO: 163 
                 APKGTLDQMVDQVTRSVVFLQRRYPSNEGIYLCGHSAGAHL 
               
               
                   
                 AAMVLLARWTKHGVTPNLQGFLLVSGIYDLEPLIATSQNDPL 
               
               
                   
                 RMTLEDAQRNSPQRHLDVVPAQPVAPACPVLVLVGQHDSPE 
               
               
                   
                 FHRQSKEFYETLLRVGWKASFQQLRGVDHFDIIENLTREDDV 
               
               
                   
                 LTQIILKTVFQKL 
               
               
                   
               
               
                 BNA3: kynurenine-- 
                 MKQRFIRQFTNLMSTSRPKVVANKYFTSNTAKDVWSLTNEA 
               
               
                 oxoglutarate 
                 AAKAANNSKNQGRELINLGQGFFSYSPPQFAIKEAQKALDIPM 
               
               
                 transaminase from S. 
                 VNQYSPTRGRPSLINSLIKLYSPIYNTELKAENVTVTTGANEGI 
               
               
                 cerevisae 
                 LSCLMGLLNAGDEVIVFEPFFDQYIPNIELCGGKVVYVPINPPK 
               
               
                 SEQ ID NO: 164 
                 ELDQRNTRGEEWTIDFEQFEKAITSKTKAVIINTPHNPIGKVFT 
               
               
                   
                 REELTTLGNICVKHNVVIISDEVYEHLYFTDSFTRIATLSPEIGQ 
               
               
                   
                 LTLTVGSAGKSFAATGWRIGWVLSLNAELLSYAAKAHTRICF 
               
               
                   
                 ASPSPLQEACANSINDALKIGYFEKMRQEYINKFKIFTSIFDEL 
               
               
                   
                 GLPYTAPEGTYFVLVDFSKVKIPEDYPYPEEILNKGKDFRISH 
               
               
                   
                 WLINELGVVAIPPTEFYIKEHEKAAENLLRFAVCKDDAYLEN 
               
               
                   
                 AVERLKLLKDYL 
               
               
                   
               
               
                 GOT2: Aspartate 
                 MALLHSGRVLPGIAAAFHPGLAAAASARASSWWTHVEMGPP 
               
               
                 aminotransferase, 
                 DPILGVTEAFKRDTNSKKMNLGVGAYRDDNGKPYVLPSVRK 
               
               
                 mitochondrial from 
                 AEAQIAAKNLDKEYLPIGGLAEFCKASAELALGENSEVLKSG 
               
               
                 homo sapiens 
                 RFVTVQTISGTGALRIGASFLQRFFKFSRDVFLPKPTWGNHTPI 
               
               
                 SEQ ID NO: 165 
                 FRDAGMQLQGYRYYDPKTCGFDFTGAVEDISKIPEQSVLLLH 
               
               
                   
                 ACAHNPTGVDPRPEQWKEIATVVKKRNLFAFFDMAYQGFAS 
               
               
                   
                 GDGDKDAWAVRHFIEQGINVCLCQSYAKNMGLYGERVGAFT 
               
               
                   
                 MVCKDADEAKRVESQLKILIRPMYSNPPLNGARIAAAILNTPD 
               
               
                   
                 LRKQWLQEVKVMADRIIGMRTQLVSNLKKEGSTHNWQHITD 
               
               
                   
                 QIGMFCFTGLKPEQVERLIKEFSIYMTKDGRISVAGVTSSNVG 
               
               
                   
                 YLAHAIHQVTK 
               
               
                   
               
               
                 AADAT: 
                 MNYARFITAASAARNPSPIRTMTDILSRGPKSMISLAGGLPNP 
               
               
                 Kynurenine/alpha- 
                 NMFPFKTAVITVENGKTIQFGEEMMKRALQYSPSAGIPELLSW 
               
               
                 aminoadipate 
                 LKQLQIKLHNPPTIHYPPSQGQMDLCVTSGSQQGLCKVFEMII 
               
               
                 aminotransferase, 
                 NPGDNVLLDEPAYSGTLQSLHPLGCNIINVASDESGIVPDSLR 
               
               
                 mitochondrial 
                 DILSRWKPEDAKNPQKNTPKFLYTVPNGNNPTGNSLTSERKK 
               
               
                 SEQ ID NO: 166 
                 EIYELARKYDFLIIEDDPYYFLQFNKFRVPTFLSMDVDGRVIRA 
               
               
                   
                 DSFSKIISSGLRIGFLTGPKPLIERVILHIQVSTLHPSTFNQLMIS 
               
               
                   
                 QLLHEWGEEGFMAHVDRVIDFYSNQKDAILAAADKWLTGLA 
               
               
                   
                 EWHVPAAGMFLWIKVKGINDVKELIEEKAVKMGVLMLPGN 
               
               
                   
                 AFYVDSSAPSPYLRASFSSASPEQMDVAFQVLAQLIKESL 
               
               
                   
               
               
                 CCLB1: Kynurenine- 
                 MAKQLQARRLDGIDYNPWVEFVKLASEHDVVNLGQGFPDFP 
               
               
                 -oxoglutarate 
                 PPDFAVEAFQHAVSGDFMLNQYTKTFGYPPLTKILASFFGELL 
               
               
                 transaminase 1 from 
                 GQEIDPLRNVLVTVGGYGALFTAFQALVDEGDEVIIIEPFFDC 
               
               
                 homo sapiens 
                 YEPMTMMAGGRPVFVSLKPGPIQNGELGSSSNWQLDPMELA 
               
               
                 SEQ ID NO: 167 
                 GKFTSRTKALVLNTPNNPLGKVFSREELELVASLCQQHDVVCI 
               
               
                   
                 TDEVYQWMVYDGHQHISIASLPGMWERTLTIGSAGKTFSATG 
               
               
                   
                 WKVGWVLGPDHIMKHLRTVHQNSVFHCPTQSQAAVAESFER 
               
               
                   
                 EQLLFRQPSSYFVQFPQAMQRCRDHMIRSLQSVGLLPIIPQGS 
               
               
                   
                 YFLITDISDFKRKMPDLPGAVDEPYDRRFVKWMIKNKGLVAI 
               
               
                   
                 PVSIFYSVPHQKHFDHYIRFCFVKDEATLQAMDEKLRKWKVE 
               
               
                 L 
                   
               
               
                   
               
               
                 CCLB2: kynurenine-- 
                 MFLAQRSLCSLSGRAKFLKTISSSKILGFSTSAKMSLKFTNAKR 
               
               
                 oxoglutarate 
                 IEGLDSNVWIEFTKLAADPSVVNLGQGFPDISPPTYVKEELSKI 
               
               
                   
                 AAIDSLNQYTRGFGHPSLVKALSYLYEKLYQKQIDSNKEILVT 
               
               
                 transaminase 3 from 
                 VGAYGSLFNTIQALIDEGDEVILIVPFYDCYEPMVRMAGATPV 
               
               
                 homo sapiens 
                 FIPLRSKPVYGKRWSSSDWTLDPQELESKFNSKTKAIILNTPHN 
               
               
                 SEQ ID NO: 168 
                 PLGKVYNREELQVIADLCIKYDTLCISDEVYEWLVYSGNKHL 
               
               
                   
                 KIATFPGMWERTITIGSAGKTFSVTGWKLGWSIGPNHLIKHLQ 
               
               
                   
                 TVQQNTIYTCATPLQEALAQAFWIDIKRMDDPECYFNSLPKEL 
               
               
                   
                 EVKRDRMVRLLESVGLKPIVPDGGYFIIADVSLLDPDLSDMK 
               
               
                   
                 NNEPYDYKFVKWMTKHKKLSAIPVSAFCNSETKSQFEKFVRF 
               
               
                   
                 CFIKKDSTLDAAEEIIKAWSVQKS 
               
               
                   
               
               
                 TnaA: tryptophanase 
                 MENFKHLPEPFRIRVIEPVKRTTRAYREEAIIKSGMNPFLLDSE 
               
               
                 from E. coli 
                 DVFIDLLTDSGTGAVTQSMQAAMMRGDEAYSGSRSYYALAE 
               
               
                 SEQ ID NO: 140 
                 SVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKREQEKGLDRSKM 
               
               
                   
                 VAFSNYFFDTTQGHSQINGCTVRNVYIKEAFDTGVRYDFKGN 
               
               
                   
                 FDLEGLERGIEEVGPNNVPYIVATITSNSAGGQPVSLANLKAM 
               
               
                   
                 YSIAKKYDIPVVMDSARFAENAYFIKQREAEYKDWTIEQITRE 
               
               
                   
                 TYKYADMLAMSAKKDAMVPMGGLLCMKDDSFFDVYTECRT 
               
               
                   
                 LCVVQEGFPTYGGLEGGAMERLAVGLYDGMNLDWLAYRIA 
               
               
                   
                 QVQYLVDGLEEIGVVCQQAGGHAAFVDAGKLLPHIPADQFP 
               
               
                   
                 AQALACELYKVAGIRAVEIGSFLLGRDPKTGKQLPCPAELLRL 
               
               
                   
                 TIPRATYTQTHMDFIIEAFKHVKENAANIKGLTFTYEPKVLRH 
               
               
                   
                 FTAKLKEV 
               
               
                   
               
               
                 Trp 
                 MTATTISIETVPQAPAAGTKTNGTSGKYNPRTYLSDRAKVTEI 
               
               
                 aminotransferase 
                 DGSDAGRPNPDTFPFNSITLNLKPPLGLPESSNNMPVSITIEDPD 
               
               
                 (EC 2.6.1.27); 
                 LATALQYAPSAGIPKLREWLADLQAHVHERPRGDYAISVGSG 
               
               
                 tryptophan 
                 SQDLMFKGFQAVLNPGDPVLLETPMYSGVLPALRILKADYAE 
               
               
                 aminotransferase 
                 VDVDDQGLSAKNLEKVLSEWPADKKRPRVLYTSPIGSNPSGC 
               
               
                 +Cryptococcus 
                 SASKERKLEVLKVCKKYDVLIFEDDPYYYLAQELIPSYFALEK 
               
               
                 deuterogattii R265 
                 QVYPEGGHVVRFDSFSKLLSAGMRLGFATGPKEILHAIDVSTA 
               
               
                 SEQ ID NO: 169 
                 GANLHTSAVSQGVALRLMQYWGIEGFLAHGRAVAKLYTERR 
               
               
                   
                 AQFEATAHKYLDGLATWVSPVAGMFLWIDLRPAGIEDSYELI 
               
               
                   
                 RHEALAKGVLGVPGMAFYPTGRKSSHVRVSFSIVDLEDESDL 
               
               
                   
                 GFQRLAEAIKDKRKALGLA 
               
               
                   
               
               
                 Tryptophan 
                 MSQVIKKKRNTFMIGTEYILNSTQLEEAIKSFVHDFCAEKHEIH 
               
               
                 Decarboxylase (EC 
                 DQPVVVEAKEHQEDKIKQIKIPEKGRPVNEVVSEMMNEVYRY 
               
               
                 4.1.1.28) Chain A, 
                 RGDANHPRFFSFVPGPASSVSWLGDIMTSAYNIHAGGSKLAP 
               
               
                 Ruminococcus 
                 MVNCIEQEVLKWLAKQVGFTENPGGVFVSGGSMANITALTA 
               
               
                 Gnavus 
                 ARDNKLTDINLHLGTAYISDQTHSSVAKGLRIIGITDSRIRRIPT 
               
               
                 SEQ ID NO: 170 
                 NSHFQMDTTKLEEAIETDKKSGYIPFVVIGTAGTTNTGSIDPLT 
               
               
                   
                 EISALCKKHDMWFHIDGAYGASVLLSPKYKSLLTGTGLADSIS 
               
               
                   
                 WDAHKWLFQTYGCAMVLVKDIRNLFHSFHVNPEYLKDLEN 
               
               
                   
                 DIDNVNTWDIGMELTRPARGLKLWLTLQVLGSDLIGSAIEHG 
               
               
                   
                 FQLAVWAEEALNPKKDWEIVSPAQMAMINFRYAPKDLTKEE 
               
               
                   
                 QDILNEKISHRILESGYAAIFTTVLNGKTVLRICAIHPEATQED 
               
               
                   
                 MQHTIDLLDQYGREIYTEMKKa 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11B 
               
             
            
               
                   
               
               
                 Tryptophan Pathway Catabolic Enzymes 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 Trp 
                 ATGACGGCAACTACAATTTCTATTGAGAGCCGTACCTC 
               
               
                 aminotransferase 
                 AGGCCCCGGCGGCGGGGACCAAAACTAATGGGACTT 
               
               
                 (EC 2.6.1.27); 
                 CAGGAAAATACAACCCCCGCACTTACCTGTCCGACC 
               
               
                 tryptophan 
                 GCGCCAAAGTCACTGAGATTGATGGATCTGACGCCG 
               
               
                 aminotransferase 
                 GTCGCCCCAATCCCGATACTTTCCCATTTAACTCGAT 
               
               
                 [Cryptococcusdeuterogattii 
                 TACCTTAAATTTGAAACCACCTTTAGGCTTGCCCGAG 
               
               
                 R265], codon optimized for 
                 AGTTCAAATAACATGCCGGTCTCTATCACGATTGAA 
               
               
                 expression in E. coli  
                 GACCCCGATTTAGCGACGGCCTTACAATATGCACCT 
               
               
                   
                 AGCGCCGGTATTCCTAAGCTGCGCGAATGGCTGGCT 
               
               
                   
                 GACTTACAAGCTCACGTTCATGAGCGCCCCCGTGGC 
               
               
                   
                 GATTATGCCATCTCGGTCGGGTCGGGGTCACAGGAT 
               
               
                   
                 TTGATGTTTAAGGGCTTCCAAGCTGTCTTGAATCCAG 
               
               
                   
                 GTGATCCAGTCCTTCTGGAAACCCCAATGTATTCAGG 
               
               
                   
                 TGTTCTGCCAGCGCTGCGCATTCTGAAGGCGGATTAT 
               
               
                   
                 GCAGAAGTTGATGTAGACGACCAGGGGTTATCTGCT 
               
               
                   
                 AAAAACCTTGAAAAAGTTTTATCAGAGTGGCCCGCA 
               
               
                   
                 GATAAGAAGCGTCCTCGTGTCCTGTATACGTCGCCA 
               
               
                   
                 ATCGGCTCCAATCCTTCCGGATGTTCAGCATCCAAGG 
               
               
                   
                 AACGCAAGTTAGAGGTACTGAAAGTCTGTAAGAAGT 
               
               
                   
                 ACGATGTGCTGATCTTCGAAGACGATCCGTATTATTA 
               
               
                   
                 CCTTGCTCAAGAGCTTATTCCATCCTATTTTGCGTTG 
               
               
                   
                 GAAAAACAAGTTTATCCGGAGGGTGGGCACGTTGTA 
               
               
                   
                 CGCTTTGACTCATTTAGTAAATTGCTTTCTGCTGGGA 
               
               
                   
                 TGCGCTTGGGATTTGCTACAGGGCCGAAGGAAATTC 
               
               
                   
                 TTCATGCGATTGACGTCAGTACAGCAGGCGCAAATT 
               
               
                   
                 TACATACTTCAGCGGTCTCTCAAGGTGTCGCTCTTCG 
               
               
                   
                 CCTGATGCAGTATTGGGGGATCGAGGGATTCCTTGC 
               
               
                   
                 ACATGGCCGCGCGGTGGCCAAACTTTACACGGAGCG 
               
               
                   
                 CCGCGCTCAGTTCGAGGCAACCGCACATAAGTACCT 
               
               
                   
                 GGACGGGCTGGCCACTTGGGTATCTCCCGTAGCGGG 
               
               
                   
                 AATGTTTTTATGGATCGATCTTCGTCCAGCAGGAATC 
               
               
                   
                 GAAGATTCTTACGAATTAATTCGCCATGAAGCATTA 
               
               
                   
                 GCCAAAGGCGTTTTAGGCGTTCCAGGGATGGCGTTTT 
               
               
                   
                 ATCCGACAGGCCGTAAGTCTTCCCATGTTCGTGTCAG 
               
               
                   
                 TTTCAGTATCGTCGACCTGGAAGACGAATCTGACCTT 
               
               
                   
                 GGTTTTCAACGCCTGGCTGAAGCTATTAAGGATAAA 
               
               
                   
                 CGCAAGGCTTTAGGGCTGGCT 
               
               
                   
               
               
                 Tryptophan 
                 ATGAGTCAAGTGATTAAGAAGAAACGTAACACCTTT 
               
               
                 Decarboxylase (EC 
                 ATGATCGGAACGGAGTACATTCTTAACAGTACACAA 
               
               
                 4.1.1.28) Chain A, 
                 TTGGAGGAAGCGATTAAATCATTCGTACATGATTTCT 
               
               
                 Ruminococcus 
                 GCGCAGAGAAGCATGAGATCCATGATCAACCTGTGG 
               
               
                 Gnavus Tryptophan 
                 TAGTAGAAGCTAAAGAACATCAGGAGGACAAAATC 
               
               
                 Decarboxylase Rum 
                 AAACAAATCAAAATCCCGGAAAAGGGACGTCCTGTA 
               
               
                 gna_01526 (alpha- 
                 AATGAAGTCGTTTCTGAGATGATGAATGAAGTGTAT 
               
               
                 fmt); codon 
                 CGCTACCGCGGAGACGCCAACCATCCTCGCTTTTTTT 
               
               
                 optimized for the 
                 CTTTTGTGCCCGGACCTGCAAGCAGTGTGTCGTGGTT 
               
               
                 expression in  
                 GGGGGATATTATGACGTCCGCCTACAATATTCATGCT 
               
               
                 E. coli 
                 GGAGGCTCAAAGCTGGCACCGATGGTTAACTGCATT 
               
               
                 SEQ ID NO: 172 
                 GAGCAGGAAGTTCTGAAGTGGTTAGCAAAGCAAGTG 
               
               
                   
                 GGGTTCACAGAAAATCCAGGTGGCGTATTTGTGTCG 
               
               
                   
                 GGCGGTTCAATGGCGAATATTACGGCACTTACTGCG 
               
               
                   
                 GCTCGTGACAATAAACTGACCGACATTAACCTTCATT 
               
               
                   
                 TGGGAACTGCTTATATTAGTGACCAGACTCATAGTTC 
               
               
                   
                 AGTTGCGAAAGGATTACGCATTATTGGAATCACTGA 
               
               
                   
                 CAGTCGCATCCGTCGCATTCCCACTAACTCCCACTTC 
               
               
                   
                 CAGATGGATACCACCAAGCTGGAGGAAGCCATCGAG 
               
               
                   
                 ACCGACAAGAAGTCTGGCTACATTCCGTTCGTCGTTA 
               
               
                   
                 TCGGAACAGCAGGTACCACCAACACTGGITCGATTG 
               
               
                   
                 ACCCCCTGACAGAAATCTCTGCGTTATGTAAGAAGC 
               
               
                   
                 ATGACATGTGGTTTCATATCGACGGAGCGTATGGAG 
               
               
                   
                 CTAGTGTTCTGCTGTCACCTAAGTACAAGAGCCTTCT 
               
               
                   
                 TACCGGAACCGGCTTGGCTGACAGTATTTCGTGGGA 
               
               
                   
                 TGCTCATAAATGGTTGTTCCAAACGTACGGCTGTGCA 
               
               
                   
                 ATGGTACTTGTCAAAGATATCCGTAATTTATTCCACT 
               
               
                   
                 CTTTTCATGTGAATCCCGAGTATCTTAAGGATCTGGA 
               
               
                   
                 AAACGACATCGATAACGTTAATACATGGGACATCGG 
               
               
                   
                 CATGGAGCTGACGCGCCCTGCACGCGGTCTTAAATT 
               
               
                   
                 GTGGCTTACTTTACAGGTCCTTGGATCTGACTTGATT 
               
               
                   
                 GGGAGTGCCATTGAACACGGTTTCCAGCTGGCAGTT 
               
               
                   
                 TGGGCTGAGGAAGCATTGAATCCAAAGAAAGACTGG 
               
               
                   
                 GAGATCGTTTCTCCAGCTCAGATGGCTATGATTAATT 
               
               
                   
                 TCCGTTATGCCCCTAAGGATTTAACCAAAGAGGAAC 
               
               
                   
                 AGGATATTCTGAATGAAAAGATCTCCCACCGCATTTT 
               
               
                   
                 AGAGAGCGGATACGCTGCAATTTTCACTACTGTATTA 
               
               
                   
                 AACGGCAAGACCGTTTTACGCATCTGTGCAATTCACC 
               
               
                   
                 CGGAGGCAACTCAAGAGGATATGCAACACACAATCG 
               
               
                   
                 ACTTATTAGACCAATACGGTCGTGAAATCTATACCG 
               
               
                   
                 AGATGAAGAAAGCG 
               
               
                   
               
               
                 Tdc (tdc from C. roseus) 
                 ATGGGTTCTATTGACTCGACGAATGTGGCCATGTCT 
               
               
                 SEQ ID NO: 260 
                 AATTCTCCTGTTGGCGAGTTTAAGCCCCTTGAAGCA 
               
               
                   
                 GAAGAGTTCCGTAAACAGGCACACCGCATGGTGGA 
               
               
                   
                 TTTTATTGCGGATTATTACAAGAACGTAGAAACATA 
               
               
                   
                 CCCGGTCCTTTCCGAGGTTGAACCCGGCTATCTGCG 
               
               
                   
                 CAAACGTATTCCCGAAACCGCACCATACCTGCCGG 
               
               
                   
                 AGCCACTTGATGATATTATGAAGGATATTCAAAAG 
               
               
                   
                 GACATTATCCCCGGAATGACGAACTGGATGTCCCCG 
               
               
                   
                 AACTTTTACGCCTTCTTCCCGGCCACAGTTAGCTCA 
               
               
                   
                 GCAGCTTTCTTGGGGGAAATGCTTTCAACGGCCCTT 
               
               
                   
                 AACAGCGTAGGATTTACCTGGGTCAGTTCCCCGGCA 
               
               
                   
                 GCGACTGAATTAGAGATGATCGTTATGGATTGGCTT 
               
               
                   
                 GCGCAAATTTTGAAACTTCCAAAAAGCTTTATGTTC 
               
               
                   
                 TCCGGAACCGGGGGTGGTGTCATCCAAAACACTAC 
               
               
                   
                 GTCAGAGTCGATCTTGTGCACTATTATCGCGGCCCG 
               
               
                   
                 TGAACGCGCCTTGGAAAAATTGGGCCCTGATTCAAT 
               
               
                   
                 TGGTAAGCTTGTCTGCTATGGGTCCGATCAAACGCA 
               
               
                   
                 CACAATGTTTCCGAAAACCTGTAAGTTAGCAGGAAT 
               
               
                   
                 TTATCCGAATAATATCCGCCTTATCCCTACCACGGT 
               
               
                   
                 AGAAACCGACTTTGGCATCTCACCGCAGGTACTTCG 
               
               
                   
                 CAAGATGGTCGAAGACGACGTCGCTGCGGGGTACG 
               
               
                   
                 TTCCCTTATTTTTGTGTGCCACCTTGGGAACGACATC 
               
               
                   
                 AACTACGGCAACAGATCCTGTAGATTCGCTGTCCGA 
               
               
                   
                 AATCGCAAACGAGTTTGGTATCTGGATTCATGTCGA 
               
               
                   
                 CGCCGCATATGCTGGATCGGCTTGCATCTGCCCAGA 
               
               
                   
                 ATTTCGTCACTACCTTGATGGCATCGAACGTGTGGA 
               
               
                   
                 TTCCTTATCGCTGTCTCCCCACAAATGGCTTTTAGCA 
               
               
                   
                 TATCTGGATTGCACGTGCTTGTGGGTAAAACAACCT 
               
               
                   
                 CACCTGCTGCTTCGCGCTTTAACGACTAATCCCGAA 
               
               
                   
                 TACTTGAAGAATAAACAGAGTGATTTAGATAAGGT 
               
               
                   
                 CGTGGATTTTAAGAACTGGCAGATCGCAACAGGAC 
               
               
                   
                 GTAAGTTCCGCTCTTTAAAACTTTGGTTAATTCTGC 
               
               
                   
                 GTTCCTACGGGGTAGTTAACCTGCAAAGTCATATCC 
               
               
                   
                 GTAGTGATGTAGCGATGGGGAAGATGTTTGAGGAA 
               
               
                   
                 TGGGTCCGTTCCGATAGCCGCTTTGAAATCGTCGTG 
               
               
                   
                 CCACGTAATTTTTCGCTTGTATGCTTTCGCTTGAAAC 
               
               
                   
                 CGGATGTATCTAGTTTACATGTCGAGGAGGTCAACA 
               
               
                   
                 AGAAGTTGTTGGATATGCTTAACTCCACCGGTCGCG 
               
               
                   
                 TATATATGACGCATACAATTGTTGGCGGAATCTATA 
               
               
                   
                 TGTTACGTTTGGCTGTAGGTAGCAGCTTGACAGAGG 
               
               
                   
                 AACATCACGTGCGCCGCGTTTGGGACTTGATCCAGA 
               
               
                   
                 AGCTTACGGACGACCTGCTTAAAGAGGCGTGA 
               
               
                   
               
               
                 Tdc (tdc from 
                 ATGAAATTTTGGCGCAAGTATACGCAACAGGAGAT 
               
               
                 Clostridium 
                 GGATGAGAAAATCACAGAATCGCTTGAGAAGACAT 
               
               
                 sporogenes) 
                 TAAATTACGATAACACGAAAACCATCGGCATCCCA 
               
               
                 SEQ ID NO: 262 
                 GGTACTAAGCTGGATGATACTGTATTTTATGACGAT 
               
               
                   
                 CACTCCTTCGTTAAGCACTCTCCCTATTTACGTACGT 
               
               
                   
                 TCATCCAAAACCCTAATCACATTGGTTGTCACACGT 
               
               
                   
                 ACGATAAAGCAGACATCTTGTTTGGCGGCACGTTTG 
               
               
                   
                 ACATCGAACGCGAACTGATTCAGCTTTTGGCCATCG 
               
               
                   
                 ATGTCTTAAACGGAAATGATGAGGAATTCGATGGA 
               
               
                   
                 TATGTGACACAGGGGGGAACCGAGGCGAATATTCA 
               
               
                   
                 GGCAATGTGGGTTTATCGTAACTATTTCAAAAAAGA 
               
               
                   
                 ACGTAAAGCAAAACATGAGGAAATCGCAATCATCA 
               
               
                   
                 CGAGCGCGGATACCCATTACAGTGCATATAAGGGG 
               
               
                   
                 AGCGACTTGCTGAACATTGATATTATCAAGGTCCCA 
               
               
                   
                 GTAGACTTCTATTCGCGTAAGATCCAGGAGAACAC 
               
               
                   
                 GTTAGACTCGATTGTCAAGGAGGCGAAGGAAATTG 
               
               
                   
                 GAAAGAAGTACTTCATTGTCATCTCAAACATGGGTA 
               
               
                   
                 CGACTATGTTTGGCAGTGTAGACGACCCTGATCTTT 
               
               
                   
                 ATGCTAACATTTTTGATAAGTATAACTTAGAATACA 
               
               
                   
                 AAATCCACGTCGATGGAGCTTTTGGGGGTTTCATTT 
               
               
                   
                 ATCCTATCGATAATAAGGAGTGCAAAACAGATTTCT 
               
               
                   
                 CGAACAAGAACGTCTCATCCATCACGCTTGACGGTC 
               
               
                   
                 ACAAAATGCTTCAAGCCCCCTATGGGACTGGTATCT 
               
               
                   
                 TCGTGTCACGTAAGAACTTGATCCATAACACCCTGA 
               
               
                   
                 CAAAGGAAGCAACGTATATTGAAAACCTGGACGTT 
               
               
                   
                 ACCCTGAGTGGGTCCCGCTCCGGATCCAACGCCGTT 
               
               
                   
                 GCGATCTGGATGGTTTTAGCCTCTTATGGCCCCTAC 
               
               
                   
                 GGGTGGATGGAGAAGATTAACAAGTTGCGCAATCG 
               
               
                   
                 CACTAAGTGGCTTTGCAAGCAGCTTAACGACATGCG 
               
               
                   
                 CATCAAATACTATAAGGAGGATAGCATGAATATCG 
               
               
                   
                 TCACGATTGAAGAGCAATACGTAAATAAAGAGATT 
               
               
                   
                 GCAGAGAAATACTTCCTTGTGCCTGAAGTACACAAT 
               
               
                   
                 CCTACCAACAATTGGTACAAGATTGTAGTCATGGAA 
               
               
                   
                 CATGTTGAACTTGACATCTTGAACTCCCTTGTTTATG 
               
               
                   
                 ATTTACGTAAATTCAACAAGGAGCACCTGAAGGCA 
               
               
                   
                 ATGTGA 
               
               
                   
               
               
                 ipdC 
                 ATGCGTACACCCTACTGTGTCGCCGATTATCTTTTA 
               
               
                 SEQ ID NO: 265 
                 GATCGTCTGACGGACTGCGGGGCCGATCACCTGTTT 
               
               
                   
                 GGCGTACCGGGCGATTACAACTTGCAGTTTCTGGAC 
               
               
                   
                 CACGTCATTGACTCACCAGATATCTGCTGGGTAGGG 
               
               
                   
                 TGTGCGAACGAGCTTAACGCGAGCTACGCTGCTGA 
               
               
                   
                 CGGATATGCGCGTTGTAAAGGCTTTGCTGCACTTCT 
               
               
                   
                 TACTACCTTCGGGGTCGGTGAGTTATCGGCGATGAA 
               
               
                   
                 CGGTATCGCAGGCTCGTACGCTGAGCACGTCCCGGT 
               
               
                   
                 ATTACACATTGTGGGAGCTCCGGGTACCGCAGCTCA 
               
               
                   
                 ACAGCGCGGAGAACTGTTACACCACACGCTGGGCG 
               
               
                   
                 ACGGAGAATTCCGCCACTTTTACCATATGTCCGAGC 
               
               
                   
                 CAATTACTGTAGCCCAGGCTGTACTTACAGAGCAAA 
               
               
                   
                 ATGCCTGTTACGAGATCGACCGTGTTTTGACCACGA 
               
               
                   
                 TGCTTCGCGAGCGCCGTCCCGGGTATTTGATGCTGC 
               
               
                   
                 CAGCCGATGTTGCCAAAAAAGCTGCGACGCCCCCA 
               
               
                   
                 GTGAATGCCCTGACGCATAAACAAGCTCATGCCGA 
               
               
                   
                 TTCCGCCTGTTTAAAGGCTTTTCGCGATGCAGCTGA 
               
               
                   
                 AAATAAATTAGCCATGTCGAAACGCACCGCCTTGTT 
               
               
                   
                 GGCGGACTTTCTGGTCCTGCGCCATGGCCTTAAACA 
               
               
                   
                 CGCCCTTCAGAAATGGGTCAAAGAAGTCCCGATGG 
               
               
                   
                 CCCACGCTACGATGCTTATGGGTAAGGGGATTTTTG 
               
               
                   
                 ATGAACGTCAAGCGGGATTTTATGGAACTTATTCCG 
               
               
                   
                 GTTCGGCGAGTACGGGGGCGGTAAAGGAAGCGATT 
               
               
                   
                 GAGGGAGCCGACACAGTTCTTTGCGTGGGGACACG 
               
               
                   
                 TTTCACCGATACACTGACCGCTGGATTCACACACCA 
               
               
                   
                 ACTTACTCCGGCACAAACGATTGAGGTGCAACCCC 
               
               
                   
                 ATGCGGCTCGCGTGGGGGATGTATGGTTTACGGGC 
               
               
                   
                 ATTCCAATGAATCAAGCCATTGAGACTCTTGTCGAG 
               
               
                   
                 CTGTGCAAACAGCACGTCCACGCAGGACTGATGAG 
               
               
                   
                 TTCGAGCTCTGGGGCGATTCCTTTTCCACAACCAGA 
               
               
                   
                 TGGTAGTTTAACTCAAGAAAACTTCTGGCGCACATT 
               
               
                   
                 GCAAACCTTTATCCGCCCAGGTGATATCATCTTAGC 
               
               
                   
                 AGACCAGGGTACTTCAGCCTTTGGAGCAATTGACCT 
               
               
                   
                 GCGCTTACCAGCAGACGTGAACTTTATTGTGCAGCC 
               
               
                   
                 GCTGTGGGGGTCTATTGGTTATACTTTAGCTGCGGC 
               
               
                   
                 CTTCGGAGCGCAGACAGCGTGTCCAAACCGTCGTGT 
               
               
                   
                 GATCGTATTGACAGGAGATGGAGCAGCGCAGTTGA 
               
               
                   
                 CCATTCAGGAGTTAGGCTCGATGTTACGCGATAAGC 
               
               
                   
                 AGCACCCCATTATCCTGGTCCTGAACAATGAGGGGT 
               
               
                   
                 ATACAGTTGAACGCGCCATTCATGGTGCGGAACAA 
               
               
                   
                 CGCTACAATGACATCGCTTTATGGAATTGGACGCAC 
               
               
                   
                 ATCCCCCAAGCCTTATCGTTAGATCCCCAATCGGAA 
               
               
                   
                 TGTTGGCGTGTGTCTGAAGCAGAGCAACTGGCTGAT 
               
               
                   
                 GTTCTGGAAAAAGTTGCTCATCATGAACGCCTGTCG 
               
               
                   
                 TTGATCGAGGTAATGTTGCCCAAGGCCGATATCCCT 
               
               
                   
                 CCGTTACTGGGAGCCTTGACCAAGGCTTTAGAAGCC 
               
               
                   
                 TGCAACAACGCTTAA 
               
               
                   
               
               
                 Iad1 
                 ATGCCCACCTTGAACTTGGACTTACCCAACGGTATT 
               
               
                 SEQ ID NO: 266 
                 AAGAGCACGATTCAGGCAGACCTTTTCATCAATAAT 
               
               
                   
                 AAGTTTGTGCCGGCGCTTGATGGGAAAACGTTCGCA 
               
               
                   
                 ACTATTAATCCGTCTACGGGGAAAGAGATCGGACA 
               
               
                   
                 GGTGGCAGAGGCTTCGGCGAAGGATGTGGATCTTG 
               
               
                   
                 CAGTTAAGGCCGCGCGTGAGGCGTTTGAAACTACTT 
               
               
                   
                 GGGGGGAAAACACGCCAGGTGATGCTCGTGGCCGT 
               
               
                   
                 TTACTGATTAAGCTTGCTGAGTTGGTGGAAGCGAAT 
               
               
                   
                 ATTGATGAGTTAGCGGCAATTGAATCACTGGACAAT 
               
               
                   
                 GGGAAAGCGTTCTCTATTGCTAAGTCATTCGACGTA 
               
               
                   
                 GCTGCTGTGGCCGCAAACTTACGTTACTACGGCGGT 
               
               
                   
                 TGGGCTGATAAAAACCACGGTAAAGTCATGGAGGT 
               
               
                   
                 AGACACAAAGCGCCTGAACTATACCCGCCACGAGC 
               
               
                   
                 CGATCGGGGTTTGCGGACAAATCATTCCGTGGAATT 
               
               
                   
                 TCCCGCTTTTGATGTTTGCATGGAAGCTGGGTCCCG 
               
               
                   
                 CTTTAGCCACAGGGAACACAATTGTGTTAAAGACTG 
               
               
                   
                 CCGAGCAGACTCCCTTAAGTGCTATCAAGATGTGTG 
               
               
                   
                 AATTAATCGTAGAAGCCGGCTTTCCGCCCGGAGTAG 
               
               
                   
                 TTAATGTGATCTCGGGATTCGGACCGGTGGCGGGG 
               
               
                   
                 GCCGCGATCTCGCAACACATGGACATCGATAAGAT 
               
               
                   
                 TGCCTTTACAGGATCGACATTGGTTGGCCGCAACAT 
               
               
                   
                 TATGAAGGCAGCTGCGTCGACTAACTTAAAAAAGG 
               
               
                   
                 TTACACTTGAGTTAGGAGGAAAATCCCCGAATATCA 
               
               
                   
                 TTTTCAAAGATGCCGACCTTGACCAAGCTGTTCGCT 
               
               
                   
                 GGAGCGCCTTCGGTATCATGTTTAACCACGGACAAT 
               
               
                   
                 GCTGCTGCGCTGGATCGCGCGTATATGTGGAAGAAT 
               
               
                   
                 CCATCTATGACGCCTTCATGGAAAAAATGACTGCGC 
               
               
                   
                 ATTGTAAGGCGCTTCAAGTTGGAGATCCTTTCAGCG 
               
               
                   
                 CGAACACCTTCCAAGGACCACAAGTCTCGCAGTTAC 
               
               
                   
                 AATACGACCGTATCATGGAATACATCGAATCAGGG 
               
               
                   
                 AAAAAAGATGCAAATCTTGCTTTAGGCGGCGTTCGC 
               
               
                   
                 AAAGGGAATGAGGGGTATTTCATTGAGCCAACTAT 
               
               
                   
                 TTTTACAGACGTGCCGCACGACGCGAAGATTGCCA 
               
               
                   
                 AAGAGGAGATCTTCGGTCCAGTGGTTGTTGTGTCGA 
               
               
                   
                 AATTTAAGGACGAAAAAGATCTGATCCGTATCGCA 
               
               
                   
                 AATGATTCTATTTATGGTTTAGCTGCGGCAGTCTTTT 
               
               
                   
                 CCCGCGACATCAGCCGCGCGATCGAGACAGCACAC 
               
               
                   
                 AAACTGAAAGCAGGCACGGTCTGGGTCAACTGCTA 
               
               
                   
                 TAATCAGCTTATTCCGCAGGTGCCATTCGGAGGGTA 
               
               
                   
                 TAAGGCTTCCGGTATCGGCCGTGAGTTGGGGGAAT 
               
               
                   
                 ATGCCTTGTCTAATTACACAAATATCAAGGCCGTCC 
               
               
                   
                 ACGTTAACCTTTCTCAACCGGCGCCCATTTGA 
               
               
                   
               
               
                 fldA 
                 ATGGAAAACAACACCAATATGTTCTCTGGAGTGAA 
               
               
                 SEQ ID NO: 276 
                 GGTGATCGAACTGGCCAACTTTATCGCTGCTCCGGC 
               
               
                   
                 GGCAGGTCGCTTCTTTGCTGATGGGGGAGCAGAAG 
               
               
                   
                 TAATTAAGATCGAATCTCCAGCAGGCGACCCGCTGC 
               
               
                   
                 GCTACACGGCCCCATCAGAAGGACGCCCGCTTTCTC 
               
               
                   
                 AAGAGGAAAACACAACGTATGATTTGGAAAACGCG 
               
               
                   
                 AATAAGAAAGCAATTGTTCTGAACTTAAAATCGGA 
               
               
                   
                 AAAAGGAAAGAAAATTCTTCACGAGATGCTTGCTG 
               
               
                   
                 AGGCAGACATCTTGTTAACAAATTGGCGCACGAAA 
               
               
                   
                 GCGTTAGTCAAACAGGGGTTAGATTACGAAACACT 
               
               
                   
                 GAAAGAGAAGTATCCAAAATTGGTATTTGCACAGA 
               
               
                   
                 TTACAGGATACGGGGAGAAAGGACCCGACAAAGAC 
               
               
                   
                 CTGCCTGGTTTCGACTACACGGCGTTTTTCGCCCGC 
               
               
                   
                 GGAGGAGTCTCCGGTACATTATATGAAAAAGGAAC 
               
               
                   
                 TGTCCCTCCTAATGTGGTACCGGGTCTGGGTGACCA 
               
               
                   
                 CCAGGCAGGAATGTTCTTAGCTGCCGGTATGGCTGG 
               
               
                   
                 TGCGTTGTATAAGGCCAAAACCACCGGACAAGGCG 
               
               
                   
                 ACAAAGTCACCGTTAGTCTGATGCATAGCGCAATGT 
               
               
                   
                 ACGGCCTGGGAATCATGATTCAGGCAGCCCAGTAC 
               
               
                   
                 AAGGACCATGGGCTGGTGTACCCGATCAACCGTAA 
               
               
                   
                 TGAAACGCCTAATCCTTTCATCGTTTCATACAAGTC 
               
               
                   
                 CAAAGATGATTACTTTGTCCAAGTTTGCATGCCTCC 
               
               
                   
                 CTATGATGTGTTTTATGATCGCTTTATGACGGCCTTA 
               
               
                   
                 GGACGTGAAGACTTGGTAGGTGACGAACGCTACAA 
               
               
                   
                 TAAGATCGAGAACTTGAAGGATGGTCGCGCAAAAG 
               
               
                   
                 AAGTCTATTCCATCATCGAACAACAAATGGTAACG 
               
               
                   
                 AAGACGAAGGACGAATGGGACAAGATTTTTCGTGA 
               
               
                   
                 TGCAGACATTCCATTCGCTATTGCCCAAACGTGGGA 
               
               
                   
                 AGATCTTTTAGAAGACGAGCAGGCATGGGCCAACG 
               
               
                   
                 ACTACCTGTATAAAATGAAGTATCCCACAGGCAAC 
               
               
                   
                 GAACGTGCCCTGGTACGTTTACCTGTGTTCTTCAAA 
               
               
                   
                 GAAGCTGGACTTCCTGAATACAACCAGTCGCCACA 
               
               
                   
                 GATTGCTGAGAATACCGTGGAAGTGTTAAAGGAGA 
               
               
                   
                 TGGGATATACCGAGCAAGAAATTGAGGAGCTTGAG 
               
               
                   
                 AAAGACAAAGACATCATGGTACGTAAAGAGAAATG 
               
               
                   
                 A 
               
               
                   
               
               
                 fldB 
                 ATGTCAGACCGCAACAAAGAAGTGAAAGAAAAGA 
               
               
                 SEQ ID NO: 277 
                 AGGCTAAACACTATCTGCGCGAGATCACAGCTAAA 
               
               
                   
                 CACTACAAGGAAGCGTTAGAGGCTAAAGAGCGTGG 
               
               
                   
                 GGAGAAAGTGGGTTGGTGTGCCTCTAACTTCCCCCA 
               
               
                   
                 AGAGATTGCAACCACGTTGGGTGTAAAGGTTGTTTA 
               
               
                   
                 TCCCGAAAACCACGCCGCCGCCGTAGCGGCACGTG 
               
               
                   
                 GCAATGGGCAAAATATGTGCGAACACGCGGAGGCT 
               
               
                   
                 ATGGGATTCAGTAATGATGTGTGTGGATATGCACGT 
               
               
                   
                 GTAAATTTAGCCGTAATGGACATCGGCCATAGTGA 
               
               
                   
                 AGATCAACCTATTCCAATGCCTGATTTCGTTCTGTG 
               
               
                   
                 CTGTAATAATATCTGCAATCAGATGATTAAATGGTA 
               
               
                   
                 TGAACACATTGCAAAAACGTTGGATATTCCTATGAT 
               
               
                   
                 CCTTATCGATATTCCATATAATACTGAGAACACGGT 
               
               
                   
                 GTCTCAGGACCGCATTAAGTACATCCGCGCCCAGTT 
               
               
                   
                 CGATGACGCTATCAAGCAACTGGAAGAAATCACTG 
               
               
                   
                 GCAAAAAGTGGGACGAGAATAAATTCGAAGAAGTG 
               
               
                   
                 ATGAAGATTTCGCAAGAATCGGCCAAGCAATGGTT 
               
               
                   
                 ACGCGCCGCGAGCTACGCGAAATACAAACCATCAC 
               
               
                   
                 CGTTTTCGGGCTTTGACCTTTTTAATCACATGGCTGT 
               
               
                   
                 AGCCGTTTGTGCTCGCGGCACCCAGGAAGCCGCCG 
               
               
                   
                 ATGCATTCAAAATGTTAGCAGATGAATATGAAGAG 
               
               
                   
                 AACGTTAAGACAGGAAAGTCTACTTATCGCGGCGA 
               
               
                   
                 GGAGAAGCAGCGTATCTTGTTCGAGGGCATCGCTTG 
               
               
                   
                 TTGGCCTTATCTGCGCCACAAGTTGACGAAACTGAG 
               
               
                   
                 TGAATATGGAATGAACGTCACAGCTACGGTGTACG 
               
               
                   
                 CCGAAGCTTTTGGGGTTATTTACGAAAACATGGATG 
               
               
                   
                 AACTGATGGCCGCTTACAATAAAGTGCCTAACTCAA 
               
               
                   
                 TCTCCTTCGAGAACGCGCTGAAGATGCGTCTTAATG 
               
               
                   
                 CCGTTACAAGCACCAATACAGAAGGGGCTGTTATC 
               
               
                   
                 CACATTAATCGCAGTTGTAAGCTGTGGTCAGGATTC 
               
               
                   
                 TTATACGAACTGGCCCGTCGTTTGGAAAAGGAGAC 
               
               
                   
                 GGGGATCCCTGTTGTTTCGTTCGACGGAGATCAAGC 
               
               
                   
                 GGATCCCCGTAACTTCTCCGAGGCTCAATATGACAC 
               
               
                   
                 TCGCATCCAAGGTTTAAATGAGGTGATGGTCGCGA 
               
               
                   
                 AAAAAGAAGCAGAGTGA 
               
               
                   
               
               
                 fldC 
                 ATGTCGAATAGTGACAAGTTTTTTAACGACTTCAAG 
               
               
                 SEQ ID NO: 278 
                 GACATTGTGGAAAACCCAAAGAAGTATATCATGAA 
               
               
                   
                 GCATATGGAACAAACGGGACAAAAAGCCATCGGTT 
               
               
                   
                 GCATGCCTTTATACACCCCAGAAGAGCTTGTCTTAG 
               
               
                   
                 CGGCGGGTATGTTTCCTGTTGAGTATGGGGCTCGA 
               
               
                   
                 ATACTGAGTTGTCAAAAGCCAAGACCTACTTTCCGG 
               
               
                   
                 CTTTTATCTGTTCTATCTTGCAAACTACTTTAGAAAA 
               
               
                   
                 CGCATTGAATGGGGAGTATGACATGCTGTCTGGTAT 
               
               
                   
                 GATGATCACAAACTATTGCGATTCGCTGAAATGTAT 
               
               
                   
                 GGGACAAAACTTCAAACTTACAGTGGAAAATATCG 
               
               
                   
                 AATTCATCCCGGTTACGGTTCCACAAAACCGCAAGA 
               
               
                   
                 TGGAGGCGGGTAAAGAATTTCTGAAATCCCAGTAT 
               
               
                   
                 AAAATGAATATCGAACAACTGGAAAAAATCTCAGG 
               
               
                   
                 GAATAAGATCACTGACGAGAGCTTGGAGAAGGCTA 
               
               
                   
                 TTGAAATTTACGATGAGCACCGTAAAGTCATGAAC 
               
               
                   
                 GATTTCTCTATGCTTGCGTCCAAGTACCCTGGTATC 
               
               
                   
                 ATTACGCCAACGAAACGTAACTACGTGATGAAGTC 
               
               
                   
                 AGCGTATTATATGGACAAGAAAGAACATACAGAGA 
               
               
                   
                 AGGTACGTCAGTTGATGGATGAAATCAAGGCCATT 
               
               
                   
                 GAGCCTAAACCATTCGAAGGAAAACGCGTGATTAC 
               
               
                   
                 CACTGGGATCATTGCAGATTCGGAGGACCTTTTGAA 
               
               
                   
                 AATCTTGGAGGAGAATAACATTGCTATCGTGGGAG 
               
               
                   
                 ATGATATTGCACACGAGTCTCGCCAATACCGCACTT 
               
               
                   
                 TGACCCCGGAGGCCAACACACCTATGGACCGTCTTG 
               
               
                   
                 CTGAACAATTTGCGAACCGCGAGTGTTCGACGTTGT 
               
               
                   
                 ATGACCCTGAAAAAAAACGTGGACAGTATATTGTC 
               
               
                   
                 GAGATGGCAAAAGAGCGTAAGGCCGACGGAATCAT 
               
               
                   
                 CTTCTTCATGACAAAATTCTGCGATCCCGAAGAATA 
               
               
                   
                 CGATTACCCTCAGATGAAAAAAGACTTCGAAGAAG 
               
               
                   
                 CCGGTATTCCCCACGTTCTGATTGAGACAGACATGC 
               
               
                   
                 AAATGAAGAACTACGAACAAGCTCGCACCGCTATT 
               
               
                   
                 CAAGCATTTTCAGAAACCCTTTG 
               
               
                   
               
               
                 Acu1 
                 ATGCGTGCTGTCTTAATCGAGAAGTCAGATGACACC 
               
               
                 SEQ ID NO: 279 
                 CAGAGTGTTTCAGTTACGGAGTTGGCTGAAGACCA 
               
               
                   
                 ATTACCCGAAGGTGACGTCCTTGTGGATGTCGCGTA 
               
               
                   
                 CAGCACATTGAATTACAAGGATGCTCTTGCGATTAC 
               
               
                   
                 TGGAAAAGCACCCGTTGTACGCCGTTTTCCTATGGT 
               
               
                   
                 CCCCGGAATTGACTTTACTGGGACTGTCGCACAGAG 
               
               
                   
                 TTCCCATGCTGATTTCAAGCCAGGCGACCGCGTAAT 
               
               
                   
                 TCTGAACGGATGGGGAGTTGGTGAGAAACACTGGG 
               
               
                   
                 GCGGTCTTGCAGAACGCGCACGCGTACGTGGGGAC 
               
               
                   
                 TGGCTTGTCCCGTTGCCAGCCCCCTTAGACTTGCGC 
               
               
                   
                 CAGGCTGCAATGATTGGCACTGCGGGGTACACAGC 
               
               
                   
                 TATGCTGTGCGTGCTTGCCCTTGAGCGCCATGGAGT 
               
               
                   
                 CGTACCTGGGAACGGCGAGATTGTCGTCTCAGGCG 
               
               
                   
                 CAGCAGGAGGGGTAGGTTCTGTAGCAACCACACTG 
               
               
                   
                 TTAGCAGCCAAAGGCTACGAAGTGGCCGCCGTGAC 
               
               
                   
                 CGGGCGCGCAAGCGAGGCCGAATATTTACGCGGAT 
               
               
                   
                 TAGGCGCCGCGTCGGTCATTGATCGCAATGAATTAA 
               
               
                   
                 CGGGGAAGGTGCGTCCATTAGGGCAGGAACGCTGG 
               
               
                   
                 GCAGGAGGAATCGATGTAGCAGGATCAACCGTACT 
               
               
                   
                 TGCTAATATGTTGAGCATGATGAAATACCGTGGCGT 
               
               
                   
                 GGTGGCGGCCTGTGGCCTGGCGGCTGGAATGGACT 
               
               
                   
                 TGCCCGCGTCTGTCGCCCCTTTTATTCTGCGTGGTAT 
               
               
                   
                 GACTTTGGCAGGGGTAGATTCAGTCATGTGCCCCAA 
               
               
                   
                 AACTGATCGTCTGGCTGCTTGGGCACGCCTGGCATC 
               
               
                   
                 CGACCTGGACCCTGCAAAGCTGGAAGAGATGACAA 
               
               
                   
                 CTGAATTACCGTTCTCTGAGGTGATTGAAACGGCTC 
               
               
                   
                 CGAAGTTCTTGGATGGAACAGTGCGTGGGCGTATTG 
               
               
                   
                 TCATTCCGGTAACACCTTGA 
               
               
                   
               
               
                 fldH1 
                 ATGAAAATCTTGGCATACTGCGTCCGCCCAGACGA 
               
               
                 SEQ ID NO: 280 
                 GGTAGACTCCTTTAAGAAATTTAGTGAAAAGTACG 
               
               
                   
                 GGCATACAGTTGATCTTATTCCAGACTCTTTTGGAC 
               
               
                   
                 CTAATGTCGCTCATTTGGCGAAGGGTTACGATGGGA 
               
               
                   
                 TTTCTATTCTGGGCAACGACACGTGTAACCGTGAGG 
               
               
                   
                 CTGGCAACCCGTACAGCCGGAGTGAACAACATTGA 
               
               
                   
                 CTTCGATGCAGCAAAGGAGTTCGGTATTAACGTGGC 
               
               
                   
                 TAATGTTCCCGCATATTCCCCCAACTCGGTCAGCGA 
               
               
                   
                 ATTTACCATTGGATTGGCATTAAGTCTGACGCGTAA 
               
               
                   
                 GATTCCATTTGCCCTGAAACGCGTGGAACTGAACAA 
               
               
                   
                 TTTTGCGCTTGGCGGCCTTATTGGTGTGGAATTGCG 
               
               
                   
                 TAACTTAACTTTAGGAGTCATCGGTACTGGTCGCAT 
               
               
                   
                 CGGATTGAAAGTGATTGAGGGCTTCTCTGGGTTTGG 
               
               
                   
                 AATGAAAAAAATGATCGGTTATGACATTTTTGAAA 
               
               
                   
                 ATGAAGAAGCAAAGAAGTACATCGAATACAAATCA 
               
               
                   
                 TTAGACGAAGTTTTTAAAGAGGCTGATATTATCACT 
               
               
                   
                 CTGCATGCGCCTCTGACAGACGACAACTATCATATG 
               
               
                   
                 ATTGGTAAAGAATCCATTGCTAAAATGAAGGATGG 
               
               
                   
                 GGTATTTATTATCAACGCAGCGCGTGGAGCCTTAAT 
               
               
                   
                 CGATAGTGAGGCCCTGATTGAAGGGTTAAAATCGG 
               
               
                   
                 GGAAGATTGCGGGCGCGGCTCTGGATAGCTATGAG 
               
               
                   
                 TATGAGCAAGGTGTCTTTCACAACAATAAGATGAAT 
               
               
                   
                 GAAATTATGCAGGATGATACCTTGGAACGTCTGAA 
               
               
                   
                 ATCTTTTCCCAACGTCGTGATCACGCCGCATTTGGG 
               
               
                   
                 TTTTTATACTGATGAGGCGGTTTCCAATATGGTAGA 
               
               
                   
                 GATCACACTGATGAACCTTCAGGAATTCGAGTTGAA 
               
               
                   
                 AGGAACCTGTAAGAACCAGCGTGTTTGTAAATGA 
               
               
                   
               
               
                 FldD 
                 ATGTTCTTTACGGAGCAACACGAACTTATTCGCAAA 
               
               
                 SEQ ID NO: 282 
                 CTGGCGCGTGACTTTGCCGAACAGGAAATCGAGCC 
               
               
                   
                 TATCGCAGACGAAGTAGATAAAACCGCAGAGTTCC 
               
               
                   
                 CAAAAGAAATCGTGAAGAAGATGGCTCAAAATGGA 
               
               
                   
                 TTTTTCGGCATTAAAATGCCTAAAGAATACGGAGGG 
               
               
                   
                 GCGGGTGCGGATAACCGCGCTTATGTCACTATTATG 
               
               
                   
                 GAGGAAATTTCACGTGCTTCCGGGGTAGCGGGTATC 
               
               
                   
                 TACCTGAGCTCGCCGAACAGTTTGTTAGGAACTCCC 
               
               
                   
                 TTCTTATTGGTCGGAACCGATGAGCAAAAAGAAAA 
               
               
                   
                 GTACCTTAAGCCTATGATCCGCGGCGAGAAGACTCT 
               
               
                   
                 GGCGTTCGCCCTGACAGAGCCTGGTGCTGGCTCTGA 
               
               
                   
                 TGCGGGTGCGTTGGCTACTACTGCCCGTGAAGAGG 
               
               
                   
                 GCGACTATTATATCTTAAATGGCCGCAAGACGTTTA 
               
               
                   
                 TTACAGGGGCTCCTATTAGCGACAATATTATTGTGT 
               
               
                   
                 TCGCAAAAACCGATATGAGCAAAGGGACCAAAGGT 
               
               
                   
                 ATCACCACTTTCATTGTGGACTCAAAGCAGGAAGG 
               
               
                   
                 GGTAAGTTTTGGTAAGCCAGAGGACAAAATGGGAA 
               
               
                   
                 TGATTGGTTGTCCGACAAGCGACATCATCTTGGAAA 
               
               
                   
                 ACGTTAAAGTTCATAAGTCCGACATCTTGGGAGAA 
               
               
                   
                 GTCAATAAGGGGTTTATTACCGCGATGAAAACACTT 
               
               
                   
                 TCCGTTGGTCGTATCGGAGTGGCGTCACAGGCGCTT 
               
               
                   
                 GGAATTGCACAGGCCGCCGTAGATGAGGCGGTAAA 
               
               
                   
                 GTACGCCAAGCAACGTAAACAATTCAATCGCCCAA 
               
               
                   
                 TCGCGAAATTTCAGGCCATTCAATTTAAACTTGCCA 
               
               
                   
                 ATATGGAGACTAAATTAAATGCCGCTAAACTTCTTG 
               
               
                   
                 TTTATAACGCAGCGTACAAAATGGATTGTGGAGAA 
               
               
                   
                 AAAGCCGACAAGGAAGCCTCTATGGCTAAATACTT 
               
               
                   
                 TGCTGCTGAATCAGCGATCCAAATCGTTAACGACGC 
               
               
                   
                 GCTGCAAATCCATGGCGGGTATGGCTATATCAAAG 
               
               
                   
                 ACTACAAGATTGAACGTTTGTACCGCGATGTGCGTG 
               
               
                   
                 TGATCGCTATTTATGAGGGCACTTCCGAGGTCCAAC 
               
               
                   
                 AGATGGTTATCGCGTCCAATCTGCTGAAGTAA 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 11B or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as listed in Table 11A or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of one or more nucleic acid sequence of Table 11B or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide the polypeptide sequences listed in Table 11A or a functional fragment thereof. 
     In one embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 80% identity with the entire sequence of SEQ ID NO: 141. In another embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 85% identity with the entire sequence of SEQ ID NO: 141. In one embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 90% identity with the entire sequence of SEQ ID NO: 141. In one embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 95% identity with the entire sequence of SEQ ID NO: 141. In another embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 141. Accordingly, in one embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 141. In another embodiment, the Tryptophan Decarboxylase gene encodes a polypeptide which comprises the sequence of SEQ ID NO: 141. In yet another embodiment the Tryptophan Decarboxylase gene encodes a polypeptide which consists of the sequence of SEQ ID NO: 141. 
     In one embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 80% identity with the entire sequence of SEQ ID NO: 149. In another embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 85% identity with the entire sequence of SEQ ID NO: 149. In one embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 90% identity with the entire sequence of SEQ ID NO: 149. In one embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 95% identity with the entire sequence of SEQ ID NO: 149. In another embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 149. Accordingly, in one embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 149. In another embodiment, the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which comprises the sequence of SEQ ID NO: 149. In yet another embodiment the Indole-3-pyruvate decarboxylase gene encodes a polypeptide which consists of the sequence of SEQ ID NO: 149. 
     In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 80% identity with the entire sequence of SEQ ID NO: 150. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 85% identity with the entire sequence of SEQ ID NO: 150. In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 90% identity with the entire sequence of SEQ ID NO: 150. In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 95% identity with the entire sequence of SEQ ID NO: 150. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 150. Accordingly, in one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 150. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which comprises the sequence of SEQ ID NO: 150. In yet another embodiment the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which consists of the sequence of SEQ ID NO: 150. 
     In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 80% identity with the entire sequence of SEQ ID NO: 154. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 85% identity with the entire sequence of SEQ ID NO: 154. In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 90% identity with the entire sequence of SEQ ID NO: 154. In one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 95% identity with the entire sequence of SEQ ID NO: 154. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 154. Accordingly, in one embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 154. In another embodiment, the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which comprises the sequence of SEQ ID NO: 154. In yet another embodiment the Indole-3-acetaldehyde dehydrogenase gene encodes a polypeptide which consists of the sequence of SEQ ID NO: 154. 
     In one embodiment, genetically engineered bacteria comprise one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 80% identity with the entire sequence of one or more sequence(s) of Table 11A. In another embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 85% identity with the entire sequence of one or more sequence(s) of Table 11A. In one embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 90% identity with the entire sequence of one or more sequence(s) of Table 11A. In one embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 95% identity with the entire sequence of one or more sequence(s) of Table 11A. In another embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of one or more sequence(s) of Table 11A. Accordingly, in one embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of one or more sequence(s) of Table 11A. In another embodiment, the one or more gene sequence(s) which encode one or more polypeptide(s) which comprises the entire sequence of one or more sequence(s) of Table 11A. 
     In some embodiments, the genetically engineered bacteria comprise a gene cassette for the production of tryptamine from tryptophan. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter as described above herein. In some embodiments the bacteria further produce tryptamine from tryptophan. In some embodiments, the genetically engineered bacteria optionally comprise a tryptamine exporter. In some embodiments, the genetically engineered bacteria comprise an exporter of one or more indole metabolites, in order to increase the export of indole metabolites produced. 
     Table 12 depicts non-limiting examples of contemplated polypeptide sequences, which are encoded by indole-3-propionate producing bacteria. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Non-limiting Examples of Sequences for indole-3-propionate Production 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 FldA: indole-3- 
                 MENNTNMFSGVKVIELANFIAAPAAGRFFADGGAEVIKIESPA 
               
               
                 propionyl- 
                 GDPLRYTAPSEGRPLSQEENTTYDLENANKKAIVLNLKSEKGK 
               
               
                 CoA:indole-3- 
                 KILHEMLAEADILLTNWRTKALVKQGLDYETLKEKYPKLVFA 
               
               
                 lactate CoA 
                 QITGYGEKGPDKDLPGFDYTAFFARGGVSGTLYEKGTVPPNV 
               
               
                 transferase from 
                 VPGLGDHQAGMFLAAGMAGALYKAKTTGQGDKVTVSLMHS 
               
               
                 Clostridium 
                 AMYGLGIMIQAAQYKDHGLVYPINRNETPNPFIVSYKSKDDYF 
               
               
                 sporogenes 
                 VQVCMPPYDVFYDRFMTALGREDLVGDERYNKIENLKDGRA 
               
               
                 SEQ ID NO: 173 
                 KEVYSIIEQQMVTKTKDEWDKIFRDADIPFAIAQTWEDLLEDE 
               
               
                   
                 QAWANDYLYKMKYPTGNERALVRLPVFFKEAGLPEYNQSPQI 
               
               
                   
                 AENTVEVLKEMGYTEQEIEELEKDKDIMVRKEK 
               
               
                   
               
               
                 FldB: subunit of 
                 MSDRNKEVKEKKAKHYLREITAKHYKEALEAKERGEKVGWC 
               
               
                 indole-3-lactate 
                 ASNFPQEIATTLGVKVVYPENHAAAVAARGNGQNMCEHAEA 
               
               
                 dehydratase from 
                 MGFSNDVCGYARVNLAVMDIGHSEDQPIPMPDFVLCCNNICN 
               
               
                   
                 QMIKWYEHIAKTLDIPMILIDIPYNTENTVSQDRIKYIRAQFDD 
               
               
                 Clostridium 
                 AIKQLEEITGKKWDENKFEEVMKISQESAKQWLRAASYAKYK 
               
               
                 sporogenes 
                 PSPFSGFDLFNHMAVAVCARGTQEAADAFKMLADEYEENVKT 
               
               
                 SEQ ID NO: 174 
                 GKSTYRGEEKQRILFEGIACWPYLRHKLTKLSEYGMNVTATV 
               
               
                   
                 YAEAFGVIYENMDELMAAYNKVPNSISFENALKMRLNAVTST 
               
               
                   
                 NTEGAVIHINRSCKLWSGFLYELARRLEKETGIPVVSFDGDQA 
               
               
                   
                 DPRNFSEAQYDTRIQGLNEVMVAKKEAE 
               
               
                   
               
               
                 FldC: subunit of 
                 MSNSDKFFNDFKDIVENPKKYIMKHMEQTGQKAIGCMPLYTP 
               
               
                 indole-3 -lactate 
                 EELVLAAGMFPVGVWGSNTELSKAKTYFPAFICSILQTTLENA 
               
               
                 dehydratase from 
                 LNGEYDMLSGMMITNYCDSLKCMGQNFKLTVENIEFIPVTVPQ 
               
               
                 Clostridium 
                 NRKMEAGKEFLKSQYKMNIEQLEKISGNKITDESLEKAIEIYDE 
               
               
                 sporogenes 
                 HRKVMNDFSMLASKYPGIITPTKRNYVMKSAYYMDKKEHTE 
               
               
                 SEQ ID NO: 175 
                 KVRQLMDEIKAIEPKPFEGKRVITTGIIADSEDLLKILEENNIAIV 
               
               
                   
                 GDDIAHESRQYRTLTPEANTPMDRLAEQFANRECSTLYDPEKK 
               
               
                   
                 RGQYIVEMAKERKADGIIFFMTKFCDPEEYDYPQMKKDFEEA 
               
               
                   
                 GIPHVLIETDMQMKNYEQARTAIQAFSETL 
               
               
                   
               
               
                 FldD: indole-3- 
                 MFFTEQHELIRKLARDFAEQEIEPIADEVDKTAEFPKEIVKKMA 
               
               
                 acrylyl-CoA 
                 QNGFFGIKMPKEYGGAGADNRAYVTIMEEISRASGVAGIYLSS 
               
               
                 reductase from 
                 PNSLLGTPFLLVGTDEQKEKYLKPMIRGEKTLAFALTEPGAGS 
               
               
                 Clostridium 
                 DAGALATTAREEGDYYILNGRKTFITGAPISDNIIVFAKTDMSK 
               
               
                 sporogenes 
                 GTKGITTFIVDSKQEGVSFGKPEDKMGMIGCPTSDIILENVKVH 
               
               
                 SEQ ID NO: 176 
                 KSDILGEVNKGFITAMKTLSVGRIGVASQALGIAQAAVDEAVK 
               
               
                   
                 YAKQRKQFNRPIAKFQAIQFKLANMETKLNAAKLLVYNAAYK 
               
               
                   
                 MDCGEKADKEASMAKYFAAESAIQIVNDALQIHGGYGYIKDY 
               
               
                   
                 KIERLYRDVRVIAIYEGTSEVQQMVIASNLLK 
               
               
                   
               
               
                 FldH1: indole-3- 
                 MKILAYCVRPDEVDSFKKFSEKYGHTVDLIPDSFGPNVAHLAK 
               
               
                 lactate 
                 GYDGISILGNDTCNREALEKIKDCGIKYLATRTAGVNNIDFDA 
               
               
                 dehydrogenase 
                 AKEFGINVANVPAYSPNSVSEFTIGLALSLTRKIPFALKRVELN 
               
               
                 from Clostridium 
                 NFALGGLIGVELRNLTLGVIGTGRIGLKVIEGFSGFGMKKMIGY 
               
               
                 sporogenes 
                 DIFENEEAKKYIEYKSLDEVFKEADIITLHAPLTDDNYHMIGKE 
               
               
                 SEQ ID NO: 177 
                 SIAKMKDGVFIINAARGALIDSEALIEGLKSGKIAGAALDSYEY 
               
               
                   
                 EQGVFHNNKMNEIMQDDTLERLKSFPNVVITPHLGFYTDEAVS 
               
               
                   
                 NMVEITLMNLQEFELKGTCKNQRVCK 
               
               
                   
               
               
                 FldH2: indole-3- 
                 MKILMYSVREHEKPAIKKWLEANPGVQIDLCNNALSEDTVCK 
               
               
                 lactate 
                 AKEYDGIAIQQTNSIGGKAVYSTLKEYGIKQIASRTAGVDMIDL 
               
               
                 dehydrogenase 
                 KMASDSNILVTNVPAYSPNAIAELAVTHTMNLLRNIKTLNKRI 
               
               
                 from Clostridium 
                 AYGDYRWSADLIAREVRSVTVGVVGTGKIGRTSAKLFKGLGA 
               
               
                 sporogenes 
                 NVIGYDAYPDKKLEENNLLTYKESLEDLLREADVVTLHTPLLE 
               
               
                 SEQ ID NO: 178 
                 STKYMINKNNLKYMKPDAFIVNTGRGGIINTEDLIEALEQNKIA 
               
               
                   
                 GAALDTFENEGLFLNKVVDPTKLPDSQLDKLLKMDQVLITHH 
               
               
                   
                 VGFFTTTAVQNIVDTSLDSVVEVLKTNNSVNKVN 
               
               
                   
               
               
                 AcuI: acrylyl- 
                 MRAVLIEKSDDTQSVSVTELAEDQLPEGDVLVDVAYSTLNYK 
               
               
                 CoA reductase 
                 DALAITGKAPVVRRFPMVPGIDFTGTVAQSSHADFKPGDRVIL 
               
               
                 from Rhodobacter 
                 NGWGVGEKHWGGLAERARVRGDWLVPLPAPLDLRQAAMIG 
               
               
                 sphaeroides 
                 TAGYTAMLCVLALERHGVVPGNGEIVVSGAAGGVGSVATTLL 
               
               
                 SEQ ID NO: 179 
                 AAKGYEVAAVTGRASEAEYLRGLGAASVIDRNELTGKVRPLG 
               
               
                   
                 QERWAGGIDVAGSTVLANMLSMMKYRGVVAACGLAAGMDL 
               
               
                   
                 PASVAPFILRGMTLAGVDSVMCPKTDRLAAWARLASDLDPAK 
               
               
                   
                 LEEMTTELPFSEVIETAPKFLDGTVRGRIVIPVTP 
               
               
                   
               
            
           
         
       
     
     In one embodiment, the tryptophan pathway catabolic enzyme encoded by the genetically engineered bacteria has at least about 80% identity with the entire sequence of one or more of SEQ ID NO: 173 through SEQ ID NO: 179. In another embodiment, the tryptophan pathway catabolic enzyme has at least about 85% identity with the entire sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. In one embodiment, the tryptophan pathway catabolic enzyme has at least about 90% identity with the entire sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. In one embodiment, the tryptophan pathway catabolic enzyme has at least about 95% identity with the entire sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. In another embodiment, the tryptophan pathway catabolic enzyme has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. Accordingly, in one embodiment, the tryptophan pathway catabolic enzyme has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the entire sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. In another embodiment, the tryptophan pathway catabolic enzyme comprises the sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. In yet another embodiment the tryptophan pathway catabolic enzyme consists of the sequence of one or more SEQ ID NO: 173 through SEQ ID NO: 179. 
     In some embodiments, the genetically engineered bacteria comprise a gene cassette for the production of one or more indole pathway metabolites described herein from tryptophan or a tryptophan metabolite. In some embodiments, the genetically engineered bacteria take up tryptophan through an endogenous or exogenous transporter as described above herein. In some embodiments, the genetically engineered bacteria additionally produce tryptophan and/or chorismate through any of the pathways described herein, e.g.  FIG. 43 ,  FIG. 49A  and  FIG. 49B . In some embodiments, the genetically engineered bacteria comprise an exporter of one or more indole metabolites, in order to increase the export of indole metabolites produced. 
     In some embodiments, the genetically engineered bacteria are capable of expressing any one or more of the described circuits in low-oxygen conditions, in the presence of disease or tissue specific molecules or metabolites, in the presence of molecules or metabolites associated with inflammation or an inflammatory response or immune suppression or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose or tetracycline. In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the bacterial chromosome. In some embodiments, the tryptophan synthesis and/or tryptophan catabolism cassette(s) is under control of an inducible promoter. Exemplary inducible promoters which may control the expression of the at least one sequence(s) include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. 
     Also, in some embodiments, the genetically engineered bacteria are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more exporters for exporting biological molecules or substrates, such any of the exporters described herein or otherwise known in the art, (6) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, and (7) combinations of one or more of such additional circuits. 
     Trypophan Repressor (TrpR) 
     In any of these embodiments, the tryptophan repressor (trpR) optionally may be deleted, mutated, or modified so as to diminish or obliterate its repressor function. Also, in any of these embodiments, the genetically engineered bacteria optionally comprise gene sequence(s) to produce the tryptophan precursor, Chorismate, e.g., sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC. 
     In some embodiments, the expression of the gene sequences(s) is controlled by an inducible promoter. In some embodiments, the expression of the gene sequences(s) is controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed in vivo, e.g., in the gut. 
     Trypophan and Tryptophan Metabolite Transport 
     Metabolite transporters may further be expressed or modified in the genetically engineered bacteria of the invention in order to enhance tryptophan or KP metabolite transport into the cell. 
     The inner membrane protein YddG of  E. coli , encoded by the yddG gene, is a homologue of the known amino acid exporters RhtA and YdeD. Studies have shown that YddG is capable of exporting aromatic amino acids, including tryptophan. Thus, YddG can function as a tryptophan exporter or a tryptophan secretion system (or tryptophan secretion protein). Other aromatic amino acid exporters are described in Doroshenko et al., FEMS Microbiol. Lett., 275:312-318 (2007). Thus, in some embodiments, the engineered bacteria optionally further comprise gene sequence(s) encoding YddG. In some embodiments, the engineered bacteria can over-express YddG. In some embodiments, the engineered bacteria optionally comprise one or more copies of yddG gene. 
     In some embodiments, the engineered microbe has a mechanism for importing (transporting) Kynurenine from the local environment into the cell. Thus, in some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding a kynureninase secreter. In some embodiments, the genetically engineered bacteria comprise one or more copies of aroP, tnaB or mir gene. 
     In some embodiments the genetically engineered bacteria comprise a transporter to facilitate uptake of tryptophan into the cell. Three permeases, Mtr, TnaB, and AroP, are involved in the uptake of L-tryptophan in  Escherichia coli . In some embodiments, the genetically engineered bacteria comprise one or more copies of one or more of Mtr, TnaB, and AroP. 
     In some embodiments, the genetically engineered bacteria of the invention also comprise multiple copies of the the transporter gene. In some embodiments, the genetically engineered bacteria of the invention also comprise a transport gene from a different bacterial species. In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of a transporter gene from a different bacterial species. In some embodiments, the native transporter gene in the genetically engineered bacteria of the invention is not modified. In some embodiments, the genetically engineered bacteria of the invention comprise a transporter gene that is controlled by its native promoter, an inducible promoter, or a promoter that is stronger than the native promoter, e.g., a GlnRS promoter, a P(Bla) promoter, or a constitutive promoter. 
     In some embodiments, the native transporter gene in the genetically engineered bacteria is not modified, and one or more additional copies of the native transporter gene are inserted into the genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In alternate embodiments, the native transporter gene is not modified, and a copy of a non-native transporter gene from a different bacterial species is inserted into the genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. 
     In some embodiments, the expression of the gene sequences(s) is controlled by an inducible promoter. In some embodiments, the expression of the gene sequences(s) is controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed in vivo, e.g., in the gut. 
     In some embodiments, the expression of the gene sequences(s) is controlled by an inducible promoter. In some embodiments, the expression of the gene sequences(s) is controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed in vivo, e.g., in the gut. 
     In some embodiments, the native transporter gene in the genetically engineered bacteria is not modified, and one or more additional copies of the native transporter gene are present in the bacteria on a plasmid and under the control of the same inducible promoter that controls expression of the payload e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In alternate embodiments, the native transporter gene is not modified, and a copy of a non-native transporter gene from a different bacterial species is present in the bacteria on a plasmid and under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. 
     In some embodiments, the expression of the gene sequences(s) is controlled by an inducible promoter. In some embodiments, the expression of the gene sequences(s) is controlled by a constitutive promoter. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. In some embodiments, the gene sequences(s) are controlled by an inducible and/or constitutive promoter, and are expressed in vivo, e.g., in the gut. 
     In some embodiments, the native transporter gene is mutagenized, the mutants exhibiting increased ammonia transport are selected, and the mutagenized transporter gene is isolated and inserted into the genetically engineered bacteria. In some embodiments, the native transporter gene is mutagenized, mutants exhibiting increased ammonia transport are selected, and those mutants are used to produce the bacteria of the invention. The transporter modifications described herein may be present on a plasmid or chromosome. 
     In some embodiments, the genetically engineered bacterium is  E. coli  Nissle, and the native transporter gene in  E. coli  Nissle is not modified; one or more additional copies the native  E. coli  Nissle transporter genes are inserted into the  E. coli  Nissle genome under the control of the same inducible promoter that controls expression of the payload e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. In an alternate embodiment, the native transporter gene in  E. coli  Nissle is not modified, and a copy of a non-native transporter gene from a different bacterium, e.g.,  Lactobacillus plantarum , is inserted into the  E. coli  Nissle genome under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload or a constitutive promoter. 
     In some embodiments, the expression of the gene sequences(s) encoding the transporter is controlled by an inducible promoter. In some embodiments, the expression of the gene sequences(s) encoding the transporter is controlled by a constitutive promoter. In some embodiments, the expression of the gene sequences(s) encoding the transporter is controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. In some embodiments, the expression of the gene sequences(s) encoding the transporter is controlled by an inducible and/or constitutive promoter, and are expressed in vivo, e.g., in the gut. 
     In some embodiments, the genetically engineered bacterium is  E. coli  Nissle, and the native transporter gene in  E. coli  Nissle is not modified; one or more additional copies the native  E. coli  Nissle transporter genes are present in the bacterium on a plasmid and under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload, or a constitutive promoter. In an alternate embodiment, the native transporter gene in  E. coli  Nissle is not modified, and a copy of a non-native transporter gene from a different bacterium, e.g.,  Lactobacillus plantarum , are present in the bacterium on a plasmid and under the control of the same inducible promoter that controls expression of the payload, e.g., a FNR promoter, or a different inducible promoter than the one that controls expression of the payload, or a constitutive promoter. 
     In some embodiments, the expression of the gene sequences(s) encoding the transporter is controlled by an inducible promoter. In some embodiments, the expression of the gene sequences(s) encoding the transporter is controlled by a constitutive promoter. In some embodiments, the expression of the gene sequences(s) encoding the transporter is controlled by an inducible and/or constitutive promoter, and are expressed during bacterial culture in vitro, e.g., for bacterial expansion, production and/or manufacture, as described herein. In some embodiments, the expression of the gene sequences(s) encoding the transporter is controlled by an inducible and/or constitutive promoter, and are expressed in vivo, e.g., in the gut. 
     Secreted Polypeptides 
     IL-10 
     In some embodiments, the genetically engineered bacteria of the invention are capable of producing IL-10. Interleukin-10 (IL-10) is a class 2 cytokine, a category which includes cytokines, interferons, and interferon-like molecules, such as IL-19, IL-20, IL-22, IL-24, IL-26, IL-28A, IL-28B, IL-29, IFN-α, IFN-β, IFN-δ, IFN-ε, IFN-κ, IFN-τ, IFN-Ω, and limiting. IL-10 is an anti-inflammatory cytokine that signals through two receptors, IL-10R1 and IL-10R2. Anti-inflammatory properties of human IL-10 include down-regulation of pro-inflammatory cytokines, inhibition of antigen presentation on dendritic cells or suppression of major histocompatibility complex expression. Deficiencies in IL-10 and/or its receptors are associated with IBD and intestinal sensitivity (Nielsen, 2014). Bacteria expressing IL-10 or protease inhibitors may ameliorate conditions such as Crohn&#39;s disease and ulcerative colitis (Simpson et al., 2014). The genetically engineered bacteria may comprise any suitable gene encoding IL-10, e.g., human IL-10. In some embodiments, the gene encoding IL-10 is modified and/or mutated, e.g., to enhance stability, increase IL-10 production, and/or increase anti-inflammatory potency under inducing conditions. In some embodiments, the genetically engineered bacteria are capable of producing IL-10 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing IL-10 in low-oxygen conditions. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that encodes IL-10. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence comprising SEQ ID NO: 134 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence comprising SEQ ID NO: 49 or a functional fragment thereof. 
     
       
         
           
               
             
               
                 TABLE 13 
               
               
                   
               
             
            
               
                 IL-10 (SEQ ID NO: 134) 
               
               
                 ATGAGCCCCGGACAGGGAACTCAAAGCGAGAACAGCTGCACACATTTTC 
               
               
                   
               
               
                 CAGGTAATCTTCCAAATATGCTTCGTGACTTGCGTGACGTTTCTCTCGC 
               
               
                   
               
               
                 GTGAAAACCTTTTTTCAGATGAAGGATCAGTTAGATAATCTGCTGCTGA 
               
               
                   
               
               
                 AAGAATCGCTTCTTGAGGACTTCAAGGGATATCTGGGATGTCAGGCGTT 
               
               
                   
               
               
                 ATCTGAGATGATTCAGTTTTATTTGGAAGAAGTTATGCCCCAGGCTGAG 
               
               
                   
               
               
                 AATCAAGACCCTGACATCAAAGCGCATGTGAATAGCCTGGGCGAGAATC 
               
               
                   
               
               
                 TGAAGACACTGCGCCTGCGTCTTCGCCGCTGTCACCGTTTTCTGCCTTG 
               
               
                   
               
               
                 CGAAAATAAGAGTAAGGCCGTTGAGCAAGTGAAAAATGCTTTCAACAAG 
               
               
                   
               
               
                 TTACAAGAAAAAGGGATTTACAAAGCTATGTCTGAGTTTGACATTTTCA 
               
               
                   
               
               
                 TTAATTACATTGAGGCCTACATGACTATGAAGATTCGCAAT 
               
               
                   
               
            
           
         
       
     
     Wild type IL-10 (wtIL-10) is a domain swapped dimer whose structural integrity depends on the dimerization of two peptide chains. wtIL-10 was converted to a monomeric isomer by inserting 6 amino acids into the loop connecting the swapped secondary structural elements (see, e.g., Josephson, K. et al. Design and analysis of an engineered human interleukin-10 monomer. J. Biol. Chem. 275, 13552-13557 (2000), and Yoon, S. I. et al. Epstein-Barr Virus IL-10 Engages IL-10R1 by a Two-step Mechanism Leading to Altered Signaling Properties. J. Biol. Chem. 287, 26586-26595 (2012). Monomoerized IL-10 therefore comprises a small linker which deviates from the wild-type human IL-10 sequence. This linker causes the IL10 to become active as a monomer rather than a dimer (see, e.g., Josephson, K. et al. Design and analysis of an engineered human interleukin-10 monomer. J. Biol. Chem. 275, 13552-13557 (2000), and Yoon, S I. et al. Epstein-Barr Virus IL-10 Engages IL-10R1 by a Two-step Mechanism Leading to Altered Signaling Properties. J. Biol. Chem. 287, 26586-26595 (2012)). 
     Secretion of a monomeric protein may have advantages, avoiding the extra step of dimerization in the periplasmic space. Moreover, there is more flexibility in the selection of appropriate secretion systems. For example, the tat-dependent secretion system secretes polypeptides in a folded fashion. Dimers cannot fold correctly without the formation of disulfide bonds. Disulfide bonds, however, cannot form in the reducing intracellular environment and require the oxidizing environment of the periplasm to form. Therefore, the tat-dependent system may no be appropriate for the secretion of proteins which require dimerization to function properly. 
     In some embodiments, the genetically engineered bacteria of the invention are capable of producing monomerized human IL-10. In some embodiments, the genetically engineered bacteria are capable of producing monomerized IL-10 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing monomerized IL-10 in low-oxygen conditions. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that encodes monomerized IL-10. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence comprising SEQ ID NO: 198 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence comprising SEQ ID NO: 198 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria comprise a sequence which encodes the polypeptide encoded by SEQ ID NO: 198 or a fragment or functional variant thereof. In some embodiments, the monomerized IL-10 expressed by the bacteria stimulates IL-10R1 and IL-10R2 and initiates signal transduction. Signaling includes Stat signaling, e.g. through the phosphorylation of Tyr705 and/or Ser727. 
     In some embodiments, the genetically engineered bacteria of the invention are capable of producing viral IL-10. Exemplary viral IL-10 homologues encoded by the bacteria include human cytomegalo- (HCMV) and Epstein-Barr virus (EBV) IL-10. Apart from its anti-inflammatory effects, human IL-10 also possesses pro-inflammatory activity, e.g., stimulation of B-cell maturation and proliferation of natural killer cells (Foerster et al., Secretory expression of biologically active human Herpes virus interleukin-10 analogues in  Escherichia coli  via a modified Sec-dependent transporter construct, BMC Biotechnol. 2013; 13: 82, and references therein). In contrast, viral IL-10 homologues share many biological activities of hIL-10 but, due to selective pressure during virus evolution and the need to escape the host immune system, also display unique traits, including increased stability and lack of immunostimulatory functions (Foerster et al, and references therein). As such, viral counterparts may be useful and possibly more effective than hIL-10 with respect to anti-inflammatory and/or immune suppressing effects. 
     In some embodiments, the genetically engineered bacteria are capable of producing viral IL-10 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing viral IL-10 in low-oxygen conditions. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence that encodes viral IL-10. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence comprising SEQ ID NO: 193 and/or SEQ ID NO: 194 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence comprising SEQ ID NO: 193 and/or SEQ ID NO: 194 or a functional fragment thereof. In some embodiments, the viral d IL-10 expressed by the bacteria stimulates IL-10R1 and IL-10R2 and initiates signal transduction. Signaling includes Stat signaling, e.g. through the phosphorylation of Tyr705 and/or Ser727. 
     To improve acetate production, while maintaining high levels of IL-10 secretion, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10-polypeptides for secretion described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-10, OmpF-IL-10, and TorA-IL-10 and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-10, OmpF-IL-10, and TorA-IL-10 and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-10, OmpF-IL-10, and TorA-IL-10 and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-10, OmpF-IL-10, and TorA-IL-10 and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-10, OmpF-IL-10, and TorA-IL-10 and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-10, OmpF-IL-10, and TorA-IL-O1 and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-10, OmpF-IL-10, and TorA-IL-10 and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more IL-10 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more IL-10 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more IL-10 than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle, while maintaining high levels of IL-10 secretion. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for IL-10 secretion. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for IL-10 synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-10 polypeptides for secretion and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or IL-10 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-10, OmpF-IL-10, and TorA-IL-10 and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-10, OmpF-IL-10, and TorA-IL-10 and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-10, OmpF-IL-10, and TorA-IL-10 and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-10, OmpF-IL-10, and TorA-IL-10 and further comprise a mutation in the endogenous pta and ldhA genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-10, OmpF-IL-10, and TorA-IL-10 and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-10, OmpF-IL-10, and TorA-IL-10 and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-10, OmpF-IL-10, and TorA-IL-10 and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-10, OmpF-IL-10, and TorA-IL-10 and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-10, OmpF-IL-10, and TorA-IL-10 and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more IL-0 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more IL-10 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more IL-10 than unmodified bacteria of the same bacterial subtype under the same conditions. 
     IL-2 
     In some embodiments, the genetically engineered bacteria are capable of producing IL-2. Interleukin 2 (IL-2) mediates autoimmunity by preserving health of regulatory T cells (Treg). Treg cells, including those expressing Foxp3, typically suppress effector T cells that are active against self-antigens, and in doing so, can dampen autoimmune activity. IL-2 functions as a cytokine to enhance Treg cell differentiation and activity while diminished IL-2 activity can promote autoimmunity events. IL-2 is generated by activated CD4+ T cells, and by other immune mediators including activated CD8+ T cells, activated dendritic cells, natural killer cells, and NK T cells. IL-2 binds to IL-2R, which is composed of three chains including CD25, CD122, and CD132. IL-2 promotes growth of Treg cells in the thymus, while preserving their function and activity in systemic circulation. Treg cell activity plays an intricate role in the IBD setting, with murine studies suggesting a protective role in disease pathogenesis. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 135 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence encoding SEQ ID NO: 135 or a functional fragment thereof. 
     In some embodiments, the genetically engineered bacteria are capable of producing IL-2 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing IL-2 in low-oxygen conditions. 
     
       
         
           
               
             
               
                 TABLE 14 
               
               
                   
               
               
                  SEQ ID NO: 135 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 SEQ ID NO: 135 
               
               
                   
                 MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKLTRM 
               
               
                   
                 LTFKFYMPKK ATELKHLQCL EEELKPLEEV LNLAQSKNFH 
               
               
                   
                 LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN 
               
               
                   
                 RWITFCQSII STLT 
               
               
                   
                   
               
            
           
         
       
     
     To improve acetate production, while maintaining high levels of IL-2 secretion, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2-polypeptides for secretion described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-2, OmpF-IL-2, and TorA-IL-2 and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-2, OmpF-IL-2, and TorA-IL-2 and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-2, OmpF-IL-2, and TorA-IL-2 and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-2, OmpF-IL-2, and TorA-IL-2 and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-2, OmpF-IL-2, and TorA-IL-2 and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-2, OmpF-IL-2, and TorA-IL-2 and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-2, OmpF-IL-2, and TorA-IL-2 and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more IL-2 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more IL-2 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more IL-2 than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle, while maintaining high levels of IL-2 secretion. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for IL-2 secretion. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for IL-2 synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-2 polypeptides for secretion and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or IL-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-2, OmpF-IL-2, and TorA-IL-2 and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-2, OmpF-IL-2, and TorA-IL-2 and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-2, OmpF-IL-2, and TorA-IL-2 and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-2, OmpF-IL-2, and TorA-IL-2 and further comprise a mutation in the endogenous pta and ldhA genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-2, OmpF-IL-2, and TorA-IL-2 and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-2, OmpF-IL-2, and TorA-IL-2 and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-2, OmpF-IL-2, and TorA-IL-2 and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-2, OmpF-IL-2, and TorA-IL-2 and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-2, OmpF-IL-2, and TorA-IL-2 and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more IL-2 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more IL-2 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more IL-2 than unmodified bacteria of the same bacterial subtype under the same conditions. 
     IL-22 
     In some embodiments, the genetically engineered bacteria are capable of producing IL-22. Interleukin 22 (IL-22) cytokine can be produced by dendritic cells, lymphoid tissue inducer-like cells, natural killer cells and expressed on adaptive lymphocytes. Through initiation of Jak-STAT signaling pathways, IL-22 expression can trigger expression of antimicrobial compounds as well as a range of cell growth related pathways, both of which enhance tissue repair mechanisms. IL-22 is critical in promoting intestinal barrier fidelity and healing, while modulating inflammatory states. Murine models have demonstrated improved intestinal inflammation states following administration of IL-22. Additionally, IL-22 activates STAT3 signaling to promote enhanced mucus production to preserve barrier function. IL-22&#39;s association with IBD susceptibility genes may modulate phenotypic expression of disease as well. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 136 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence encoding SEQ ID NO: 136 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing IL-22 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing IL-22 in low-oxygen conditions. 
     
       
         
           
               
             
               
                 TABLE 15 
               
               
                   
               
               
                  SEQ ID NO: 136 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 SEQ ID NO: 136 
               
               
                 MAALQKSVSS FLMGTLATSC LLLLALLVQG GAAAPISSHC RLDKSNFQQP 
               
               
                 YITNRTFMLA KEASLADNNT DVRLIGEKLF HGVSMSERCY LMKQVLNFTL 
               
               
                 EEVLFPQSDR FQPYMQEVVP FLARLSNRLS TCHIEGDDLH IQRNVQKLKD 
               
               
                 TVKKLGESGE IKAIGELDLL FMSLRNACI 
               
               
                   
               
            
           
         
       
     
     To improve acetate production, while maintaining high levels of IL-22 secretion, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22-polypeptides for secretion described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-22, OmpF-IL-22, and TorA-IL-22 and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-22, OmpF-IL-22, and TorA-IL-22 and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-22, OmpF-IL-22, and TorA-IL-22 and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-22, OmpF-IL-22, and TorA-IL-22 and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-22, OmpF-IL-22, and TorA-IL-22 and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-22, OmpF-IL-22, and TorA-IL-22 and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-22, OmpF-IL-22, and TorA-IL-22 and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more IL-22 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more IL-22 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more IL-22 than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle, while maintaining high levels of IL-22 secretion. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for IL-22 secretion. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for IL-22 synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-22 polypeptides for secretion and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or IL-22 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-22, OmpF-IL-22, and TorA-IL-22 and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-22, OmpF-IL-22, and TorA-IL-22 and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-22, OmpF-IL-22, and TorA-IL-22 and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-22, OmpF-IL-22, and TorA-IL-22 and further comprise a mutation in the endogenous pta and ldhA genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-22, OmpF-IL-22, and TorA-IL-22 and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-22, OmpF-IL-22, and TorA-IL-22 and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-22, OmpF-IL-22, and TorA-IL-22 and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-22, OmpF-IL-22, and TorA-IL-22 and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-22, OmpF-IL-22, and TorA-IL-22 and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more IL-22 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more IL-22 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more IL-22 than unmodified bacteria of the same bacterial subtype under the same conditions. 
     IL-27 
     In some embodiments, the genetically engineered bacteria are capable of producing IL-27. Interleukin 27 (IL-27) cytokine is predominately expressed by activated antigen presenting cells, while IL-27 receptor is found on a range of cells including T cells, NK cells, among others. In particular, IL-27 suppresses development of pro-inflammatory T helper 17 (Th17) cells, which play a critical role in IBD pathogenesis. Further, IL-27 can promote differentiation of IL-10 producing Tr1 cells and enhance IL-10 output, both of which have anti-inflammatory effects. IL-27 has protective effects on epithelial barrier function via activation of MAPK and STAT signaling within intestinal epithelial cells. Additionally, IL-27 enhances production of antibacterial proteins that curb bacterial growth. Improvement in barrier function and reduction in bacterial growth suggest a favorable role for IL-27 in IBD pathogenesis. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 137 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence encoding SEQ ID NO: 137 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing IL-27 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing IL-27 in low-oxygen conditions. 
     
       
         
           
               
             
               
                 TABLE 16 
               
               
                   
               
               
                  SEQ ID NO: 137 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 SEQ ID NO: 137 
               
               
                 MGQTAGDLGW RLSLLLLPLL LVQAGVWGFP RPPGRPQLSL QELRREFTVS 
               
               
                 LHLARKLLSE VRGQAHRFAE SHLPGVNLYL LPLGEQLPDV SLTFQAWRRL 
               
               
                 SDPERLCFIS TTLQPFHALL GGLGTQGRWT NMERMQLWAM RLDLRDLQRH 
               
               
                 LRFQVLAAGF NLPEEEEEEE EEEEEERKGL LPGALGSALQ GPAQVSWPQL 
               
               
                 LSTYRLLHSL ELVLSRAVRE LLLLSKAGHS VWPLGFPTLS PQP 
               
               
                   
               
            
           
         
       
     
     To improve acetate production, while maintaining high levels of IL-27 secretion, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27-polypeptides for secretion described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-27, OmpF-IL-27, and TorA-IL-27 and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-27, OmpF-IL-27, and TorA-IL-27 and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-27, OmpF-IL-27, and TorA-IL-27 and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-27, OmpF-IL-27, and TorA-IL-27 and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-27, OmpF-IL-27, and TorA-IL-27 and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-27, OmpF-IL-27, and TorA-IL-27 and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-27, OmpF-IL-27, and TorA-IL-27 and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more IL-27 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more IL-27 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more IL-27 than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle, while maintaining high levels of IL-27 secretion. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for IL-27 secretion. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for IL-27 synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-27 polypeptides for secretion and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or IL-27 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-27, OmpF-IL-27, and TorA-IL-27 and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-27, OmpF-IL-27, and TorA-IL-27 and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-27, OmpF-IL-27, and TorA-IL-27 and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-27, OmpF-IL-27, and TorA-IL-27 and further comprise a mutation in the endogenous pta and ldhA genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-27, OmpF-IL-27, and TorA-IL-27 and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-27, OmpF-IL-27, and TorA-IL-27 and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-27, OmpF-IL-27, and TorA-IL-27 and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-27, OmpF-IL-27, and TorA-IL-27 and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-27, OmpF-IL-27, and TorA-IL-27 and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more IL-27 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more IL-27 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more IL-27 than unmodified bacteria of the same bacterial subtype under the same conditions. 
     SOD 
     In some embodiments, the genetically engineered bacteria of the invention are capable of producing SOD. Increased ROS levels contribute to pathophysiology of inflammatory bowel disease. Increased ROS levels may lead to enhanced expression of vascular cell adhesion molecule 1 (VCAM-1), which can facilitate translocation of inflammatory mediators to disease affected tissue, and result in a greater degree of inflammatory burden. Antioxidant systems including superoxide dismutase (SOD) can function to mitigate overall ROS burden. However, studies indicate that the expression of SOD in the setting of IBD may be compromised, e.g., produced at lower levels in IBD, thus allowing disease pathology to proceed. Further studies have shown that supplementation with SOD to rats within a colitis model is associated with reduced colonic lipid peroxidation and endothelial VCAM-1 expression as well as overall improvement in inflammatory environment. Thus, in some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 138 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence encoding SEQ ID NO: 138 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing SOD under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing SOD in low-oxygen conditions. 
     
       
         
           
               
             
               
                 TABLE 17 
               
               
                   
               
               
                  SEQ ID NO: 138 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 SEQ ID NO: 138 
               
               
                 MATKAVCVLK GDGPVQGIIN FEQKESNGPV KVWGSIKGLT EGLHGFHVHE 
               
               
                 FGDNTAGCTS AGPHFNPLSR KHGGPKDEER HVGDLGNVTA DKDGVADVSI 
               
               
                 EDSVISLSGD HCIIGRTLVV HEKADDLGKG GNEESTKTGN AGSRLACGVI 
               
               
                 GIAQ 
               
               
                   
               
            
           
         
       
     
     GLP2 
     In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 or proglucagon. Glucagon-like peptide 2 (GLP-2) is produced by intestinal endocrine cells and stimulates intestinal growth and enhances gut barrier function. GLP-2 administration has therapeutic potential in treating IBD, short bowel syndrome, and small bowel enteritis (Yazbeck et al., 2009). The genetically engineered bacteria may comprise any suitable gene encoding GLP-2 or proglucagon, e.g., human GLP-2 or proglucagon. In some embodiments, a protease inhibitor, e.g., an inhibitor of dipeptidyl peptidase, is also administered to decrease GLP-2 degradation. In some embodiments, the genetically engineered bacteria express a degradation resistant GLP-2 analog, e.g., Teduglutide (Yazbeck et al., 2009). In some embodiments, the gene encoding GLP-2 or proglucagon is modified and/or mutated, e.g., to enhance stability, increase GLP-2 production, and/or increase gut barrier enhancing potency under inducing conditions. In some embodiments, the genetically engineered bacteria of the invention are capable of producing GLP-2 or proglucagon under inducing conditions. GLP-2 administration in a murine model of IBD is associated with reduced mucosal damage and inflammation, as well as a reduction in inflammatory mediators, such as TNF-α and IFN-y. Further, GLP-2 supplementation may also lead to reduced mucosal myeloperoxidase in colitis/ileitis models. In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 139 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence encoding SEQ ID NO: 139 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 in low-oxygen conditions. 
     
       
         
           
               
             
               
                 TABLE 18 
               
               
                   
               
               
                 SEQ ID NO: 139 GLP-2 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 SEQ ID NO: 139 
               
               
                   
                 HADGSFSDEMNTILDNLAARDFINWLIQTKITD 
               
               
                   
                   
               
            
           
         
       
     
     In some embodiments, the genetically engineered bacteria are capable of producing GLP-2 analogs, including but not limited to, Gattex and teduglutide. Teduglutide is a protease resistant analog of GLP-2. It is made up of 33 amino acids and differs from GLP-2 by one amino acid (alanine is substituted by glycine). The significance of this substitution is that teduglutide is longer acting than endogenous GLP-2 as it is more resistant to proteolysis from dipeptidyl peptidase-4. 
     
       
         
           
               
             
               
                 TABLE 19 
               
               
                   
               
               
                 SEQ ID NO: 140 Teduglutide\ 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 SEQ ID NO: 140 
               
               
                   
                 HGDGSFSDEMNTILDNLAARDFINWLIQTKITD 
               
               
                   
                   
               
            
           
         
       
     
     In some embodiments, the genetically engineered bacteria comprise a nucleic acid sequence encoding SEQ ID NO: 140 or a functional fragment thereof. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to a nucleic acid sequence encoding SEQ ID NO: 140 or a functional fragment thereof. In some embodiments, the genetically engineered bacteria are capable of producing Teduglutide under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing Teduglutide in low-oxygen conditions. 
     To improve acetate production, while maintaining high levels of GLP-2 secretion, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2-polypeptides for secretion described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-GLP-2, OmpF-GLP-2, and TorA-GLP-2 and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-GLP-2, OmpF-GLP-2, and TorA-GLP-2 and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-GLP-2, OmpF-GLP-2, and TorA-GLP-2 and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-GLP-2, OmpF-GLP-2, and TorA-GLP-2 and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-GLP-2, OmpF-GLP-2, and TorA-GLP-2 and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-GLP-2, OmpF-GLP-2, and TorA-GLP-2 and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-GLP-2, OmpF-GLP-2, and TorA-GLP-2 and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more GLP-2 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more GLP-2 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more GLP-2 than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle, while maintaining high levels of GLP-2 secretion. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for GLP-2 secretion. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for GLP-2 synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more GLP-2 polypeptides for secretion and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or GLP-2 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-GLP-2, OmpF-GLP-2, and TorA-GLP-2 and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-GLP-2, OmpF-GLP-2, and TorA-GLP-2 and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-GLP-2, OmpF-GLP-2, and TorA-GLP-2 and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-GLP-2, OmpF-GLP-2, and TorA-GLP-2 and further comprise a mutation in the endogenous pta and ldhA genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-GLP-2, OmpF-GLP-2, and TorA-GLP-2 and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-GLP-2, OmpF-GLP-2, and TorA-GLP-2 and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-GLP-2, OmpF-GLP-2, and TorA-GLP-2 and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-GLP-2, OmpF-GLP-2, and TorA-GLP-2 and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-GLP-2, OmpF-GLP-2, and TorA-GLP-2 and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more GLP-2 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more GLP-2 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more GLP-2 than unmodified bacteria of the same bacterial subtype under the same conditions. 
     IL-19, IL-20, and/or IL-24 
     In some embodiments, the genetically engineered bacteria are capable of producing IL-19, IL-20, and/or IL-24. In some embodiments, the genetically engineered bacteria are capable of producing IL-19, IL-20, and/or IL-24 under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing IL-19, IL-20 and/or IL-24 in low-oxygen conditions. 
     To improve acetate production, while maintaining high levels of IL-19, IL-20, AND/OR IL-24 secretion, targeted one or more deletions can be introduced in competing metabolic arms of mixed acid fermentation to prevent the production of alternative metabolic fermentative byproducts (thereby increasing acetate production). 
     Non-limiting examples of competing such competing metabolic arms are frdA (converts phosphoenolpyruvate to succinate), ldhA (converts pyruvate to lactate) and adhE (converts Acetyl-CoA to Ethanol). Deletions which may be introduced therefore include deletion of adhE, ldh, and frd. Thus, in certain embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise mutations and/or deletions in one or more of frdA, ldhA, and adhE. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24-polypeptides for secretion described herein and one or more mutation(s) and/or deletion(s) in one or more genes selected from the ldhA gene, the frdA gene and the adhE gene. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA and rdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation and/or deletion in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-19, IL-20, AND/OR IL-24, OmpF-IL-19, IL-20, AND/OR IL-24, and TorA-IL-19, IL-20, AND/OR IL-24 and further comprise a mutation and/or deletion in the endogenous ldhA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-19, IL-20, AND/OR IL-24, OmpF-IL-19, IL-20, AND/OR IL-24, and TorA-IL-19, IL-20, AND/OR IL-24 and further comprise a mutation and/or deletion in the endogenous adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-19, IL-20, AND/OR IL-24, OmpF-IL-19, IL-20, AND/OR IL-24, and TorA-IL-19, IL-20, AND/OR IL-24 and further comprise a mutation and/or deletion in the endogenous frdA gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-19, IL-20, AND/OR IL-24, OmpF-IL-19, IL-20, AND/OR IL-24, and TorA-IL-19, IL-20, AND/OR IL-24 and further comprise a mutation and/or deletion in the endogenous ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-19, IL-20, AND/OR IL-24, OmpF-IL-19, IL-20, AND/OR IL-24, and TorA-IL-19, IL-20, AND/OR IL-24 and further comprise a mutation and/or deletion in the endogenous ldhA genes and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-19, IL-20, AND/OR IL-24, OmpF-IL-19, IL-20, AND/OR IL-24, and TorA-IL-19, IL-20, AND/OR IL-24 and further comprise a mutation and/or deletion in the endogenous frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-19, IL-20, AND/OR IL-24, OmpF-IL-19, IL-20, AND/OR IL-24, and TorA-IL-19, IL-20, AND/OR IL-24 and further comprise a mutation and/or deletion in the endogenous ldhA, the frdA, and adhE genes. In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more IL-19, IL-20, AND/OR IL-24 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more IL-19, IL-20, AND/OR IL-24 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more IL-19, IL-20, AND/OR IL-24 than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In certain situations, the need may arise to prevent and/or reduce acetate production by of an engineered or naturally occurring strain, e.g.,  E. coli  Nissle, while maintaining high levels of IL-19, IL-20, AND/OR IL-24 secretion. Without wishing to be bound by theory, one or more mutations and/or deletions in one or more gene(s) encoding in one or more enzymes which function in the acetate producing metabolic arm of fermentation should reduce and/or prevent production of acetate. A non-limiting example of such an enzyme is phosphate acetyltransferase (Pta), which is the first enzyme in the metabolic arm converting acetyl-CoA to acetate. Deletion and/or mutation of the Pta gene or a gene encoding another enzyme in this metabolic arm may also allow for more acetyl-CoA to be used for IL-19, IL-20, AND/OR IL-24 secretion. Additionally, one or more mutations preventing or reducing the flow through other metabolic arms of mixed acid fermentation, such as those which produce succinate, lactate, and/or ethanol can increase the production of acetyl-CoA, which is available for IL-19, IL-20, AND/OR IL-24 synthesis. Such mutations and/or deletions, include but are not limited to mutations and/or deletions in the frdA, ldhA, and/or adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation in the endogenous pta and ldhA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or more IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding one or IL-19, IL-20, AND/OR IL-24 polypeptides for secretion and further comprise a mutation and/or deletion in the endogenous pta, ldhA, frdA, and adhE genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-19, IL-20, AND/OR IL-24, OmpF-IL-19, IL-20, AND/OR IL-24, and TorA-IL-19, IL-20, AND/OR IL-24 and further comprise a mutation and/or deletion in the endogenous pta gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-19, IL-20, AND/OR IL-24, OmpF-IL-19, IL-20, AND/OR IL-24, and TorA-IL-19, IL-20, AND/OR IL-24 and further comprise a mutation and/or deletion in the endogenous pta gene and in one or more endogenous genes selected from in the ldhA gene, the frdA gene and the adhE gene. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-19, IL-20, AND/OR IL-24, OmpF-IL-19, IL-20, AND/OR IL-24, and TorA-IL-19, IL-20, AND/OR IL-24 and further comprise a mutation in the endogenous pta and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-19, IL-20, AND/OR IL-24, OmpF-IL-19, IL-20, AND/OR IL-24, and TorA-IL-19, IL-20, AND/OR IL-24 and further comprise a mutation in the endogenous pta and ldhA genes. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-19, IL-20, AND/OR IL-24, OmpF-IL-19, IL-20, AND/OR IL-24, and TorA-IL-19, IL-20, AND/OR IL-24 and further comprise a mutation in the endogenous pta and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-19, IL-20, AND/OR IL-24, OmpF-IL-19, IL-20, AND/OR IL-24, and TorA-IL-19, IL-20, AND/OR IL-24 and further comprise a mutation and/or deletion in the endogenous pta, ldhA and frdA genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-19, IL-20, AND/OR IL-24, OmpF-1-19, IL-20, AND/OR IL-24, and TorA-IL-19, IL-20, AND/OR IL-24 and further comprise a mutation in the endogenous pta, ldhA, and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-19, IL-20, AND/OR IL-24, OmpF-IL-19, IL-20, AND/OR IL-24, and TorA-IL-19, IL-20, AND/OR IL-24 and further comprise a mutation in the endogenous pta, frdA and adhE genes. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) selected from PhoA-IL-19, IL-20, AND/OR IL-24, OmpF-IL-19, IL-20, AND/OR IL-24, and TorA-IL-19, IL-20, AND/OR IL-24 and further comprise a mutation in the endogenous pta, ldhA, frdA, and adhE genes 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, less acetate than unmodified bacteria of the same bacterial subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce 0% to to 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 18%, 18% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45% 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70% to 80%, 80% to 90%, or 90% to 100% more IL-19, IL-20, AND/OR IL-24 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the genetically engineered bacteria produce 1.0-1.2-fold, 1.2-1.4-fold, 1.4-1.6-fold, 1.6-1.8-fold, 1.8-2-fold, or two-fold more IL-19, IL-20, AND/OR IL-24 than unmodified bacteria of the same bacterial subtype under the same conditions. In yet another embodiment, the the genetically engineered bacteria produce three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold, forty-fold, or fifty-fold, more IL-19, IL-20, AND/OR IL-24 than unmodified bacteria of the same bacterial subtype under the same conditions. 
     Inhibition of Pro-Inflammatory Molecules 
     In some embodiments, the genetically engineered bacteria of the invention are capable of producing a molecule that is capable of inhibiting a pro-inflammatory molecule. The genetically engineered bacteria may express any suitable inhibitory molecule, e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA, that is capable of neutralizing one or more pro-inflammatory molecules, e.g., TNF, IFN-γ, IL-1β, IL-6, IL-8, IL-17, IL-18, IL-21, IL-23, IL-26, IL-32, Arachidonic acid, prostaglandins (e.g., PGE 2 ), PGI 2 , serotonin, thromboxanes (e.g., TXA 2 ), leukotrienes (e.g., LTB 4 ), hepoxillin A 3 , or chemokines (Keates et al., 2008; Ahmad et al., 2012). The genetically engineered bacteria may inhibit one or more pro-inflammatory molecules, e.g., TNF, IL-17. In some embodiments, the genetically engineered bacteria are capable of modulating one or more molecule(s) shown in Table 20. In some embodiments, the genetically engineered bacteria are capable of inhibiting, removing, degrading, and/or metabolizing one or more inflammatory molecules. 
     
       
         
           
               
               
               
             
               
                 TABLE 20 
               
               
                   
               
               
                 Metabolites 
                 Related bacteria 
                 Potential biological functions 
               
               
                   
               
             
            
               
                 Bile acids: cholate, hyocholate, 
                   Lactobacillus , 
                 Absorb dietary fats and lipid-soluble 
               
               
                 deoxycholate, chenodeoxycholate, 
                   Bifidobacteria , 
                 vitamins, facilitate lipid absorption, 
               
               
                 a-muricholate, b-muricholate, w- 
                   Enterobacter , 
                 maintain intestinal barrier function, 
               
               
                 muricholate, taurocholate, 
                   Bacteroides , 
                 signal systemic endocrine functions to 
               
               
                 glycocholate, taurochenoxycholate, 
                 
                   Clostridium 
                 
                 regulate triglycerides, cholesterol, 
               
               
                 glycochenodeoxycholate, 
                   
                 glucose and energy homeostasis. 
               
               
                 taurocholate, tauro-a-muricholate, 
               
               
                 tauro-b-muricholate, lithocholate, 
               
               
                 ursodeoxycholate, 
               
               
                 hyodeoxycholate, 
               
               
                 glycodeoxylcholate 
               
               
                 Choline metabolites: methylamine, 
                 
                   Faecalibacterium 
                 
                 Modulate lipid metabolism and glucose 
               
               
                 dimethylamine, trimethylamine, 
                   prausnitzii , 
                 homeostasis. Involved in nonalcoholic 
               
               
                 trimethylamine-N-oxide, 
                 
                   Bifidobacterium 
                 
                 fatty liver disease, dietary induced 
               
               
                 dimethylglycine, betaine 
                   
                 obesity, diabetes, and cardiovascular 
               
               
                   
                   
                 disease. 
               
               
                 Phenolic, benzoyl, and phenyl 
                   Clostridium difficile , 
                 Detoxification of xenobiotics; indicate gut 
               
               
                 derivatives: benzoic acid, hippuric 
                   F. prausnitzii , 
                 microbial composition and activity; utilize 
               
               
                 acid, 2-hydroxyhippuric acid, 2- 
                   Bifidobacterium , 
                 polyphenols. Urinary hippuric acid may 
               
               
                 hydroxybenzoic acid, 3- 
                   Subdoligranulum , 
                 be a biomarker of hypertension and 
               
               
                 hydroxyhippuric acid, 3- 
                 
                   Lactobacillus 
                 
                 obesity in humans. Urinary 4- 
               
               
                 hydroxybenzoic acid, 4 
                   
                 hydroxyphenylacetate, 4-cresol, and 
               
               
                 hydroxybenzoic acid, 
                   
                 phenylacetate are elevated in colorectal 
               
               
                 3hydroxyphenylpropionate, 4- 
                   
                 cancer. Urinary 4-cresyl sulfate is 
               
               
                 hydroxyphenylpropionate, 3- 
                   
                 elevated in children with severe autism. 
               
               
                 hydroxycinnamate, 4- 
               
               
                 methylphenol, tyrosine, 
               
               
                 phenylalanine, 4-cresol, 4-cresyl 
               
               
                 sulfate, 4-cresyl glucuronide, 4- 
               
               
                 hydroxyphenylacetate 
               
               
                 Indole derivatives: N- 
                 
                   Clostridium 
                 
                 Protect against stress-induced lesions in 
               
               
                 acetyltryptophan, indoleacetate, 
                   sporogenes , 
                 the GI tract; modulate expression of 
               
               
                 indoleacetylglycine (IAG), indole, 
                 
                   E. coli 
                 
                 proinflammatory genes, increase 
               
               
                 indoxyl sulfate, indole-3- 
                   
                 expression of anti-inflammatory genes, 
               
               
                 propionate, melatonin, melatonin 
                   
                 strengthen epithelial cell barrier 
               
               
                 6-sulfate, serotonin, 5- 
                   
                 properties. Implicated in GI pathologies, 
               
               
                 hydroxyindole 
                   
                 brain-gut axis, and a few neurological 
               
               
                   
                   
                 conditions. 
               
               
                 Vitamins: vitamin K, vitamin B12, 
                 
                   Bifidobacterium 
                 
                 Provide complementary endogenous 
               
               
                 biotin, folate, 
                   
                 sources of vitamins, strengthen immune 
               
               
                 thiamine, riboflavin, pyridoxine 
                   
                 function, exert epigenetic effects to 
               
               
                   
                   
                 regulate cell proliferation. 
               
               
                 Polyamines: putrescine, 
                 
                   Campylobacter 
                 
                 Exert genotoxic effects on the host, anti- 
               
               
                 cadaverine, 
                   jejuni , 
                 inflammatory and antitumoral effects. 
               
               
                 spermidine, spermine 
                 
                   Clostridium 
                 
                 Potential tumor markers. 
               
               
                   
                 
                   saccharolyticum 
                 
               
               
                 Lipids: conjugated fatty acids, LPS, 
                   Bifidobacterium , 
                 Impact intestinal permeability, activate 
               
               
                 peptidoglycan, acylglycerols, 
                   Roseburia , 
                 intestinebrain- liver neural axis to 
               
               
                 sphingomyelin, cholesterol, 
                   Lactobacillus , 
                 regulate glucose homeostasis; LPS 
               
               
                 phosphatidylcholines, 
                   Klebsiella , 
                 induces chronic systemic inflammation; 
               
               
                 phosphoethanolamines, 
                   Enterobacter , 
                 conjugated fatty acids improve 
               
               
                 triglycerides 
                   Citrobacter , 
                 hyperinsulinemia, enhance the immune 
               
               
                   
                 
                   Clostridium 
                 
                 system and alter lipoprotein profiles. 
               
               
                 Others: D-lactate, formate, 
                   Bacteroides , 
                 Direct or indirect synthesis or utilization 
               
               
                 methanol, ethanol, succinate, 
                   Pseudobutyrivibrio , 
                 of 
               
               
                 lysine, glucose, urea, a- 
                   Ruminococcus , 
                 compounds or modulation of linked 
               
               
                 ketoisovalerate, creatine, 
                 
                   Faecalibacterium 
                 
                 pathways including endocannabinoid 
               
               
                 creatinine, endocannabinoids, 2- 
                   
                 system. 
               
               
                 arachidonoylglycerol 
               
               
                 (2-AG), N- 
               
               
                 arachidonoylethanolamide, LPS 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the genetically engineered bacteria are capable of producing an anti-inflammation and/or gut barrier enhancer molecule and further producing a molecule that is capable of inhibiting an inflammatory molecule. In some embodiments, the genetically engineered bacteria of the invention are capable of producing an anti-inflammation and/or gut barrier enhancer molecule and further producing an enzyme that is capable of degrading an inflammatory molecule. For example, the genetically engineered bacteria of the invention are capable of expressing a gene cassette for producing butyrate, as well as a molecule or biosynthetic pathway for inhibiting, removing, degrading, and/or metabolizing an inflammatory molecule, e.g., PGE 2 . 
     RNAi, scFV, Other Mechanisms 
     RNA interference (RNAi) is a post-transcriptional gene silencing mechanism in plants and animals. RNAi is activated when microRNA (miRNA), double-stranded RNA (dsRNA), or short hairpin RNA (shRNA) is processed into short interfering RNA (siRNA) duplexes (Keates et al., 2008). RNAi can be “activated in vitro and in vivo by non-pathogenic bacteria engineered to manufacture and deliver shRNA to target cells” such as mammalian cells (Keates et al., 2008). In some embodiments, the genetically engineered bacteria of the invention induce RNAi-mediated gene silencing of one or more pro-inflammatory molecules in low-oxygen conditions. In some embodiments, the genetically engineered bacteria produce siRNA targeting TNF in low-oxygen conditions. 
     Single-chain variable fragments (scFv) are “widely used antibody fragments . . . produced in prokaryotes” (Frenzel et al., 2013). scFv lacks the constant domain of a traditional antibody and expresses the antigen-binding domain as a single peptide. Bacteria such as  Escherichia coli  are capable of producing scFv that target pro-inflammatory cytokines, e.g., TNF (Hristodorov et al., 2014). In some embodiments, the genetically engineered bacteria of the invention express a binding protein for neutralizing one or more pro-inflammatory molecules in low-oxygen conditions. In some embodiments, the genetically engineered bacteria produce scFv targeting TNF in low-oxygen conditions. In some embodiments, the genetically engineered bacteria produce both scFv and siRNA targeting one or more pro-inflammatory molecules in low-oxygen conditions (see, e.g., Xiao et al., 2014). 
     One of skill in the art would appreciate that additional genes and gene cassettes capable of producing anti-inflammation and/or gut barrier function enhancer molecules are known in the art and may be expressed by the genetically engineered bacteria of the invention. In some embodiments, the gene or gene cassette for producing a therapeutic molecule also comprises additional transcription and translation elements, e.g., a ribosome binding site, to enhance expression of the therapeutic molecule. 
     In some embodiments, the genetically engineered bacteria produce two or more anti-inflammation and/or gut barrier function enhancer molecules. In certain embodiments, the two or more molecules behave synergistically to reduce gut inflammation and/or enhance gut barrier function. In some embodiments, the genetically engineered bacteria express at least one anti-inflammation molecule and at least one gut barrier function enhancer molecule. In certain embodiments, the genetically engineered bacteria express IL-10 and GLP-2. In alternate embodiments, the genetically engineered bacteria express IL-10 and butyrate. 
     In some embodiments, the genetically engineered bacteria are capable of producing IL-2, IL-10, IL-22, IL-27, propionate, and butyrate. In some embodiments, the genetically engineered bacteria are capable of producing IL-10, IL-27, GLP-2, and butyrate. In some embodiments, the genetically engineered bacteria are capable of producing GLP-2, IL-10, IL-22, SOD, butyrate, and propionate. In some embodiments, the genetically engineered bacteria are capable of GLP-2, IL-2, IL-10, IL-22, IL-27, SOD, butyrate, and propionate. Any suitable combination of therapeutic molecules may be produced by the genetically engineered bacteria. 
     Generation of Bacterial Strains with Enhance Ability to Transport Amino Acids 
     Due to their ease of culture, short generation times, very high population densities and small genomes, microbes can be evolved to unique phenotypes in abbreviated timescales. Adaptive laboratory evolution (ALE) is the process of passaging microbes under selective pressure to evolve a strain with a preferred phenotype. Most commonly, this is applied to increase utilization of carbon/energy sources or adapting a strain to environmental stresses (e.g., temperature, pH), whereby mutant strains more capable of growth on the carbon substrate or under stress will outcompete the less adapted strains in the population and will eventually come to dominate the population. 
     This same process can be extended to any essential metabolite by creating an auxotroph. An auxotroph is a strain incapable of synthesizing an essential metabolite and must therefore have the metabolite provided in the media to grow. In this scenario, by making an auxotroph and passaging it on decreasing amounts of the metabolite, the resulting dominant strains should be more capable of obtaining and incorporating this essential metabolite. 
     For example, if the biosynthetic pathway for producing an amino acid is disrupted a strain capable of high-affinity capture of said amino acid can be evolved via ALE. First, the strain is grown in varying concentrations of the auxotrophic amino acid, until a minimum concentration to support growth is established. The strain is then passaged at that concentration, and diluted into lowering concentrations of the amino acid at regular intervals. Over time, cells that are most competitive for the amino acid—at growth-limiting concentrations—will come to dominate the population. These strains will likely have mutations in their amino acid-transporters resulting in increased ability to import the essential and limiting amino acid. 
     Similarly, by using an auxotroph that cannot use an upstream metabolite to form an amino acid, a strain can be evolved that not only can more efficiently import the upstream metabolite, but also convert the metabolite into the essential downstream metabolite. These strains will also evolve mutations to increase import of the upstream metabolite, but may also contain mutations which increase expression or reaction kinetics of downstream enzymes, or that reduce competitive substrate utilization pathways. 
     A metabolite innate to the microbe can be made essential via mutational auxotrophy and selection applied with growth-limiting supplementation of the endogenous metabolite. However, phenotypes capable of consuming non-native compounds can be evolved by tying their consumption to the production of an essential compound. For example, if a gene from a different organism is isolated which can produce an essential compound or a precursor to an essential compound this gene can be recombinantly introduced and expressed in the heterologous host. This new host strain will now have the ability to synthesize an essential nutrient from a previously non-metabolizable substrate. 
     Hereby, a similar ALE process can be applied by creating an auxotroph incapable of converting an immediately downstream metabolite and selecting in growth-limiting amounts of the non-native compound with concurrent expression of the recombinant enzyme. This will result in mutations in the transport of the non-native substrate, expression and activity of the heterologous enzyme and expression and activity of downstream native enzymes. It should be emphasized that the key requirement in this process is the ability to tether the consumption of the non-native metabolite to the production of a metabolite essential to growth. 
     Once the basis of the selection mechanism is established and minimum levels of supplementation have been established, the actual ALE experimentation can proceed. Throughout this process several parameters must be vigilantly monitored. It is important that the cultures are maintained in an exponential growth phase and not allowed to reach saturation/stationary phase. This means that growth rates must be check during each passaging and subsequent dilutions adjusted accordingly. If growth rate improves to such a degree that dilutions become large, then the concentration of auxotrophic supplementation should be decreased such that growth rate is slowed, selection pressure is increased and dilutions are not so severe as to heavily bias subpopulations during passaging. In addition, at regular intervals cells should be diluted, grown on solid media and individual clones tested to confirm growth rate phenotypes observed in the ALE cultures. 
     Predicting when to halt the stop the ALE experiment also requires vigilance. As the success of directing evolution is tied directly to the number of mutations “screened” throughout the experiment and mutations are generally a function of errors during DNA replication, the cumulative cell divisions (CCD) acts as a proxy for total mutants which have been screened. Previous studies have shown that beneficial phenotypes for growth on different carbon sources can be isolated in about 1011.2 CCD1. This rate can be accelerated by the addition of chemical mutagens to the cultures—such as N-methyl-N-nitro-N-nitrosoguanidine (NTG)—which causes increased DNA replication errors. However, when continued passaging leads to marginal or no improvement in growth rate the population has converged to some fitness maximum and the ALE experiment can be halted. 
     At the conclusion of the ALE experiment, the cells should be diluted, isolated on solid media and assayed for growth phenotypes matching that of the culture flask. Best performers from those selected are then prepped for genomic DNA and sent for whole genome sequencing. Sequencing with reveal mutations occurring around the genome capable of providing improved phenotypes, but will also contain silent mutations (those which provide no benefit but do not detract from desired phenotype). In cultures evolved in the presence of NTG or other chemical mutagen, there will be significantly more silent, background mutations. If satisfied with the best performing strain in its current state, the user can proceed to application with that strain. Otherwise the contributing mutations can be deconvoluted from the evolved strain by reintroducing the mutations to the parent strain by genome engineering techniques. See Lee, D.-H., Feist, A. M., Barrett, C. L. &amp; Palsson, B. Ø. Cumulative Number of Cell Divisions as a Meaningful Timescale for Adaptive Laboratory Evolution of  Escherichia coli . PLoS ONE 6, e26172 (2011). 
     Similar methods can be used to generate  E. coli  Nissle mutants that consume or import tryptophan. 
     Inducible Regulatory Regions 
     In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the gene(s) encoding payload (s), such that the payload(s) can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. In some embodiments, bacterial cell comprises two or more distinct payloads or operons, e.g., two or more payload genes. In some embodiments, bacterial cell comprises three or more distinct transporters or operons, e.g., three or more payload genes. In some embodiments, bacterial cell comprises 4, 5, 6, 7, 8, 9, 10, or more distinct payloads or operons, e.g., 4, 5, 6, 7, 8, 9, 10, or more payload genes. 
     Herein the terms “payload” “polypeptide of interest” or “polypeptides of interest”, “protein of interest”, “proteins of interest”, “payloads” “effector molecule”, “effector” refers to one or more effector molecules described herein and/or one or more enzyme(s) or polypeptide(s) function as enzymes for the production of such effector molecules. Non-limiting examples of payloads include anti-inflammation and/or gut barrier function enhancer molecule(s), including but not limited to, butyrate, propionate, acetate, IL10, IL-2, IL-22, IL-27, IL-20, IL-24, IL-19, SOD, GLP2, and/or tryptophan and/or its metabolites. As used herein, the term “polypeptide of interest” or “polypeptides of interest”, “protein of interest”, “proteins of interest”, “payload”, “payloads” further includes any or a plurality of any of the anti-inflammation and/or gut barrier function enhancer molecule(s). As used herein, the term “gene of interest” or “gene sequence of interest” includes any or a plurality of any of the gene(s) an/or gene sequence(s) and or gene cassette(s) encoding one or more anti-inflammation and/or gut barrier function enhancer molecule(s) described herein. 
     In some embodiments, the genetically engineered bacteria comprise multiple copies of the same payload gene(s). In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene encoding the payload is present on plasmid and operably linked to a promoter that is induced by exposure to tetracycline or arabinose, or another chemical or nutritional inducer described herein. 
     In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene encoding the payload is present on chromosome and operably linked to a promoter that is induced by exposure to tetracycline or arabinose, or another chemical or nutritional inducer described herein. 
     In some embodiments, the genetically engineered bacteria comprise two or more payloads, all of which are present on the chromosome. In some embodiments, the genetically engineered bacteria comprise two or more payloads, all of which are present on one or more same or different plasmids. In some embodiments, the genetically engineered bacteria comprise two or more payloads, some of which are present on the chromosome and some of which are present on one or more same or different plasmids. 
     In any of the nucleic acid embodiments described above, the one or more payload(s) for producing the anti-inflammation and/or gut barrier function enhancer molecule combinations are operably linked to one or more directly or indirectly inducible promoter(s). In some embodiments, the one or more payload(s) are operably linked to a directly or indirectly inducible promoter that is induced under exogeneous environmental conditions, e.g., conditions found in the gut. In some embodiments, the one or more payload(s) are operably linked to a directly or indirectly inducible promoter that is induced by metabolites found in the gut, or other specific conditions. In some embodiments, the one or more payload(s) are operably linked to a directly or indirectly inducible promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the one or more payload(s) are operably linked to a directly or indirectly inducible promoter that is induced under inflammatory conditions (e.g., RNS, ROS), as described herein. In some embodiments, the one or more payload(s) are operably linked to a directly or indirectly inducible promoter that is induced under immunosuppressive conditions, e.g., as found in the tumor, as described herein. In some embodiments, the two or more gene sequence(s) are linked to a directly or indirectly inducible promoter that is induced by exposure a chemical or nutritional inducer, which may or may not be present under in vivo conditions and which may be present during in vitro conditions (such as strain culture, expansion, manufacture), such as tetracycline or arabinose, or others described herein. In some embodiments, the two or more payloads are all linked to a constitutive promoter. Such constitutive promoters are described in the tables herein. 
     In some embodiments, the promoter is induced under in vivo conditions, e.g., the gut, as described herein. In some embodiments, the promoters is induced under in vitro conditions, e.g., various cell culture and/or cell manufacturing conditions, as described herein. In some embodiments, the promoter is induced under in vivo conditions, e.g., the gut, as described herein, and under in vitro conditions, e.g., various cell culture and/or cell production and/or manufacturing conditions, as described herein. 
     In some embodiments, the promoter that is operably linked to the gene encoding the payload is directly induced by exogenous environmental conditions (e.g., in vivo and/or in vitro and/or production/manufacturing conditions). In some embodiments, the promoter that is operably linked to the gene encoding the payload is indirectly induced by exogenous environmental conditions (e.g., in vivo and/or in vitro and/or production/manufacturing conditions). 
     In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the hypoxic environment of a tumor and/or the small intestine of a mammal. In some embodiments, the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions such as the environment of the mammalian gut. In some embodiments, the promoter is directly or indirectly induced by molecules or metabolites that are specific to the tumor, a particular tissue or the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the bacterial cell. 
     FNR Dependent Regulation 
     The genetically engineered bacteria of the invention comprise a gene or gene cassette for producing an anti-inflammation and/or gut barrier function enhancer molecule(s), wherein the gene or gene cassette is operably linked to a directly or indirectly inducible promoter that is controlled by exogenous environmental condition(s). In some embodiments, the inducible promoter is an oxygen level-dependent promoter and the anti-inflammation and/or gut barrier function enhancer molecule(s) is expressed in low-oxygen, microaerobic, or anaerobic conditions. For example, in low oxygen conditions, the oxygen level-dependent promoter is activated by a corresponding oxygen level-sensing transcription factor, thereby driving production of the anti-inflammation and/or gut barrier function enhancer molecule(s). 
     Bacteria have evolved transcription factors that are capable of sensing oxygen levels. Different signaling pathways may be triggered by different oxygen levels and occur with different kinetics. An oxygen level-dependent promoter is a nucleic acid sequence to which one or more oxygen level-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression. In one embodiment, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload under the control of an oxygen level-dependent promoter. In a more specific aspect, the genetically engineered bacteria comprise a gene or gene cassette for producing a payload under the control of an oxygen level-dependent promoter that is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. 
     In certain embodiments, the bacterial cell comprises a gene encoding a payload expressed under the control of a fumarate and nitrate reductase regulator (FNR) responsive promoter. In  E. coli , FNR is a major transcriptional activator that controls the switch from aerobic to anaerobic metabolism (Unden et al., 1997). In the anaerobic state, FNR dimerizes into an active DNA binding protein that activates hundreds of genes responsible for adapting to anaerobic growth. In the aerobic state, FNR is prevented from dimerizing by oxygen and is inactive. FNR responsive promoters include, but are not limited to, the FNR responsive promoters listed in Table 21 and Table 22 below. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning. 
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 FNR Promoter Sequences 
               
            
           
           
               
               
            
               
                 FNR Responsive 
                   
               
               
                 Promoter 
                 Sequence 
               
               
                   
               
               
                 SEQ ID NO: 563 
                 GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGGCA 
               
               
                   
                 CTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATCTATTT 
               
               
                   
                 CTATAAATCCGTTCAATTTGTCTGTTTTTTGCACAAACATGAAATATCA 
               
               
                   
                 GACAATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATATACCCC 
               
               
                   
                 TTAAGGAGTATATAAAGGTGAATTTGATTTACATCAATAAGCGGGGTT 
               
               
                   
                 GCTGAATCGTTAAGGTAGGCGGTAATAG AAAAGAAATCGAGGCAAAA   
               
               
                   
               
               
                 SEQ ID NO: 564 
                 ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATGG 
               
               
                   
                 CTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACAAAAAA 
               
               
                   
                 TATTTCACTCGACAGGAGTATTTATATTGCGCCCGTTACGTGGGCTTCG 
               
               
                   
                 ACTGTAAATC AGAAAGGAGAAAACACCT   
               
               
                   
               
               
                 SEQ ID NO: 565 
                 GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGGCA 
               
               
                   
                 CTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATCTATTT 
               
               
                   
                 CTATAAATCCGTTCAATTTGTCTGTTTTTTGCACAAACATGAAATATCA 
               
               
                   
                 GACAATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATATACCCC 
               
               
                   
                 TTAAGGAGTATATAAAGGTGAATTTGATTTACATCAATAAGCGGGGTT 
               
               
                   
                 GCTGAATCGTTAAGGATCC CTCTAGAAATAATTTTGTTTAACTTTAAG   
               
               
                   
                 
                   AAGGAGATATACAT 
                 
               
               
                   
               
               
                 SEQ ID NO: 566 
                 CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATG 
               
               
                   
                 GCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACAAAAA 
               
               
                   
                 ATATTTCACTCGACAGGAGTATTTATATTGCGCCCGGATCC CTCTAGA   
               
               
                   
                 
                   AATAATTTTGTTTAACTTTAAGAAGGAGATATACAT 
                 
               
               
                   
               
               
                 SEQ ID NO: 567 
                 AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGG 
               
               
                   
                 TTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGTA 
               
               
                   
                 AAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGCAATATCT 
               
               
                   
                 CTCTTGGATCC CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGAT   
               
               
                   
                 
                   ATACAT 
                 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 FNR Promoter sequences 
               
            
           
           
               
               
            
               
                 FNR-responsive 
                   
               
               
                 regulatory region 
                 12345678901234567890123456789012345678901234567890 
               
               
                   
               
               
                 SEQ ID NO: 568 
                 ATCCCCATCACTCTTGATGGAGATCAATTCCCCAAGCTGCTAGAGC 
               
               
                   
                 GTTACCTTGCCCTTAAACATTAGCAATGTCGATTTATCAGAGGGCC 
               
               
                   
                 GACAGGCTCCCACAGGAGAAAACCG 
               
               
                   
               
               
                 SEQ ID NO: 569 
                 CTCTTGATCGTTATCAATTCCCACGCTGTTTCAGAGCGTTACCTTGC 
               
               
                   
                 CCTTAAACATTAGCAATGTCGATTTATCAGAGGGCCGACAGGCTCC 
               
               
                   
                 CACAGGAGAAAACCG 
               
               
                   
               
               
                 nirB1 
                 GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGG 
               
               
                 SEQ ID NO: 570 
                 CACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATC 
               
               
                   
                 TATTTCTATAAATCCGTTCAATTTGTCTGTTTTTTGCACAAACATGA 
               
               
                   
                 AATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGC 
               
               
                   
                 AATATACCCCTTAAGGAGTATATAAAGGTGAATTTGATTTACATCA 
               
               
                   
                 ATAAGCGGGGTTGCTGAATCGTTAAGGTAGGCGGTAATAG AAAAG   
               
               
                   
                 
                   AAATCGAGGCAAAA 
                 
               
               
                   
               
               
                 nirB2 
                 CGGCCCGATCGTTGAACATAGCGGTCCGCAGGCGGCACTGCTTAC 
               
               
                 SEQ ID NO: 571 
                 AGCAAACGGTCTGTACGCTGTCGTCTTTGTGATGTGCTTCCTGTTA 
               
               
                   
                 GGTTTCGTCAGCCGTCACCGTCAGCATAACACCCTGACCTCTCATT 
               
               
                   
                 AATTGCTCATGCCGGACGGCACTATCGTCGTCCGGCCGGCCTTTTCCTCT 
               
               
                   
                 CTTCCCCCGCTACGTGCATCTATTTCTATAAACCCGCTCATTTTGTC 
               
               
                   
                 TATTTTTTGCACAAACATGAAATATCAGACAATTCCGTGACTTAAG 
               
               
                   
                 AAAATTTATACAAATCAGCAATATACCCATTAAGGAGTATATAAA 
               
               
                   
                 GGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTAAG 
               
               
                   
                 GTAGGCGGTAATAGAAAAGAAATCGAGGCAAAAatgtttgtttaactttaagaa 
               
               
                   
                 ggagatatacat 
               
               
                   
               
               
                 nirB3 
                 GTCAGCATAACACCCTGACCTCTCATTAATTGCTCATGCCGGACGG 
               
               
                 SEQ ID NO: 572 
                 CACTATCGTCGTCCGGCCTTTTCCTCTCTTCCCCCGCTACGTGCATC 
               
               
                   
                 TATTTCTATAAACCCGCTCATTTTGTCTATTTTTTGCACAAACATGA 
               
               
                   
                 AATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGC 
               
               
                   
                 AATATACCCATTAAGGAGTATATAAAGGTGAATTTGATTTACATCA 
               
               
                   
                 ATAAGCGGGGTTGCTGAATCGTTAAGGTAGGCGGTAATAGAAAAG 
               
               
                   
                 AAATCGAGGCAAAA 
               
               
                   
               
               
                 ydfZ 
                 ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTAT 
               
               
                 SEQ ID NO: 573 
                 GGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACAA 
               
               
                   
                 AAAATATTTCACTCGACAGGAGTATTTATATTGCGCCCGTTACGTG 
               
               
                   
                 GGCTTCGACTGTAAATC AGAAAGGAGAAAACACCT   
               
               
                   
               
               
                 nirB+RBS 
                 GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGG 
               
               
                 SEQ ID NO: 574 
                 CACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATC 
               
               
                   
                 TATTTCTATAAATCCGTTCAATTTGTCTGTTTTTTGCACAAACATGA 
               
               
                   
                 AATATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGC 
               
               
                   
                 AATATACCCCTTAAGGAGTATATAAAGGTGAATTTGATTTACATCA 
               
               
                   
                 ATAAGCGGGGTTGCTGAATCGTTAAGGATCC CTCTAGAAATAATT   
               
               
                   
                 
                   TTGTTTAACTTTAAGAAGGAGATATACAT 
                 
               
               
                   
               
               
                 ydfZ+RBS 
                 CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTA 
               
               
                 SEQ ID NO: 575 
                 TGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACA 
               
               
                   
                 AAAAATATTTCACTCGACAGGAGTATTTATATTGCGCCCGGATCC 
               
               
                   
                 
                   CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT 
                 
               
               
                   
               
               
                 fnrS1 
                 AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAAT 
               
               
                 SEQ ID NO: 576 
                 GGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGC 
               
               
                   
                 CGTAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGC 
               
               
                   
                 AATATCTCTCTTGGATCCCTCTAGAAATAATTTTGTTTAACTTTAA 
               
               
                   
                 GAAGGAGATATACAT 
               
               
                   
               
               
                 fnrS2 
                 AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAAT 
               
               
                 SEQ ID NO: 577 
                 GGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGC 
               
               
                   
                 CGCAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGC 
               
               
                   
                 AATATCTCTCTT GGATCCAAAGTGAACTCTAGAAATAATTTTGTTT   
               
               
                   
                 
                   AACTTTAAGAAGGAGATATACAT 
                 
               
               
                   
               
               
                 nirB+crp 
                 TCGTCTTTGTGATGTGCTTCCTGTTAGGTTTCGTCAGCCGTCACCGT 
               
               
                 SEQ ID NO: 578 
                 CAGCATAACACCCTGACCTCTCATTAATTGCTCATGCCGGACGGCA 
               
               
                   
                 CTATCGTCGTCCGGCCTTTTCCTCTCTTCCCCCGCTACGTGCATCTA 
               
               
                   
                 TTTCTATAAACCCGCTCATTTTGTCTATTTTTTGCACAAACATGAAA 
               
               
                   
                 TATCAGACAATTCCGTGACTTAAGAAAATTTATACAAATCAGCAAT 
               
               
                   
                 ATACCCATTAAGGAGTATATAAAGGTGAATTTGATTTACATCAATA 
               
               
                   
                 AGCGGGGTTGCTGAATCGTTAAGGTAGaaatgtgatctagttcacatttGCGGTA 
               
               
                   
                 ATAGAAAAGAAATCGAGGCAAAA atgtttgtttaactttaagaaggagatatacat   
               
               
                   
               
               
                 fnrS+crp 
                 AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAAT 
               
               
                 SEQ ID NO: 579 
                 GGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGC 
               
               
                   
                 CGCAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGC 
               
               
                   
                 AATATCTCTCaaatgtgatctagttcacatttt ttgtttaactttaagaaggagatatacat   
               
               
                   
               
            
           
         
       
     
     FNR promoter sequences are known in the art, and any suitable FNR promoter sequence(s) may be used in the genetically engineered bacteria of the invention. Any suitable FNR promoter(s) may be combined with any suitable payload. 
     Non-limiting FNR promoter sequences are provided in Table 21 and Table 22. Table 21 and Table 22 depicts the nucleic acid sequences of exemplary regulatory region sequences comprising a FNR-responsive promoter sequence. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning. In some embodiments, the genetically engineered bacteria of the invention comprise one or more of: SEQ ID NO: 563, SEQ ID NO: 564, SEQ ID NO: 565, SEQ ID NO: 566, SEQ ID NO: 567, SEQ ID NO: 568, SEQ ID NO: 569, nirB1 promoter (SEQ ID NO: 570), nirB2 promoter (SEQ ID NO: 571), nirB3 promoter (SEQ ID NO: 572), ydfZ promoter (SEQ ID NO: 573), nirB promoter fused to a strong ribosome binding site (SEQ ID NO: 574), ydfZ promoter fused to a strong ribosome binding site (SEQ ID NO: 575), fnrS, an anaerobically induced small RNA gene (fnrS1 promoter SEQ ID NO: 576 or fnrS2 promoter SEQ ID NO: 577), nirB promoter fused to a crp binding site (SEQ ID NO: 578), and fnrS fused to a crp binding site (SEQ ID NO: 579). In some embodiments, the FNR-responsive promoter is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the sequence of any one of SEQ ID NOs: 563-579. 
     In some embodiments, multiple distinct FNR nucleic acid sequences are inserted in the genetically engineered bacteria. In alternate embodiments, the genetically engineered bacteria comprise a gene encoding a payload expressed under the control of an alternate oxygen level-dependent promoter, e.g., DNR (Trunk et al., 2010) or ANR (Ray et al., 1997). In these embodiments, expression of the payload gene is particularly activated in a low-oxygen or anaerobic environment, such as in the gut. In some embodiments, gene expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites and/or increasing mRNA stability. In one embodiment, the mammalian gut is a human mammalian gut. 
     In another embodiment, the genetically engineered bacteria comprise the gene or gene cassette for producing an anti-inflammation and/or gut barrier function enhancer molecule(s) expressed under the control of anaerobic regulation of arginine deiminiase and nitrate reduction transcriptional regulator (ANR). In  P. aeruginosa , ANR is “required for the expression of physiological functions which are inducible under oxygen-limiting or anaerobic conditions” (Winteler et al., 1996; Sawers 1991).  P. aeruginosa  ANR is homologous with  E. coli  FNR, and “the consensus FNR site (TTGAT-ATCAA) was recognized efficiently by ANR and FNR” (Winteler et al., 1996). Like FNR, in the anaerobic state, ANR activates numerous genes responsible for adapting to anaerobic growth. In the aerobic state, ANR is inactive.  Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae , and  Pseudomonas mendocina  all have functional analogs of ANR (Zimmermann et al., 1991). Promoters that are regulated by ANR are known in the art, e.g., the promoter of the arcDABC operon (see, e.g., Hasegawa et al., 1998). 
     In other embodiments, the one or more gene sequence(s) for producing a payload are expressed under the control of an oxygen level-dependent promoter fused to a binding site for a transcriptional activator, e.g., CRP. CRP (cyclic AMP receptor protein or catabolite activator protein or CAP) plays a major regulatory role in bacteria by repressing genes responsible for the uptake, metabolism, and assimilation of less favorable carbon sources when rapidly metabolizable carbohydrates, such as glucose, are present (Wu et al., 2015). This preference for glucose has been termed glucose repression, as well as carbon catabolite repression (Deutscher, 2008; Görke and Stülke, 2008). In some embodiments, the gene or gene cassette for producing an anti-inflammation and/or gut barrier function enhancer molecule(s) is controlled by an oxygen level-dependent promoter fused to a CRP binding site. In some embodiments, the one or more gene sequence(s) for a payload are controlled by a FNR promoter fused to a CRP binding site. In these embodiments, cyclic AMP binds to CRP when no glucose is present in the environment. This binding causes a conformational change in CRP, and allows CRP to bind tightly to its binding site. CRP binding then activates transcription of the gene or gene cassette by recruiting RNA polymerase to the FNR promoter via direct protein-protein interactions. In the presence of glucose, cyclic AMP does not bind to CRP and transcription of the gene or gene cassette for producing an payload is repressed. In some embodiments, an oxygen level-dependent promoter (e.g., an FNR promoter) fused to a binding site for a transcriptional activator is used to ensure that the gene or gene cassette for producing a payload is not expressed under anaerobic conditions when sufficient amounts of glucose are present, e.g., by adding glucose to growth media in vitro. 
     In some embodiments, the genetically engineered bacteria comprise an oxygen level-dependent promoter from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise an oxygen level-sensing transcription factor, e.g., FNR, ANR or DNR, from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise an oxygen level-sensing transcription factor and corresponding promoter from a different species, strain, or substrain of bacteria. The heterologous oxygen-level dependent transcriptional regulator and/or promoter increases the transcription of genes operably linked to said promoter, e.g., one or more gene sequence(s) for producing the payload(s) in a low-oxygen or anaerobic environment, as compared to the native gene(s) and promoter in the bacteria under the same conditions. In certain embodiments, the non-native oxygen-level dependent transcriptional regulator is an FNR protein from  N. gonorrhoeae  (see, e.g., Isabella et al., 2011). In some embodiments, the corresponding wild-type transcriptional regulator is left intact and retains wild-type activity. In alternate embodiments, the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild-type activity. 
     In some embodiments, the genetically engineered bacteria comprise a wild-type oxygen-level dependent transcriptional regulator, e.g., FNR, ANR, or DNR, and corresponding promoter that is mutated relative to the wild-type promoter from bacteria of the same subtype. The mutated promoter enhances binding to the wild-type transcriptional regulator and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the payload, in a low-oxygen or anaerobic environment, as compared to the wild-type promoter under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type oxygen-level dependent promoter, e.g., FNR, ANR, or DNR promoter, and corresponding transcriptional regulator that is mutated relative to the wild-type transcriptional regulator from bacteria of the same subtype. The mutated transcriptional regulator enhances binding to the wild-type promoter and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the payload, in a low-oxygen or anaerobic environment, as compared to the wild-type transcriptional regulator under the same conditions. In certain embodiments, the mutant oxygen-level dependent transcriptional regulator is an FNR protein comprising amino acid substitutions that enhance dimerization and FNR activity (see, e.g., Moore et al., (2006). In some embodiments, both the oxygen level-sensing transcriptional regulator and corresponding promoter are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in low-oxygen conditions. 
     In some embodiments, the bacterial cells comprise multiple copies of the endogenous gene encoding the oxygen level-sensing transcriptional regulator, e.g., the FNR gene. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a plasmid. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on different plasmids. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on the same plasmid. 
     In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a chromosome. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on different chromosomes. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the payload are present on the same chromosome. In some instances, it may be advantageous to express the oxygen level-sensing transcriptional regulator under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the transcriptional regulator is controlled by a different promoter than the promoter that controls expression of the gene encoding the payload. In some embodiments, expression of the transcriptional regulator is controlled by the same promoter that controls expression of the payload. In some embodiments, the transcriptional regulator and the payload are divergently transcribed from a promoter region 
     RNS-Dependent Regulation 
     In some embodiments, the genetically engineered bacteria or genetically engineered virus comprise a gene encoding a payload that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium or genetically engineered virus that expresses a payload under the control of a promoter that is activated by inflammatory conditions. In one embodiment, the gene for producing the payload is expressed under the control of an inflammatory-dependent promoter that is activated in inflammatory environments, e.g., a reactive nitrogen species or RNS promoter. 
     As used herein, “reactive nitrogen species” and “RNS” are used interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular nitrogen. RNS can cause deleterious cellular effects such as nitrosative stress. RNS includes, but is not limited to, nitric oxide (NO.), peroxynitrite or peroxynitrite anion (ONOO—), nitrogen dioxide (.NO2), dinitrogen trioxide (N2O3), peroxynitrous acid (ONOOH), and nitroperoxycarbonate (ONOOCO2-) (unpaired electrons denoted by .). Bacteria have evolved transcription factors that are capable of sensing RNS levels. Different RNS signaling pathways are triggered by different RNS levels and occur with different kinetics. 
     As used herein, “RNS-inducible regulatory region” refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of RNS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the RNS-inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; in the presence of RNS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The RNS-inducible regulatory region may be operatively linked to a gene or genes, e.g., a payload gene sequence(s), e.g., any of the payloads described herein. For example, in the presence of RNS, a transcription factor senses RNS and activates a corresponding RNS-inducible regulatory region, thereby driving expression of an operatively linked gene sequence. Thus, RNS induces expression of the gene or gene sequences. 
     As used herein, “RNS-derepressible regulatory region” refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the RNS-derepressible regulatory region comprises a promoter sequence. The RNS-derepressible regulatory region may be operatively linked to a gene or genes, e.g., a payload gene sequence(s). For example, in the presence of RNS, a transcription factor senses RNS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, RNS derepresses expression of the gene or genes. 
     As used herein, “RNS-repressible regulatory region” refers to a nucleic acid sequence to which one or more RNS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor binds to and represses the regulatory region. In some embodiments, the RNS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the transcription factor that senses RNS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The RNS-repressible regulatory region may be operatively linked to a gene sequence or gene cassette. For example, in the presence of RNS, a transcription factor senses RNS and binds to a corresponding RNS-repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, RNS represses expression of the gene or gene sequences. 
     As used herein, a “RNS-responsive regulatory region” refers to a RNS-inducible regulatory region, a RNS-repressible regulatory region, and/or a RNS-derepressible regulatory region. In some embodiments, the RNS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding RNS-sensing transcription factor. Examples of transcription factors that sense RNS and their corresponding RNS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 23. 
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 Examples of RNS-sensing transcription 
               
               
                 factors and RNS-responsive genes 
               
            
           
           
               
               
               
            
               
                 RNS-sensing 
                 Primarily 
                 Examples of responsive genes, 
               
               
                 transcription 
                 capable 
                 promoters, and/or regulatory 
               
               
                 factor: 
                 of sensing: 
                 regions: 
               
               
                   
               
               
                 NsrR 
                 NO 
                 norB, aniA, nsrR, hmpA, ytfE, ygbA, 
               
               
                   
                   
                 hcp, hcr, nrfA, aox 
               
               
                 NorR 
                 NO 
                 norVW, norR 
               
               
                 DNR 
                 NO 
                 norCB, nir, nor, nos 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the genetically engineered bacteria of the invention comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive nitrogen species. The tunable regulatory region is operatively linked to a gene or genes capable of directly or indirectly driving the expression of a payload, thus controlling expression of the payload relative to RNS levels. For example, the tunable regulatory region is a RNS-inducible regulatory region, and the payload is a payload, such as any of the payloads provided herein; when RNS is present, e.g., in an inflamed tissue, a RNS-sensing transcription factor binds to and/or activates the regulatory region and drives expression of the payload gene or genes. Subsequently, when inflammation is ameliorated, RNS levels are reduced, and production of the payload is decreased or eliminated. 
     In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region; in the presence of RNS, a transcription factor senses RNS and activates the RNS-inducible regulatory region, thereby driving expression of an operatively linked gene or genes. In some embodiments, the transcription factor senses RNS and subsequently binds to the RNS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the RNS-inducible regulatory region in the absence of RNS; when the transcription factor senses RNS, it undergoes a conformational change, thereby inducing downstream gene expression. 
     In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is NorR. NorR “is an NO-responsive transcriptional activator that regulates expression of the norVW genes encoding flavorubredoxin and an associated flavoprotein, which reduce NO to nitrous oxide” (Spiro 2006). The genetically engineered bacteria of the invention may comprise any suitable RNS-responsive regulatory region from a gene that is activated by NorR. Genes that are capable of being activated by NorR are known in the art (see, e.g., Spiro 2006; Vine et al., 2011; Karlinsey et al., 2012). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norVW that is operatively linked to a gene or genes, e.g., one or more payload gene sequence(s). In the presence of RNS, a NorR transcription factor senses RNS and activates to the norVW regulatory region, thereby driving expression of the operatively linked gene(s) and producing the payload(s). 
     In some embodiments, the tunable regulatory region is a RNS-inducible regulatory region, and the transcription factor that senses RNS is DNR. DNR (dissimilatory nitrate respiration regulator) “promotes the expression of the nir, the nor and the nos genes” in the presence of nitric oxide (Castiglione et al., 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS-responsive regulatory region from a gene that is activated by DNR. Genes that are capable of being activated by DNR are known in the art (see, e.g., Castiglione et al., 2009; Giardina et al., 2008). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-inducible regulatory region from norCB that is operatively linked to a gene or gene cassette, e.g., a butyrogenic gene cassette. In the presence of RNS, a DNR transcription factor senses RNS and activates to the norCB regulatory region, thereby driving expression of the operatively linked gene or genes and producing one or more payloads. In some embodiments, the DNR is  Pseudomonas aeruginosa  DNR. 
     In another embodiment, the genetically engineered bacteria comprise the gene or gene cassette for producing an anti-inflammation and/or gut barrier function enhancer molecule(s) expressed under the control of the dissimilatory nitrate respiration regulator (DNR). DNR is a member of the FNR family (Arai et al., 1995) and is a transcriptional regulator that is required in conjunction with ANR for “anaerobic nitrate respiration of  Pseudomonas aeruginosa ” (Hasegawa et al., 1998). For certain genes, the FNR-binding motifs “are probably recognized only by DNR” (Hasegawa et al., 1998). Any suitable transcriptional regulator that is controlled by exogenous environmental conditions and corresponding regulatory region may be used. Non-limiting examples include ArcA/B, ResD/E, NreA/B/C, and AirSR, and others are known in the art. 
     In some embodiments, the tunable regulatory region is a RNS-derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette. 
     In some embodiments, the tunable regulatory region is a RNS-derepressible regulatory region, and the transcription factor that senses RNS is NsrR. NsrR is “an Rrf2-type transcriptional repressor [that] can sense NO and control the expression of genes responsible for NO metabolism” (Isabella et al., 2009). The genetically engineered bacteria of the invention may comprise any suitable RNS-responsive regulatory region from a gene that is repressed by NsrR. In some embodiments, the NsrR is  Neisseria gonorrhoeae  NsrR. Genes that are capable of being repressed by NsrR are known in the art (see, e.g., Isabella et al., 2009; Dunn et al., 2010). In certain embodiments, the genetically engineered bacteria of the invention comprise a RNS-derepressible regulatory region from norB that is operatively linked to a gene or genes, e.g., a payload gene or genes. In the presence of RNS, an NsrR transcription factor senses RNS and no longer binds to the norB regulatory region, thereby derepressing the operatively linked a payload gene or genes and producing the encoding a payload(s). 
     In some embodiments, it is advantageous for the genetically engineered bacteria to express a RNS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some embodiments, the genetically engineered bacterium of the invention expresses a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention. In some embodiments, the genetically engineered bacterium of the invention is  Escherichia coli , and the RNS-sensing transcription factor is NsrR, e.g., from is  Neisseria gonorrhoeae , wherein the  Escherichia coli  does not comprise binding sites for said NsrR. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria. 
     In some embodiments, the tunable regulatory region is a RNS-repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of RNS, the transcription factor senses RNS and binds to the RNS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the RNS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. 
     In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a payload. The two repressor activation regulatory circuit comprises a first RNS-sensing repressor and a second repressor, which is operatively linked to a gene or gene cassette, e.g., encoding a payload. In one aspect of these embodiments, the RNS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these embodiments, include, but are not limited to, TetR, C1, and LexA. In the absence of binding by the first repressor (which occurs in the absence of RNS), the second repressor is transcribed, which represses expression of the gene or genes. In the presence of binding by the first repressor (which occurs in the presence of RNS), expression of the second repressor is repressed, and the gene or genes, e.g., a payload gene or genes is expressed. 
     A RNS-responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. One or more types of RNS-sensing transcription factors and corresponding regulatory region sequences may be present in genetically engineered bacteria. In some embodiments, the genetically engineered bacteria comprise one type of RNS-sensing transcription factor, e.g., NsrR, and one corresponding regulatory region sequence, e.g., from norB. In some embodiments, the genetically engineered bacteria comprise one type of RNS-sensing transcription factor, e.g., NsrR, and two or more different corresponding regulatory region sequences, e.g., from norB and aniA. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors, e.g., NsrR and NorR, and two or more corresponding regulatory region sequences, e.g., from norB and norR, respectively. One RNS-responsive regulatory region may be capable of binding more than one transcription factor. In some embodiments, the genetically engineered bacteria comprise two or more types of RNS-sensing transcription factors and one corresponding regulatory region sequence. Nucleic acid sequences of several RNS-regulated regulatory regions are known in the art (see, e.g., Spiro 2006; Isabella et al., 2009; Dunn et al., 2010; Vine et al., 2011; Karlinsey et al., 2012). 
     In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a RNS-sensing transcription factor, e.g., the nsrR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the RNS-sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the RNS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the therapeutic molecule. In some embodiments, expression of the RNS-sensing transcription factor is controlled by the same promoter that controls expression of the therapeutic molecule. In some embodiments, the RNS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region. 
     In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a RNS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS-responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a RNS-sensing transcription factor and corresponding RNS-responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous RNS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of RNS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria comprise a RNS-sensing transcription factor, NsrR, and corresponding regulatory region, nsrR, from  Neisseria gonorrhoeae . In some embodiments, the native RNS-sensing transcription factor, e.g., NsrR, is left intact and retains wild-type activity. In alternate embodiments, the native RNS-sensing transcription factor, e.g., NsrR, is deleted or mutated to reduce or eliminate wild-type activity. 
     In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the RNS-sensing transcription factor, e.g., the nsrR gene. In some embodiments, the gene encoding the RNS-sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same plasmid. In some embodiments, the gene encoding the RNS-sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the RNS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same chromosome. 
     In some embodiments, the genetically engineered bacteria comprise a wild-type gene encoding a RNS-sensing transcription factor, e.g., the NsrR gene, and a corresponding regulatory region, e.g., a norB regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype. The mutated regulatory region increases the expression of the payload in the presence of RNS, as compared to the wild-type regulatory region under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type RNS-responsive regulatory region, e.g., the norB regulatory region, and a corresponding transcription factor, e.g., NsrR, that is mutated relative to the wild-type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the payload in the presence of RNS, as compared to the wild-type transcription factor under the same conditions. In some embodiments, both the RNS-sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in the presence of RNS. 
     In some embodiments, the gene or gene cassette for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is present on a plasmid and operably linked to a promoter that is induced by RNS. In some embodiments, expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability. 
     In some embodiments, any of the gene(s) of the present disclosure may be integrated into the bacterial chromosome at one or more integration sites. For example, one or more copies of one or more encoding a payload gene(s) may be integrated into the bacterial chromosome. Having multiple copies of the gene or gen(s) integrated into the chromosome allows for greater production of the payload(s) and also permits fine-tuning of the level of expression. Alternatively, different circuits described herein, such as any of the secretion or exporter circuits, in addition to the therapeutic gene(s) or gene cassette(s) could be integrated into the bacterial chromosome at one or more different integration sites to perform multiple different functions. 
     In some embodiments, the genetically engineered bacteria of the invention produce at least one payload in the presence of RNS to reduce local gut inflammation by at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold as compared to unmodified bacteria of the same subtype under the same conditions. Inflammation may be measured by methods known in the art, e.g., counting disease lesions using endoscopy; detecting T regulatory cell differentiation in peripheral blood, e.g., by fluorescence activated sorting; measuring T regulatory cell levels; measuring cytokine levels; measuring areas of mucosal damage; assaying inflammatory biomarkers, e.g., by qPCR; PCR arrays; transcription factor phosphorylation assays; immunoassays; and/or cytokine assay kits (Mesoscale, Cayman Chemical, Qiagen). 
     In some embodiments, the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of payload in the presence of RNS than unmodified bacteria of the same subtype under the same conditions. Certain unmodified bacteria will not have detectable levels of the payload. In embodiments using genetically modified forms of these bacteria, payload will be detectable in the presence of RNS 
     ROS-Dependent Regulation 
     In some embodiments, the genetically engineered bacteria or genetically engineered virus comprise a gene for producing a payload that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium or genetically engineered virus that expresses a payload under the control of a promoter that is activated by conditions of cellular damage. In one embodiment, the gene for producing the payload is expressed under the control of an cellular damaged-dependent promoter that is activated in environments in which there is cellular or tissue damage, e.g., a reactive oxygen species or ROS promoter. 
     As used herein, “reactive oxygen species” and “ROS” are used interchangeably to refer to highly active molecules, ions, and/or radicals derived from molecular oxygen. ROS can be produced as byproducts of aerobic respiration or metal-catalyzed oxidation and may cause deleterious cellular effects such as oxidative damage. ROS includes, but is not limited to, hydrogen peroxide (H2O2), organic peroxide (ROOH), hydroxyl ion (OH—), hydroxyl radical (.OH), superoxide or superoxide anion (.O2-), singlet oxygen (1O2), ozone (O3), carbonate radical, peroxide or peroxyl radical (.O2-2), hypochlorous acid (HOCl), hypochlorite ion (OCl—), sodium hypochlorite (NaOCl), nitric oxide (NO.), and peroxynitrite or peroxynitrite anion (ONOO—) (unpaired electrons denoted by .). Bacteria have evolved transcription factors that are capable of sensing ROS levels. Different ROS signaling pathways are triggered by different ROS levels and occur with different kinetics (Marinho et al., 2014). 
     As used herein, “ROS-inducible regulatory region” refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression; in the presence of ROS, the transcription factor binds to and/or activates the regulatory region. In some embodiments, the ROS-inducible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS; in the presence of ROS, the transcription factor undergoes a conformational change, thereby activating downstream gene expression. The ROS-inducible regulatory region may be operatively linked to a gene sequence or gene sequence, e.g., a sequence or sequences encoding one or more payload(s). For example, in the presence of ROS, a transcription factor, e.g., OxyR, senses ROS and activates a corresponding ROS-inducible regulatory region, thereby driving expression of an operatively linked gene sequence or gene sequences. Thus, ROS induces expression of the gene or genes. 
     As used herein, “ROS-derepressible regulatory region” refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor does not bind to and does not repress the regulatory region. In some embodiments, the ROS-derepressible regulatory region comprises a promoter sequence. The ROS-derepressible regulatory region may be operatively linked to a gene or genes, e.g., one or more genes encoding one or more payload(s). For example, in the presence of ROS, a transcription factor, e.g., OhrR, senses ROS and no longer binds to and/or represses the regulatory region, thereby derepressing an operatively linked gene sequence or gene cassette. Thus, ROS derepresses expression of the gene or gene cassette. 
     As used herein, “ROS-repressible regulatory region” refers to a nucleic acid sequence to which one or more ROS-sensing transcription factors is capable of binding, wherein the binding of the corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor binds to and represses the regulatory region. In some embodiments, the ROS-repressible regulatory region comprises a promoter sequence. In some embodiments, the transcription factor that senses ROS is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the transcription factor that senses ROS is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. The ROS-repressible regulatory region may be operatively linked to a gene sequence or gene sequences. For example, in the presence of ROS, a transcription factor, e.g., PerR, senses ROS and binds to a corresponding ROS-repressible regulatory region, thereby blocking expression of an operatively linked gene sequence or gene sequences. Thus, ROS represses expression of the gene or genes. 
     As used herein, a “ROS-responsive regulatory region” refers to a ROS-inducible regulatory region, a ROS-repressible regulatory region, and/or a ROS-derepressible regulatory region. In some embodiments, the ROS-responsive regulatory region comprises a promoter sequence. Each regulatory region is capable of binding at least one corresponding ROS-sensing transcription factor. Examples of transcription factors that sense ROS and their corresponding ROS-responsive genes, promoters, and/or regulatory regions include, but are not limited to, those shown in Table 24. 
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 Examples of ROS-sensing transcription 
               
               
                 factors and ROS-responsive genes 
               
            
           
           
               
               
               
            
               
                 ROS-sensing 
                 Primarily 
                 Examples of responsive genes, 
               
               
                 transcription 
                 capable 
                 promoters, and/or regulatory 
               
               
                 factor: 
                 of sensing: 
                 regions: 
               
               
                   
               
               
                 OxyR 
                 H 2 O 2   
                 ahpC; ahpF; dps; dsbG; fhuF; flu; 
               
               
                   
                   
                 fur; gor; grxA; hemH; katG; oxyS; 
               
               
                   
                   
                 sufA; sufB; sufC; sufD; sufE; sufS; 
               
               
                   
                   
                 trxC; uxuA; yaaA; yaeH; yaiA; ybjM; 
               
               
                   
                   
                 ydcH; ydeN; ygaQ; yljA; ytfK 
               
               
                 PerR 
                 H 2 O 2   
                 katA; ahpCF; mrgA; zoaA; fur; 
               
               
                   
                   
                 hemAXCDBL; srfA 
               
               
                 OhrR 
                 Organic peroxides 
                 ohrA 
               
               
                   
                 NaOCl 
               
               
                 SoxR 
                 •O 2   −   
                 soxS 
               
               
                   
                 NO• 
               
               
                   
                 (also capable of 
               
               
                   
                 sensing H 2 O 2 ) 
               
               
                 RosR 
                 H 2 O 2   
                 rbtT; tnp16a; rluC1; tnp5a; mscL; 
               
               
                   
                   
                 tnp2d; phoD; tnp15b; pstA; tnp5b; 
               
               
                   
                   
                 xylC; gabD1; rluC2; cgtS9; azlC; 
               
               
                   
                   
                 narKGHJI; rosR 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the genetically engineered bacteria comprise a tunable regulatory region that is directly or indirectly controlled by a transcription factor that is capable of sensing at least one reactive oxygen species. The tunable regulatory region is operatively linked to a gene or gene cassette capable of directly or indirectly driving the expression of a payload, thus controlling expression of the payload relative to ROS levels. For example, the tunable regulatory region is a ROS-inducible regulatory region, and the molecule is a payload; when ROS is present, e.g., in an inflamed tissue, a ROS-sensing transcription factor binds to and/or activates the regulatory region and drives expression of the gene sequence for the payload, thereby producing the payload. Subsequently, when inflammation is ameliorated, ROS levels are reduced, and production of the payload is decreased or eliminated. 
     In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region; in the presence of ROS, a transcription factor senses ROS and activates the ROS-inducible regulatory region, thereby driving expression of an operatively linked gene or gene cassette. In some embodiments, the transcription factor senses ROS and subsequently binds to the ROS-inducible regulatory region, thereby activating downstream gene expression. In alternate embodiments, the transcription factor is bound to the ROS-inducible regulatory region in the absence of ROS; when the transcription factor senses ROS, it undergoes a conformational change, thereby inducing downstream gene expression. 
     In some embodiments, the tunable regulatory region is a ROS-inducible regulatory region, and the transcription factor that senses ROS is OxyR. OxyR “functions primarily as a global regulator of the peroxide stress response” and is capable of regulating dozens of genes, e.g., “genes involved in H2O2 detoxification (katE, ahpCF), heme biosynthesis (hemH), reductant supply (grxA, gor, trxC), thiol-disulfide isomerization (dsbG), Fe—S center repair (sufA-E, sufS), iron binding (yaaA), repression of iron import systems (fur)” and “OxyS, a small regulatory RNA” (Dubbs et al., 2012). The genetically engineered bacteria may comprise any suitable ROS-responsive regulatory region from a gene that is activated by OxyR. Genes that are capable of being activated by OxyR are known in the art (see, e.g., Zheng et al., 2001; Dubbs et al., 2012). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-inducible regulatory region from oxyS that is operatively linked to a gene, e.g., a payload gene. In the presence of ROS, e.g., H2O2, an OxyR transcription factor senses ROS and activates to the oxyS regulatory region, thereby driving expression of the operatively linked payload gene and producing the payload. In some embodiments, OxyR is encoded by an  E. coli  oxyR gene. In some embodiments, the oxyS regulatory region is an  E. coli  oxyS regulatory region. In some embodiments, the ROS-inducible regulatory region is selected from the regulatory region of katG, dps, and ahpC. 
     In alternate embodiments, the tunable regulatory region is a ROS-inducible regulatory region, and the corresponding transcription factor that senses ROS is SoxR. When SoxR is “activated by oxidation of its [2Fe-2S] cluster, it increases the synthesis of SoxS, which then activates its target gene expression” (Koo et al., 2003). “SoxR is known to respond primarily to superoxide and nitric oxide” (Koo et al., 2003), and is also capable of responding to H2O2. The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is activated by SoxR. Genes that are capable of being activated by SoxR are known in the art (see, e.g., Koo et al., 2003). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-inducible regulatory region from soxS that is operatively linked to a gene, e.g., a payload. In the presence of ROS, the SoxR transcription factor senses ROS and activates the soxS regulatory region, thereby driving expression of the operatively linked a payload gene and producing the a payload. 
     In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor no longer binds to the regulatory region, thereby derepressing the operatively linked gene or gene cassette. 
     In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and the transcription factor that senses ROS is OhrR. OhrR “binds to a pair of inverted repeat DNA sequences overlapping the ohrA promoter site and thereby represses the transcription event,” but oxidized OhrR is “unable to bind its DNA target” (Duarte et al., 2010). OhrR is a “transcriptional repressor [that] . . . senses both organic peroxides and NaOCl” (Dubbs et al., 2012) and is “weakly activated by H 2 O 2  but it shows much higher reactivity for organic hydroperoxides” (Duarte et al., 2010). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by OhrR. Genes that are capable of being repressed by OhrR are known in the art (see, e.g., Dubbs et al., 2012). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from ohrA that is operatively linked to a gene or gene cassette, e.g., a payload gene. In the presence of ROS, e.g., NaOCl, an OhrR transcription factor senses ROS and no longer binds to the ohrA regulatory region, thereby derepressing the operatively linked payload gene and producing the a payload. 
     OhrR is a member of the MarR family of ROS-responsive regulators. “Most members of the MarR family are transcriptional repressors and often bind to the −10 or −35 region in the promoter causing a steric inhibition of RNA polymerase binding” (Bussmann et al., 2010). Other members of this family are known in the art and include, but are not limited to, OspR, MgrA, RosR, and SarZ. In some embodiments, the transcription factor that senses ROS is OspR, MgRA, RosR, and/or SarZ, and the genetically engineered bacteria of the invention comprises one or more corresponding regulatory region sequences from a gene that is repressed by OspR, MgRA, RosR, and/or SarZ. Genes that are capable of being repressed by OspR, MgRA, RosR, and/or SarZ are known in the art (see, e.g., Dubbs et al., 2012). 
     In some embodiments, the tunable regulatory region is a ROS-derepressible regulatory region, and the corresponding transcription factor that senses ROS is RosR. RosR is “a MarR-type transcriptional regulator” that binds to an “18-bp inverted repeat with the consensus sequence TTGTTGAYRYRTCAACWA” and is “reversibly inhibited by the oxidant H2O2” (Bussmann et al., 2010). RosR is capable of repressing numerous genes and putative genes, including but not limited to “a putative polyisoprenoid-binding protein (cg1322, gene upstream of and divergent from rosR), a sensory histidine kinase (cgtS9), a putative transcriptional regulator of the Crp/FNR family (cg3291), a protein of the glutathione S-transferase family (cg1426), two putative FMN reductases (cg1150 and cg1850), and four putative monooxygenases (cg0823, cg1848, cg2329, and cg3084)” (Bussmann et al., 2010). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by RosR. Genes that are capable of being repressed by RosR are known in the art (see, e.g., Bussmann et al., 2010). In certain embodiments, the genetically engineered bacteria of the invention comprise a ROS-derepressible regulatory region from cgtS9 that is operatively linked to a gene or gene cassette, e.g., a payload. In the presence of ROS, e.g., H2O2, a RosR transcription factor senses ROS and no longer binds to the cgtS9 regulatory region, thereby derepressing the operatively linked payload gene and producing the payload. 
     In some embodiments, it is advantageous for the genetically engineered bacteria to express a ROS-sensing transcription factor that does not regulate the expression of a significant number of native genes in the bacteria. In some embodiments, the genetically engineered bacterium of the invention expresses a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria, wherein the transcription factor does not bind to regulatory sequences in the genetically engineered bacterium of the invention. In some embodiments, the genetically engineered bacterium of the invention is  Escherichia coli , and the ROS-sensing transcription factor is RosR, e.g., from  Corynebacterium glutamicum , wherein the  Escherichia coli  does not comprise binding sites for said RosR. In some embodiments, the heterologous transcription factor minimizes or eliminates off-target effects on endogenous regulatory regions and genes in the genetically engineered bacteria. 
     In some embodiments, the tunable regulatory region is a ROS-repressible regulatory region, and binding of a corresponding transcription factor represses downstream gene expression; in the presence of ROS, the transcription factor senses ROS and binds to the ROS-repressible regulatory region, thereby repressing expression of the operatively linked gene or gene cassette. In some embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that overlaps with part of the promoter sequence. In alternate embodiments, the ROS-sensing transcription factor is capable of binding to a regulatory region that is upstream or downstream of the promoter sequence. 
     In some embodiments, the tunable regulatory region is a ROS-repressible regulatory region, and the transcription factor that senses ROS is PerR. In  Bacillus subtilis , PerR “when bound to DNA, represses the genes coding for proteins involved in the oxidative stress response (katA, ahpC, and mrgA), metal homeostasis (hemAXCDBL, fur, and zoaA) and its own synthesis (perR)” (Marinho et al., 2014). PerR is a “global regulator that responds primarily to H2O2” (Dubbs et al., 2012) and “interacts with DNA at the per box, a specific palindromic consensus sequence (TTATAATNATTATAA) residing within and near the promoter sequences of PerR-controlled genes” (Marinho et al., 2014). PerR is capable of binding a regulatory region that “overlaps part of the promoter or is immediately downstream from it” (Dubbs et al., 2012). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by PerR. Genes that are capable of being repressed by PerR are known in the art (see, e.g., Dubbs et al., 2012). 
     In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express a payload. The two repressor activation regulatory circuit comprises a first ROS-sensing repressor, e.g., PerR, and a second repressor, e.g., TetR, which is operatively linked to a gene or gene cassette, e.g., a payload. In one aspect of these embodiments, the ROS-sensing repressor inhibits transcription of the second repressor, which inhibits the transcription of the gene or gene cassette. Examples of second repressors useful in these embodiments include, but are not limited to, TetR, C1, and LexA. In some embodiments, the ROS-sensing repressor is PerR. In some embodiments, the second repressor is TetR. In this embodiment, a PerR-repressible regulatory region drives expression of TetR, and a TetR-repressible regulatory region drives expression of the gene or gene cassette, e.g., a payload. In the absence of PerR binding (which occurs in the absence of ROS), tetR is transcribed, and TetR represses expression of the gene or gene cassette, e.g., a payload. In the presence of PerR binding (which occurs in the presence of ROS), tetR expression is repressed, and the gene or gene cassette, e.g., a payload, is expressed. 
     A ROS-responsive transcription factor may induce, derepress, or repress gene expression depending upon the regulatory region sequence used in the genetically engineered bacteria. For example, although “OxyR is primarily thought of as a transcriptional activator under oxidizing conditions . . . OxyR can function as either a repressor or activator under both oxidizing and reducing conditions” (Dubbs et al., 2012), and OxyR “has been shown to be a repressor of its own expression as well as that of fhuF (encoding a ferric ion reductase) and flu (encoding the antigen 43 outer membrane protein)” (Zheng et al., 2001). The genetically engineered bacteria of the invention may comprise any suitable ROS-responsive regulatory region from a gene that is repressed by OxyR. In some embodiments, OxyR is used in a two repressor activation regulatory circuit, as described above. Genes that are capable of being repressed by OxyR are known in the art (see, e.g., Zheng et al., 2001). Or, for example, although RosR is capable of repressing a number of genes, it is also capable of activating certain genes, e.g., the narKGHJI operon. In some embodiments, the genetically engineered bacteria comprise any suitable ROS-responsive regulatory region from a gene that is activated by RosR. In addition, “PerR-mediated positive regulation has also been observed . . . and appears to involve PerR binding to distant upstream sites” (Dubbs et al., 2012). In some embodiments, the genetically engineered bacteria comprise any suitable ROS-responsive regulatory region from a gene that is activated by PerR. 
     One or more types of ROS-sensing transcription factors and corresponding regulatory region sequences may be present in genetically engineered bacteria. For example, “OhrR is found in both Gram-positive and Gram-negative bacteria and can coreside with either OxyR or PerR or both” (Dubbs et al., 2012). In some embodiments, the genetically engineered bacteria comprise one type of ROS-sensing transcription factor, e.g., OxyR, and one corresponding regulatory region sequence, e.g., from oxyS. In some embodiments, the genetically engineered bacteria comprise one type of ROS-sensing transcription factor, e.g., OxyR, and two or more different corresponding regulatory region sequences, e.g., from oxyS and katG. In some embodiments, the genetically engineered bacteria comprise two or more types of ROS-sensing transcription factors, e.g., OxyR and PerR, and two or more corresponding regulatory region sequences, e.g., from oxyS and katA, respectively. One ROS-responsive regulatory region may be capable of binding more than one transcription factor. In some embodiments, the genetically engineered bacteria comprise two or more types of ROS-sensing transcription factors and one corresponding regulatory region sequence. 
     Nucleic acid sequences of several exemplary OxyR-regulated regulatory regions are shown in Table 25. OxyR binding sites are underlined and bolded. In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 580, SEQ ID NO: 581, SEQ ID NO: 582, or SEQ ID NO: 583, or a functional fragment thereof. 
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 Nucleotide sequences of exemplary OxyR-regulated regulatory regions  
               
            
           
           
               
               
            
               
                 Regulatory 
                   
               
               
                 sequence 
                 Sequence 
               
               
                   
               
               
                 katG 
                 TGTGGCTTTTATGAAAATCACACAGTGATCACAAATTTTAAACA 
               
               
                 (SEQ ID NO: 580) 
                 GAGCACAAAATGCTGCCTCGAAATGAGGGCGGGAAAATAAGGT 
               
               
                   
                 TATCAGCCTTGTTTTCTCCCTCATTACTTGAAGGATATGAAGCTA 
               
               
                   
                 AAACCCTTTTTTATAAAGCATTTGTCCGAATTCGGACATAATCA 
               
               
                   
                 AAAAAGCTTAATTAAGATCAATTTGATCTACATCTCTTTAACCA 
               
               
                   
                 ACAATAT GTAAGATCTCAACTATC GCATC CGTGGATTAATTCAA   
               
               
                   
                   TT ATAACTTCTCTCTAACGCTGTGTATCGTAACGGTAACACTGTA 
               
               
                   
                 GAGGGGAGCACATTGATGCGAATTCATTAAAGAGGAGAAAGGT 
               
               
                   
                 ACC 
               
               
                   
               
               
                 dps 
                 TTCCGAAAATTCCTGGCGAGCAGATAAATAAGAATTGTTCTTAT 
               
               
                 (SEQ ID NO: 581) 
                 CAATATATCTAACTCATTGAATCTTTATTAGTTTTGTTTTTCA CG   
               
               
                   
                   CTTGTTACCACTATT AGTGT GATAGGAACAGCCAGAA TAGCGGA 
               
               
                   
                 ACACATAGCCGGTGCTATACTTAATCTCGTTAATTACTGGGACA 
               
               
                   
                 TAACATCAAGAGGATATGAAATTCGAATTCATTAAAGAGGAGA 
               
               
                   
                 AAGGTACC 
               
               
                   
               
               
                 ahpC 
                 GCTTAGATCAGGTGATTGCCCTTTGTTTATGAGGGTGTTGTAATC 
               
               
                 (SEQ ID NO: 582) 
                 CATGTCGTTGTTGCATTTGTAAGGGCAACACCTCAGCCTGCAGG 
               
               
                   
                 CAGGCACTGAAGATACCAAAGGGTAGTTCAGATTACACGGTCA 
               
               
                   
                 CCTGGAAAGGGGGCCATTTTACTTTTTATCGCCGCTGGCGGTGC 
               
               
                   
                 AAAGTTCACAAAGTTGTCTTACGAAGGTT GTAAGGTAAAACTTA   
               
               
                   
                   TC GATTT GATAATGGAAACGCATT AGCCGAATCGGCAAAAATTG 
               
               
                   
                 GTTACCTTACATCTCATCGAAAACACGGAGGAAGTATAGATGCG 
               
               
                   
                 AATTCATTAAAGAGGAGAAAGGTACC 
               
               
                   
               
               
                 oxyS 
                 CTCGAGTTCATTATCCATCCTCCATCGCCAC GATAGTTCATGGCG   
               
               
                 (SEQ ID NO: 583) 
                   ATA GGTAG AATAGCAATGAACGATT ATCCCTATCAAGCATTCTG 
               
               
                   
                 ACTGATAATTGCTCACACGAATTCATTAAAGAGGAGAAAGGTA 
               
               
                   
                 CC 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the regulatory region sequence is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the sequence of SEQ ID NO: 580, SEQ ID NO: 581, SEQ ID NO: 582, and/or SEQ ID NO: 583. 
     In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a ROS-sensing transcription factor, e.g., the oxyR gene, that is controlled by its native promoter, an inducible promoter, a promoter that is stronger than the native promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a constitutive promoter. In some instances, it may be advantageous to express the ROS-sensing transcription factor under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the ROS-sensing transcription factor is controlled by a different promoter than the promoter that controls expression of the therapeutic molecule. In some embodiments, expression of the ROS-sensing transcription factor is controlled by the same promoter that controls expression of the therapeutic molecule. In some embodiments, the ROS-sensing transcription factor and therapeutic molecule are divergently transcribed from a promoter region. 
     In some embodiments, the genetically engineered bacteria of the invention comprise a gene for a ROS-sensing transcription factor from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS-responsive regulatory region from a different species, strain, or substrain of bacteria. In some embodiments, the genetically engineered bacteria comprise a ROS-sensing transcription factor and corresponding ROS-responsive regulatory region from a different species, strain, or substrain of bacteria. The heterologous ROS-sensing transcription factor and regulatory region may increase the transcription of genes operatively linked to said regulatory region in the presence of ROS, as compared to the native transcription factor and regulatory region from bacteria of the same subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria comprise a ROS-sensing transcription factor, OxyR, and corresponding regulatory region, oxyS, from  Escherichia coli . In some embodiments, the native ROS-sensing transcription factor, e.g., OxyR, is left intact and retains wild-type activity. In alternate embodiments, the native ROS-sensing transcription factor, e.g., OxyR, is deleted or mutated to reduce or eliminate wild-type activity. 
     In some embodiments, the genetically engineered bacteria of the invention comprise multiple copies of the endogenous gene encoding the ROS-sensing transcription factor, e.g., the oxyR gene. In some embodiments, the gene encoding the ROS-sensing transcription factor is present on a plasmid. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different plasmids. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same. In some embodiments, the gene encoding the ROS-sensing transcription factor is present on a chromosome. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on different chromosomes. In some embodiments, the gene encoding the ROS-sensing transcription factor and the gene or gene cassette for producing the therapeutic molecule are present on the same chromosome. 
     In some embodiments, the genetically engineered bacteria comprise a wild-type gene encoding a ROS-sensing transcription factor, e.g., the soxR gene, and a corresponding regulatory region, e.g., a soxS regulatory region, that is mutated relative to the wild-type regulatory region from bacteria of the same subtype. The mutated regulatory region increases the expression of the payload in the presence of ROS, as compared to the wild-type regulatory region under the same conditions. In some embodiments, the genetically engineered bacteria comprise a wild-type ROS-responsive regulatory region, e.g., the oxyS regulatory region, and a corresponding transcription factor, e.g., OxyR, that is mutated relative to the wild-type transcription factor from bacteria of the same subtype. The mutant transcription factor increases the expression of the payload in the presence of ROS, as compared to the wild-type transcription factor under the same conditions. In some embodiments, both the ROS-sensing transcription factor and corresponding regulatory region are mutated relative to the wild-type sequences from bacteria of the same subtype in order to increase expression of the payload in the presence of ROS. 
     In some embodiments, the gene or gene cassette for producing the payload is present on a plasmid and operably linked to a promoter that is induced by ROS. In some embodiments, the gene or gene cassette for producing the payload is present in the chromosome and operably linked to a promoter that is induced by ROS. In some embodiments, the gene or gene cassette for producing the payload is present on a chromosome and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, the gene or gene cassette for producing the payload is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability. 
     In some embodiments, the genetically engineered bacteria may comprise multiple copies of the gene(s) capable of producing a payload(s). In some embodiments, the gene(s) capable of producing a payload(s) is present on a plasmid and operatively linked to a ROS-responsive regulatory region. In some embodiments, the gene(s) capable of producing a payload is present in a chromosome and operatively linked to a ROS-responsive regulatory region. 
     Thus, in some embodiments, the genetically engineered bacteria or genetically engineered virus produce one or more payloads under the control of an oxygen level-dependent promoter, a reactive oxygen species (ROS)-dependent promoter, or a reactive nitrogen species (RNS)-dependent promoter, and a corresponding transcription factor. 
     In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying a gene for producing a payload, such that the payload can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo. In some embodiments, a bacterium may comprise multiple copies of the gene encoding the payload. In some embodiments, the gene encoding the payload is expressed on a low-copy plasmid. In some embodiments, the low-copy plasmid may be useful for increasing stability of expression. In some embodiments, the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions. In some embodiments, the gene encoding the payload is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the payload. In some embodiments, the gene encoding the payload is expressed on a chromosome. 
     Propionate and Other Promoters 
     In some embodiments, the genetically engineered bacteria comprise the gene or gene cassette for producing an anti-inflammation and/or gut barrier function enhancer molecule(s) expressed under the control of an inducible promoter that is responsive to specific molecules or metabolites in the environment, e.g., the tumor microenvironment, a specific tissue, or the mammalian gut. For example, the short-chain fatty acid propionate is a major microbial fermentation metabolite localized to the gut (Hosseini et al., 2011). In one embodiment, the gene or gene cassette for producing an anti-inflammation and/or gut barrier function enhancer molecule(s) is under the control of a propionate-inducible promoter. In a more specific embodiment, the gene or gene cassette for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is under the control of a propionate-inducible promoter that is activated by the presence of propionate in the mammalian gut. Any molecule or metabolite found in the mammalian gut, in a healthy and/or disease state, may be used to induce payload expression. Non-limiting examples of inducers include propionate, bilirubin, aspartate aminotransferase, alanine aminotransferase, blood coagulation factors II, VII, IX, and X, alkaline phosphatase, gamma glutamyl transferase, hepatitis antigens and antibodies, alpha fetoprotein, anti-mitochondrial, smooth muscle, and anti-nuclear antibodies, iron, transferrin, ferritin, copper, ceruloplasmin, ammonia, and manganese. In alternate embodiments, the gene or gene cassette for producing an anti-inflammation and/or gut barrier function enhancer molecule(s) is under the control of a pBAD promoter, which is activated in the presence of the sugar arabinose. 
     In some embodiments, the gene or gene cassette for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene or gene cassette for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene or gene cassette for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is present on a plasmid and operably linked to a promoter that is induced by molecules or metabolites that are specific to the mammalian gut. In some embodiments, the gene or gene cassette for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is present on a chromosome and operably linked to a promoter that is induced by molecules or metabolites that are specific to the tumor and/or the mammalian gut. In some embodiments, the gene or gene cassette for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is present on a chromosome and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, the gene or gene cassette for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, expression is further optimized by methods known in the art, e.g., by optimizing ribosomal binding sites, manipulating transcriptional regulators, and/or increasing mRNA stability. 
     In some embodiments, the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying the gene or gene cassette for producing the anti-inflammation and/or gut barrier function enhancer molecule(s), such that the gene or gene cassette can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. In some embodiments, a bacterium may comprise multiple copies of the gene or gene cassette for producing the anti-inflammation and/or gut barrier function enhancer molecule(s). In some embodiments, gene or gene cassette for producing the payload is expressed on a low-copy plasmid. In some embodiments, the low-copy plasmid may be useful for increasing stability of expression. In some embodiments, the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions. In some embodiments, gene or gene cassette for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing gene or gene cassette expression. In some embodiments, gene or gene cassette for producing the anti-inflammation and/or gut barrier function enhancer molecule(s) is expressed on a chromosome. 
     Table 26 lists a propionate promoter sequence. In some embodiments, the propionate promoter is induced in the mammalian gut. In some embodiments, the propionate promoter sequence is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the sequence of SEQ ID NO: 584. 
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 Propionate promoter sequence 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 Prp (Propionate) 
                 
                   TTACCCGTCTGGATTTTCAGTACGCGCTTTTAAACGACGCCA 
                 
               
               
                 promoter Bold: prpR 
                 
                   CAGCGTGGTACGGCTGATCCCCAAATAACGTGCGGCGGCGCG 
                 
               
               
                   
                 
                   CTTATCGCCATTAAAGCGTGCGAGCACCTCCTGCAATGGAAG 
                 
               
               
                   
                 
                   CGCTTCTGCTGACGAGGGCGTGATTTCTGCTGTGGTCCCCAC 
                 
               
               
                   
               
               
                 Lower case: 
                 CAGTTCAGGTAATAATTGCCGCATAAATTGTCTGTCCAGTGT 
               
               
                 ribosome binding 
                 TGGTGCGGGATCGACGCTTAAAAAAAGCGCCAGGCGTTCCAT 
               
               
                 site ATG underlined: 
                 
                   CATATTCCGCAGTTCGCGAATATTACCGGGCCAATGATAGTT 
                 
               
               
                 start of gene of 
                 
                   CAGTAGAAGCGGCTGACACTGCGTCAGCCCATGACGCACCGA 
                 
               
               
                 interest SEQ ID NO: 584 
                 
                   TTCGGTAAAAGGGATCTCCATCGCGGCCAGCGATTGTTTTAA 
                 
               
               
                   
                 
                   AAAGTTTTCCGCCAGAGGCAGAATATCAGGCTGTCGCTCGCG 
                 
               
               
                   
                 
                   CAAGGGGGGAAGCGGCAGACGCAGAATGCTCAAACGGTAAAA 
                 
               
               
                   
                 
                   CAGATCGGTACGAAAACGTCCTTGCGTTATCTCCCGATCCAG 
                 
               
               
                   
                 
                   ATCGCAATGCGTGGCGCTGATCACCCGGACATCTACCGGGAT 
                 
               
               
                   
                 
                   CGGCTGATGCCCGCCAACGCGGGTGACGGCTTTTTCCTCCAG 
                 
               
               
                   
                 
                   TACGCGTAGAAGGCGGGTTTGTAACGGCAGCGGCATTTCGCC 
                 
               
               
                   
                 
                   AATTTCGTCAAGAAACAGCGTGCCGCCGTGGGCGACCTCAAA 
                 
               
               
                   
                 
                   CAGCCCCGCACGTCCACCTCGTCTTGAGCCGGTAAACGCTCC 
                 
               
               
                   
                 
                   CTCCTCATAGCCAAACAGTTCAGCCTCCAGCAACGACTCGGT 
                 
               
               
                   
                 
                   AATCGCGCCGCAATTAACGGCGACAAAGGGCGGAGAAGGCTT 
                 
               
               
                   
                 
                   GTTCTGACGGTGGGGCTGACGGTTAAACAACGCCTGATGAAT 
                 
               
               
                   
                 
                   CGCTTGCGCCGCCAGCTCTTTCCCGGTCCCTGTTTCCCCCTG 
                 
               
               
                   
                 
                   AATCAGCACTGCCGCGCGGGAACGGGCATAGAGTGTAATCGT 
                 
               
               
                   
                 
                   ATGGCGAACCTGCTCCATTTGTGGTGAATCGCCGAGGATATC 
                 
               
               
                   
                 
                   GCTCAGCGCATAACGGGTCTGTAATCCCTTGCTGGAGGTATG 
                 
               
               
                   
                 
                   CTGGCTATACTGACGCCGTGTCAGGCGGGTCATATCCAGCGC 
                 
               
               
                   
                 
                   ATCATGGAAAGCCTGACGTACGGTGGCCGCTGAATAAATAAA 
                 
               
               
                   
                 
                   GATGGCGGTCATTCCTGCCTCTTCCGCCAGGTCGGTAATTAG 
                 
               
               
                   
                 
                   TCCTGCCCCAATTACAGCCTCAATGCCGTTAGCTTTGAGCTC 
                 
               
               
                   
                 
                   GTTAATTTGCCCGCGAGCATCCTCTTCAGTGATATAGCTTCG 
                 
               
               
                   
                 
                   CTGTTCAAGACGGAGGTGAAACGTTTTCTGAAAGGCGACCAG 
                 
               
               
                   
                 
                   AGCCGGAATGGTCTCCTGATAGGTCACGATTCCCATTGAGGA 
                 
               
               
                   
                 
                   AGTCAGCTTTCCCGCTTTTGCCAGAGCCTGTAATACATCGAA 
                 
               
               
                   
                 
                   TCCGCTGGGTTTGATGAGGATGACAGGTACCGACAGTCGGCT 
                 
               
               
                   
                 
                   TTTTAAATAAGCGCCGTTGGAACCTGCCGCGATAATCGCGTC 
                 
               
               
                   
                 
                   GCAGCGTTCGGTTGCCAGTTTTTTGCGAATGTAGGCTACTGC 
                 
               
               
                   
                 
                   CTTTTCAAAACCGAGCTGAATAGGCGTGATCGTCGCCAGATG 
                 
               
               
                   
                 
                   ATCAAACTCCAGGCTGATATCCCGAAATAGTTCGAACAGGCG 
                 
               
               
                   
                 
                   CGTTACCGAGACCGTCCAGATCACCGGTTTATCGCTATTATC 
                 
               
               
                   
                   GCGCGAAGCGCTATGCACAGTAACCAT CGTCGTAGATTCATG 
               
               
                   
                 TTTAAGGAACGAATTCTTGTTTTATAGATGTTTCGTTAATGT 
               
               
                   
                 TGCAATGAAACACAGGCCTCCGTTTCATGAAACGTTAGCTGA 
               
               
                   
                 CTCGTTTTTCTTGTGACTCGTCTGTCAGTATTAAAAAAGATT 
               
               
                   
                 TTTCATTTAACTGATTGTTTTTAAATTGAATTTTATTTAATG 
               
               
                   
                 GTTTCTCGGTTTTTGGGTCTGGCATATCCCTTGCTTTAATGA 
               
               
                   
                 GTGCATCTTAATTAACAATTCAATAACAAGAGGGCTGAATag 
               
               
                   
                 taatttcaacaaaataacgagcattcga atg   
               
               
                   
               
            
           
         
       
     
     Other Inducible Promoters 
     In some embodiments, the gene encoding the anti-inflammation and/or gut barrier function enhancer molecule(s) is present on a plasmid and operably linked to a promoter that is induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the gene encoding the anti-inflammation and/or gut barrier function enhancer molecule(s) is present in the chromosome and operably linked to a promoter that is induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). 
     In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the one or more gene sequences(s), inducible by one or more nutritional and/or chemical inducer(s) and/or metabolite(s), encoding the anti-inflammation and/or gut barrier function enhancer molecule(s), such that the anti-inflammation and/or gut barrier function enhancer molecule(s) can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. In some embodiments, bacterial cell comprises two or more distinct copies of the one or more gene sequences(s) encoding the anti-inflammation and/or gut barrier function enhancer molecule(s), which is controlled by a promoter inducible one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the genetically engineered bacteria comprise multiple copies of the same one or more gene sequences(s) encoding the anti-inflammation and/or gut barrier function enhancer molecule(s), which is controlled by a promoter inducible one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the one or more gene sequences(s) encoding the anti-inflammation and/or gut barrier function enhancer molecule(s), is present on a plasmid and operably linked to a directly or indirectly inducible promoter inducible by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the one or more gene sequences(s) encoding the anti-inflammation and/or gut barrier function enhancer molecule(s), is present on a chromosome and operably linked to a directly or indirectly inducible by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). 
     In some embodiments, one or more gene sequence(s) encoding polypeptides of interest described herein is present on a plasmid and operably linked to promoter a directly or indirectly inducible by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the gene encoding the anti-inflammation and/or gut barrier function enhancer molecule(s), which is induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s), such that the anti-inflammation and/or gut barrier function enhancer molecule(s) can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., under culture conditions, and/or in vivo, e.g., in the gut and/or the tumor microenvironment. In some embodiments, bacterial cell comprises two or more gene sequence(s) for the production of a polypeptide of interest, one or more of which are induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the genetically engineered bacteria comprise multiple copies of the same gene sequence(s) for the production of a polypeptide of interest which are induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, the genetically engineered bacteria comprise multiple copies of different gene sequence(s) for the production of a polypeptide of interest, one or more of which are induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). 
     In some embodiments, the gene sequence(s) for the production of a polypeptide of interest is present on a plasmid and operably linked to a promoter that is induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). In some embodiments, gene sequence(s) for the production of a polypeptide of interest is present in the chromosome and operably linked to a promoter that is induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). 
     In some embodiments, the promoter that is operably linked to the gene encoding the polypeptide of interest is directly or indirectly induced by one or more nutritional and/or chemical inducer(s) and/or metabolite(s). 
     In some embodiments, one or more inducible promoter(s) are useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, the promoters are induced during in vivo expression of one or more anti-inflammation and/or gut barrier function enhancer molecule(s) and/or other polypeptide(s) of interest. In some embodiments, expression of one or more anti-inflammation and/or gut barrier function enhancer molecule(s) and/or other polypeptide(s) of interest is driven directly or indirectly by one or more arabinose inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a chemical and/or nutritional inducer and/or metabolite which is co-administered with the genetically engineered bacteria of the invention. 
     In some embodiments, expression of one or more anti-inflammation and/or gut barrier function enhancer molecule(s) and/or other polypeptide(s) of interest, is driven directly or indirectly by one or more promoter(s) induced by a chemical and/or nutritional inducer and/or metabolite during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the promoter(s) induced by a chemical and/or nutritional inducer and/or metabolite are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the anti-inflammation and/or gut barrier function enhancer molecule(s) and/or other polypeptide(s) of interest prior to administration. In some embodiments, the cultures, which are induced by a chemical and/or nutritional inducer and/or metabolite, are grown aerobically. In some embodiments, the cultures, which are induced by a chemical and/or nutritional inducer and/or metabolite, are grown anaerobically. 
     The genes of arabinose metabolism are organized in one operon, AraBAD, which is controlled by the PAraBAD promoter. The PAraBAD (or Para) promoter suitably fulfills the criteria of inducible expression systems. PAraBAD displays tighter control of payload gene expression than many other systems, likely due to the dual regulatory role of AraC, which functions both as an inducer and as a repressor. Additionally, the level of ParaBAD-based expression can be modulated over a wide range of L-arabinose concentrations to fine-tune levels of expression of the payload. However, the cell population exposed to sub-saturating L-arabinose concentrations is divided into two subpopulations of induced and uninduced cells, which is determined by the differences between individual cells in the availability of L-arabinose transporter (Zhang et al., Development and Application of an Arabinose-Inducible Expression System by Facilitating Inducer Uptake in  Corynebacterium glutamicum ; Appl. Environ. Microbiol. August 2012 vol. 78 no. 16 5831-5838). Alternatively, inducible expression from the ParaBad can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein. An exemplary construct is depicted in the figures and examples. 
     In one embodiment, expression of one or more anti-inflammation and/or gut barrier function enhancer molecule(s) of interest, e.g., one or more therapeutic polypeptide(s), is driven directly or indirectly by one or more arabinose inducible promoter(s). 
     In some embodiments, the arabinose inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more anti-inflammation and/or gut barrier function enhancer molecule(s) of interest is driven directly or indirectly by one or more arabinose inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., arabinose. 
     In some embodiments, expression of one or more protein(s) of interest, is driven directly or indirectly by one or more arabinose inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the arabinose inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., arabinose. In some embodiments, the cultures, which are induced by arabinose, are grown aerobically. In some embodiments, the cultures, which are induced by arabinose, are grown anaerobically. 
     In one embodiment, the arabinose inducible promoter drives the expression of a construct comprising one or more protein(s) of interest, jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the arabinose inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., arabinose and IPTG). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including arabinose presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more arabinose promoters drive expression of one or more protein(s) of interest, in combination with the FNR promoter driving the expression of the same gene sequence(s). 
     In some embodiments, the arabinose inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the arabinose inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein. 
     In some embodiments, one or more protein(s) of interest are knocked into the arabinose operon and are driven by the native arabinose inducible promoter 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 585. In some embodiments, the arabinose inducible construct further comprises a gene encoding AraC, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 586. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 587. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through a rhamnose inducible system. The genes rhaBAD are organized in one operon which is controlled by the rhaP BAD promoter. The rhaP BAD promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which divergently transcribed in the opposite direction of rhaBAD. In the presence of L-rhamnose, RhaR binds to the rhaP RS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose then bind to the rhaP BAD and the rhaP T promoter and activate the transcription of the structural genes. In contrast to the arabinose system, in which AraC is provided and divergently transcribed in the gene sequence(s), it is not necessary to express the regulatory proteins in larger quantities in the rhamnose expression system because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaP BAD promoter is cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression. Alternatively, inducible expression from the rhaBAD can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein. 
     In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more rhamnose inducible promoter(s). In one embodiment, expression of the payload is driven directly or indirectly by a rhamnose inducible promoter. 
     In some embodiments, the rhamnose inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more rhamnose inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., rhamnose 
     In some embodiments, expression of one or more protein(s) of interest, is driven directly or indirectly by one or more rhamnose inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the rhamnose inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., rhamnose. In some embodiments, the cultures, which are induced by rhamnose, are grown aerobically. In some embodiments, the cultures, which are induced by rhamnose, are grown anaerobically. 
     In one embodiment, the rhamnose inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the rhamnose inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., rhamnose and arabinose). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including rhamnose presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more rhamnose promoters drive expression of one or more protein(s) of interest and/or transcriptional regulator(s), e.g., FNRS24Y, in combination with the FNR promoter driving the expression of the same gene sequence(s). 
     In some embodiments, the rhamnose inducible promoter drives the expression of one or more protein(s) of interest, from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the rhamnose inducible promoter drives the expression of one or more protein(s) of interest, from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 588. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through an Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible system or other compound which induced transcription from the Lac Promoter. IPTG is a molecular mimic of allolactose, a lactose metabolite that activates transcription of the lac operon. In contrast to allolactose, the sulfur atom in IPTG creates a non-hydrolyzable chemical blond, which prevents the degradation of IPTG, allowing the concentration to remain constant. IPTG binds to the lac repressor and releases the tetrameric repressor (lacI) from the lac operator in an allosteric manner, thereby allowing the transcription of genes in the lac operon. Since IPTG is not metabolized by  E. coli , its concentration stays constant and the rate of expression of Lac promoter-controlled is tightly controlled, both in vivo and in vitro. IPTG intake is independent on the action of lactose permease, since other transport pathways are also involved. Inducible expression from the PLac can be controlled or fine-tuned through the optimization of the ribosome binding site (RBS), as described herein. Other compounds which inactivate LacI, can be used instead of IPTG in a similar manner. 
     In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s). 
     In some embodiments, the IPTG inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., IPTG. 
     In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more IPTG inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the IPTG inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., IPTG. In some embodiments, the cultures, which are induced by IPTG, are grown aerobically. In some embodiments, the cultures, which are induced by IPTG, are grown anaerobically. 
     In one embodiment, the IPTG inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the IPTG inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., arabinose and IPTG). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including IPTG presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more IPTG inducible promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s). 
     In some embodiments, the IPTG inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the IPTG inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 589. In some embodiments, the IPTG inducible construct further comprises a gene encoding lacI, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 590. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 591. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are inducible through a tetracycline inducible system. The initial system Gossen and Bujard (Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Gossen M &amp; Bujard H.  PNAS,  1992 Jun. 15; 89(12):5547-51) developed is known as tetracycline off: in the presence of tetracycline, expression from a tet-inducible promoter is reduced. Tetracycline-controlled transactivator (tTA) was created by fusing tetR with the C-terminal domain of VP16 (virion protein 16) from herpes simplex virus. In the absence of tetracycline, the tetR portion of tTA will bind tetO sequences in the tet promoter, and the activation domain promotes expression. In the presence of tetracycline, tetracycline binds to tetR, precluding tTA from binding to the tetO sequences. Next, a reverse Tet repressor (rTetR), was developed which created a reliance on the presence of tetracycline for induction, rather than repression. The new transactivator rtTA (reverse tetracycline-controlled transactivator) was created by fusing rTetR with VP16. The tetracycline on system is also known as the rtTA-dependent system. 
     In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more tetracycline inducible promoter(s). 
     In some embodiments, the tetracycline inducible promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest and/or transcriptional regulator(s), e.g., FNRS24Y, is driven directly or indirectly by one or more tetracycline inducible promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., tetracycline 
     In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more tetracycline inducible promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, the tetracycline inducible promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the promoter is directly or indirectly induced by a molecule that is added to in the bacterial culture to induce expression and pre-load the bacterium with the payload prior to administration, e.g., tetracycline. In some embodiments, the cultures, which are induced by tetracycline, are grown arerobically. In some embodiments, the cultures, which are induced by tetracycline, are grown anaerobically. 
     In one embodiment, the tetracycline inducible promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the tetracycline inducible promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., tetracycline and IPTG). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., including tetracycline presence, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more tetracycline promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s). 
     In some embodiments, the tetracycline inducible promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the tetracycline inducible promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the bolded sequences of SEQ ID NO: 596 (tet promoter is in bold). In some embodiments, the tetracycline inducible construct further comprises a gene encoding AraC, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 596 in italics (Tet repressor is in italics). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 596 in italics (Tet repressor is in italics). 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) whose expression is controlled by a temperature sensitive mechanism. Thermoregulators are advantageous because of strong transcriptional control without the use of external chemicals or specialized media (see, e.g., Nemani et al., Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated  E. coli  for enzyme-prodrug therapy; J Biotechnol. 2015 Jun. 10; 203: 32-40, and references therein). Thermoregulated protein expression using the mutant cI857 repressor and the pL and/or pR phage λ promoters have been used to engineer recombinant bacterial strains. The gene of interest cloned downstream of the λ promoters can then be efficiently regulated by the mutant thermolabile cI857 repressor of bacteriophage λ. At temperatures below 37° C., cI857 binds to the oL or oR regions of the pR promoter and blocks transcription by RNA polymerase. At higher temperatures, the functional cI857 dimer is destabilized, binding to the oL or oR DNA sequences is abrogated, and mRNA transcription is initiated. An exemplary construct is depicted in in the figures and examples. Inducible expression from the ParaBad can be controlled or further fine-tuned through the optimization of the ribosome binding site (RBS), as described herein. 
     In one embodiment, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s). 
     In some embodiments, the thermoregulated promoter is useful for or induced during in vivo expression of the one or more protein(s) of interest. In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s) in vivo. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the genetically engineered bacteria of the invention, e.g., temperature. 
     In some embodiments, expression of one or more protein(s) of interest is driven directly or indirectly by one or more thermoregulated promoter(s) during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, it may be advantageous to shup off production of the one or more protein(s) of interest. This can be done in a thermoregulated system by growing the strain at lower temperatures, e.g., 30 C. Expression can then be induced by elevating the temperature to 37 C and/or 42 C. In some embodiments, the thermoregulated promoter(s) are induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In some embodiments, the cultures, which are induced by temperatures between 37 C and 42 C, are grown arerobically. In some embodiments, the cultures, which are induced by induced by temperatures between 37 C and 42 C, are grown anaerobically. 
     In one embodiment, the thermoregulated promoter drives the expression of a construct comprising one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter. In some embodiments, two promoters are positioned proximally to the construct and drive its expression, wherein the thermoregulated promoter drives expression under a first set of exogenous conditions, and the second promoter drives the expression under a second set of exogenous conditions. In a non-limiting example, the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., thermoregulation and arabinose). In another non-limiting example, the first inducing conditions may be culture conditions, e.g., permissive temperature, and the second inducing conditions may be in vivo conditions. Such in vivo conditions include low-oxygen, microaerobic, or anaerobic conditions, presence of gut metabolites, and/or metabolites administered in combination with the bacterial strain. In some embodiments, the one or more thermoregulated promoters drive expression of one or more protein(s) of interest in combination with the FNR promoter driving the expression of the same gene sequence(s). 
     In some embodiments, the thermoregulated promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the thermoregulated promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 592. In some embodiments, the thermoregulated construct further comprises a gene encoding mutant cI857 repressor, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 593. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 595. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) which are indirectly inducible through a system driven by the PssB promoter. The Pssb promoter is active under aerobic conditions, and shuts off under anaerobic conditions. 
     This promoter can be used to express a gene of interest under aerobic conditions. This promoter can also be used to tightly control the expression of a gene product such that it is only expressed under anaerobic conditions. In this case, the oxygen induced PssB promoter induces the expression of a repressor, which represses the expression of a gene of interest. As a result, the gene of interest is only expressed in the absence of the repressor, i.e., under anaerobic conditions. This strategy has the advantage of an additional level of control for improved fine-tuning and tighter control.  FIG. 84A  depicts a schematic of the gene organization of a PssB promoter. 
     In one embodiment, expression of one or more protein(s) of interest is indirectly regulated by a repressor expressed under the control of one or more PssB promoter(s). 
     In some embodiments, induction of the RssB promoter(s) indirectly drives the in vivo expression of one or more protein(s) of interest. In some embodiments, induction of the RssB promoter(s) indirectly drives the expression of one or more protein(s) of interest during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration. In some embodiments, conditions for induction of the RssB promoter(s) are provided in culture, e.g., in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. 
     In some embodiments, the PssB promoter indirectly drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein. In some embodiments, the PssB promoter indirectly drives the expression of one or more protein(s) of interest from a construct which is integrated into the bacterial chromosome. Exemplary insertion sites are described herein. 
     In another non-limiting example, this strategy can be used to control expression of thyA and/or dapA, e.g., to make a conditional auxotroph. The chromosomal copy of dapA or ThyA is knocked out. Under anaerobic conditions, dapA or thyA—as the case may be—are expressed, and the strain can grow in the absence of dap or thymidine. Under aerobic conditions, dapA or thyA expression is shut off, and the strain cannot grow in the absence of dap or thymidine. Such a strategy can, for example be employed to allow survival of bacteria under anaerobic conditions, e.g., the gut, but prevent survival under aerobic conditions (biosafety switch). In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 597. 
     Sequences useful for expression from inducible promoters are listed in Table 27. 
     
       
         
           
               
             
               
                 TABLE 27 
               
             
            
               
                   
               
               
                 Inducible promoter construct sequences 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 Arabinose 
                 CAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGC 
               
               
                 Promoter region 
                 TAACCCAACCGGTAACCCCGCTTATTAAAAGCATTCTGTA 
               
               
                 SEQ ID NO: 
                 ACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAA 
               
               
                 585 
                 AAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTAT 
               
               
                   
                 TTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATC 
               
               
                   
                 CATAAGATTAGCGGATCCAGCCTGACGCTTTTTTTCGCAA 
               
               
                   
                 CTCTCTACTGTTTCTCCATACCTCTAGAAATAATTTTGTTT 
               
               
                   
                 AACTTTAAGAAGGAGATATACAT 
               
               
                   
               
               
                 AraC (reverse 
                 TTATTCACAACCTGCCCTAAACTCGCTCGGACTCGCCCCG 
               
               
                 orientation) 
                 GTGCATTTTTTAAATACTCGCGAGAAATAGAGTTGATCGT 
               
               
                 SEQ ID NO: 
                 CAAAACCGACATTGCGACCGACGGTGGCGATAGGCATCC 
               
               
                 586 
                 GGGTGGTGCTCAAAAGCAGCTTCGCCTGACTGATGCGCTG 
               
               
                   
                 GTCCTCGCGCCAGCTTAATACGCTAATCCCTAACTGCTGG 
               
               
                   
                 CGGAACAAATGCGACAGACGCGACGGCGACAGGCAGACA 
               
               
                   
                 TGCTGTGCGACGCTGGCGATATCAAAATTACTGTCTGCCA 
               
               
                   
                 GGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATT 
               
               
                   
                 ATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATG 
               
               
                   
                 CGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAGCA 
               
               
                   
                 ATTCCGAATAGCGCCCTTCCCCTTGTCCGGCATTAATGATT 
               
               
                   
                 TGCCCAAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCAT 
               
               
                   
                 CCGGGCGAAAGAAACCGGTATTGGCAAATATCGACGGCC 
               
               
                   
                 AGTTAAGCCATTCATGCCAGTAGGCGCGCGGACGAAAGT 
               
               
                   
                 AAACCCACTGGTGATACCATTCGTGAGCCTCCGGATGACG 
               
               
                   
                 ACCGTAGTGATGAATCTCTCCAGGCGGGAACAGCAAAAT 
               
               
                   
                 ATCACCCGGTCGGCAGACAAATTCTCGTCCCTGATTTTTCA 
               
               
                   
                 CCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACC 
               
               
                   
                 TTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGAGATAA 
               
               
                   
                 CCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGG 
               
               
                   
                 CGTTAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGC 
               
               
                   
                 TTCAGCCATACTTTTCATACTCCCGCCATTCAGAGAAGAA 
               
               
                   
                 ACCAATTGTCCATATTGCAT 
               
               
                   
               
               
                 AraC 
                 MQYGQLVSSLNGGSMKSMAEAQNDPLLPGYSFNAHLVAGL 
               
               
                 polypeptide 
                 TPIEANGYLDFFIDRPLGMKGYILNLTIRGQGVVKNQGREFV 
               
               
                 SEQ ID NO: 
                 CRPGDILLFPPGEIHHYGRHPEAHEWYHQWVYFRPRAYWHE 
               
               
                 587 
                 WLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYS 
               
               
                   
                 ELLAINLLEQLLLRRMEAINESLHPPMDNRVREACQYISDHL 
               
               
                   
                 ADSNFDIASVAQHVCLSPSRLSHLFRQQLGISVLSWREDQRIS 
               
               
                   
                 QAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSE 
               
               
                   
                 FRAGCE* 
               
               
                   
               
               
                 Region 
                 CGGTGAGCATCACATCACCACAATTCAGCAAATTGTGAAC 
               
               
                 comprising 
                 ATCATCACGTTCATCTTTCCCTGGTTGCCAATGGCCCATTT 
               
               
                 rhamnose 
                 TCCTGTCAGTAACGAGAAGGTCGCGAATCAGGCGCTTTTT 
               
               
                 inducible 
                 AGACTGGTCGTAATGAAATTCAGCTGTCACCGGATGTGCT 
               
               
                 promoter 
                 TTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTAC 
               
               
                 SEQ ID NO: 
                 AAATAATTTTGTTTAAAACAACACCCACTAAGATAACTCT 
               
               
                 588 
                 AGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT 
               
               
                   
               
               
                 Lac Promoter 
                 ATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATG 
               
               
                 region 
                 CCATACCGCGAAAGGTTTTGCGCCATTCGATGGCGCGCCG 
               
               
                 SEQ ID NO: 
                 CTTCGTCAGGCCACATAGCTTTCTTGTTCTGATCGGAACGA 
               
               
                 589 
                 TCGTTGGCTGTGTTGACAATTAATCATCGGCTCGTATAATG 
               
               
                   
                 TGTGGAATTGTGAGCGCTCACAATTAGCTGTCACCGGATG 
               
               
                   
                 TGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCT 
               
               
                   
                 CTACAAATAATTTTGTTTAAAACAACACCCACTAAGATAA 
               
               
                   
                 CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA 
               
               
                   
                 CAT 
               
               
                   
               
               
                 LacO 
                 GGAATTGTGAGCGCTCACAATT 
               
               
                   
               
               
                 LacI (in reverse 
                 TCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGC 
               
               
                 orientation) 
                 TGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTT 
               
               
                 SEQ ID NO: 
                 GCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGA 
               
               
                 590 
                 GACTGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGA 
               
               
                   
                 GAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCA 
               
               
                   
                 GGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATA 
               
               
                   
                 ACATGAGCTATCTTCGGTATCGTCGTATCCCACTACCGAG 
               
               
                   
                 ATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGC 
               
               
                   
                 GCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCAT 
               
               
                   
                 CGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTT 
               
               
                   
                 TGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTT 
               
               
                   
                 CCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATG 
               
               
                   
                 CCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAA 
               
               
                   
                 TGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCG 
               
               
                   
                 ACCAGATGCTCCACGCCCAGTCGCGTACCGTCCTCATGGG 
               
               
                   
                 AGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATC 
               
               
                   
                 AAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCAC 
               
               
                   
                 AGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATC 
               
               
                   
                 AGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCG 
               
               
                   
                 CTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACC 
               
               
                   
                 ACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCG 
               
               
                   
                 CCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGG 
               
               
                   
                 AGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAG 
               
               
                   
                 TTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCC 
               
               
                   
                 ATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTG 
               
               
                   
                 GCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAG 
               
               
                   
                 ACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTT 
               
               
                   
                 TCAT 
               
               
                   
               
               
                 LacI 
                 MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEA 
               
               
                 polypeptide 
                 AMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAA 
               
               
                 sequence 
                 IKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLI 
               
               
                 SEQ ID NO: 
                 INYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGT 
               
               
                 591 
                 RLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRN 
               
               
                   
                 QIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQ 
               
               
                   
                 MALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIK 
               
               
                   
                 QDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLA 
               
               
                   
                 PNTQTASPRALADSLMQLARQVSRLESGQ 
               
               
                   
               
               
                 Region 
                 ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACC 
               
               
                 comprising 
                 GTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTG 
               
               
                 Temperature 
                 CATAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCC 
               
               
                 sensitive 
                 GTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAAA 
               
               
                 promoter 
                 CAACACCCACTAAGATAACTCTAGAAATAATTTTGTTTAA 
               
               
                 SEQ ID NO: 
                 CTTTAAGAAGGAGATATACAT 
               
               
                 592 
                   
               
               
                   
               
               
                 mutant cI857 
                 TCAGCCAAACGTCTCTTCAGGCCACTGACTAGCGATAACT 
               
               
                 repressor 
                 TTCCCCACAACGGAACAACTCTCATTGCATGGGATCATTG 
               
               
                 SEQ ID NO: 
                 GGTACTGTGGGTTTAGTGGTTGTAAAAACACCTGACCGCT 
               
               
                 593 
                 ATCCCTGATCAGTTTCTTGAAGGTAAACTCATCACCCCCA 
               
               
                   
                 AGTCTGGCTATGCAGAAATCACCTGGCTCAACAGCCTGCT 
               
               
                   
                 CAGGGTCAACGAGAATTAACATTCCGTCAGGAAAGCTTGG 
               
               
                   
                 CTTGGAGCCTGTTGGTGCGGTCATGGAATTACCTTCAACC 
               
               
                   
                 TCAAGCCAGAATGCAGAATCACTGGCTTTTTTGGTTGTGC 
               
               
                   
                 TTACCCATCTCTCCGCATCACCTTTGGTAAAGGTTCTAAGC 
               
               
                   
                 TTAGGTGAGAACATCCCTGCCTGAACATGAGAAAAAACA 
               
               
                   
                 GGGTACTCATACTCACTTCTAAGTGACGGCTGCATACTAA 
               
               
                   
                 CCGCTTCATACATCTCGTAGATTTCTCTGGCGATTGAAGG 
               
               
                   
                 GCTAAATTCTTCAACGCTAACTTTGAGAATTTTTGTAAGCA 
               
               
                   
                 ATGCGGCGTTATAAGCATTTAATGCATTGATGCCATTAAA 
               
               
                   
                 TAAAGCACCAACGCCTGACTGCCCCATCCCCATCTTGTCT 
               
               
                   
                 GCGACAGATTCCTGGGATAAGCCAAGTTCATTTTTCTTTTT 
               
               
                   
                 TTCATAAATTGCTTTAAGGCGACGTGCGTCCTCAAGCTGC 
               
               
                   
                 TCTTGTGTTAATGGTTTCTTTTTTGTGCTCAT 
               
               
                   
               
               
                 RBS and leader 
                 CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA 
               
               
                 region 
                 CAT 
               
               
                 SEQ ID NO: 
                   
               
               
                 594 
                   
               
               
                   
               
               
                 mutant cI857 
                 MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKM 
               
               
                 repressor 
                 GMGQSGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAR 
               
               
                 polypeptide 
                 EIYEMYEAVSMQPSLRSEYEYPVFSHVQAGMFSPKLRTFTKG 
               
               
                 sequence 
                 DAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPDGML 
               
               
                 SEQ ID NO: 
                 ILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNP 
               
               
                 595 
                 QYPMIPCNESCSVVGKVIASQWPEETFG 
               
               
                   
               
               
                 TetR-Tet 
                 
                   Ttaagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaaggccgaataa 
                 
               
               
                 promoter 
                 
                   gaaggctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcata 
                 
               
               
                 construct 
                 
                   ctatcagtagtaggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaacct 
                 
               
               
                 SEQ ID NO: 
                 
                   aaagtaaaatgccccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttgg 
                 
               
               
                 596 
                 
                   cataaaaaggctaattgattttcgagagtttcatactttttctgtaggccgtgtacctaaatgta 
                 
               
               
                   
                 
                   cttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaaaaaat 
                 
               
               
                   
                 
                   cttgccagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggct 
                 
               
               
                   
                 
                   aaggcgtcgagcaaagcccgcttattttttacatgccaatacaatgtaggctgctctacaccta 
                 
               
               
                   
                 
                   gcttctgggcgagtttacgggttgttaaaccttcgattccgacctcattaagcagctctaatgcg 
                 
               
               
                   
                 
                   atgttaatcactttacttttatctaatctagacatcattaattcctaattttt 
                   gttgacactctatcattg 
                 
               
               
                   
                 
                   atagagttattttaccactccctatcagtgatagagaaaagtgaa 
                   
                     ctctagaaataatttt 
                     gttt 
                   
                 
               
               
                   
                 
                   
                     aactttaagaaggagatatacat 
                   
                 
               
               
                   
               
               
                 PssB promoter 
                 tcacctttcccggattaaacgcttttttgcccggtggcatggtgctaccggcgatcacaaacggtta 
               
               
                 SEQ ID NO: 
                 attatgacacaaattgacctgaatgaatatacagtattggaatgcattacccggagtgttgtgtaac 
               
               
                 597 
                 aatgtctggccaggtttgtttcccggaaccgaggtcacaacatagtaaaagcgctattggtaatgg 
               
               
                   
                 tacaatcgcgcgtttacacttattc 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the anti-inflammation and/or gut barrier enhancer molecule is butyrate. Methods of measuring butyrate levels, e.g., by mass spectrometry, gas chromatography, high-performance liquid chromatography (HPLC), are known in the art (see, e.g., Aboulnaga et al., 2013). In some embodiments, butyrate is measured as butyrate level/bacteria optical density (OD). In some embodiments, measuring the activity and/or expression of one or more gene products in the butyrogenic gene cassette serves as a proxy measurement for butyrate production. In some embodiments, the bacterial cells of the invention are harvested and lysed to measure butyrate production. In alternate embodiments, butyrate production is measured in the bacterial cell medium. In some embodiments, the genetically engineered bacteria produce at least about 1 nM/OD, at least about 10 nM/OD, at least about 100 nM/OD, at least about 500 nM/OD, at least about 1 μM/OD, at least about 10 μM/OD, at least about 100 μM/OD, at least about 500 μM/OD, at least about 1 mM/OD, at least about 2 mM/OD, at least about 3 mM/OD, at least about 5 mM/OD, at least about 10 mM/OD, at least about 20 mM/OD, at least about 30 mM/OD, or at least about 50 mM/OD of butyrate in the presence of ROS. 
     Constitutive Promoters 
     In some embodiments, the gene encoding the payload is present on a plasmid and operably linked to a constitutive promoter. In some embodiments, the gene encoding the payload is present on a chromosome and operably linked to a constitutive promoter. 
     In some embodiments, the constitutive promoter is active under in vivo conditions, e.g., the gut, as described herein. In some embodiments, the promoters is active under in vitro conditions, e.g., various cell culture and/or cell manufacturing conditions, as described herein. In some embodiments, the constitutive promoter is active under in vivo conditions, e.g., the gut, as described herein, and under in vitro conditions, e.g., various cell culture and/or cell production and/or manufacturing conditions, as described herein. 
     In some embodiments, the constitutive promoter that is operably linked to the gene encoding the payload is active in various exogenous environmental conditions (e.g., in vivo and/or in vitro and/or production/manufacturing conditions). 
     In some embodiments, the constitutive promoter is active in exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the constitutive promoter is active in exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the constitutive promoter is active in low-oxygen or anaerobic conditions such as the environment of the mammalian gut. In some embodiments, the constitutive promoter is active in the presence of molecules or metabolites that are specific to the gut of a mammal. In some embodiments, the constitutive promoter is directly or indirectly induced by a molecule that is co-administered with the bacterial cell. In some embodiments, the constitutive promoter is active in the presence of molecules or metabolites or other conditions, that are present during in vitro culture, cell production and/or manufacturing conditions. 
     Bacterial constitutive promoters are known in the art. Examplary constitutive promoters are listed in the following Tables. 
     
       
         
           
               
             
               
                 TABLE 28A 
               
             
            
               
                   
               
               
                 Constitutive E. coli σ70 promoters 
               
            
           
           
               
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
                 Length 
               
               
                   
               
               
                 BBa_I14018 
                 P(Bla) 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                   
                 gtttatacataggcgagtactctgttatgg 
                   
               
               
                 598 
                   
                   
                   
               
               
                   
               
               
                 BBa_I14033 
                 P(Cat) 
                 . . . 
                 38 
               
               
                 SEQ ID NO: 
                   
                 agaggttccaactttcaccataatgaaaca 
                   
               
               
                 599 
                   
                   
                   
               
               
                   
               
               
                 BBa_I14034 
                 P(Kat) 
                 . . . 
                 45 
               
               
                 SEQ ID NO: 
                   
                 taaacaactaacggacaattctacctaaca 
                   
               
               
                 600 
                   
                   
                   
               
               
                   
               
               
                 BBa_I732021 
                 Template for Building 
                 . . . 
                 159 
               
               
                 SEQ ID NO: 
                 Primer Family Member 
                 acatcaagccaaattaaacaggattaacac 
                   
               
               
                 601 
                   
                   
                   
               
               
                   
               
               
                 BBa_I742126 
                 Reverse lambda cI- 
                 . . . 
                 49 
               
               
                   
                 regulated promoter 
                 gaggtaaaatagtcaacacgcacggtgtta 
                   
               
               
                 SEQ ID NO: 
                   
                   
                   
               
               
                 602 
                   
                   
                   
               
               
                   
               
               
                 BBa_J01006 
                 Key Promoter absorbs 3 
                 . . . 
                 59 
               
               
                 SEQ ID NO: 
                   
                 caggccggaataactccctataatgcgcca 
                   
               
               
                 603 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23100 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 ggctagctcagtcctaggtacagtgctagc 
                   
               
               
                 604 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23101 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 agctagctcagtcctaggtattatgctagc 
                   
               
               
                 605 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23102 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 agctagctcagtcctaggtactgtgctagc 
                   
               
               
                 606 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23103 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 agctagctcagtcctagggattatgctagc 
                   
               
               
                 607 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23104 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 agctagctcagtcctaggtattgtgctagc 
                   
               
               
                 608 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23105 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 ggctagctcagtectaggtactatgctagc 
                   
               
               
                 609 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23106 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 ggctagctcagtcctaggtactatgctagc 
                   
               
               
                 610 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23107 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 ggctagctcagccctaggtattatgctagc 
                   
               
               
                 611 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23108 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 agctagctcagtcctaggtataatgctagc 
                   
               
               
                 612 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23109 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 agctagctcagtcctagggactgtgctagc 
                   
               
               
                 613 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23110 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 ggctagctcagtcctaggtacaatgctagc 
                   
               
               
                 614 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23111 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 ggctagctcagtcctaggtatagtgctagc 
                   
               
               
                 615 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23112 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 agctagctcagtcctagggattatgctagc 
                   
               
               
                 616 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23113 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 ggctagctcagtcctagggattatgctagc 
                   
               
               
                 617 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23114 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 ggctagctcagtcctaggtacaatgctagc 
                   
               
               
                 618 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23115 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 agctagctcagcccttggtacaatgctagc 
                   
               
               
                 619 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23116 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 agctagctcagtcctagggactatgctagc 
                   
               
               
                 620 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23117 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 agctagctcagtcctagggattgtgctagc 
                   
               
               
                 621 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23118 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 ggctagctcagtcctaggtattgtgctagc 
                   
               
               
                 622 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23119 
                 constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 family member 
                 agctagctcagtcctaggtataatgctagc 
                   
               
               
                 623 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23150 
                 l bp mutant from J23107 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                   
                 ggctagctcagtcctaggtattatgctagc 
                   
               
               
                 624 
                   
                   
                   
               
               
                   
               
               
                 BBa_J23151 
                 l bp mutant from J23114 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                   
                 ggctagctcagtcctaggtacaatgctagc 
                   
               
               
                 625 
                   
                   
                   
               
               
                   
               
               
                 BBa_J44002 
                 pBAD reverse 
                 . . . 
                 130 
               
               
                 SEQ ID NO: 
                   
                 aaagtgtgacgccgtgcaaataatcaatgt 
                   
               
               
                 626 
                   
                   
                   
               
               
                   
               
               
                 BBa_J48104 
                 NikR promoter, a protein 
                 . . . 
                 40 
               
               
                 SEQ ID NO: 
                 of the ribbon helix-helix 
                 gacgaatacttaaaatcgtcatacttattt 
                   
               
               
                 627 
                 family of trancription 
                   
                   
               
               
                   
                 factors that repress expre 
                   
                   
               
               
                   
               
               
                 BBa_J54200 
                 lacq_Promoter 
                 . . . 
                 50 
               
               
                 SEQ ID NO: 
                   
                 aaacctttcgcggtatggcatgatagcgcc 
                   
               
               
                 628 
                   
                   
                   
               
               
                   
               
               
                 BBa_J56015 
                 lacIQ - promoter sequence 
                 . . . 
                 57 
               
               
                 SEQ ID NO: 
                   
                 tgatagcgcccggaagagagtcaattcagg 
                   
               
               
                 629 
                   
                   
                   
               
               
                   
               
               
                 BBa_J64951 
                 E. coli CreABCD 
                 . . . 
                 81 
               
               
                 SEQ ID NO: 
                 phosphate sensing operon 
                 ttatttaccgtgacgaactaattgctcgtg 
                   
               
               
                 630 
                 promoter 
                   
                   
               
               
                   
               
               
                 BBa_K088007 
                 GlnRS promoter 
                 . . . 
                 38 
               
               
                 SEQ ID NO: 
                   
                 catacgccgttatacgttgtttacgctttg 
                   
               
               
                 631 
                   
                   
                   
               
               
                   
               
               
                 BBa_K119000 
                 Constitutive weak 
                 . . . 
                 38 
               
               
                 SEQ ID NO: 
                 promoter of lacZ 
                 ttatgatccggctcgtatgttgtgtggac 
                   
               
               
                 632 
                   
                   
                   
               
               
                   
               
               
                 BBa_K119001 
                 Mutated LacZ promoter 
                 . . . 
                 38 
               
               
                 SEQ ID NO: 
                   
                 ttatgcttccggctcgtatggtgtgtggac 
                   
               
               
                 633 
                   
                   
                   
               
               
                   
               
               
                 BBa_K1330002 
                 Constitutive promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 (J23105) 
                 ggctagctcagtcctaggtactatgctagc 
                   
               
               
                 634 
                   
                   
                   
               
               
                   
               
               
                 BBa_K137029 
                 constitutive promoter with 
                 . . .  
                 39 
               
               
                 SEQ ID NO: 
                 (TA)10 between -10 and - 
                 atatatatatatatataatggaagcgtttt 
                   
               
               
                 635 
                 35 elements 
                   
                   
               
               
                   
               
               
                 BBa_K137030 
                 constitutive promoter with 
                 . . .  
                 37 
               
               
                 SEQ ID NO: 
                 (TA)9 between -10 and - 
                 atatatatatatatataatggaagcgtttt 
                   
               
               
                 636 
                 35 elements 
                   
                   
               
               
                   
               
               
                 BBa_K137031 
                 constitutive promoter with 
                 . . . 
                 62 
               
               
                 SEQ ID NO: 
                 (C)10 between -10 and - 
                 ccccgaaagcttaagaatataattgtaagc 
                   
               
               
                 637 
                 35 elements 
                   
                   
               
               
                   
               
               
                 BBa_K137032 
                 constitutive promoter with 
                 . . . 
                 64 
               
               
                 SEQ ID NO: 
                 (C)12 between -10 and - 
                 ccccgaaagcttaagaatataattgtaagc 
                   
               
               
                 638 
                 35 elements 
                   
                   
               
               
                   
               
               
                 BBa_K137085 
                 optimized (TA) repeat 
                 . . . 
                 31 
               
               
                 SEQ ID NO: 
                 constitutive promoter with 
                 tgacaatatatatatatatataatgctagc 
                   
               
               
                 639 
                 13 bp between -10 and - 
                   
                   
               
               
                   
                 35 elements 
                   
                   
               
               
                   
               
               
                 BBa_K137086 
                 optimized (TA) repeat 
                 . . . 
                 33 
               
               
                 SEQ ID NO: 
                 constitutive promoter with 
                 acaatatatatatatatatataatgctagc 
                   
               
               
                 640 
                 15 bp between -10 and - 
                   
                   
               
               
                   
                 35 elements 
                   
                   
               
               
                   
               
               
                 BBa_K137087 
                 optimized (TA) repeat 
                 . . .  
                 35 
               
               
                 SEQ ID NO: 
                 constitutive promoter with 
                 aatatatatatatatatatataatgctagc 
                   
               
               
                 641 
                 17 bp between -10 and - 
                   
                   
               
               
                   
                 35 elements 
                   
                   
               
               
                   
               
               
                 BBa_K137088 
                 optimized (TA) repeat 
                 . . .  
                 37 
               
               
                 SEQ ID NO: 
                 constitutive promoter with 
                 tatatatatatatatatatataatgctagc 
                   
               
               
                 642 
                 19 bp between -10 and - 
                   
                   
               
               
                   
                 35 elements 
                   
                   
               
               
                   
               
               
                 BBa_K137089 
                 optimized (TA) repeat 
                 . . .  
                 39 
               
               
                 SEQ ID NO: 
                 constitutive promoter with 
                 tatatatatatatatatatataatgctagc 
                   
               
               
                 643 
                 21 bp between -10 and - 
                   
                   
               
               
                   
                 35 elements 
                   
                   
               
               
                   
               
               
                 BBa_K137090 
                 optimized (A) repeat 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 
                 constitutive promoter with 
                 aaaaaaaaaaaaaaaaaatataatgctagc 
                   
               
               
                 644 
                 17 bp between -10 and - 
                   
                   
               
               
                   
                 35 elements 
                   
                   
               
               
                   
               
               
                 BBa_K137091 
                 optimized (A) repeat 
                 . . . 
                 36 
               
               
                 SEQ ID NO: 
                 constitutive promoter with 
                 aaaaaaaaaaaaaaaaaatataatgctagc 
                   
               
               
                 645 
                 18 bp between -10 and - 
                   
                   
               
               
                   
                 35 elements 
                   
                   
               
               
                   
               
               
                 BBa_K1585100 
                 Anderson Promoter with 
                 . . . 
                 78 
               
            
           
           
               
               
               
               
               
            
               
                 SEQ ID NO: 
                 lacI binding site 
                 ggaattgtgagcggataacaatttcacaca 
                   
                   
               
               
                 646 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K1585101 
                 Anderson Promoter with 
                 . . . 
                 78 
                   
               
               
                 SEQ ID NO: 
                 lacI binding site 
                 ggaattgtgagcggataacaatttcacaca 
                   
                   
               
               
                 647 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K1585102 
                 Anderson Promoter with 
                 . . . 
                 78 
                   
               
               
                 SEQ ID NO: 
                 lacI binding site 
                 ggaattgtgagcggataacaatttcacaca 
                   
                   
               
               
                 648 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K1585103 
                 Anderson Promoter with 
                 . . . 
                 78 
                   
               
               
                 SEQ ID NO: 
                 lacI binding site 
                 ggaattgtgagcggataacaatttcacaca 
                   
                   
               
               
                 649 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K1585104 
                 Anderson Promoter with 
                 . . . 
                 78 
                   
               
               
                 SEQ ID NO: 
                 lacI binding site 
                 ggaattgtgagcggataacaatttcacaca 
                   
                   
               
               
                 650 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K1585105 
                 Anderson Promoter with 
                 . . . 
                 78 
                   
               
               
                 SEQ ID NO: 
                 lacI binding site 
                 ggaattgtgagcggataacaatttcacaca 
                   
                   
               
               
                 651 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K1585106 
                 Anderson Promoter with 
                 . . . 
                 78 
                   
               
               
                 SEQ ID NO: 
                 lacI binding site 
                 ggaattgtgagcggataacaatttcacaca 
                   
                   
               
               
                 652 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K1585110 
                 Anderson Promoter with 
                 . . . 
                 78 
                   
               
               
                 SEQ ID NO: 
                 lacI binding site 
                 ggaattgtgagcggataacaatttcacaca 
                   
                   
               
               
                 653 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K1585113 
                 Anderson Promoter with 
                 . . . 
                 78 
                   
               
               
                 SEQ ID NO: 
                 lacI binding site 
                 ggaattgtgagcggataacaatttcacaca 
                   
                   
               
               
                 654 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K1585115 
                 Anderson Promoter with 
                 . . . 
                 78 
                   
               
               
                 SEQ ID NO: 
                 lacI binding site 
                 ggaattgtgagcggataacaatttcacaca 
                   
                   
               
               
                 655 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K1585116 
                 Anderson Promoter with 
                 . . . 
                 78 
                   
               
               
                 SEQ ID NO: 
                 lacI binding site 
                 ggaattgtgagcggataacaatttcacaca 
                   
                   
               
               
                 656 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K1585117 
                 Anderson Promoter with 
                 . . . 
                 78 
                   
               
               
                 SEQ ID NO: 
                 lacI binding site 
                 ggaattgtgagcggataacaatttcacaca 
                   
                   
               
               
                 657 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K1585118 
                 Anderson Promoter with 
                 . . . 
                 78 
                   
               
               
                 SEQ ID NO: 
                 lacI binding site 
                 ggaattgtgagcggataacaatttcacaca 
                   
                   
               
               
                 658 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K1585119 
                 Anderson Promoter with 
                 . . . 
                 78 
                   
               
               
                 SEQ ID NO: 
                 lacI binding site 
                 ggaattgtgagcggataacaatttcacaca 
                   
                   
               
               
                 659 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K1824896 
                 J23100 + RBS 
                 . . . 
                 88 
                   
               
               
                 SEQ ID NO: 
                   
                 gattaaagaggagaaatactagagtactag 
                   
                   
               
               
                 660 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K256002 
                 J23101:GFP 
                 . . . 
                 918 
                   
               
               
                 SEQ ID NO: 
                   
                 caccttcgggtgggcctttctgcgtttata 
                   
                   
               
               
                 661 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K2560I8 
                 J23119:IFP 
                 . . . 
                 1167 
                   
               
               
                 SEQ ID NO: 
                   
                 caccttcgggtgggcctttctgcgtttata 
                   
                   
               
               
                 662 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K256020 
                 J23119:HOI 
                 . . . 
                 949 
                   
               
               
                 SEQ ID NO: 
                   
                 caccttcgggtgggcctttctgcgtttata 
                   
                   
               
               
                 663 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K256033 
                 Infrared signal reporter 
                 . . . 
                 2124 
                   
               
               
                 SEQ ID NO: 
                 (J23119:IFP:J23119:HOI) 
                 caccttcgggtgggcctttctgcgtttata 
                   
                   
               
               
                 664 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K292000 
                 Double terminator + 
                 . . . 
                 138 
                   
               
               
                 SEQ ID NO: 
                 constitutive promoter 
                 ggctagctcagtcctaggtacagtgctagc 
                   
                   
               
               
                 665 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K292001 
                 Double terminator + 
                 . . . 
                 161 
                   
               
               
                 SEQ ID NO: 
                 Constitutive promoter + 
                 tgctagctactagagattaaagaggagaaa 
                   
                   
               
               
                 666 
                 Strong RBS 
                   
                   
                   
               
               
                   
               
               
                 BBa_K418000 
                 IPTG inducible Lac 
                 . . . 
                 1416 
                   
               
               
                 SEQ ID NO: 
                 promoter cassette 
                 ttgtgagcggataacaagatactgagcaca 
                   
                   
               
               
                 667 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K418002 
                 IPTG inducible Lac 
                 . . . 
                 1414 
                   
               
               
                 SEQ ID NO: 
                 promoter cassette 
                 ttgtgagcggataacaagatactgagcaca 
                   
                   
               
               
                 668 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K418003 
                 IPTG inducible Lac 
                 . . . 
                 1416 
                   
               
               
                 SEQ ID NO: 
                 promoter cassette 
                 ttgtgagcggataacaagatactgagcaca 
                   
                   
               
               
                 669 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K823004 
                 Anderson promoter 
                 . . . 
                 35 
                   
               
               
                 SEQ ID NO: 
                 J23100 
                 ggctagctcagtcctaggtacagtgctagc 
                   
                   
               
               
                 670 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K823005 
                 Anderson promoter 
                 . . . 
                 35 
                   
               
               
                 SEQ ID NO: 
                 J23101 
                 agctagctcagtcctaggtattatgctagc 
                   
                   
               
               
                 671 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K823006 
                 Anderson promoter 
                 . . . 
                 35 
                   
               
               
                 SEQ ID NO: 
                 J23102 
                 agctagctcagtcctaggtactgtgctagc 
                   
                   
               
               
                 672 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K823007 
                 Anderson promoter 
                 . . . 
                 35 
                   
               
               
                 SEQ ID NO: 
                 J23103 
                 agctagctcagtcctagggattatgctagc 
                   
                   
               
               
                 673 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K823008 
                 Anderson promoter 
                 . . . 
                 35 
                   
               
               
                 SEQ ID NO: 
                 J23106 
                 ggctagctcagtcctaggtatagtgctagc 
                   
                   
               
               
                 674 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K823010 
                 Anderson promoter 
                 . . . 
                 35 
                   
               
               
                 SEQ ID NO: 
                 J23113 
                 ggctagctcagtcctagggattatgctagc 
                   
                   
               
               
                 675 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K823011 
                 Anderson promoter 
                 . . . 
                 35 
                   
               
               
                 SEQ ID NO: 
                 J23114 
                 ggctagctcagtcctaggtacaatgctagc 
                   
                   
               
               
                 676 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K823013 
                 Anderson promoter 
                 . . . 
                 35 
                   
               
               
                 SEQ ID NO: 
                 J23117 
                 agctagctcagtcctagggattgtgctagc 
                   
                   
               
               
                 677 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_K823014 
                 Anderson promoter 
                 . . . 
                 35 
                   
               
               
                 SEQ ID NO: 
                 J23118 
                 ggctagctcagtcctaggtattgtgctagc 
                   
                   
               
               
                 678 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_M13101 
                 M13K07 gene I promoter 
                 . . .  
                 47 
                   
               
               
                 SEQ ID NO: 
                   
                 cctgtttttatgttattctctctgtaaagg 
                   
                   
               
               
                 679 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_M13102 
                 M13K07 gene II promoter 
                 . . .  
                 48 
                   
               
               
                 SEQ ID NO: 
                   
                 aaatatttgcttatacaatcttcctgtttt 
                   
                   
               
               
                 680 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_M13103 
                 M13K07 gene III 
                 . . . 
                 48 
                   
               
               
                 SEQ ID NO: 
                 promoter 
                 gctgataaaccgatacaattaaaggctcct 
                   
                   
               
               
                 681 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_M13104 
                 M13K07 gene IV 
                 . . . 
                 49 
                   
               
               
                 SEQ ID NO: 
                 promoter 
                 ctcttctcagcgtcttaatctaagctatcg 
                   
                   
               
               
                 682 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_M13105 
                 M13K07 gene V promoter 
                 . . . 
                 50 
                   
               
               
                 SEQ ID NO: 
                   
                 atgagccagttcttaaaatcgcataaggta 
                   
                   
               
               
                 683 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_M13106 
                 M13K07 gene VI 
                 . . . 
                 49 
                   
               
               
                 SEQ ID NO: 
                 promoter 
                 ctattgattgtgacaaaataaacttattcc 
                   
                   
               
               
                 684 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_M13108 
                 M13K07 gene VIII 
                 . . . 
                 47 
                   
               
               
                 SEQ ID NO: 
                 promoter 
                 gtttcgcgcttggtataatcgctgggggtc 
                   
                   
               
               
                 685 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_M13110 
                 M13110 
                 . . . 
                 48 
                   
               
               
                 SEQ ID NO: 
                   
                 ctttgcttctgactataatagtcagggtaa 
                   
                   
               
               
                 686 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_M31519 
                 Modified promoter 
                 . . . 
                 60 
                   
               
               
                 SEQ ID NO: 
                 sequence of g3. 
                 aaaccgatacaattaaaggctcctgctagc 
                   
                   
               
               
                 687 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_R1074 
                 Constitutive Promoter I 
                 . . . 
                 74 
                   
               
               
                 SEQ ID NO: 
                   
                 caccacactgatagtgctagtgtagatcac 
                   
                   
               
               
                 688 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_R1075 
                 Constitutive Promoter II 
                 . . . 
                 49 
                   
               
               
                 SEQ ID NO: 
                   
                 gccggaataactccctataatgcgccacca 
                   
                   
               
               
                 689 
                   
                   
                   
                   
               
               
                   
               
               
                 BBa_S03331 
                 --Specify Parts List-- 
                 ttgacaagcttttcctcagctccgtaaact 
                   
                   
               
               
                 SEQ ID NO: 
                   
                   
                   
                   
               
               
                 690 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 28B 
               
             
            
               
                   
               
               
                 Constitutive E. coli σ S  promoters 
               
            
           
           
               
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
                 Length 
               
               
                   
               
               
                 BBa_J45992 
                 Full-length stationary phase 
                 . . .  
                 199 
               
               
                 SEQ ID NO: 
                 osmY promoter 
                 ggtttcaaaattgtgatctatatttaacaa 
                   
               
               
                 691 
                   
                   
                   
               
               
                   
               
               
                 BBa_J45993 
                 Minimal stationary phase 
                 . . .  
                 57 
               
               
                 SEQ ID NO: 
                 osmY promoter 
                 ggtttcaaaattgtgatctatatttaacaa 
                   
               
               
                 692 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 28C 
               
             
            
               
                   
               
               
                 Constitutive E. coli σ 32  promoters 
               
            
           
           
               
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
                 Length 
               
               
                   
               
               
                 BBa_J45504 
                 htpG Heat Shock Promoter 
                 . . .  
                 405 
               
               
                 SEQ ID NO: 693 
                   
                 tctattccaataaagaaatcttcctgcgtg 
                   
               
               
                   
               
               
                 BBa_K1895002 
                 dnaK Promoter 
                 . . .  
                 182 
               
               
                 SEQ ID NO: 694 
                   
                 gaccgaatatatagtggaaacgtttagatg 
                   
               
               
                   
               
               
                 BBa_K1895003 
                 htpG Promoter 
                 . . .  
                 287 
               
               
                 SEQ ID NO: 695 
                   
                 ccacatcctgtttttaaccttaaaatggca 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 28D 
               
             
            
               
                   
               
               
                 Constitutive B. subtilis σ A  promoters 
               
            
           
           
               
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
                 Length 
               
               
                   
               
               
                 BBa_K143012 
                 Promoter veg a constitutive 
                 . . . 
                 97 
               
               
                 SEQ ID NO: 696 
                 promoter for  B. subtilis   
                 aaaaatgggctcgtgttgtacaataaatgt 
                   
               
               
                   
               
               
                 BBa_K143013 
                 Promoter 43 a constitutive 
                 . . . 
                 56 
               
               
                 SEQ ID NO: 697 
                 promoter for  B. subtilis   
                 aaaaaaagcgcgcgattatgtaaaatataa 
                   
               
               
                   
               
               
                 BBa_K780003 
                 Strong constitutive promoter 
                 . . . 
                 36 
               
               
                 SEQ ID NO: 698 
                 for Bacillus subtilis 
                 aattgcagtaggcatgacaaaatggactca 
                   
               
               
                   
               
               
                 BBa_K823000 
                 P liaG   
                 . . .  
                 121 
               
               
                 SEQ ID NO: 699 
                   
                 caagcttttcctttataatagaatgaatga 
                   
               
               
                   
               
               
                 BBa_K823002 
                 P lepA   
                 . . .  
                 157 
               
               
                 SEQ ID NO: 700 
                   
                 tctaagctagtgtattttgcgtttaatagt 
                   
               
               
                   
               
               
                 BBa_K823003 
                 P veg   
                 . . . 
                 237 
               
               
                 SEQ ID NO: 701 
                   
                 aatgggctcgtgttgtacaataaatgtagt 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 28E 
               
             
            
               
                   
               
               
                 Constitutive B. subtilis σ B  promoters 
               
            
           
           
               
               
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
                 Length 
                   
               
               
                   
               
               
                 BBa_K143010 
                 Promoter ctc for B. subtilis 
                 . . .  
                 56 
                   
               
               
                 SEQ ID NO: 702 
                   
                 atccttatcgttatgggtattgtttgtaat 
                   
                   
               
               
                   
               
               
                 BBa_K143011 
                 Promoter gsiB for B. 
                 . . . 
                 38 
                   
               
               
                 SEQ ID NO: 703 
                 subtilis 
                 taaaagaattgtgagcgggaatacaacaac 
                   
                   
               
               
                   
               
               
                 BBa_K143013 
                 Promoter 43 a constitutive 
                 . . . 
                 56 
                   
               
               
                 SEQ ID NO: 704 
                 promoter for B. subtilis 
                 aaaaaaagcgcgcgattatgtaaaatataa 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 28F 
               
             
            
               
                   
               
               
                 Constitutive promoters from miscellaneous prokaryotes 
               
            
           
           
               
               
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
                 Length 
                   
               
               
                   
               
               
                 BBa_K112706 
                 Pspv2 from Salmonella 
                 . . .  
                 474 
                   
               
               
                 SEQ ID NO: 705 
                   
                 tacaaaataattcccctgcaaacattatca 
                   
                   
               
               
                   
               
               
                 BBa_K112707 
                 Pspv from Salmonella 
                 . . .  
                 1956 
                   
               
               
                 SEQ ID NO: 706 
                   
                 tacaaaataattcccctgcaaacattatcg 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 28G 
               
             
            
               
                   
               
               
                 Constitutive promoters from bacteriophage T7 
               
            
           
           
               
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
                 Length 
               
               
                   
               
               
                 BBa_I712074 
                 T7 promoter (strong 
                 . . . 
                 46 
               
               
                 SEQ ID NO: 707 
                 promoter from T7 
                 agggaatacaagctacttgttctttttgca 
                   
               
               
                   
                 bacteriophage) 
                   
                   
               
               
                   
               
               
                 BBa_I719005 
                 T7 Promoter 
                 taatacgactcactatagggaga 
                 23 
               
               
                 SEQ ID NO: 708 
                   
                   
                   
               
               
                   
               
               
                 BBa_J34814 
                 T7 Promoter 
                 gaatttaatacgactcactatagggaga 
                 28 
               
               
                 SEQ ID NO: 709 
                   
                   
                   
               
               
                   
               
               
                 BBa_J64997 
                 T7 consensus -10 and 
                 taatacgactcactatagg 
                 19 
               
               
                 SEQ ID NO: 710 
                 rest 
                   
                   
               
               
                   
               
               
                 BBa_K113010 
                 overlapping T7 
                 . . . 
                 40 
               
               
                 SEQ ID NO: 711 
                 promoter 
                 gagtcgtattaatacgactcactatagggg 
                   
               
               
                   
               
               
                 BBa_K113011 
                 more overlapping T7 
                 . . . 
                 37 
               
               
                 SEQ ID NO: 712 
                 promoter 
                 agtgagtcgtactacgactcactatagggg 
                   
               
               
                   
               
               
                 BBa_K113012 
                 weaken overlapping T7 
                 . . . 
                 40 
               
               
                 SEQ ID NO: 713 
                 promoter 
                 gagtcgtattaatacgactctctatagggg 
                   
               
               
                   
               
               
                 BBa_K1614000 
                 T7 promoter for 
                 taatacgactcactatag 
                 18 
               
               
                 SEQ ID NO: 714 
                 expression of functional 
                   
                   
               
               
                   
                 RNA 
                   
                   
               
               
                   
               
               
                 BBa_R0085 
                 T7 Consensus Promoter 
                 taatacgactcactatagggaga 
                 23 
               
               
                 SEQ ID NO: 715 
                 Sequence 
                   
                   
               
               
                   
               
               
                 BBa_R0180 
                 T7 RNAP promoter 
                 ttatacgactcactatagggaga 
                 23 
               
               
                 SEQ ID NO: 716 
                   
                   
                   
               
               
                   
               
               
                 BBa_R0181 
                 T7 RNAP promoter 
                 gaatacgactcactatagggaga 
                 23 
               
               
                 SEQ ID NO: 717 
                   
                   
                   
               
               
                   
               
               
                 BBa_R0182 
                 T7 RNAP promoter 
                 taatacgtctcactatagggaga 
                 23 
               
               
                 SEQ ID NO: 718 
                   
                   
                   
               
               
                   
               
               
                 BBa_R0183 
                 T7 RNAP promoter 
                 tcatacgactcactatagggaga 
                 23 
               
               
                 SEQ ID NO: 719 
                   
                   
                   
               
               
                   
               
               
                 BBa_Z0251 
                 T7 strong promoter 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 720 
                   
                 taatacgactcactatagggagaccacaac 
                   
               
               
                   
               
               
                 BBa_Z0252 
                 T7 weak binding and 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 721 
                 processivity 
                 taattgaactcactaaagggagaccacagc 
                   
               
               
                   
               
               
                 BBa_Z0253 
                 T7 weak binding 
                 . . . 
                 35 
               
               
                 SEQ ID NO: 722 
                 promoter 
                 cgaagtaatacgactcactattagggaaga 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 28H 
               
             
            
               
                   
               
               
                 Constitutive promoters from bacteriophage SP6 
               
            
           
           
               
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
                 Length 
               
               
                   
               
               
                 BBa_J64998 
                 consensus -10 and rest from SP6 
                 atttaggtgacactataga 
                 19 
               
               
                 SEQ ID NO: 723 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 28I 
               
             
            
               
                   
               
               
                 Constitutive promoters from yeast 
               
            
           
           
               
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
                 Length 
               
               
                   
               
               
                 BBa_I766555 
                 pCyc (Medium) Promoter 
                 . . . 
                 244 
               
               
                 SEQ ID NO: 724 
                   
                 acaaacacaaatacacacactaaattaata 
                   
               
               
                   
               
               
                 BBa_I766556 
                 pAdh (Strong) Promoter 
                 . . . 
                 1501 
               
               
                 SEQ ID NO: 725 
                   
                 ccaagcatacaatcaactatctcatataca 
                   
               
               
                   
               
               
                 BBa_I766557 
                 pSte5 (Weak) Promoter 
                 . . . 
                 601 
               
               
                 SEQ ID NO: 726 
                   
                 gatacaggatacagcggaaacaacttttaa 
                   
               
               
                   
               
               
                 BBa_J63005 
                 yeast ADH1 promoter 
                 . . . 
                 1445 
               
               
                 SEQ ID NO: 727 
                   
                 tttcaagctataccaagcatacaatcaact 
                   
               
               
                   
               
               
                 BBa_K105027 
                 cycl00 minimal promoter 
                 . . .  
                 103 
               
               
                 SEQ ID NO: 728 
                   
                 cctttgcagcataaattactatacttctat 
                   
               
               
                   
               
               
                 BBa_K105028 
                 cyc70 minimal promoter 
                 . . .  
                 103 
               
               
                 SEQ ID NO: 729 
                   
                 cctttgcagcataaattactatacttctat 
                   
               
               
                   
               
               
                 BBa_K105029 
                 cyc43 minimal promoter 
                 . . .  
                 103 
               
               
                 SEQ ID NO: 730 
                   
                 cctttgcagcataaattactatacttctat 
                   
               
               
                   
               
               
                 BBa_K105030 
                 cyc28 minimal promoter 
                 . . .  
                 103 
               
               
                 SEQ ID NO: 731 
                   
                 cctttgcagcataaattactatacttctat 
                   
               
               
                   
               
               
                 BBa_K105031 
                 cyc16 minimal promoter 
                 . . .  
                 103 
               
               
                 SEQ ID NO: 732 
                   
                 cctttgcagcataaattactatacttctat 
                   
               
               
                   
               
               
                 BBa_K122000 
                 pPGK1 
                 . . .  
                 1497 
               
               
                 SEQ ID NO: 733 
                   
                 ttatctactttttacaacaaatataaaaca 
                   
               
               
                   
               
               
                 BBa_K124000 
                 pCYC Yeast Promoter 
                 . . . 
                 288 
               
               
                 SEQ ID NO: 734 
                   
                 acaaacacaaatacacacactaaattaata 
                   
               
               
                   
               
               
                 BBa_K124002 
                 Yeast GPD (TDH3) 
                 . . . 
                 681 
               
               
                 SEQ ID NO: 735 
                 Promoter 
                 gtttcgaataaacacacataaacaaacaaa 
                   
               
               
                   
               
               
                 BBa_K319005 
                 yeast mid-length ADH1 
                 . . . 
                 720 
               
               
                 SEQ ID NO: 736 
                 promoter 
                 ccaagcatacaatcaactatctcatataca 
                   
               
               
                   
               
               
                 BBa_M31201 
                 Yeast CLB1 promoter 
                 . . . 
                 500 
               
               
                 SEQ ID NO: 737 
                 region, G2/M cell cycle 
                 accatcaaaggaagctttaatcttctcata 
                   
               
               
                   
                 specific 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 28J 
               
             
            
               
                   
               
               
                 Constitutive promoters from miscellaneous eukaryotes 
               
            
           
           
               
               
               
               
            
               
                 Name 
                 Description 
                 Promoter Sequence 
                 Length 
               
               
                   
               
               
                 BBa_I712004 
                 CMV promoter 
                 . . .  
                 654 
               
               
                 SEQ ID NO: 738 
                   
                 agaacccactgcttactggcttatcgaaat 
                   
               
               
                   
               
               
                 BBa_K076017 
                 Ubc Promoter 
                 . . .  
                 1219 
               
               
                 SEQ ID NO: 739 
                   
                 ggccgtttttggcttttttgttagacgaag 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 28K 
               
             
            
               
                   
               
               
                 Promoters 
               
            
           
           
               
               
               
            
               
                 Name 
                 Sequence 
                 Description 
               
               
                   
               
               
                 Plpp 
                 ataagtgccttcccatcaaaaaaatattctc 
                 The Plpp promoter is a natural promoter 
               
               
                 SEQ ID 
                 aacataaaaaactttgtgtaatacttgtaac 
                 taken from the Nissle genome. In situ it is 
               
               
                 NO: 740 
                 gcta 
                 used to drive production of lpp, which is 
               
               
                   
                   
                 known to be the most abundant protein in the 
               
               
                   
                   
                 cell. Also, in some previous RNAseq 
               
               
                   
                   
                 experiments I was able to confirm that the 
               
               
                   
                   
                 lpp mRNA is one of the most abundant 
               
               
                   
                   
                 mRNA in Nissle during exponential growth. 
               
               
                   
               
               
                 PapFAB46 
                 AAAAAGAGTATTGACTTC 
                 See, e.g., Kosuri, S., Goodman, D. B. &amp; 
               
               
                 SEQ ID 
                 GCATCTTTTTGTACCTATA 
                 Cambray, G. Composability of regulatory 
               
               
                 NO: 741 
                 ATAGATTCATTGCTA 
                 sequences controlling transcription and 
               
               
                   
                   
                 translation in Escherichia coli. in 1-20 
               
               
                   
                   
                 (2013). doi:10.1073/pnas. 
               
               
                   
               
               
                 PJ23101+ 
                 ggaaaatttttttaaaaaaaaaactttacag 
                 UP element helps recruit RNA polymerase 
               
               
                 UP element 
                 ctagctcagtcctaggtattatgctagc 
                 (ggaaaatttttttaaaaaaaaaac) 
               
               
                 SEQ ID 
                   
                   
               
               
                 NO: 742 
                   
                   
               
               
                   
               
               
                 PJ23107+ 
                 ggaaaatttttttaaaaaaaaaactttacgg 
                 UP element helps recruit RNA polymerase 
               
               
                 UP element 
                 ctagctcagccctaggtattatgctagc 
                 (ggaaaatttttttaaaaaaaaaac) 
               
               
                 SEQ ID 
                   
                   
               
               
                 NO: 743 
                   
                   
               
               
                   
               
               
                 PSYN2311 
                 ggaaaatttttttaaaaaaaaaacTTGA 
                 UP element at 5&#39; end; consensus -10 region 
               
               
                 9 
                 CAGCTAGCTCAGTCCTTG 
                 is TATAAT; the consensus -35 is TTGACA; 
               
               
                 SEQ ID 
                 GTATAATGCTAGCACGAA 
                 the extended -10 region is generally 
               
               
                 NO: 744 
                   
                 TGNTATAAT (TGGTATAAT in this 
               
               
                   
                   
                 sequence) 
               
               
                   
               
            
           
         
       
     
     Bacterial constitutive promoters are known in the art. Examplary constitutive promoters are listed in the following Tables. In some embodiments, the constitutive promoter is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the sequence of any one of SEQ ID NOs: 598-744. 
     Ribosome Binding Sites 
     In some embodiments, ribosome binding sites are added, switched out or replaced. By testing a few ribosome binding sites, expression levels can be fine-tuned to the desired level. Table A and Table B lists a number RBS which are suitable for prokaryotic expression and can be used to achieve the desired expression levels (See, e.g., Registry of standard biological parts). 
     
       
         
           
               
             
               
                 TABLE 29A 
               
             
            
               
                   
               
               
                 Selected Ribosome Binding Sites 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 Identifier 
                 Sequence a   
                 NO 
               
               
                   
               
               
                 Master Sequence 
                 TCTAGAGAAAGANNNGANNNACTAGATG 
                 1018 
               
               
                   
               
               
                 BBa_J61100 
                 TCTAGAGAAAGAGGGGACAAACTAGATG 
                 1019 
               
               
                   
               
               
                 BBa_J61101 
                 TCTAGAGAAAGACAGGACCCACTAGATG 
                 1020 
               
               
                   
               
               
                 BBa_J61102 
                 TCTAGAGAAAGATCCGATGTACTAGATG 
                 1021 
               
               
                   
               
               
                 BBa_J61103 
                 TCTAGAGAAAGATTAGACAAACTAGATG 
                 1022 
               
               
                   
               
               
                 BBa_J61104 
                 TCTAGAGAAAGAAGGGACAGACTAGATG 
                 1023 
               
               
                   
               
               
                 BBa_J61105 
                 TCTAGAGAAAGACATGACGTACTAGATG 
                 1024 
               
               
                   
               
               
                 BBa_J61106 
                 TCTAGAGAAAGATAGGAGACACTAGATG 
                 1025 
               
               
                   
               
               
                 BBa_J61107 
                 TCTAGAGAAAGAAGAGACTCACTAGATG 
                 1026 
               
               
                   
               
               
                 BBa_J61108 
                 TCTAGAGAAAGACGAGATATACTAGATG 
                 1027 
               
               
                   
               
               
                 BBa_J61109 
                 TCTAGAGAAAGACTGGAGACACTAGATG 
                 1028 
               
               
                   
               
               
                 BBa_J61110 
                 TCTAGAGAAAGAGGCGAATTACTAGATG 
                 1029 
               
               
                   
               
               
                 BBa_J61111 
                 TCTAGAGAAAGAGGCGATACACTAGATG 
                 1030 
               
               
                   
               
               
                 BBa_J61112 
                 TCTAGAGAAAGAGGTGACATACTAGATG 
                 1031 
               
               
                   
               
               
                 BBa_J61113 
                 TCTAGAGAAAGAGTGGAAAAACTAGATG 
                 1032 
               
               
                   
               
               
                 BBa_J61114 
                 TCTAGAGAAAGATGAGAAGAACTAGATG 
                 1033 
               
               
                   
               
               
                 BBa_J61115 
                 TCTAGAGAAAGAAGGGATACACTAGATG 
                 1034 
               
               
                   
               
               
                 BBa_J61116 
                 TCTAGAGAAAGACATGAGGCACTAGATG 
                 1035 
               
               
                   
               
               
                 BBa_J61117 
                 TCTAGAGAAAGACATGAGTTACTAGATG 
                 1036 
               
               
                   
               
               
                 BBa_J61118 
                 TCTAGAGAAAGAGACGAATCACTAGATG 
                 1037 
               
               
                   
               
               
                 BBa_J61119 
                 TCTAGAGAAAGATTTGATATACTAGATG 
                 1038 
               
               
                   
               
               
                 BBa_J61120 
                 TCTAGAGAAAGACGCGAGAAACTAGATG 
                 1039 
               
               
                   
               
               
                 BBa_J61121 
                 TCTAGAGAAAGAGACGAGTCACTAGATG 
                 1040 
               
               
                   
               
               
                 BBa_J61122 
                 TCTAGAGAAAGAGAGGAGCCACTAGATG 
                 1041 
               
               
                   
               
               
                 BBa_J61123 
                 TCTAGAGAAAGAGATGACTAACTAGATG 
                 1042 
               
               
                   
               
               
                 BBa_J61124 
                 TCTAGAGAAAGAGCCGACATACTAGATG 
                 1043 
               
               
                   
               
               
                 BBa_J61125 
                 TCTAGAGAAAGAGCCGAGTTACTAGATG 
                 1044 
               
               
                   
               
               
                 BBa_J61126 
                 TCTAGAGAAAGAGGTGACTCACTAGATG 
                 1045 
               
               
                   
               
               
                 BBa_J61127 
                 TCTAGAGAAAGAGTGGAACTACTAGATG 
                 1046 
               
               
                   
               
               
                 BBa_J61128 
                 TCTAGAGAAAGATAGGACTCACTAGATG 
                 1047 
               
               
                   
               
               
                 BBa_J61129 
                 TCTAGAGAAAGATTGGACGTACTAGATG 
                 1048 
               
               
                   
               
               
                 BBa_J61130 
                 TCTAGAGAAAGAAACGACATACTAGATG 
                 1049 
               
               
                   
               
               
                 BBa_J61131 
                 TCTAGAGAAAGAACCGAATTACTAGATG 
                 1050 
               
               
                   
               
               
                 BBa_J61132 
                 TCTAGAGAAAGACAGGATTAACTAGATG 
                 873 
               
               
                   
               
               
                 BBa_J61133 
                 TCTAGAGAAAGACCCGAGACACTAGATG 
                 869 
               
               
                   
               
               
                 BBa_J61134 
                 TCTAGAGAAAGACCGGAAATACTAGATG 
                 870 
               
               
                   
               
               
                 BBa_J61135 
                 TCTAGAGAAAGACCGGAGACACTAGATG 
                 871 
               
               
                   
               
               
                 BBa_J61136 
                 TCTAGAGAAAGAGCTGAGCAACTAGATG 
                 874 
               
               
                   
               
               
                 BBa_J61137 
                 TCTAGAGAAAGAGTAGATCAACTAGATG 
                 875 
               
               
                   
               
               
                 BBa_J61138 
                 TCTAGAGAAAGATATGAATAACTAGATG 
                 876 
               
               
                   
               
               
                 BBa_J61139 
                 TCTAGAGAAAGATTAGAGTCACTAGATG 
                 877 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 29B 
               
             
            
               
                   
               
               
                 Selected Ribosome Binding Sites 
               
            
           
           
               
               
               
            
               
                 Identifier 
                 Sequence a   
                 SEQ ID NO 
               
               
                   
               
               
                 BBa_B0029 
                 TCTAGAGTTCACACAGGAAACCTACTAGATG 
                 880 
               
               
                   
               
               
                 BBa_B0030 
                 TCTAGAGATTAAAGAGGAGAAATACTAGATG 
                 881 
               
               
                   
               
               
                 BBa_B0031 
                 TCTAGAGTCACACAGGAAACCTACTAGATG 
                 882 
               
               
                   
               
               
                 BBa_B0032 
                 TCTAGAGTCACACAGGAAAGTACTAGATG 
                 883 
               
               
                   
               
               
                 BBa_B0033 
                 TCTAGAGTCACACAGGACTACTAGATG 
                 884 
               
               
                   
               
               
                 BBa_B0034 
                 TCTAGAGAAAGAGGAGAAATACTAGATG 
                 885 
               
               
                   
               
               
                 BBa_B0035 
                 TCTAGAGATTAAAGAGGAGAATACTAGATG 
                 886 
               
               
                   
               
               
                 BBa_B0064 
                 TCTAGAGAAAGAGGGGAAATACTAGATG 
                 887 
               
               
                   
               
            
           
         
       
     
     Multiple Mechanisms of Action 
     In some embodiments, the bacteria are genetically engineered to include multiple mechanisms of action (MOAs), e.g., circuits producing multiple copies of the same product (e.g., to enhance copy number) or circuits performing multiple different functions. Examples of insertion sites include, but are not limited to, malE/K, insB/I, araC/BAD, lacZ, dapA, cea, and other shown in  FIG. 52 . For example, the genetically engineered bacteria may include four copies of GLP-2 inserted at four different insertion sites, e.g., malE/K, insB/I, arac/BAD, and lacZ. Alternatively, the genetically engineered bacteria may include three copies of GLP-2 inserted at three different insertion sites, e.g., malE/K, insB/I, and lacZ, and three copies of a butyrogenic gene cassette inserted at three different insertion sites, e.g., dapA, cea, and araC/BA 
     In some embodiments, the bacteria are genetically engineered to include multiple mechanisms of action (MOAs), e.g., circuits producing multiple copies of the same product (e.g., to enhance copy number) or circuits performing multiple different functions. For example, the genetically engineered bacteria may include four copies of the gene, gene(s), or gene cassettes for producing the payload(s) inserted at four different insertion sites. Alternatively, the genetically engineered bacteria may include three copies of the gene, gene(s), or gene cassettes for producing the payload(s) inserted at three different insertion sites and three copies of the gene, gene(s), or gene cassettes for producing the payload(s) inserted at three different insertion sites. 
     In some embodiments, the genetically engineered bacteria comprise one or more of (1) one or more gene(s) or gene cassette(s) for the production of propionate, as described herein (2) one or more gene(s) or gene cassette(s) for the production of butyrate, as described herein (3) one or more gene(s) or gene cassette(s) for the production of acetate, as described herein (4) one or more gene(s) or gene cassette(s) for the production of tryptophan and/or its metabolites (including but not limited to kynurenine, indole, indole-3-acetic acid, indole-3 aldehyde, and IPA), as described herein (5) one or more gene(s) or gene cassette(s) for the production of one or more of GLP-2 and GLP-2 analogs, as described herein (6) one or more gene(s) or gene cassette(s) for the production of human or viral or monomerized IL-10, as described herein (7) one or more gene(s) or gene cassette(s) for the production of human IL-22, as described herein (8) one or more gene(s) or gene cassette(s) for the production of IL-2, and/or SOD, and/or IL-27 and other interleukins, as described herein (9) one or more gene(s) or gene cassette(s) for the production of one or more transporters, e.g. for the import of tryptophan and/or metabolites as described herein (10) one or more polypeptides for secretion, including but not limited to GLP-2 and its analogs, IL-10, and/or IL-22, SCFA and/or tryptophan synthesis and/or catabolic enzymes in wild type or in mutated form (for increased stability or metabolic activity) (11) one or more components of secretion machinery, as described herein (12) one or more auxotrophies, e.g., deltaThyA (13) one more more antibiotic resistances, including but not limited to, kanamycin or chloramphenicol resistance (14) one or more mutations/deletions to increase the flux through a metabolic pathway encoded by one or more genes or gene cassette(s), e.g mutations/deletions in genes in NADH consuming pathways, genes involved in feedback inhibition of a metabolic pathway encoded by the gene(s) or gene cassette(s) genes, as described herein (15) one or more mutations/deletions in one or more genes of the endogenous metabolic pathways, e.g., tryptophan synthesis pathway. 
     In some embodiments, the genetically engineered bacteria promote one or more of the following effector functions: (1) neutralizes TNF-α, IFN-γ, IL-1β, IL-6, IL-8, IL-17, and/or chemokines, e.g., CXCL-8 and CCL2 (2) activates include AHR (e.g., which result in IL-22 production) and (3) activates PXR, (4) inhibits HDACs, (5) activates GPR41 and/or GPR43 and/or GPR109A, (6) inhibits NF-kappaB signaling, (7) modulators of PPARgamma, (8) activates of AMPK signaling, (9) modulates GLP-1 secretion and/or (10). scavenges hydroxyl radicals and functions as antioxidants. 
     In some embodiments, under conditions where the gene, gene(s), or gene cassettes for producing the payload(s) is expressed, the genetically engineered bacteria of the disclosure produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of the payload(s) as compared to unmodified bacteria of the same subtype under the same conditions. 
     In some embodiments, the genetically engineered bacteria produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of a payload under inducing conditions than unmodified bacteria of the same subtype under the same conditions. Certain unmodified bacteria will not have detectable levels of the payload. In embodiments using genetically modified forms of these bacteria, the payload will be detectable under inducing conditions. 
     In certain embodiments, the immune modulator molecule is butyrate. Methods of measuring butyrate levels, e.g., by mass spectrometry, gas chromatography, high-performance liquid chromatography (HPLC), are known in the art (see, e.g., Aboulnaga et al., 2013). In some embodiments, butyrate is measured as butyrate level/bacteria optical density (OD). In some embodiments, measuring the activity and/or expression of one or more gene products in the butyrogenic gene cassette serves as a proxy measurement for butyrate production. In some embodiments, the bacterial cells of the invention are harvested and lysed to measure butyrate production. In alternate embodiments, butyrate production is measured in the bacterial cell medium. In some embodiments, the genetically engineered bacteria produce at least about 1 nM/OD, at least about 10 nM/OD, at least about 100 nM/OD, at least about 500 nM/OD, at least about 1 μM/OD, at least about 10 μM/OD, at least about 100 μM/OD, at least about 500 μM/OD, at least about 1 mM/OD, at least about 2 mM/OD, at least about 3 mM/OD, at least about 5 mM/OD, at least about 10 mM/OD, at least about 20 mM/OD, at least about 30 mM/OD, or at least about 50 mM/OD of butyrate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. 
     In certain embodiments, the immune modulator molecule is propionate. Methods of measuring propionate levels, e.g., by mass spectrometry, gas chromatography, high-performance liquid chromatography (HPLC), are known in the art (see, e.g., Hillman 1978; Lukovac et al., 2014). In some embodiments, measuring the activity and/or expression of one or more gene products in the propionate gene cassette serves as a proxy measurement for propionate production. In some embodiments, the bacterial cells of the invention are harvested and lysed to measure propionate production. In alternate embodiments, propionate production is measured in the bacterial cell medium. In some embodiments, the genetically engineered bacteria produce at least about 1 PM, at least about 10 μM, at least about 100 μM, at least about 500 μM, at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 5 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, or at least about 50 mM of propionate in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or in the presence of some other metabolite that may or may not be present in the gut, such as arabinose. 
     In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the gene, gene(s), or gene cassettes for producing the payload(s). Primers may be designed and used to detect mRNA in a sample according to methods known in the art. In some embodiments, a fluorophore is added to a sample reaction mixture that may contain payload RNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. The reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100° C., 60-70° C., and 30-50° C. for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97° C., 55-65° C., and 35-45° C. for a predetermined number of cycles. In some embodiments, the accumulating amplicon is quantified after each cycle of the qPCR. The number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT). At least one CT result for each sample is generated, and the CT result(s) may be used to determine mRNA expression levels of the payload(s). 
     In some embodiments, quantitative PCR (qPCR) is used to amplify, detect, and/or quantify mRNA expression levels of the payload(s). Primers may be designed and used to detect mRNA in a sample according to methods known in the art. In some embodiments, a fluorophore is added to a sample reaction mixture that may contain payload mRNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. The reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods. In certain embodiments, the heating and cooling is repeated for a predetermined number of cycles. In some embodiments, the reaction mixture is heated and cooled to 90-100° C., 60-70° C., and 30-50° C. for a predetermined number of cycles. In a certain embodiment, the reaction mixture is heated and cooled to 93-97° C., 55-65° C., and 35-45° C. for a predetermined number of cycles. In some embodiments, the accumulating amplicon is quantified after each cycle of the qPCR. The number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT). At least one CT result for each sample is generated, and the CT result(s) may be used to determine mRNA expression levels of the payload(s). 
     In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding short chain fatty acid production enzymes described herein and/or one or more gene sequence(s) encoding tryptophan catabolism enzyme(s) described herein and one or more gene sequence(s) encoding metabolite transporters described herein, and/or one or more gene sequence(s) encoding one or more therapeutic peptides for secretion, as described herein. 
     In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate. In some embodiments, the genetically engineered bacteria comprise a propionate gene cassette and are capable of producing propionate. In some embodiments, the genetically engineered bacteria comprise a acetate gene cassette and are capable of producing acetate. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding IL-10. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding IL-2. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding IL-22. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding IL-27. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding SOD. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding GLP-2. In some embodiments, the genetically engineered bacteria are capable of producing kyurenine. 
     In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding IL-10. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding IL-2. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding IL-22. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding IL-27. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding SOD. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding GLP-2. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and are capable of producing kyurenine. 
     In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding IL-10 and one or more gene sequences encoding IL-2, IL-22, IL-27, GLP-2, and SOD. In any of these embodiments the bacteria comprise a propionate gene cassette and can produce propionate. In any of these embodiments, the bacteria can produce kyuernine. 
     In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding IL-2 and one or more gene sequences encoding IL-10, IL-22, IL-27, GLP-2, and SOD. In any of these embodiments the bacteria comprise a propionate gene cassette and can produce propionate. In any of these embodiments, the bacteria can produce kyuernine. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding IL-22 and one or more gene sequences encoding IL-2, IL-10, IL-27, GLP-2, and SOD. In any of these embodiments the bacteria comprise a propionate gene cassette and can produce propionate. In any of these embodiments, the bacteria can produce kyuernine. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding IL-27 and one or more gene sequences encoding IL-2, IL-22, IL-10, GLP-2, and SOD. In any of these embodiments the bacteria comprise a propionate gene cassette and can produce propionate. In any of these embodiments, the bacteria can produce kyuernine. In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding GLP-2 and one or more gene sequences encoding IL-2, IL-22, IL-27, IL-10, and SOD. In any of these embodiments the bacteria comprise a propionate gene cassette and can produce propionate. In any of these embodiments, the bacteria can produce kyuernine. 
     In some embodiments, the genetically engineered bacteria comprise a butyrate gene cassette and are capable of producing butyrate and comprise a gene sequence encoding SOD and one or more gene sequences encoding IL-2, IL-22, IL-27, GLP-2, and IL-10. In any of these embodiments the bacteria comprise a propionate gene cassette and can produce propionate. In any of these embodiments, the bacteria can produce kyuernine. 
     In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding IL-10 and a gene sequence(s) encoding one or more molecules selected from IL-2, IL-22, IL-27, GLP-2, and SOD. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding IL-2 and a gene sequence(s) encoding one or more molecules selected from IL-10, IL-22, IL-27, GLP-2, and SOD. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding IL-22 and a gene sequence(s) encoding one or more molecules selected from IL-2, IL-27, IL-10, GLP-2, and SOD. In some embodiments, the genetically engineered bacteria comprise a gene sequence(s) encoding IL-27 and a gene sequence encoding one or more molecules selected from IL-2, IL-22, IL-10, GLP-2, and SOD. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding SOD and a gene sequence(s) encoding one or more molecules selected from IL-2, IL-22, IL-27, GLP-2, and IL-10. In some embodiments, the genetically engineered bacteria comprise a gene sequence encoding GLP-2 and a gene sequence(s) encoding one or more molecules selected from IL-2, IL-22, IL-27, IL-10, and SOD. In any of these embodiments, the genetically engineered bacteria are capable of producing kyurenine. In any of these embodiments, the genetically engineered bacteria are capable of producing butyrate. In any of these embodiments, the genetically engineered bacteria are capable of producing propionate. In any of these embodiments, the genetically engineered bacteria are capable of producing acetate. 
     In some embodiments, the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are expressed under the control of a constitutive promoter. In another embodiment, the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are expressed under the control of an inducible promoter. In some embodiments, the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. In some embodiments, the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are expressed under the control of a promoter that is directly or indirectly induced by inflammatory conditions. Exemplary inducible promoters described herein include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose and tetracycline. Examples of inducible promoters include, but are not limited to, an FNR responsive promoter, a P araC  promoter, a P araBAD  promoter, and a P TetR  promoter, each of which are described in more detail herein. Inducible promoters are described in more detail infra. 
     The at least one gene encoding the at least one short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are located on a plasmid in the bacterial cell. In another embodiment, the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are located in the chromosome of the bacterial cell, and at least one gene encoding at least one short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion from a different species of bacteria are located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are located on a plasmid in the bacterial cell, and at least one gene encoding the at least one one short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion from a different species of bacteria are located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are located in the chromosome of the bacterial cell, and at least one gene encoding the at least one one short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion from a different species of bacteria are located in the chromosome of the bacterial cell. 
     In some embodiments, the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are expressed on a low-copy plasmid. In some embodiments, the gene sequence(s) encoding the one or more short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the at least one short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion. 
     In some embodiments, a recombinant bacterial cell of the invention comprising at least one gene encoding at least one short chain fatty acid production enzyme(s) and/or tryptophan catabolism enzyme(s) and/or tryptophan biosynthesis enzyme(s) and/or metabolite transporters and/or therapeutic peptides for secretion are expressed on a high-copy plasmid do not increase tryptophan catabolism as compared to a recombinant bacterial cell comprising the same gene expressed on a low-copy plasmid in the absence of a heterologous importer of tryptophan and/or its metabolites and additional copies of a native importer of tryptophan and/or its metabolites. In alternate embodiments, the importer of tryptophan and/or its metabolites is used in conjunction with a high-copy plasmid. 
     In some embodiments, the genetically engineered bacteria described above further comprise one or more of the modifications, mutations, and/or deletions in endogenous genes described herein. 
     In some embodiments, the the genetically engineered microorganism further comprises a mutation and/or deletion in ldhA. In some embodiments, the genetically engineered microorganism further comprises a mutation and/or deletion in frdA. In some embodiments, the genetically engineered microorganism further comprises a mutation and/or deletion in adhE. In some embodiments, the the genetically engineered microorganism further comprises a mutation and/or deletion in one or more of ldhA, frdA, and adhE. 
     In some embodiments, surface display could be used to display a protein of interest on the surface of the genetically modified bacterium. In some embodiments, the genetically engineered bacteria and/or microorganisms encode one or more gene(s) and/or gene cassette(s) encoding a protein of interest, e.g., an anti-inflammation and/or gut barrier function enhancer molecule, which is anchored or displayed on the surface of the bacteria and/or microorganisms. 
     Induction of Payloads During Strain Culture 
     In some embodiments, it is desirable to pre-induce payload or protein of interest expression and/or payload activity prior to administration. Such payload or protein of interest may be an effector intended for secretion or may be an enzyme which catalyzes a metabolic reaction to produce an effector. In other embodiments, the protein of interest is an enzyme which catabolizes a harmful metabolite. In such situations, the strains are pre-loaded with active payload or protein of interest. In such instances, the genetically engineered bacteria of the invention express one or more protein(s) of interest, under conditions provided in bacterial culture during cell growth, expansion, purification, fermentation, and/or manufacture prior to administration in vivo. Such culture conditions can be provided in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. As used herein, the term “bacterial culture” or bacterial cell culture” or “culture” refers to bacterial cells or microorganisms, which are maintained or grown in vitro during several production processes, including cell growth, cell expansion, recovery, purification, fermentation, and/or manufacture. As used herein, the term “fermentation” refers to the growth, expansion, and maintenance of bacteria under defined conditions. Fermentation may occur under a number of cell culture conditions, including anaerobic or low oxygen or oxygenated conditions, in the presence of inducers, nutrients, at defined temperatures, and the like. 
     Culture conditions are selected to achieve optimal activity and viability of the cells, while maintaining a high cell density (high biomass) yield. A number of cell culture conditions and operating parameters are monitored and adjusted to achieve optimal activity, high yield and high viability, including oxygen levels (e.g., low oxygen, microaerobic, aerobic), temperature of the medium, and nutrients and/or different growth media, chemical and/or nutritional inducers and other components provided in the medium. 
     In some embodiments, the one or more protein(s) of interest and are directly or indirectly induced, while the strains is grown up for in vivo administration. Without wishing to be bound by theory, pre-induction may boost in vivo activity. This is particularly important in proximal regions of the gut which are reached first by the bacteria, e.g., the small intestine. If the bacterial residence time in this compartment is relatively short, the bacteria may pass through the small intestine without reaching full in vivo induction capacity. In contrast, if a strain is pre-induced and preloaded, the strains are already fully active, allowing for greater activity more quickly as the bacteria reach the intestine. Ergo, no transit time is “wasted”, in which the strain is not optimally active. As the bacteria continue to move through the intestine, in vivo induction occurs under environmental conditions of the gut (e.g., low oxygen, or in the presence of gut metabolites). 
     In one embodiment, expression of one or more payload(s), is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of several different proteins of interest is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s), is driven from the same promoter as a multicistronic message. In one embodiment, expression of one or more payload(s) is driven from the same promoter as two or more separate messages. In one embodiment, expression of one or more payload(s) is driven from the one or more different promoters. 
     In some embodiments, the strains are administered without any pre-induction protocols during strain growth prior to in vivo administration. 
     Anaerobic Induction 
     In some embodiments, cells are induced under anaerobic or low oxygen conditions in culture. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1×10{circumflex over ( )}8 to 1×10{circumflex over ( )}11, and exponential growth and are then switched to anaerobic or low oxygen conditions for approximately 3 to 5 hours. In some embodiments, strains are induced under anaerobic or low oxygen conditions, e.g. to induce FNR promoter activity and drive expression of one or more payload(s) under the control of one or more FNR promoters. 
     In one embodiment, expression of one or more payload(s), is under the control of one or more anaerobic or low oxygen inducible promoter(s), e.g., FNR promoter(s), and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic or low oxygen conditions. In one embodiment, expression of several different proteins of interest is under the control of one or more anaerobic or low oxygen inducible promoter(s), e.g., FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic or low oxygen conditions. 
     In one embodiment, expression of two or more payload(s), is under the control of one or more anaerobic or low oxygen inducible promoter(s), e.g., FNR promoter(s), and is driven from the same promoter in the form of a multicistronic message under anaerobic or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more anaerobic or low oxygen inducible promoter(s), e.g., FNR promoter(s), and is driven from the same promoter as two or more separate messages under anaerobic or low oxygen conditions. In one embodiment, expression of one or more payload(s) under the control of one or more anaerobic or low oxygen inducible promoter(s), e.g., FNR promoter(s), and is driven from the one or more different promoters under anaerobic or low oxygen conditions. 
     Without wishing to be bound by theory, strains that comprise one or more payload(s) under the control of an FNR promoter, may allow expression of payload(s) from these promoters in vitro, under anaerobic or low oxygen culture conditions, and in vivo, under the low oxygen conditions found in the gut. 
     In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced under anaerobic or low oxygen conditions in the presence of the chemical and/or nutritional inducer. In some embodiments, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s) and/or under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) under the control of a one or more constitutive promoter(s) described herein. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more thermoregulated promoter(s) described herein. 
     In one embodiment, expression of one or more payload gene sequence(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic and/or low oxygen conditions. In one embodiment, the chemical and/or nutritional inducer is arabinose and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline. 
     In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under anaerobic and/or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under anaerobic and/or low oxygen conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under anaerobic and/or low oxygen conditions. 
     In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, under anaerobic or low oxygen conditions. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In some embodiments, the strains comprise gene sequence(s) under the control of a a third inducible promoter, e.g., an anaerobic/low oxygen promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced promoter or a low oxygen promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload sequence(s) under the control of one or more constitutive promoter(s) active under low oxygen conditions. 
     Aerobic Induction 
     In some embodiments, it is desirable to prepare, pre-load and pre-induce the strains under aerobic conditions. This allows more efficient growth and viability, and, in some cases, reduces the build-up of toxic metabolites. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1×10{circumflex over ( )}8 to 1×10{circumflex over ( )}11, and exponential growth and are then induced through the addition of the inducer or through other means, such as shift to a permissive temperature, for approximately 3 to 5 hours. 
     In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art can be induced under aerobic conditions in the presence of the chemical and/or nutritional inducer during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under aerobic conditions. 
     In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under aerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under aerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under aerobic conditions. 
     In one embodiment, the chemical and/or nutritional inducer is arabinose and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline. 
     In some embodiments, promoters regulated by temperature are induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under aerobic conditions. 
     In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is driven from the same promoter in the form of a multicistronic message under aerobic conditions. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is driven from the same promoter as two or more separate messages under aerobic conditions. In one embodiment, expression of one or more payload(s) is driven directly or indirectly by one or more thermoregulated promoter(s) and is driven from the one or more different promoters under aerobic conditions. 
     In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced under aerobic conditions. In some embodiments, a strain comprises three or more different promoters which are induced under aerobic culture conditions. 
     In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g. a chemically inducible promoter, and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter under aerobic culture conditions. In some embodiments two or more chemically induced promoter gene sequence(s) are combined with a thermoregulated construct described herein. In one embodiment, the chemical and/or nutritional inducer is arabinose and the promoter is inducible by arabinose. In one embodiment, the chemical and/or nutritional inducer is IPTG and the promoter is inducible by IPTG. In one embodiment, the chemical and/or nutritional inducer is rhamnose and the promoter is inducible by rhamnose. In one embodiment, the chemical and/or nutritional inducer is tetracycline and the promoter is inducible by tetracycline. 
     In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload sequences under the control of one or more constitutive promoter(s) active under aerobic conditions. 
     In some embodiments, genetically engineered strains comprise gene sequence(s) which are induced under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene sequence(s) for in vivo activation in the gut. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut. 
     In some embodiments, genetically engineered strains comprise gene sequence(s), which are arabinose inducible under aerobic culture conditions. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut. 
     In some embodiments, genetically engineered strains comprise gene sequence(s), which are IPTG inducible under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene sequence(s) for in vivo activation in the gut. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut. 
     In some embodiments, genetically engineered strains comprise gene sequence(s) which are arabinose inducible under aerobic culture conditions. In some embodiments, such a strain further comprises sequence(s) which are IPTG inducible under aerobic culture conditions. In some embodiments, these strains further comprise FNR inducible gene payload sequence(s) for in vivo activation in the gut. In some embodiments, these strains do not further comprise FNR inducible gene sequence(s) for in vivo activation in the gut. 
     As evident from the above non-limiting examples, genetically engineered strains comprise inducible gene sequence(s) which can be induced numerous combinations. For example, rhamnose or tetracycline can be used as an inducer with the appropriate promoters in addition or in lieu of arabinose and/or IPTG or with thermoregulation. Additionally, such bacterial strains can also be induced with the chemical and/or nutritional inducers under anaerobic conditions. 
     Microaerobic Induction 
     In some embodiments, viability, growth, and activity are optimized by pre-inducing the bacterial strain under microaerobic conditions. In some embodiments, microaerobic conditions are best suited to “strike a balance” between optimal growth, activity and viability conditions and optimal conditions for induction; in particular, if the expression of the one or more payload(s) are driven by an anaerobic and/or low oxygen promoter, e.g., a FNR promoter. In such instances, cells are grown (e.g., for 1.5 to 3 hours) until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1×10{circumflex over ( )}8 to 1×10{circumflex over ( )}11, and exponential growth and are then induced through the addition of the inducer or through other means, such as shift to at a permissive temperature, for approximately 3 to 5 hours. 
     In one embodiment, expression of one or more payload(s) is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under microaerobic conditions. 
     In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter in the form of a multicistronic message under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the one or more different promoters under microaerobic conditions. 
     Without wishing to be bound by theory, strains that comprise one or more payload(s) under the control of an FNR promoter, may allow expression of payload(s) from these promoters in vitro, under microaerobic culture conditions, and in vivo, under the low oxygen conditions found in the gut. 
     In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced under microaerobic conditions in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) under the control of a one or more constitutive promoter(s) described herein. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more thermoregulated promoter(s) described herein. 
     In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under microaerobic conditions. 
     In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages under microaerobic conditions. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters under microaerobic conditions. 
     In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, under microaerobic conditions. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers. In some embodiments, the strains comprise gene sequence(s) under the control of a third inducible promoter, e.g., an anaerobic/low oxygen promoter or microaerobic promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced promoter or a low oxygen or microaerobic promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload under the control of one or more constitutive promoter(s) active under low oxygen conditions. 
     Induction of Strains Using Phasing, Pulsing and/or Cycling 
     In some embodiments, cycling, phasing, or pulsing techniques are employed during cell growth, expansion, recovery, purification, fermentation, and/or manufacture to efficiently induce and grow the strains prior to in vivo administration. This method is used to “strike a balance” between optimal growth, activity, cell health, and viability conditions and optimal conditions for induction; in particular, if growth, cell health or viability are negatively affected under inducing conditions. In such instances, cells are grown (e.g., for 1.5 to 3 hours) in a first phase or cycle until they have reached a certain OD, e.g., ODs within the range of 0.1 to 10, indicating a certain density e.g., ranging from 1×10{circumflex over ( )}8 to 1×10{circumflex over ( )}11, and are then induced through the addition of the inducer or through other means, such as shift to a permissive temperature (if a promoter is thermoregulated), or change in oxygen levels (e.g., reduction of oxygen level in the case of induction of an FNR promoter driven construct) for approximately 3 to 5 hours. In a second phase or cycle, conditions are brought back to the original conditions which support optimal growth, cell health and viability. Alternatively, if a chemical and/or nutritional inducer is used, then the culture can be spiked with a second dose of the inducer in the second phase or cycle. 
     In some embodiments, two cycles of optimal conditions and inducing conditions are employed (i.e, growth, induction, recovery and growth, induction). In some embodiments, three cycles of optimal conditions and inducing conditions are employed. In some embodiments, four or more cycles of optimal conditions and inducing conditions are employed. In a non-liming example, such cycling and/or phasing is used for induction under anaerobic and/or low oxygen conditions (e.g., induction of FNR promoters). In one embodiment, cells are grown to the optimal density and then induced under anaerobic and/or low oxygen conditions. Before growth and/or viability are negatively impacted due to stressful induction conditions, cells are returned to oxygenated conditions to recover, after which they are then returned to inducing anaerobic and/or low oxygen conditions for a second time. In some embodiments, these cycles are repeated as needed. 
     In some embodiments, growing cultures are spiked once with the chemical and/or nutritional inducer. In some embodiments, growing cultures are spiked twice with the chemical and/or nutritional inducer. In some embodiments, growing cultures are spiked three or more times with the chemical and/or nutritional inducer. In a non-limiting example, cells are first grown under optimal growth conditions up to a certain density, e.g., for 1.5 to 3 hour) to reached an of 0.1 to 10, until the cells are at a density ranging from 1×10{circumflex over ( )}8 to 1×10{circumflex over ( )}11. Then the chemical inducer, e.g., arabinose or IPTG, is added to the culture. After 3 to 5 hours, an additional dose of the inducer is added to re-initiate the induction. Spiking can be repeated as needed. 
     In some embodiments, phasing or cycling changes in temperature in the culture. In another embodiment, adjustment of temperature may be used to improve the activity of a payload. For example, lowering the temperature during culture may improve the proper folding of the payload. In such instances, cells are first grown at a temperature optimal for growth (e.g., 37 C). In some embodiments, the cells are then induced, e.g., by a chemical inducer, to express the payload. Concurrently or after a set amount of induction time, the temperature in the media is lowered, e.g., between 25 and 35 C, to allow improved folding of the expressed payload. 
     In some embodiments, payload(s) are under the control of different inducible promoters, for example two different chemical inducers. In other embodiments, the payload is induced under low oxygen conditions or microaerobic conditions and a second payload is induced by a chemical inducer. 
     In one embodiment, expression of one or more payload(s) is under the control of one or more FNR promoter(s) and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture by using phasing or cycling or pulsing or spiking techniques. 
     In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter in the form of a multicistronic message through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the same promoter as two or more separate messages through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more FNR promoter(s) and is driven from the one or more different promoters through the employment of phasing or cycling or pulsing or spiking techniques. 
     In some embodiments, promoters inducible by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers can be induced through the employment of phasing or cycling or pulsing or spiking techniques in the presence of the chemical and/or nutritional inducer. In particular, strains may comprise a combination of gene sequence(s), some of which are under control of FNR promoters and others which are under control of promoters induced by chemical and/or nutritional inducers. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s) and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of one or more FNR promoter(s), and one or more payload gene sequence(s) under the control of a one or more constitutive promoter(s) described herein and are induced through the employment of phasing or cycling or pulsing or spiking techniques. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more thermoregulated promoter(s) described herein, and are induced through the employment of phasing or cycling or pulsing or spiking techniques. 
     Any of the strains described herein can be grown through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s) is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is induced during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture under anaerobic and/or low oxygen conditions. 
     In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter in the form of a multicistronic message and which are induced through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the same promoter as two or more separate messages and is grown through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, expression of one or more payload(s), is under the control of one or more promoter(s) regulated by chemical and/or nutritional inducers and is driven from the one or more different promoters, all of which are induced through the employment of phasing or cycling or pulsing or spiking techniques. 
     In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers, through the employment of phasing or cycling or pulsing or spiking techniques. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter and others which are under control of a second inducible promoter, both induced by chemical and/or nutritional inducers through the employment of phasing or cycling or pulsing or spiking techniques. In some embodiments, the strains comprise gene sequence(s) under the control of a a third inducible promoter, e.g., an anaerobic/low oxygen promoter, e.g., FNR promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced promoter or a low oxygen promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a FNR promoter and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In one embodiment, strains may comprise a combination of gene sequence(s), some of which are under control of a first inducible promoter, e.g., a chemically induced and others which are under control of a second inducible promoter, e.g. a temperature sensitive promoter. In some embodiments, strains may comprise one or more payload gene sequence(s) under the control of an FNR promoter and one or more payload gene sequence(s) under the control of a one or more promoter(s) which are induced by a one or more chemical and/or nutritional inducer(s), including, but not limited to, by arabinose, IPTG, rhamnose, tetracycline, and/or other chemical and/or nutritional inducers described herein or known in the art. Additionally the strains may comprise a construct which is under thermoregulatory control. In some embodiments, the bacteria strains further comprise payload sequence(s) under the control of one or more constitutive promoter(s) active under low oxygen conditions. Any of the strains described in these embodiments may be induced through the employment of phasing or cycling or pulsing or spiking techniques. 
     Aerobic Induction of the FNR Promoter 
     FNRS24Y is a mutated form of FNR which is more resistant to inactivation by oxygen, and therefore can activate FNR promoters under aerobic conditions (see e.g., Jervis A J The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion, Proc Natl Acad Sci USA. 2009 Mar. 24; 106(12):4659-64, the contents of which is herein incorporated by reference in its entirety). In some embodiments, an oxygen bypass system shown and described in figures and examples is used. In this oxygen bypass system, FNRS24Y is induced by addition of arabinose and then drives the expression of the protein of interest (e.g., one or more anti-cancer effector(s) described herein) by binding and activating the FNR promoter under aerobic conditions. Thus, strains can be grown, produced or manufactured efficiently under aerobic conditions, while being effectively pre-induced and pre-loaded, as the system takes advantage of the strong FNR promoter resulting in of high levels of expression of the protein of interest. This system does not interfere with or compromise in vivo activation, since the mutated FNRS24Y is no longer expressed in the absence of arabinose, and wild type FNR then binds to the FNR promoter and drives expression of the protein of interest, e.g., one or more anti-cancer effector(s) described herein. 
     In some embodiments, FNRS24Y is expressed during aerobic culture growth and induces a gene of interest. In other embodiments described herein, a second payload expression can also be induced aerobically, e.g., by arabinose. In a non-limiting example, a protein of interest and FNRS24Y can in some embodiments be induced simultaneously, e.g., from an arabinose inducible promoter. In some embodiments, FNRS24Y and the protein of interest are transcribed as a bicistronic message whose expression is driven by an arabinose promoter. In some embodiments, FNRS24Y is knocked into the arabinose operon, allowing expression to be driven from the endogenous Para promoter. 
     In some embodiments, a LacI promoter and IPTG induction are used in this system (in lieu of Para and arabinose induction). In some embodiments, a rhamnose inducible promoter is used in this system. In some embodiments, a temperature sensitive promoter is used to drive expression of FNRS24Y. 
     Secretion 
     In any of the embodiments described herein, in which the genetically engineered organism, e.g., engineered bacteria or engineered virus, produces a protein, polypeptide, or peptide, DNA, RNA, small molecule or other molecule intended to be secreted from the microorganism, the engineered microorganism may comprise a secretion mechanism and corresponding gene sequence(s) encoding the secretion system. 
     In some embodiments, the genetically engineered bacteria further comprise a native secretion mechanism or non-native secretion mechanism that is capable of secreting the molecule from the bacterial cytoplasm in the extracellular environment. Many bacteria have evolved sophisticated secretion systems to transport substrates across the bacterial cell envelope. Substrates, such as small molecules, proteins, and DNA, may be released into the extracellular space or periplasm (such as the gut lumen or other space), injected into a target cell, or associated with the bacterial membrane. 
     In Gram-negative bacteria, secretion machineries may span one or both of the inner and outer membranes. In some embodiments, the genetically engineered bacteria further comprise a non-native double membrane-spanning secretion system. Double membrane-spanning secretion systems include, but are not limited to, the type I secretion system (T1SS), the type II secretion system (T2SS), the type III secretion system (T3SS), the type IV secretion system (T4SS), the type VI secretion system (T6SS), and the resistance-nodulation-division (RND) family of multi-drug efflux pumps (Pugsley 1993; Gerlach et al., 2007; Collinson et al., 2015; Costa et al., 2015; Reeves et al., 2015; WO2014138324A1, incorporated herein by reference). Examples of such secretion systems are shown in figures and examples. Mycobacteria, which have a Gram-negative-like cell envelope, may also encode a type VII secretion system (T7SS) (Stanley et al., 2003). With the exception of the T2SS, double membrane-spanning secretions generally transport substrates from the bacterial cytoplasm directly into the extracellular space or into the target cell. In contrast, the T2SS and secretion systems that span only the outer membrane may use a two-step mechanism, wherein substrates are first translocated to the periplasm by inner membrane-spanning transporters, and then transferred to the outer membrane or secreted into the extracellular space. Outer membrane-spanning secretion systems include, but are not limited to, the type V secretion or autotransporter system or autosecreter system (T5SS), the curli secretion system, and the chaperone-usher pathway for pili assembly (Saier, 2006; Costa et al., 2015). 
     In some embodiments in which the one or more proteins of interest or therapeutic proteins are secreted or exported from the microorganism, the engineered microorganism comprises gene sequence(s) that includes a secretion tag. In some embodiments, the one or more proteins of interest or therapeutic proteins include a “secretion tag” of either RNA or peptide origin to direct the one or more proteins of interest or therapeutic proteins to specific secretion systems. For example, a secretion tag for the Type I Hemolysin secretion system is encoded in the C-terminal 53 amino acids of the alpha hemolysin protein (HlyA). 
     In some embodiments, a Hemolysin-based Secretion System is used to secrete the molecule of interest, e.g., therapeutic peptide. Type I Secretion systems offer the advantage of translocating their passenger peptide directly from the cytoplasm to the extracellular space, obviating the two-step process of other secretion types.  FIG. 57  shows the alpha-hemolysin (HlyA) of uropathogenic  Escherichia coli . This pathway uses HlyB, an ATP-binding cassette transporter; HlyD, a membrane fusion protein; and TolC, an outer membrane protein. The assembly of these three proteins forms a channel through both the inner and outer membranes. HlyB inserts into inner membrane to form a pore, HlyD aligns HlyB with TolC (outer membrane pore) thereby forming a channel through inner and outer membrane. Natively, this channel is used to secrete HlyA, however, to secrete the therapeutic peptide of the present disclosure, the secretion signal-containing C-terminal portion of HlyA is fused to the C-terminal portion of a therapeutic peptide (star) to mediate secretion of this peptide. The C-terminal secretion tag can be removed by either an autocatalytic or protease-catalyzed e.g., OmpT cleavage thereby releasing the one or more proteins of interest or therapeutic proteins into the extracellular milieu. In some embodiments the one or more proteins of interest or therapeutic proteins contain expressed as fusion protein with the 53 amino acids of the C termini of alpha-hemolysin (hlyA) of  E. coli  CFT073 (C terminal secretion tag). 
     In some embodiments, a Type V Autotransporter Secretion System is used to secrete the molecule of interest, e.g., therapeutic peptide. The Type V Auto-secretion System utilizes an N-terminal Sec-dependent peptide tag (inner membrane) and C-terminal tag (outer-membrane). This system uses the Sec-system to get from the cytoplasm to the periplasm. The C-terminal tag then inserts into the outer membrane forming a pore through which the “passenger protein” threads through. Due to the simplicity of the machinery and capacity to handle relatively large protein fluxes, the Type V secretion system is attractive for the extracellular production of recombinant proteins. As shown in  FIG. 56 , a therapeutic peptide (star) can be fused to an N-terminal secretion signal, a linker, and the beta-domain of an autotransporter. The N-terminal, Sec-dependent signal sequence directs the protein to the SecA-YEG machinery which moves the protein across the inner membrane into the periplasm, followed by subsequent cleavage of the signal sequence. The Beta-domain is recruited to the Bam complex (‘Beta-barrel assembly machinery’) where the beta-domain is folded and inserted into the outer membrane as a beta-barrel structure. The therapeutic peptide is threaded through the hollow pore of the beta-barrel structure ahead of the linker sequence. Once across the outer membrane, the passenger is released from the membrane-embedded C-terminal tag by either an autocatalytic, intein-like mechanism (left side of Bam complex) or via a membrane-bound protease (black scissors; right side of Barn complex) (i.e., OmpT). For example, a membrane-associated peptidase to a complimentary protease cut site in the linker. Thus, in some embodiments, the secreted molecule, such as a heterologous protein or peptide comprises an N-terminal secretion signal, a linker, and beta-domain of an autotransporter so as to allow the molecule to be secreted from the bacteria. 
     The N-terminal tag is removed by the Sec system. Thus, in some embodiments, the secretion system is able to remove this tag before secreting the one or more proteins of interest or therapeutic proteins, from the engineered bacteria. In the Type V auto-secretion-mediated secretion the N-terminal peptide secretion tag is removed upon translocation of the “passenger” peptide from the cytoplasm into the periplasmic compartment by the native Sec system. Further, once the auto-secretor is translocated across the outer membrane the C-terminal secretion tag can be removed by either an autocatalytic or protease-catalyzed e.g., OmpT cleavage thereby releasing the molecule(s) into the extracellular milieu. 
     In some embodiments, the genetically engineered bacteria of the invention comprise a type III or a type III-like secretion system (T3SS) from  Shigella, Salmonella, E. coli, Bivrio, Burkholderia, Yersinia, Chlamydia , or  Pseudomonas . The traditional T3SS is capable of transporting a protein from the bacterial cytoplasm to the host cytoplasm through a needle complex. In the Type III traditional secretion system, the basal body closely resembles the flagella, however, instead of a “tail”/whip, the traditional T3SS has a syringe to inject the passenger proteins into host cells. The secretion tag is encoded by an N-terminal peptide (lengths vary and there are several different tags, see PCT/US14/020972). The N-terminal tag is not removed from the polypeptides in this secretion system. 
     The T3SS may be modified to secrete the molecule from the bacterial cytoplasm, but not inject the molecule into the host cytoplasm. Thus, the molecule is secreted into the gut lumen, tumor microenvironment, or other extracellular space. In some embodiments, the genetically engineered bacteria comprise said modified T3SS and are capable of secreting the molecule of interest from the bacterial cytoplasm. In some embodiments, the secreted molecule, such as a heterologous protein or peptide comprises a type III secretion sequence that allows the molecule of interest to be secreted from the bacteria. 
     In the Flagellar modified Type III Secretion, the tag is encoded in 5′untranslated region of the mRNA and thus there is no peptide tag to cleave/remove. This modified system does not contain the “syringe” portion and instead uses the basal body of the flagella structure as the pore to translocate across both membranes and out through the forming flagella. If the fliC/fliD genes (encoding the flagella “tail”/whip) are disrupted the flagella cannot fully form and this promotes overall secretion. In some embodiments, the tail portion can be removed entirely. 
     In some embodiments, a flagellar type III secretion pathway is used to secrete the molecule of interest. In some embodiments, an incomplete flagellum is used to secrete a therapeutic peptide of interest by recombinantly fusing the peptide to an N-terminal flagellar secretion signal of a native flagellar component. In this manner, the intracellularly expressed chimeric peptide can be mobilized across the inner and outer membranes into the surrounding host environment. 
     For example, a modified flagellar type III secretion apparatus in which untranslated DNA fragment upstream of the gene fliC (encoding flagellin), e.g., a 173-bp region, is fused to the gene encoding the heterologous protein or peptide can be used to secrete polypeptides of interest (See, e.g., Majander et al., Extracellular secretion of polypeptides using a modified  Escherichia coli  flagellar secretion apparatus. Nat Biotechnol. 2005 April; 23(4):475-81). In some cases, the untranslated region from the fliC loci may not be sufficient to mediate translocation of the passenger peptide through the flagella. Here it may be necessary to extend the N-terminal signal into the amino acid coding sequence of FliC, for example, by using the 173 bp of untranslated region along with the first 20 amino acids of FliC (see, e.g., Duan et al., Secretion of Insulinotropic Proteins by Commensal Bacteria: Rewiring the Gut To Treat Diabetes, Appl. Environ. Microbiol. December 2008 vol. 74 no. 23 7437-7438). 
     In alternate embodiments, the genetically engineered bacteria further comprise a non-native single membrane-spanning secretion system. Single membrane-spanning transporters may act as a component of a secretion system, or may export substrates independently. Such transporters include, but are not limited to, ATP-binding cassette translocases, flagellum/virulence-related translocases, conjugation-related translocases, the general secretory system (e.g., the SecYEG complex in  E. coli ), the accessory secretory system in mycobacteria and several types of Gram-positive bacteria (e.g.,  Bacillus anthracis, Lactobacillus johnsonii, Corynebacterium glutamicum, Streptococcus gordonii, Staphylococcus aureus ), and the twin-arginine translocation (TAT) system (Saier, 2006; Rigel and Braunstein, 2008; Albiniak et al., 2013). It is known that the general secretory and TAT systems can both export substrates with cleavable N-terminal signal peptides into the periplasm, and have been explored in the context of biopharmaceutical production. The TAT system may offer particular advantages, however, in that it is able to transport folded substrates, thus eliminating the potential for premature or incorrect folding. In certain embodiments, the genetically engineered bacteria comprise a TAT or a TAT-like system and are capable of secreting the molecule of interest from the bacterial cytoplasm. One of ordinary skill in the art would appreciate that the secretion systems disclosed herein may be modified to act in different species, strains, and subtypes of bacteria, and/or adapted to deliver different payloads. 
     In order to translocate a protein, e.g., therapeutic polypeptide, to the extracellular space, the polypeptide must first be translated intracellularly, mobilized across the inner membrane and finally mobilized across the outer membrane. Many effector proteins (e.g., therapeutic polypeptides)—particularly those of eukaryotic origin—contain disulphide bonds to stabilize the tertiary and quaternary structures. While these bonds are capable of correctly forming in the oxidizing periplasmic compartment with the help of periplasmic chaperones, in order to translocate the polypeptide across the outer membrane the disulphide bonds must be reduced and the protein unfolded again. 
     One way to secrete properly folded proteins in gram-negative bacteria—particularly those requiring disulphide bonds—is to target the reducing-environment periplasm in conjunction with a destabilizing outer membrane. In this manner the protein is mobilized into the oxidizing environment and allowed to fold properly. In contrast to orchestrated extracellular secretion systems, the protein is then able to escape the periplasmic space in a correctly folded form by membrane leakage. These “leaky” gram-negative mutants are therefore capable of secreting bioactive, properly disulphide-bonded polypeptides. In some embodiments, the genetically engineered bacteria have a “leaky” or de-stabilized outer membrane. Destabilizing the bacterial outer membrane to induce leakiness can be accomplished by deleting or mutagenizing genes responsible for tethering the outer membrane to the rigid peptidoglycan skeleton, including for example, lpp, ompC, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl. Lpp is the most abundant polypeptide in the bacterial cell existing at ˜500,000 copies per cell and functions as the primary ‘staple’ of the bacterial cell wall to the peptidoglycan. 1. Silhavy, T. J., Kahne, D. &amp; Walker, S. The bacterial cell envelope. Cold Spring Harb Perspect Biol 2, a000414 (2010). TolA-PAL and OmpA complexes function similarly to Lpp and are other deletion targets to generate a leaky phenotype. Additionally, leaky phenotypes have been observed when periplasmic proteases are inactivated. The periplasm is very densely packed with protein and therefore encode several periplasmic proteins to facilitate protein turnover. Removal of periplasmic proteases such as degS, degP or nlpl can induce leaky phenotypes by promoting an excessive build-up of periplasmic protein. Mutation of the proteases can also preserve the effector polypeptide by preventing targeted degradation by these proteases. Moreover, a combination of these mutations may synergistically enhance the leaky phenotype of the cell without major sacrifices in cell viability. Thus, in some embodiments, the engineered bacteria have one or more deleted or mutated membrane genes. In some embodiments, the engineered bacteria have a deleted or mutated lpp gene. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from ompA, ompA, and ompF genes. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from tolA, tolB, and pal genes. in some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes. In some embodiments, the engineered bacteria have one or more deleted or mutated periplasmic protease genes selected from degS, degP, and nlpl. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from lpp, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl genes. 
     To minimize disturbances to cell viability, the leaky phenotype can be made inducible by placing one or more membrane or periplasmic protease genes, e.g., selected from lpp, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl, under the control of an inducible promoter. For example, expression of lpp or other cell wall stability protein or periplasmic protease can be repressed in conditions where the therapeutic polypeptide needs to be delivered (secreted). For instance, under inducing conditions a transcriptional repressor protein or a designed antisense RNA can be expressed which reduces transcription or translation of a target membrane or periplasmic protease gene. Conversely, overexpression of certain peptides can result in a destabilized phenotype, e.g., overexpression of colicins or the third topological domain of TolA, wherein peptide overexpression can be induced in conditions in which the therapeutic polypeptide needs to be delivered (secreted). These sorts of strategies would decouple the fragile, leaky phenotypes from biomass production. Thus, in some embodiments, the engineered bacteria have one or more membrane and/or periplasmic protease genes under the control of an inducible promoter. 
     Table 30 and Table 31A below lists secretion systems for Gram positive bacteria and Gram negative bacteria. 
     
       
         
           
               
             
               
                 TABLE 30 
               
             
            
               
                   
               
               
                 Secretion systems for gram positive bacteria 
               
            
           
           
               
               
            
               
                 Bacterial Strain 
                 Relevant Secretion System 
               
               
                   
               
               
                   C. novyi -NT (Gram+) 
                 Sec pathway 
               
               
                   
                 Twin- arginine (TAT) pathway 
               
               
                   C. butryicum  (Gram+) 
                 Sec pathway 
               
               
                   
                 Twin- arginine (TAT) pathway 
               
               
                   Listeria monocytogenes  (Gram+) 
                 Sec pathway 
               
               
                   
                 Twin- arginine (TAT) pathway 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 31A 
               
             
            
               
                   
               
               
                 Secretion Systems for Gram negative bacteria 
               
               
                 Protein secretary pathways (SP) in gram- 
               
               
                 negative bacteria and their descendants 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 # 
                   
               
               
                 Type 
                   
                   
                   
                   
                   
                 Proteins/ 
                 Energy 
               
               
                 (Abbreviation) 
                 Name 
                 TC# 2   
                 Bacteria 
                 Archaea 
                 Eukarya 
                 System 
                 Source 
               
               
                   
               
            
           
           
               
            
               
                 IMPS - Gram-negative bacterial inner membrane 
               
               
                 channel-forming translocases 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 ABC 
                 ATP binding 
                 3.A.1 
                 + 
                 + 
                 + 
                 3-4 
                 ATP 
               
               
                 (SIP) 
                 cassette 
               
               
                   
                 translocase 
               
               
                 SEC 
                 General 
                 3.A.5 
                 + 
                 + 
                 + 
                 ~12  
                 GTP OR 
               
               
                 (IISP) 
                 secretory 
                   
                   
                   
                   
                   
                 ATP + 
               
               
                   
                 translocase 
                   
                   
                   
                   
                   
                 PMF 
               
               
                 Fla/Path 
                 Flagellum/ 
                 3.A.6 
                 + 
                 − 
                 − 
                 &gt;10  
                 ATP 
               
               
                 (IIISP) 
                 virulence- 
               
               
                   
                 related 
               
               
                   
                 translocase 
               
               
                 Conj 
                 Conjugation- 
                 3.A.7 
                 + 
                 − 
                 − 
                 &gt;10  
                 ATP 
               
               
                 (IVSP) 
                 related 
               
               
                   
                 translocase 
               
               
                 Tat 
                 Twin-arginine 
                 2.A.64 
                 + 
                 + 
                 + 
                 2-4 
                 PMF 
               
               
                 (IISP) 
                 targeting 
                   
                   
                   
                 (chloroplasts) 
               
               
                   
                 translocase 
               
               
                 Oxa1 
                 Cytochrome 
                 2.A.9 
                 + 
                 + 
                 + 
                 1 
                 None or 
               
               
                 (YidC) 
                 oxidase 
                   
                   
                   
                 (mitochondria 
                   
                 PMF 
               
               
                   
                 biogenesis 
                   
                   
                   
                 chloroplasts) 
               
               
                   
                 family 
               
               
                 MscL 
                 Large 
                 1.A.22 
                 + 
                 + 
                 + 
                 1 
                 None 
               
               
                   
                 conductance 
               
               
                   
                 mechanosensitive 
               
               
                   
                 channel family 
               
               
                 Holins 
                 Holin 
                 1.E.1•21 
                 + 
                 − 
                 − 
                 1 
                 None 
               
               
                   
                 functional 
               
               
                   
                 superfamily 
               
            
           
           
               
            
               
                 Eukaryotic Organelles 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 MPT 
                 Mitochondrial 
                 3.A.B 
                 − 
                 − 
                 + 
                 &gt;20  
                 ATP 
               
               
                   
                 protein 
                   
                   
                   
                 (mitochondrial) 
               
               
                   
                 translocase 
               
               
                 CEPT 
                 Chloroplast 
                 3.A.9 
                 (+) 
                 − 
                 + 
                 ≥3   
                 GTP 
               
               
                   
                 envelope 
                   
                   
                   
                 (chloroplasts) 
               
               
                   
                 protein 
               
               
                   
                 translocase 
               
               
                 Bcl-2 
                 Eukaryotic 
                 1.A.21 
                 − 
                 − 
                 + 
                  1? 
                 None 
               
               
                   
                 Bcl-2 family 
               
               
                   
                 (programmed 
               
               
                   
                 cell death) 
               
            
           
           
               
            
               
                 Gram-negative bacterial outer membrane 
               
               
                 channel-forming translocases 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 MTB 
                 Main 
                 3.A.15 
                 + b   
                 − 
                 − 
                 ~14  
                 ATP; 
               
               
                 (IISP) 
                 terminal 
                   
                   
                   
                   
                   
                 PMF 
               
               
                   
                 branch of the 
               
               
                   
                 general 
               
               
                   
                 secretory 
               
               
                   
                 translocase 
               
               
                 FUP AT-1 
                 Fimbrial 
                 1.B.11 
                     + b   
                 − 
                 − 
                 1 
                 None 
               
               
                   
                 usher protein 
                 1.B.12 
                     + b   
                   
                 − 
                 1 
                 None 
               
               
                   
                 Autotransporter-1 
               
               
                 AT-2 OMF 
                 Autotransporter-2 
                 1.B.40 
                     + b   
                 − 
                 − 
                 1 
                 None 
               
               
                 (ISP) 
                   
                 1.B.17 
                     + b   
                   
                  +(?) 
                 1 
                 None 
               
               
                 TPS 
                   
                 1.B.20 
                 + 
                 − 
                 + 
                 1 
                 None 
               
               
                 Secretin 
                   
                 1.B.22 
                     + b   
                   
                 − 
                 1 
                 None 
               
               
                 (IISP and 
               
               
                 IISP) 
               
               
                 OmpIP 
                 Outer 
                 1.B.33 
                 + 
                 − 
                 + 
                 ≥4   
                 None? 
               
               
                   
                 membrane 
                   
                   
                   
                 (mitochondria; 
               
               
                   
                 insertion 
                   
                   
                   
                 chloroplasts) 
               
               
                   
                 porin 
               
               
                   
               
            
           
         
       
     
     The above tables for gram positive and gram negative bacteria list secretion systems that can be used to secrete polypeptides and other molecules from the engineered bacteria, which are reviewed in Milton H. Saier, Jr. Microbe/Volume 1, Number 9, 2006 “Protein Secretion Systems in Gram-Negative Bacteria Gram-negative bacteria possess many protein secretion-membrane insertion systems that apparently evolved independently”, the contents of which is herein incorporated by reference in its entirety. 
     In some embodiments, the genetically engineered bacterial comprise a native or non-native secretion system described herein for the secretion of a molecule, e.g., a cytokine, antibody (e.g., scFv), metabolic enzyme, e.g., kynureninase, an others described herein. 
     
       
         
           
               
             
               
                 TABLE 31B 
               
             
            
               
                   
               
               
                 Polypeptide Sequences of  
               
               
                 exemplary secretion tags 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 PhoA 
                 MKQSTIALALLPLLFTPVTKA 
               
               
                 SEQ ID NO: 1500 
                   
               
               
                   
               
               
                 PhoA 
                 KQSTIALALLPLLFTPVTKA 
               
               
                 SEQ ID NO: 1501 
                   
               
               
                   
               
               
                 OmpF 
                 MMKRNILAVIVPALLVAGTANA 
               
               
                 SEQ ID NO: 1502 
                   
               
               
                   
               
               
                 cvaC 
                 MRTLTLNELDSVSGG 
               
               
                 SEQ ID NO: 1503 
                   
               
               
                   
               
               
                 TorA 
                 MNNNDLFQASRRRFLAQLGGLTVAGMLGTSLLT 
               
               
                 SEQ ID NO: 1504 
                 PRRATAAQAA 
               
               
                   
               
               
                 fdnG 
                 MDVSRRQFFKICAGGMAGTTVAALGFAPKQALA 
               
               
                 SEQ ID NO: 1505 
                   
               
               
                   
               
               
                 dmsA 
                 MKTKIPDAVLAAEVSRRGLVKTTAIGGLAMASS 
               
               
                 SEQ ID NO: 1506 
                 ALTLPFSRIAHA 
               
               
                   
               
               
                 PelB 
                 KYLLPTAAAGLLLLAAQPAMA 
               
               
                 SEQ ID NO: 1507 
                   
               
               
                   
               
               
                 HlyA secretion 
                 LNPLINEISKIISAAGNFDVKEERAAASLLQLS 
               
               
                 signal 
                 GNASDFSYGRNSITLTASA 
               
               
                 SEQ ID NO: 1508 
                   
               
               
                   
               
               
                 HlyA secretion 
                 CTTAATCCATTAATTAATGAAATCAGCAAAAT 
               
               
                 signal 
                 CATTTCAGCTGCAGGTAATTTTGATGTTAAAG 
               
               
                 SEQ ID NO: 1509 
                 AGGAAAGAGCTGCAGCTTCTTTATTGCAGTTG 
               
               
                   
                 ATCCGGTAATGCCAGTGATTTTTCATATGGCG 
               
               
                   
                 GAACTCAATAACTTTGACAGCATCAGCATAA. 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 31C 
               
             
            
               
                   
               
               
                 Additionals secretion tag sequences (native to E coli.) 
               
            
           
           
               
               
            
               
                 Description 
                 Sequences 
               
               
                   
               
               
                 ECOLIN_05715 Secretion signal 
                 MKRHLNTSYRLVWNHITGAFVVASELARARGKRAGVA 
               
               
                 SEQ ID NO: 1511 
                 VALSLAAATSLPALA 
               
               
                   
               
               
                 ECOLIN_16495 Secretion signal 
                 MFWRDMTLSVWRKKTTGLKTKKRLLALVLAAALCSSPV 
               
               
                 SEQ ID NO: 1512 
                 WA 
               
               
                   
               
               
                 ECOLIN_19410 Secretion signal 
                 MGYKMNISSLRKAFIFMGAVAALSLVNAQSALA 
               
               
                 SEQ ID NO: 1513 
                   
               
               
                   
               
               
                 ECOLIN_19880 Secretion signal 
                 MNKIFKVIWNPATGSYTVASETAKSRGKKSGRSKLLISAL 
               
               
                 SEQ ID NO: 1514 
                 VAGGLLSSFGASA 
               
               
                   
               
            
           
         
       
     
     In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that encodes a polypeptide which is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 1500, SEQ ID NO: 1501, SEQ ID NO: 1502, SEQ ID NO: 1503, SEQ ID NO: 1504, SEQ ID NO: 1505, SEQ ID NO: 1506, SEQ ID NO: 1507, SEQ ID NO: 1508, SEQ ID NO: 1509, SEQ ID NO: 1511, SEQ ID NO: 1512, SEQ ID NO: 1513, and/or SEQ ID NO: 1514. 
     Any secretion tag or secretion system can be combined with any cytokine described herein, and can be used to generate a construct (plasmid based or integrated) which is driven by an directly or indirectly inducible or constitutive promoter described herein. In some embodiments, the secretion system is used in combination with one or more genomic mutations, which leads to the leaky or diffusible outer membrane phenotype (DOM), including but not limited to, lpp, nlP, tolA, PAL. 
     In some embodiments, the secretion system is selected from the type III flagellar, modified type III flagellar, type I (e.g., hemolysin system), type II, type IV, type V, type VI, and type VII secretion systems, resistance-nodulation-division (RND) multi-drug efflux pumps, a single membrane secretion system, Sec and, TAT secretion systems. 
     Any of the secretion systems described herein may according to the disclosure be employed to secrete the polypeptides of interest. In some embodiments, the therapeutic proteins secreted by the genetically engineered bacteria are modified to increase resistance to proteases, e.g. intestinal proteases. 
     In some embodiments, the genetically engineered microorganisms are capable of expressing any one or more of the described circuits in low-oxygen conditions, and/or in the presence of cancer and/or the tumor microenvironment, or tissue specific molecules or metabolites, and/or in the presence of molecules or metabolites associated with inflammation or immune suppression, and/or in the presence of metabolites that may be present in the gut, and/or in the presence of metabolites that may or may not be present in vivo, and may be present in vitro during strain culture, expansion, production and/or manufacture, such as arabinose and others described herein. In some embodiments, the gene sequences(s) are controlled by a promoter inducible by such conditions and/or inducers. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter, as described herein. In some embodiments, the gene sequences(s) are controlled by a constitutive promoter, and are expressed in in vivo conditions and/or in vitro conditions, e.g., during expansion, production and/or manufacture, as described herein. 
     In some embodiments, any one or more of the described circuits are present on one or more plasmids (e.g., high copy or low copy) or are integrated into one or more sites in the microorganisms chromosome. Also, in some embodiments, the genetically engineered microorganisms are further capable of expressing any one or more of the described circuits and further comprise one or more of the following: (1) one or more auxotrophies, such as any auxotrophies known in the art and provided herein, e.g., thyA auxotrophy, (2) one or more kill switch circuits, such as any of the kill-switches described herein or otherwise known in the art, (3) one or more antibiotic resistance circuits, (4) one or more transporters for importing biological molecules or substrates, such any of the transporters described herein or otherwise known in the art, (5) one or more secretion circuits, such as any of the secretion circuits described herein and otherwise known in the art, (6) one or more surface display circuits, such as any of the surface display circuits described herein and otherwise known in the art and (7) one or more circuits for the production or degradation of one or more metabolites described herein (8) combinations of one or more of such additional circuits. 
     Non-limiting examples of proteins of interest include GLP-2 peptides, GLP-2 analogs, IL-22, vIL-10, hIL-10, monomerized IL-10, IL-27, IL-19, IL-20, IL-24, tryptophan synthesies enzymes, SCFA biosynthesis enzymes, tryptophan catabolic enzymes, including but not limited to IDO, TDO, kynureninase, other tryptophan pathway catabolic enzymes, e.g. in the indole pathway and/or the kynurenine pathway as described herein. These polypeptides may be mutated to increase stability, resistance to protease digestion, and/or activity. 
     
       
         
           
               
             
               
                 TABLE 32 
               
             
            
               
                   
               
               
                 Comparison of Secretion systems for secretion 
               
               
                 of polypeptide from engineered bacteria 
               
            
           
           
               
               
               
               
               
            
               
                 Secretion 
                   
                   
                   
                   
               
               
                 System 
                 Tag 
                 Cleavage 
                 Advantages 
                 Other features 
               
               
                   
               
               
                 Modified 
                 mRNA 
                 No 
                 No peptide tag 
                 May not be as 
               
               
                 Type III 
                 (or N- 
                 cleavage 
                 Endogenous 
                 suited for larger 
               
               
                 (flagellar) 
                 terminal) 
                 necessary 
                   
                 proteins 
               
               
                   
                   
                   
                   
                 Deletion of 
               
               
                   
                   
                   
                   
                 flagellar genes 
               
               
                 Type V 
                 N- and 
                 Yes 
                 Large proteins 
                 2-step secretion 
               
               
                 autotransport 
                 C- 
                   
                 Endogenous 
               
               
                   
                 terminal 
                   
                 Cleavable 
               
               
                 Type I 
                 C- 
                 No 
                   
                 Tag; Exogenous 
               
               
                   
                 terminal 
                   
                   
                 Machinery 
               
               
                 Diffusible 
                 N- 
                 Yes 
                 Disulfide bond 
                 May affect cell 
               
               
                 Outer 
                 terminal 
                   
                 formation 
                 fragility/ 
               
               
                 Membrane 
                   
                   
                   
                 survivability/ 
               
               
                 (DOM) 
                   
                   
                   
                 growth/yield 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the therapeutic polypeptides of interest are secreted using components of the flagellar type III secretion system. In a non-limiting example, such a therapeutic polypeptide of interest, such as, GLP-2 peptides, GLP-2 analogs, IL-22, vIL-10, hIL-10, monomerized IL-10, IL-27, IL-19, IL-20, IL-24, are secreted via Type I Hemolysin Secretion, is assembled behind a fliC-5′UTR (e.g., 173-bp untranslated region from the fliC loci), and is driven by the native promoter. In other embodiments, the expression of the therapeutic peptide of interested secreted using components of the flagellar type III secretion system is driven by a tet-inducible promoter. In alternate embodiments, an inducible promoter such as oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by IBD specific molecules or promoters induced by inflammation or an inflammatory response (RNS, ROS promoters), and promoters induced by a metabolite that may or may not be naturally present (e.g., can be exogenously added) in the gut, e.g., arabinose is used. In some embodiments, the therapeutic polypeptide of interest is expressed from a plasmid (e.g., a medium copy plasmid). In some embodiments, the therapeutic polypeptide of interest is expressed from a construct which is integrated into fliC locus (thereby deleting fliC), where it is driven by the native FliC promoter. In some embodiments, an N terminal part of FliC (e.g., the first 20 amino acids of FliC) is included in the construct, to further increase secretion efficiency. 
     In some embodiments, the therapeutic polypeptides of interest, e.g., GLP-2 peptides, GLP-2 analogs, IL-22, vIL-10, hIL-10, monomerized IL-10, IL-27, IL-19, IL-20, IL-24, are secreted via Type I Hemolysin Secretion, are secreted using via a diffusible outer membrane (DOM) system. In some embodiments, the therapeutic polypeptide of interest is fused to a N-terminal Sec-dependent secretion signal. Non-limiting examples of such N-terminal Sec-dependent secretion signals include PhoA, OmpF, OmpA, and cvaC. In alternate embodiments, the therapeutic polypeptide of interest is fused to a Tat-dependent secretion signal. Exemplary Tat-dependent tags include TorA, FdnG, and DmsA. 
     In certain embodiments, the genetically engineered bacteria comprise deletions or mutations in one or more of the outer membrane and/or periplasmic proteins. Non-limiting examples of such proteins, one or more of which may be deleted or mutated, include lpp, pal, tolA, and/or nlpI. In some embodiments, lpp is deleted or mutated. In some embodiments, pal is deleted or mutated. In some embodiments, tolA is deleted or mutated. In other embodiments, nlpl is deleted or mutated. In yet other embodiments, certain periplasmic proteases are deleted or mutated, e.g., to increase stability of the polypeptide in the periplasm. Non-limiting examples of such proteases include degP and ompT. In some embodiments, degP is deleted or mutated. In some embodiments, ompT is deleted or mutated. In some embodiments, degP and ompT are deleted or mutated. 
     In some embodiments, the therapeutic polypeptides of interest, e.g., GLP-2 peptides, GLP-2 analogs, IL-22, vIL-10, hIL-10, monomerized IL-10, IL-27, IL-19, IL-20, IL-24, are secreted via Type I Hemolysin Secretion, are secreted via a Type V Auto-secreter (pic Protein) Secretion. In some embodiments, the therapeutic protein of interest is expressed as a fusion protein with the native Nissle auto-secreter  E. coli _01635 (where the original passenger protein is replaced with the therapeutic polypeptides of interest. 
     In some embodiments, the therapeutic polypeptides of interest, e.g., GLP-2 peptides, GLP-2 analogs, IL-22, vIL-10, hIL-10, monomerized IL-10, IL-27, IL-19, IL-20, IL-24, are secreted via Type I Hemolysin Secretion, are secreted via Type 1 Hemolysin Secretion. In one embodiment, therapeutic polypeptide of interest is expressed as fusion protein with the 53 amino acids of the C terminus of alpha-hemolysin (hlyA) of  E. coli  CFT073. 
     Essential Genes and Auxotrophs 
     As used herein, the term “essential gene” refers to a gene which is necessary to for cell growth and/or survival. Bacterial essential genes are well known to one of ordinary skill in the art, and can be identified by directed deletion of genes and/or random mutagenesis and screening (see, e.g., Zhang and Lin, 2009, DEG 5.0, a database of essential genes in both prokaryotes and eukaryotes, Nucl. Acids Res., 37:D455-D458 and Gerdes et al., Essential genes on metabolic maps, Curr. Opin. Biotechnol., 17(5):448-456, the entire contents of each of which are expressly incorporated herein by reference). 
     An “essential gene” may be dependent on the circumstances and environment in which an organism lives. For example, a mutation of, modification of, or excision of an essential gene may result in the genetically engineered bacteria of the disclosure becoming an auxotroph. An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. 
     An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In some embodiments, any of the genetically engineered bacteria described herein also comprise a deletion or mutation in a gene required for cell survival and/or growth. In one embodiment, the essential gene is a DNA synthesis gene, for example, thyA. In another embodiment, the essential gene is a cell wall synthesis gene, for example, dapA. In yet another embodiment, the essential gene is an amino acid gene, for example, serA or MetA. Any gene required for cell survival and/or growth may be targeted, including but not limited to, cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thi1, as long as the corresponding wild-type gene product is not produced in the bacteria. 
     Table 33 lists depicts exemplary bacterial genes which may be disrupted or deleted to produce an auxotrophic strain. These include, but are not limited to, genes required for oligonucleotide synthesis, amino acid synthesis, and cell wall synthesis. 
     
       
         
           
               
             
               
                 TABLE 33 
               
             
            
               
                   
               
               
                 Non-limiting Examples of Bacterial Genes 
               
               
                 Useful for Generation of an Auxotroph 
               
            
           
           
               
               
               
               
            
               
                   
                 Amino Acid 
                 Oligonucleotide 
                 Cell Wall 
               
               
                   
                   
               
               
                   
                 cysE 
                 thyA 
                 dapA 
               
               
                   
                 glnA 
                 uraA 
                 dapB 
               
               
                   
                 ilvD 
                   
                 dapD 
               
               
                   
                 leuB 
                   
                 dapE 
               
               
                   
                 lysA 
                   
                 dapF 
               
               
                   
                 serA 
               
               
                   
                 metA 
               
               
                   
                 glyA 
               
               
                   
                 hisB 
               
               
                   
                 ilvA 
               
               
                   
                 pheA 
               
               
                   
                 proA 
               
               
                   
                 thrC 
               
               
                   
                 trpC 
               
               
                   
                 tyrA 
               
               
                   
                   
               
            
           
         
       
     
     Table 34 shows the survival of various amino acid auxotrophs in the mouse gut, as detected 24 hrs and 48 hrs post-gavage. These auxotrophs were generated using BW25113, a non-Nissle strain of  E. coli . 
     
       
         
           
               
             
               
                 TABLE 34 
               
             
            
               
                   
               
               
                 Survival of amino acid auxotrophs in the mouse gut 
               
            
           
           
               
               
               
               
               
            
               
                 Gene 
                 AA Auxotroph 
                 Pre-Gavage 
                 24 hours 
                 48 hours 
               
               
                   
               
               
                 argA 
                 Arginine 
                 Present 
                 Present 
                 Absent 
               
               
                 cysE 
                 Cysteine 
                 Present 
                 Present 
                 Absent 
               
               
                 glnA 
                 Glutamine 
                 Present 
                 Present 
                 Absent 
               
               
                 glyA 
                 Glycine 
                 Present 
                 Present 
                 Absent 
               
               
                 hisB 
                 Histidine 
                 Present 
                 Present 
                 Present 
               
               
                 ilvA 
                 Isoleucine 
                 Present 
                 Present 
                 Absent 
               
               
                 leuB 
                 Leucine 
                 Present 
                 Present 
                 Absent 
               
               
                 lysA 
                 Lysine 
                 Present 
                 Present 
                 Absent 
               
               
                 metA 
                 Methionine 
                 Present 
                 Present 
                 Present 
               
               
                 pheA 
                 Phenylalanine 
                 Present 
                 Present 
                 Present 
               
               
                 proA 
                 Proline 
                 Present 
                 Present 
                 Absent 
               
               
                 serA 
                 Serine 
                 Present 
                 Present 
                 Present 
               
               
                 thrC 
                 Threonine 
                 Present 
                 Present 
                 Present 
               
               
                 trpC 
                 Tryptophan 
                 Present 
                 Present 
                 Present 
               
               
                 tyrA 
                 Tyrosine 
                 Present 
                 Present 
                 Present 
               
               
                 ilvD 
                 Valine/Isoleucine/ 
                 Present 
                 Present 
                 Absent 
               
               
                   
                 Leucine 
               
               
                 thyA 
                 Thiamine 
                 Present 
                 Absent 
                 Absent 
               
               
                 uraA 
                 Uracil 
                 Present 
                 Absent 
                 Absent 
               
               
                 flhD 
                 FlhD 
                 Present 
                 Present 
                 Present 
               
               
                   
               
            
           
         
       
     
     For example, thymine is a nucleic acid that is required for bacterial cell growth; in its absence, bacteria undergo cell death. The thyA gene encodes thimidylate synthetase, an enzyme that catalyzes the first step in thymine synthesis by converting dUMP to dTMP (Sat et al., 2003). In some embodiments, the bacterial cell of the disclosure is a thyA auxotroph in which the thyA gene is deleted and/or replaced with an unrelated gene. A thyA auxotroph can grow only when sufficient amounts of thymine are present, e.g., by adding thymine to growth media in vitro, or in the presence of high thymine levels found naturally in the human gut in vivo. In some embodiments, the bacterial cell of the disclosure is auxotrophic in a gene that is complemented when the bacterium is present in the mammalian gut. Without sufficient amounts of thymine, the thyA auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut). 
     Diaminopimelic acid (DAP) is an amino acid synthetized within the lysine biosynthetic pathway and is required for bacterial cell wall growth (Meadow et al., 1959; Clarkson et al., 1971). In some embodiments, any of the genetically engineered bacteria described herein is a dapD auxotroph in which dapD is deleted and/or replaced with an unrelated gene. A dapD auxotroph can grow only when sufficient amounts of DAP are present, e.g., by adding DAP to growth media in vitro. Without sufficient amounts of DAP, the dapD auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut). 
     In other embodiments, the genetically engineered bacterium of the present disclosure is a uraA auxotroph in which uraA is deleted and/or replaced with an unrelated gene. The uraA gene codes for UraA, a membrane-bound transporter that facilitates the uptake and subsequent metabolism of the pyrimidine uracil (Andersen et al., 1995). A uraA auxotroph can grow only when sufficient amounts of uracil are present, e.g., by adding uracil to growth media in vitro. Without sufficient amounts of uracil, the uraA auxotroph dies. In some embodiments, auxotrophic modifications are used to ensure that the bacteria do not survive in the absence of the auxotrophic gene product (e.g., outside of the gut). 
     In complex communities, it is possible for bacteria to share DNA. In very rare circumstances, an auxotrophic bacterial strain may receive DNA from a non-auxotrophic strain, which repairs the genomic deletion and permanently rescues the auxotroph. Therefore, engineering a bacterial strain with more than one auxotroph may greatly decrease the probability that DNA transfer will occur enough times to rescue the auxotrophy. In some embodiments, the genetically engineered bacteria of the invention comprise a deletion or mutation in two or more genes required for cell survival and/or growth. 
     Other examples of essential genes include, but are not limited to yhbV, yagG, hemB, secD, secF, ribD, ribE, thiL, dxs, ispA, dnaX, adk, hemH, lpxH, cysS, fold, rplT, infC, thrS, nadE, gapA, yeaZ, aspS, argS, pgsA, yefM, metG, folE, yejM, gyrA, nrdA, nrdB, folC, accD, fabB, gltX, ligA, zipA, dapE, dapA, der, hisS, ispG, suhB, tadA, acpS, era, mec, ftsB, eno, pyrG, chpR, lgt, fbaA, pgk, yqgD, metK, yqgF, plsC, ygiT, pare, ribB, cca, ygjD, tdcF, yraL, yihA, ftsN, murl, murB, birA, secE, nusG, rplJ, rplL, rpoB, rpoC, ubiA, plsB, lexA, dnaB, ssb, alsK, groS, psd, orn, yjeE, rpsR, chpS, ppa, valS, yjgP, yjgQ, dnaC, ribF, lspA, ispH, dapB, folA, imp, yabQ, ftsL, ftsI, murE, murF, mraY, murD, ftsW, murG, murC, ftsQ, ftsA, ftsZ, lpxC, secM, secA, can, folK, hemL, yadR, dapD, map, rpsB, infB, nusA, ftsH, obgE, rpmA, rplU, ispB, murA, yrbB, yrbK, yhbN, rpsl, rplM, degS, mreD, mreC, mreB, accB, accC, yrdC, def, fmt, rplQ, rpoA, rpsD, rpsK, rpsM, entD, mrdB, mrdA, nadD, hlepB, rpoE, pssA, yfiO, rplS, trmD, rpsP, ffli, grpE, yfjB, csrA, ispF, ispD, rplW, rplD, rplC, rpsJ, fusA, rpsG, rpsL, trpS, yrfF, asd, rpoH, ftsX, ftsE, ftsY, frr, dxr, ispU, rfaK, kdtA, coaD, rpmB, dfp, dut, gmk, spot, gyrB, dnaN, dnaA, rpmH, rnpA, yidC, tnaB, glmS, glmU, wzyE, hemD, hemC, yigP, ubiB, ubiD, hemG, secY, rplO, rpmD, rpsE, rplR, rplF, rpsH, rpsN, rplE, rplX, rplN, rpsQ, rpmC, rplP, rpsC, rplV, rpsS, rplB, cdsA, yaeL, yaeT, lpxD, fabZ, lpxA, lpxB, dnaE, accA, tilS, proS, yafF, tsf, pyrH, olA, rlpB, leuS, Int, ginS, fldA, cydA, infA, cydC, ftsK, lolA, serS, rpsA, msbA, lpxK, kdsB, mukF, mukE, mukB, asnS, fabA, mviN, me, yceQ, fabD, fabG, acpP, tmk, holB, lolC, lolD, lolE, purB, ymfK, minE, mind, pth, rsA, ispE, lolB, hemA, prfA, prmC, kdsA, topA, ribA, fabl, racR, dicA, ydfB, tyrS, ribC, ydiL, pheT, pheS, yhhQ, bcsB, glyQ, yibJ, and gpsA. Other essential genes are known to those of ordinary skill in the art. 
     In some embodiments, the genetically engineered bacterium of the present disclosure is a synthetic ligand-dependent essential gene (SLiDE) bacterial cell. SLiDE bacterial cells are synthetic auxotrophs with a mutation in one or more essential genes that only grow in the presence of a particular ligand (see Lopez and Anderson “Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21 (DE3 Biosafety Strain,” ACS Synthetic Biology (2015) DOI: 10.1021/acssynbio.5b00085, the entire contents of which are expressly incorporated herein by reference). 
     In some embodiments, the SLiDE bacterial cell comprises a mutation in an essential gene. In some embodiments, the essential gene is selected from the group consisting of pheS, dnaN, tyrS, metG, and adk. In some embodiments, the essential gene is dnaN comprising one or more of the following mutations: H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is dnaN comprising the mutations H191N, R240C, I317S, F319V, L340T, V347I, and S345C. In some embodiments, the essential gene is pheS comprising one or more of the following mutations: F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is pheS comprising the mutations F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is tyrS comprising one or more of the following mutations: L36V, C38A and F40G. In some embodiments, the essential gene is tyrS comprising the mutations L36V, C38A and F40G. In some embodiments, the essential gene is metG comprising one or more of the following mutations: E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is metG comprising the mutations E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is adk comprising one or more of the following mutations: I4L, L5I and L6G. In some embodiments, the essential gene is adk comprising the mutations I4L, L5I and L6G. 
     In some embodiments, the genetically engineered bacterium is complemented by a ligand. In some embodiments, the ligand is selected from the group consisting of benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid, and L-histidine methyl ester. For example, bacterial cells comprising mutations in metG (E45Q, N47R, I49G, and A51C) are complemented by benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid or L-histidine methyl ester. Bacterial cells comprising mutations in dnaN (H191N, R240C, I317S, F319V, L340T, V347I, and S345C) are complemented by benzothiazole, indole or 2-aminobenzothiazole. Bacterial cells comprising mutations in pheS (F125G, P183T, P184A, R186A, and I188L) are complemented by benzothiazole or 2-aminobenzothiazole. Bacterial cells comprising mutations in tyrS (L36V, C38A, and F40G) are complemented by benzothiazole or 2-aminobenzothiazole. Bacterial cells comprising mutations in adk (I4L, L5I and L6G) are complemented by benzothiazole or indole. 
     In some embodiments, the genetically engineered bacterium comprises more than one mutant essential gene that renders it auxotrophic to a ligand. In some embodiments, the bacterial cell comprises mutations in two essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G) and metG (E45Q, N47R, I49G, and A51C). In other embodiments, the bacterial cell comprises mutations in three essential genes. For example, in some embodiments, the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G), metG (E45Q, N47R, I49G, and A51C), and pheS (F125G, P183T, P184A, R186A, and I188L). 
     In some embodiments, the genetically engineered bacterium is a conditional auxotroph whose essential gene(s) is replaced using the arabinose system shown in  FIG. 60 . 
     In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill-switch components and systems described herein. For example, the genetically engineered bacteria may comprise a deletion or mutation in an essential gene required for cell survival and/or growth, for example, in a DNA synthesis gene, for example, thyA, cell wall synthesis gene, for example, dapA and/or an amino acid gene, for example, serA or MetA and may also comprise a toxin gene that is regulated by one or more transcriptional activators that are expressed in response to an environmental condition(s) and/or signal(s) (such as the described arabinose system) or regulated by one or more recombinases that are expressed upon sensing an exogenous environmental condition(s) and/or signal(s) (such as the recombinase systems described herein). Other embodiments are described in Wright et al., “GeneGuard: A Modular Plasmid System Designed for Biosafety,” ACS Synthetic Biology (2015) 4: 307-16, the entire contents of which are expressly incorporated herein by reference). In some embodiments, the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill-switch components and systems described herein, as well as another biosecurity system, such a conditional origin of replication (Wright et al., 2015). In other embodiments, auxotrophic modifications may also be used to screen for mutant bacteria that produce the anti-inflammatory or gut barrier enhancer molecule. 
     Genetic Regulatory Circuits 
     In some embodiments, the genetically engineered bacteria comprise multi-layered genetic regulatory circuits for expressing the constructs described herein (see, e.g., U.S. Provisional Application No. 62/184,811 and PCT/US2016/39434, both of which are incorporated herein by reference in their entireties). The genetic regulatory circuits are useful to screen for mutant bacteria that produce an anti-inflammation and/or gut barrier enhancer molecule or rescue an auxotroph. In certain embodiments, the invention provides methods for selecting genetically engineered bacteria that produce one or more genes of interest. 
     In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a therapeutic molecule (e.g., butyrate) and a T7 polymerase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a T7 polymerase, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a therapeutic molecule (e.g., butyrate), wherein the second gene or gene cassette is operably linked to a T7 promoter that is induced by the T7 polymerase; and a third gene encoding an inhibitory factor, lysY, that is capable of inhibiting the T7 polymerase. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, and the therapeutic molecule (e.g., butyrate) is not expressed. LysY is expressed constitutively (P-lac constitutive) and further inhibits T7 polymerase. In the absence of oxygen, FNR dimerizes and binds to the FNR-responsive promoter, T7 polymerase is expressed at a level sufficient to overcome lysY inhibition, and the therapeutic molecule (e.g., butyrate) is expressed. In some embodiments, the lysY gene is operably linked to an additional FNR binding site. In the absence of oxygen, FNR dimerizes to activate T7 polymerase expression as described above, and also inhibits lysY expression. 
     In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a therapeutic molecule (e.g., butyrate) and a protease-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding an mf-lon protease, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a therapeutic molecule operably linked to a Tet regulatory region (TetO); and a third gene encoding an mf-lon degradation signal linked to a Tet repressor (TetR), wherein the TetR is capable of binding to the Tet regulatory region and repressing expression of the second gene or gene cassette. The mf-lon protease is capable of recognizing the mf-ion degradation signal and degrading the TetR. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the repressor is not degraded, and the therapeutic molecule is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, thereby inducing expression of the mf-lon protease. The mf-lon protease recognizes the mf-lon degradation signal and degrades the TetR, and the therapeutic molecule is expressed. 
     In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a therapeutic molecule and a repressor-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a first repressor, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a therapeutic molecule operably linked to a first regulatory region comprising a constitutive promoter; and a third gene encoding a second repressor, wherein the second repressor is capable of binding to the first regulatory region and repressing expression of the second gene or gene cassette. The third gene is operably linked to a second regulatory region comprising a constitutive promoter, wherein the first repressor is capable of binding to the second regulatory region and inhibiting expression of the second repressor. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the first repressor is not expressed, the second repressor is expressed, and the therapeutic molecule is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the first repressor is expressed, the second repressor is not expressed, and the therapeutic molecule is expressed. 
     Examples of repressors useful in these embodiments include, but are not limited to, ArgR, TetR, ArsR, AscG, LacI, CscR, DeoR, DgoR, FruR, GalR, GatR, CI, LexA, RafR, QacR, and PtxS (US20030166191). 
     In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a therapeutic molecule and a regulatory RNA-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a regulatory RNA, wherein the first gene is operably linked to a FNR-responsive promoter, and a second gene or gene cassette for producing a therapeutic molecule. The second gene or gene cassette is operably linked to a constitutive promoter and further linked to a nucleotide sequence capable of producing an mRNA hairpin that inhibits translation of the therapeutic molecule. The regulatory RNA is capable of eliminating the mRNA hairpin and inducing translation via the ribosomal binding site. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the regulatory RNA is not expressed, and the mRNA hairpin prevents the therapeutic molecule from being translated. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the regulatory RNA is expressed, the mRNA hairpin is eliminated, and the therapeutic molecule is expressed. 
     In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a therapeutic molecule and a CRISPR-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a Cas9 protein; a first gene encoding a CRISPR guide RNA, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a therapeutic molecule, wherein the second gene or gene cassette is operably linked to a regulatory region comprising a constitutive promoter; and a third gene encoding a repressor operably linked to a constitutive promoter, wherein the repressor is capable of binding to the regulatory region and repressing expression of the second gene or gene cassette. The third gene is further linked to a CRISPR target sequence that is capable of binding to the CRISPR guide RNA, wherein said binding to the CRISPR guide RNA induces cleavage by the Cas9 protein and inhibits expression of the repressor. In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the guide RNA is not expressed, the repressor is expressed, and the therapeutic molecule is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the guide RNA is expressed, the repressor is not expressed, and the therapeutic molecule is expressed. 
     In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a therapeutic molecule and a recombinase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a recombinase, wherein the first gene is operably linked to a FNR-responsive promoter, and a second gene or gene cassette for producing a therapeutic molecule operably linked to a constitutive promoter. The second gene or gene cassette is inverted in orientation (3′ to 5′) and flanked by recombinase binding sites, and the recombinase is capable of binding to the recombinase binding sites to induce expression of the second gene or gene cassette by reverting its orientation (5′ to 3′). In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the recombinase is not expressed, the gene or gene cassette remains in the 3′ to 5′ orientation, and no functional therapeutic molecule is produced. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the recombinase is expressed, the gene or gene cassette is reverted to the 5′ to 3′ orientation, and a functional therapeutic molecule is produced. 
     In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a therapeutic molecule and a polymerase- and recombinase-regulated genetic regulatory circuit. For example, the genetically engineered bacteria comprise a first gene encoding a recombinase, wherein the first gene is operably linked to a FNR-responsive promoter; a second gene or gene cassette for producing a therapeutic molecule operably linked to a T7 promoter; a third gene encoding a T7 polymerase, wherein the T7 polymerase is capable of binding to the T7 promoter and inducing expression of the therapeutic molecule. The third gene encoding the T7 polymerase is inverted in orientation (3′ to 5′) and flanked by recombinase binding sites, and the recombinase is capable of binding to the recombinase binding sites to induce expression of the T7 polymerase gene by reverting its orientation (5′ to 3′). In the presence of oxygen, FNR does not bind the FNR-responsive promoter, the recombinase is not expressed, the T7 polymerase gene remains in the 3′ to 5′ orientation, and the therapeutic molecule is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the recombinase is expressed, the T7 polymerase gene is reverted to the 5′ to 3′ orientation, and the therapeutic molecule is expressed. 
     Synthetic gene circuits expressed on plasmids may function well in the short term but lose ability and/or function in the long term (Danino et al., 2015). In some embodiments, the genetically engineered bacteria comprise stable circuits for expressing genes of interest over prolonged periods. In some embodiments, the genetically engineered bacteria are capable of producing a therapeutic molecule and further comprise a toxin-anti-toxin system that simultaneously produces a toxin (hok) and a short-lived anti-toxin (sok), wherein loss of the plasmid causes the cell to be killed by the long-lived toxin (Danino et al., 2015). In some embodiments, the genetically engineered bacteria further comprise alp7 from  B. subtilis  plasmid pL20 and produces filaments that are capable of pushing plasmids to the poles of the cells in order to ensure equal segregation during cell division (Danino et al., 2015). 
     Host-Plasmid Mutual Dependency 
     In some embodiments, the genetically engineered bacteria of the invention also comprise a plasmid that has been modified to create a host-plasmid mutual dependency. In certain embodiments, the mutually dependent host-plasmid platform is GeneGuard (Wright et al., 2015). In some embodiments, the GeneGuard plasmid comprises (i) a conditional origin of replication, in which the requisite replication initiator protein is provided in trans; (ii) an auxotrophic modification that is rescued by the host via genomic translocation and is also compatible for use in rich media; and/or (iii) a nucleic acid sequence which encodes a broad-spectrum toxin. The toxin gene may be used to select against plasmid spread by making the plasmid DNA itself disadvantageous for strains not expressing the anti-toxin (e.g., a wild-type bacterium). In some embodiments, the GeneGuard plasmid is stable for at least 100 generations without antibiotic selection. In some embodiments, the GeneGuard plasmid does not disrupt growth of the host. The GeneGuard plasmid is used to greatly reduce unintentional plasmid propagation in the genetically engineered bacteria of the invention. 
     The mutually dependent host-plasmid platform may be used alone or in combination with other biosafety mechanisms, such as those described herein (e.g., kill switches, auxotrophies). In some embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more kill switches. In other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid and/or one or more auxotrophies. In still other embodiments, the genetically engineered bacteria comprise a GeneGuard plasmid, one or more kill switches, and/or one or more auxotrophies. 
     Synthetic gene circuits express on plasmids may function well in the short term but lose ability and/or function in the long term (Danino et al., 2015). In some embodiments, the genetically engineered bacteria comprise stable circuits for expressing genes of interest over prolonged periods. In some embodiments, the genetically engineered bacteria are capable of producing an anti-inflammation and/or gut enhancer molecule and further comprise a toxin-anti-toxin system that simultaneously produces a toxin (hok) and a short-lived anti-toxin (sok), wherein loss of the plasmid causes the cell to be killed by the long-lived toxin (Danino et al., 2015; as shown in the figures and examples). In some embodiments, the genetically engineered bacteria further comprise alp7 from  B. subtilis  plasmid pL20 and produces filaments that are capable of pushing plasmids to the poles of the cells in order to ensure equal segregation during cell division (Danino et al., 2015). 
     Kill Switch 
     In some embodiments, the genetically engineered bacteria of the invention also comprise a kill switch (see, e.g., U.S. Provisional Application Nos. 62/183,935, 62/263,329, and 62/277,654, each of which is incorporated herein by reference in their entireties). The kill switch is intended to actively kill genetically engineered bacteria in response to external stimuli. As opposed to an auxotrophic mutation where bacteria die because they lack an essential nutrient for survival, the kill switch is triggered by a particular factor in the environment that induces the production of toxic molecules within the microbe that cause cell death. 
     Bacteria comprising kill switches have been engineered for in vitro research purposes, e.g., to limit the spread of a biofuel-producing microorganism outside of a laboratory environment. Bacteria engineered for in vivo administration to treat a disease may also be programmed to die at a specific time after the expression and delivery of a heterologous gene or genes, for example, an anti-inflammation and/or gut barrier enhancer molecule, or after the subject has experienced the therapeutic effect. For example, in some embodiments, the kill switch is activated to kill the bacteria after a period of time following expression of the anti-inflammation and/or gut barrier enhancer molecule, e.g., GLP-2. In some embodiments, the kill switch is activated in a delayed fashion following expression of the anti-inflammation and/or gut barrier enhancer molecule. Alternatively, the bacteria may be engineered to die after the bacterium has spread outside of a disease site. Specifically, it may be useful to prevent long-term colonization of subjects by the microorganism, spread of the microorganism outside the area of interest (for example, outside the gut) within the subject, or spread of the microorganism outside of the subject into the environment (for example, spread to the environment through the stool of the subject). Examples of such toxins that can be used in kill-switches include, but are not limited to, bacteriocins, lysins, and other molecules that cause cell death by lysing cell membranes, degrading cellular DNA, or other mechanisms. Such toxins can be used individually or in combination. The switches that control their production can be based on, for example, transcriptional activation (toggle switches; see, e.g., Gardner et al., 2000), translation (riboregulators), or DNA recombination (recombinase-based switches), and can sense environmental stimuli such as anaerobiosis or reactive oxygen species. These switches can be activated by a single environmental factor or may require several activators in AND, OR, NAND and NOR logic configurations to induce cell death. For example, an AND riboregulator switch is activated by tetracycline, isopropyl β-D-1-thiogalactopyranoside (IPTG), and arabinose to induce the expression of lysins, which permeabilize the cell membrane and kill the cell. IPTG induces the expression of the endolysin and holin mRNAs, which are then derepressed by the addition of arabinose and tetracycline. All three inducers must be present to cause cell death. Examples of kill switches are known in the art (Callura et al., 2010). 
     Kill-switches can be designed such that a toxin is produced in response to an environmental condition or external signal (e.g., the bacteria is killed in response to an external cue) or, alternatively designed such that a toxin is produced once an environmental condition no longer exists or an external signal is ceased. 
     Thus, in some embodiments, the genetically engineered bacteria of the disclosure are further programmed to die after sensing an exogenous environmental signal, for example, in low-oxygen conditions, in the presence of ROS, or in the presence of RNS. In some embodiments, the genetically engineered bacteria of the present disclosure comprise one or more genes encoding one or more recombinase(s), whose expression is induced in response to an environmental condition or signal and causes one or more recombination events that ultimately leads to the expression of a toxin which kills the cell. In some embodiments, the at least one recombination event is the flipping of an inverted heterologous gene encoding a bacterial toxin which is then constitutively expressed after it is flipped by the first recombinase. In one embodiment, constitutive expression of the bacterial toxin kills the genetically engineered bacterium. In these types of kill-switch systems once the engineered bacterial cell senses the exogenous environmental condition and expresses the heterologous gene of interest, the recombinant bacterial cell is no longer viable. 
     In another embodiment in which the genetically engineered bacteria of the present disclosure express one or more recombinase(s) in response to an environmental condition or signal causing at least one recombination event, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to an exogenous environmental condition or signal. In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a bacterial toxin by a first recombinase. In one embodiment, the inverted heterologous gene encoding the bacterial toxin is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the anti-toxin inhibits the activity of the toxin, thereby delaying death of the genetically engineered bacterium. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin when the heterologous gene encoding the anti-toxin is no longer expressed when the exogenous environmental condition is no longer present. 
     In another embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by the flipping of an inverted heterologous gene encoding a bacterial toxin by the second recombinase. In one embodiment, the inverted heterologous gene encoding the second recombinase is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the inverted heterologous gene encoding the bacterial toxin is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the second recombinase is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the heterologous gene encoding the bacterial toxin is constitutively expressed after it is flipped by the second recombinase. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin. In one embodiment, the genetically engineered bacterium further expresses a heterologous gene encoding an anti-toxin in response to the exogenous environmental condition. In one embodiment, the anti-toxin inhibits the activity of the toxin when the exogenous environmental condition is present, thereby delaying death of the genetically engineered bacterium. In one embodiment, the genetically engineered bacterium is killed by the bacterial toxin when the heterologous gene encoding the anti-toxin is no longer expressed when the exogenous environmental condition is no longer present. 
     In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a second recombinase by a first recombinase, followed by flipping of an inverted heterologous gene encoding a third recombinase by the second recombinase, followed by flipping of an inverted heterologous gene encoding a bacterial toxin by the third recombinase. 
     In one embodiment, the at least one recombination event is flipping of an inverted heterologous gene encoding a first excision enzyme by a first recombinase. In one embodiment, the inverted heterologous gene encoding the first excision enzyme is located between a first forward recombinase recognition sequence and a first reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the first excision enzyme is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the first excision enzyme excises a first essential gene. In one embodiment, the programmed recombinant bacterial cell is not viable after the first essential gene is excised. 
     In one embodiment, the first recombinase further flips an inverted heterologous gene encoding a second excision enzyme. In one embodiment, the inverted heterologous gene encoding the second excision enzyme is located between a second forward recombinase recognition sequence and a second reverse recombinase recognition sequence. In one embodiment, the heterologous gene encoding the second excision enzyme is constitutively expressed after it is flipped by the first recombinase. In one embodiment, the genetically engineered bacterium dies or is no longer viable when the first essential gene and the second essential gene are both excised. In one embodiment, the genetically engineered bacterium dies or is no longer viable when either the first essential gene is excised or the second essential gene is excised by the first recombinase. 
     In one embodiment, the genetically engineered bacterium dies after the at least one recombination event occurs. In another embodiment, the genetically engineered bacterium is no longer viable after the at least one recombination event occurs. 
     In any of these embodiment, the recombinase can be a recombinase selected from the group consisting of: BxbI, PhiC31, TP901, BxbI, PhiC31, TP901, HK022, HP1, R4, Int1, Int2, Int3, Int4, Int5, Int6, Int7, Int8, Int9, Int10, Int11, Int12, Int13, Int14, Int15, Int16, Int17, Int18, Int19, Int20, Int21, Int22, Int23, Int24, Int25, Int26, Int27, Int28, Int29, Int30, Int31, Int32, Int33, and Int34, or a biologically active fragment thereof. 
     In the above-described kill-switch circuits, a toxin is produced in the presence of an environmental factor or signal. In another aspect of kill-switch circuitry, a toxin may be repressed in the presence of an environmental factor (not produced) and then produced once the environmental condition or external signal is no longer present. Such kill switches are called repression-based kill switches and represent systems in which the bacterial cells are viable only in the presence of an external factor or signal, such as arabinose or other sugar. Exemplary kill switch designs in which the toxin is repressed in the presence of an external factor or signal (and activated once the external signal is removed) is shown in  FIGS. 57, 60, 65 . The disclosure provides recombinant bacterial cells which express one or more heterologous gene(s) upon sensing arabinose or other sugar in the exogenous environment. In this aspect, the recombinant bacterial cells contain the araC gene, which encodes the AraC transcription factor, as well as one or more genes under the control of the araBAD promoter. In the absence of arabinose, the AraC transcription factor adopts a conformation that represses transcription of genes under the control of the araBAD promoter. In the presence of arabinose, the AraC transcription factor undergoes a conformational change that allows it to bind to and activate the araBAD promoter, which induces expression of the desired gene, for example tetR, which represses expression of a toxin gene. In this embodiment, the toxin gene is repressed in the presence of arabinose or other sugar. In an environment where arabinose is not present, the tetR gene is not activated and the toxin is expressed, thereby killing the bacteria. The arabinose system can also be used to express an essential gene, in which the essential gene is only expressed in the presence of arabinose or other sugar and is not expressed when arabinose or other sugar is absent from the environment. 
     Thus, in some embodiments in which one or more heterologous gene(s) are expressed upon sensing arabinose in the exogenous environment, the one or more heterologous genes are directly or indirectly under the control of the araBAD promoter (P araBAD ). In some embodiments, the expressed heterologous gene is selected from one or more of the following: a heterologous therapeutic gene, a heterologous gene encoding an anti-toxin, a heterologous gene encoding a repressor protein or polypeptide, for example, a TetR repressor, a heterologous gene encoding an essential protein not found in the bacterial cell, and/or a heterologous encoding a regulatory protein or polypeptide. 
     Arabinose inducible promoters are known in the art, including P ara , P araB , P araC , and P araBAD . In one embodiment, the arabinose inducible promoter is from  E. coli . In some embodiments, the P araC  promoter and the P araBAD  promoter operate as a bidirectional promoter, with the P araBAD  promoter controlling expression of a heterologous gene(s) in one direction, and the P araC  (in close proximity to, and on the opposite strand from the P araBAD  promoter), controlling expression of a heterologous gene(s) in the other direction. In the presence of arabinose, transcription of both heterologous genes from both promoters is induced. However, in the absence of arabinose, transcription of both heterologous genes from both promoters is not induced. 
     In one exemplary embodiment of the disclosure, the genetically engineered bacteria of the present disclosure contains a kill-switch having at least the following sequences: a P araBAD  promoter operably linked to a heterologous gene encoding a Tetracycline Repressor Protein (TetR), a P araC  promoter operably linked to a heterologous gene encoding AraC transcription factor, and a heterologous gene encoding a bacterial toxin operably linked to a promoter which is repressed by the Tetracycline Repressor Protein (P TetR ). In the presence of arabinose, the AraC transcription factor activates the P araBAD  promoter, which activates transcription of the TetR protein which, in turn, represses transcription of the toxin. In the absence of arabinose, however, AraC suppresses transcription from the the P araBAD  promoter and no TetR protein is expressed. In this case, expression of the heterologous toxin gene is activated, and the toxin is expressed. The toxin builds up in the recombinant bacterial cell, and the recombinant bacterial cell is killed. In one embodiment, the araC gene encoding the AraC transcription factor is under the control of a constitutive promoter and is therefore constitutively expressed. 
     In one embodiment of the disclosure, the genetically engineered bacterium further comprises an anti-toxin under the control of a constitutive promoter. In this situation, in the presence of arabinose, the toxin is not expressed due to repression by TetR protein, and the anti-toxin protein builds-up in the cell. However, in the absence of arabinose, TetR protein is not expressed, and expression of the toxin is induced. The toxin begins to build-up within the recombinant bacterial cell. The recombinant bacterial cell is no longer viable once the toxin protein is present at either equal or greater amounts than that of the anti-toxin protein in the cell, and the recombinant bacterial cell will be killed by the toxin. 
     In another embodiment of the disclosure, the genetically engineered bacterium further comprises an anti-toxin under the control of the P araBAD  promoter. In this situation, in the presence of arabinose, TetR and the anti-toxin are expressed, the anti-toxin builds up in the cell, and the toxin is not expressed due to repression by TetR protein. However, in the absence of arabinose, both the TetR protein and the anti-toxin are not expressed, and expression of the toxin is induced. The toxin begins to build-up within the recombinant bacterial cell. The recombinant bacterial cell is no longer viable once the toxin protein is expressed, and the recombinant bacterial cell will be killed by the toxin. 
     In another exemplary embodiment of the disclosure, the genetically engineered bacteria of the present disclosure contains a kill-switch having at least the following sequences: a P araBAD  promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell (and required for survival), and a P araC  promoter operably linked to a heterologous gene encoding AraC transcription factor. In the presence of arabinose, the AraC transcription factor activates the P araBAD  promoter, which activates transcription of the heterologous gene encoding the essential polypeptide, allowing the recombinant bacterial cell to survive. In the absence of arabinose, however, AraC suppresses transcription from the the P araBAD  promoter and the essential protein required for survival is not expressed. In this case, the recombinant bacterial cell dies in the absence of arabinose. In some embodiments, the sequence of P araBAD  promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin kill-switch system described directly above. In some embodiments, the sequence of P araBAD  promoter operably linked to a heterologous gene encoding an essential polypeptide not found in the recombinant bacterial cell can be present in the bacterial cell in conjunction with the TetR/toxin/anti-toxin kill-switch system described directly above. 
     In yet other embodiments, the bacteria may comprise a plasmid stability system with a plasmid that produces both a short-lived anti-toxin and a long-lived toxin. In this system, the bacterial cell produces equal amounts of toxin and anti-toxin to neutralize the toxin. However, if/when the cell loses the plasmid, the short-lived anti-toxin begins to decay. When the anti-toxin decays completely the cell dies as a result of the longer-lived toxin killing it. 
     In some embodiments, the engineered bacteria of the present disclosure further comprise the gene(s) encoding the components of any of the above-described kill-switch circuits. 
     In any of the above-described embodiments, the bacterial toxin may be selected from the group consisting of a lysin, Hok, Fst, TisB, LdrD, Kid, SymE, MazF, FlmA, Ibs, XCV2162, dinJ, CcdB, MazF, ParE, YafO, Zeta, hicB, relB, yhaV, yoeB, chpBK, hipA, microcin B, microcin B17, microcin C, microcin C7-C51, microcin J25, microcin ColV, microcin 24, microcin L, microcin D93, microcin L, microcin E492, microcin H47, microcin 147, microcin M, colicin A, colicin E1, colicin K, colicin N, colicin U, colicin B, colicin Ia, colicin Ib, colicin 5, colicin10, colicin S4, colicin Y, colicin E2, colicin E7, colicin E8, colicin E9, colicin E3, colicin E4, colicin E6, colicin E5, colicin D, colicin M, and cloacin DF13, or a biologically active fragment thereof. 
     In any of the above-described embodiments, the anti-toxin may be selected from the group consisting of an anti-lysin, Sok, RNAII, IstR, RdlD, Kis, SymR, MazE, FlmB, Sib, ptaRNA1, yafQ, CcdA, MazE, ParD, yafN, Epsilon, HicA, relE, prlF, yefM, chpBI, hipB, MccE, MccE CTD , MccF, Cai, ImmE1, Cki, Cni, Cui, Cbi, Iia, Imm, Cfi, Im10, Csi, Cyi, Im2, Im7, Im8, Im9, Im3, Im4, ImmE6, cloacin immunity protein (Cim), ImmE5, ImmD, and Cmi, or a biologically active fragment thereof. 
     In one embodiment, the bacterial toxin is bactericidal to the genetically engineered bacterium. In one embodiment, the bacterial toxin is bacteriostatic to the genetically engineered bacterium. 
     In some embodiments, the genetically engineered bacterium provided herein is an auxotroph. In one embodiment, the genetically engineered bacterium is an auxotroph selected from a cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thi1 auxotroph. In some embodiments, the engineered bacteria have more than one auxotrophy, for example, they may be a ΔthyA and ΔdapA auxotroph. 
     In some embodiments, the genetically engineered bacterium provided herein further comprises a kill-switch circuit, such as any of the kill-switch circuits provided herein. For example, in some embodiments, the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and an inverted toxin sequence. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an anti-toxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene. In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an anti-toxin. In some embodiments, the engineered bacteria further comprise one or more genes encoding a toxin under the control of a promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as P araBAD . In some embodiments, the genetically engineered bacteria further comprise one or more genes encoding an anti-toxin. 
     In some embodiments, the genetically engineered bacterium is an auxotroph comprising a therapeutic payload and further comprises a kill-switch circuit, such as any of the kill-switch circuits described herein. 
     In some embodiments of the above described genetically engineered bacteria, the gene or gene cassette for producing the anti-inflammation and/or gut barrier enhancer molecule is present on a plasmid in the bacterium and operatively linked on the plasmid to the inducible promoter. In other embodiments, the gene or gene cassette for producing the anti-inflammation and/or gut barrier enhancer molecule is present in the bacterial chromosome and is operatively linked in the chromosome to the inducible promoter. 
     Methods of Screening 
     Mutagenesis 
     In some embodiments, the inducible promoter is operably linked to a detectable product, e.g., GFP, and can be used to screen for mutants. In some embodiments, the inducible promoter is mutagenized, and mutants are selected based upon the level of detectable product, e.g., by flow cytometry, fluorescence-activated cell sorting (FACS) when the detectable product fluoresces. In some embodiments, one or more transcription factor binding sites is mutagenized to increase or decrease binding. In alternate embodiments, the wild-type binding sites are left intact and the remainder of the regulatory region is subjected to mutagenesis. In some embodiments, the mutant promoter is inserted into the genetically engineered bacteria of the invention to increase expression of the anti-inflammation and/or gut barrier enhancer molecule under inducing conditions, as compared to unmutated bacteria of the same subtype under the same conditions. In some embodiments, the inducible promoter and/or corresponding transcription factor is a synthetic, non-naturally occurring sequence. 
     In some embodiments, the gene encoding an anti-inflammation and/or gut barrier enhancer molecule is mutated to increase expression and/or stability of said molecule under inducing conditions, as compared to unmutated bacteria of the same subtype under the same conditions. In some embodiments, one or more of the genes in a gene cassette for producing an anti-inflammation and/or gut barrier enhancer molecule is mutated to increase expression of said molecule under inducing conditions, as compared to unmutated bacteria of the same subtype under the same conditions. In some embodiments, the efficacy or activity of any of the importers and exporters for metabolites of interest can be improved through mutations in any of these genes. Mutations increase uptake or export of such metabolites, including but not limited to, tryptophan, e.g., under inducing conditions, as compared to unmutated bacteria of the same subtype under the same conditions. Methods for directed mutation and screening are known in the art. 
     Generation of Bacterial Strains with Enhance Ability to Transport Metabolites of Interest 
     Due to their ease of culture, short generation times, very high population densities and small genomes, microbes can be evolved to unique phenotypes in abbreviated timescales. Adaptive laboratory evolution (ALE) is the process of passaging microbes under selective pressure to evolve a strain with a preferred phenotype. Most commonly, this is applied to increase utilization of carbon/energy sources or adapting a strain to environmental stresses (e.g., temperature, pH), whereby mutant strains more capable of growth on the carbon substrate or under stress will outcompete the less adapted strains in the population and will eventually come to dominate the population. 
     This same process can be extended to any essential metabolite by creating an auxotroph. An auxotroph is a strain incapable of synthesizing an essential metabolite and must therefore have the metabolite provided in the media to grow. In this scenario, by making an auxotroph and passaging it on decreasing amounts of the metabolite, the resulting dominant strains should be more capable of obtaining and incorporating this essential metabolite. 
     For example, if the biosynthetic pathway for producing a metabolite of interest is disrupted a strain capable of high-affinity capture of the metabolite of interest can be evolved via ALE. First, the strain is grown in varying concentrations of the auxotrophic metabolite of interest, until a minimum concentration to support growth is established. The strain is then passaged at that concentration, and diluted into lowering concentrations of the metabolite of interest at regular intervals. Over time, cells that are most competitive for the metabolite of interest—at growth-limiting concentrations—will come to dominate the population. These strains will likely have mutations in their metabolite of interest-transporters resulting in increased ability to import the essential and limiting metabolite of interest. 
     Similarly, by using an auxotroph that cannot use an upstream metabolite to form the metabolite of interest, a strain can be evolved that not only can more efficiently import the upstream metabolite, but also convert the metabolite into the essential downstream metabolite of interest. These strains will also evolve mutations to increase import of the upstream metabolite, but may also contain mutations which increase expression or reaction kinetics of downstream enzymes, or that reduce competitive substrate utilization pathways. 
     A metabolite innate to the microbe can be made essential via mutational auxotrophy and selection applied with growth-limiting supplementation of the endogenous metabolite. However, phenotypes capable of consuming non-native compounds can be evolved by tying their consumption to the production of an essential compound. For example, if a gene from a different organism is isolated which can produce an essential compound or a precursor to an essential compound this gene can be recombinantly introduced and expressed in the heterologous host. This new host strain will now have the ability to synthesize an essential nutrient from a previously non-metabolizable substrate. 
     Hereby, a similar ALE process can be applied by creating an auxotroph incapable of converting an immediately downstream metabolite and selecting in growth-limiting amounts of the non-native compound with concurrent expression of the recombinant enzyme. This will result in mutations in the transport of the non-native substrate, expression and activity of the heterologous enzyme and expression and activity of downstream native enzymes. It should be emphasized that the key requirement in this process is the ability to tether the consumption of the non-native metabolite to the production of a metabolite essential to growth. 
     Once the basis of the selection mechanism is established and minimum levels of supplementation have been established, the actual ALE experimentation can proceed. Throughout this process several parameters must be vigilantly monitored. It is important that the cultures are maintained in an exponential growth phase and not allowed to reach saturation/stationary phase. This means that growth rates must be check during each passaging and subsequent dilutions adjusted accordingly. If growth rate improves to such a degree that dilutions become large, then the concentration of auxotrophic supplementation should be decreased such that growth rate is slowed, selection pressure is increased and dilutions are not so severe as to heavily bias subpopulations during passaging. In addition, at regular intervals cells should be diluted, grown on solid media and individual clones tested to confirm growth rate phenotypes observed in the ALE cultures. 
     Predicting when to halt the stop the ALE experiment also requires vigilance. As the success of directing evolution is tied directly to the number of mutations “screened” throughout the experiment and mutations are generally a function of errors during DNA replication, the cumulative cell divisions (CCD) acts as a proxy for total mutants which have been screened. Previous studies have shown that beneficial phenotypes for growth on different carbon sources can be isolated in about 10 11.2  CCD 1 . This rate can be accelerated by the addition of chemical mutagens to the cultures—such as N-methyl-N-nitro-N-nitrosoguanidine (NTG)—which causes increased DNA replication errors. However, when continued passaging leads to marginal or no improvement in growth rate the population has converged to some fitness maximum and the ALE experiment can be halted. 
     At the conclusion of the ALE experiment, the cells should be diluted, isolated on solid media and assayed for growth phenotypes matching that of the culture flask. Best performers from those selected are then prepped for genomic DNA and sent for whole genome sequencing. Sequencing with reveal mutations occurring around the genome capable of providing improved phenotypes, but will also contain silent mutations (those which provide no benefit but do not detract from desired phenotype). In cultures evolved in the presence of NTG or other chemical mutagen, there will be significantly more silent, background mutations. If satisfied with the best performing strain in its current state, the user can proceed to application with that strain. Otherwise the contributing mutations can be deconvoluted from the evolved strain by reintroducing the mutations to the parent strain by genome engineering techniques. See Lee, D.-H., Feist, A. M., Barrett, C. L. &amp; Palsson, B. Ø. Cumulative Number of Cell Divisions as a Meaningful Timescale for Adaptive Laboratory Evolution of  Escherichia coli. PLoS ONE  6, e26172 (2011). 
     Similar methods can be used to generate  E. coli  Nissle mutants that consume or import metabolites, including, but not limited to, tryptophan. 
     Nucleic Acids 
     In some embodiments, the disclosure provides novel nucleic acids for producing butyrate In some embodiments, the nucleic acids comprises gene sequence encoding one or more butyrogenic genes. In some embodiments, the nucleic acids comprises gene sequence encoding one or more butyrogenic gene cassettes. In some embodiments, the nucleic acids comprise one or more butyrogenic genes from Table 2. In some embodiments, the nucleic acids comprises gene sequence encoding one or more butyrogenic genes selected from bcd2, et/B3, etfA3, thiA1, hbd, crt2, pbt, buk, ter, and tesB. 
     In some embodiments, the nucleic acid comprises gene sequence encoding a Bcd2 polypeptide. In some embodiments, the nucleic acid comprises a bcd2 gene sequence. In certain embodiments, the nucleic acid comprising the bcd2 gene sequence has at least about 80% identity with SEQ ID NO: 1. In certain embodiments, the nucleic acid comprising the hcd2 gene sequence has at least about 90% identity with SEQ ID NO: 1. In certain embodiments, the nucleic acid comprising the bcd2 gene sequence has at least about 95% identity with SEQ ID NO: 1. In some embodiments, the nucleic acid comprising the bcd2 gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. In some specific embodiments, the nucleic acid comprising the bcd2 gene sequence comprises SEQ ID NO: 1. In other specific embodiments the nucleic acid comprising the bcd2 gene sequence consists of SEQ ID NO: 1. 
     In some embodiments, the nucleic acid comprises gene sequence encoding a EtfB3 polypeptide. In some embodiments, the nucleic acid comprises a etfB3 gene sequence. In certain embodiments, the nucleic acid comprising the etfB3 gene sequence has at least about 80% identity with SEQ ID NO: 2. In certain embodiments, the nucleic acid comprising the etfB3 gene sequence has at least about 90% identity with SEQ ID NO: 2. In certain embodiments, the nucleic acid comprising the etfB3 gene sequence has at least about 95% identity with SEQ ID NO: 2. In some embodiments, the nucleic acid comprising the etfB3 gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2. In some specific embodiments, the nucleic acid comprising the etfB3 gene sequence comprises SEQ ID NO: 2. In other specific embodiments the nucleic acid comprising the etfB3 gene sequence consists of SEQ ID NO: 2. 
     In some embodiments, the nucleic acid comprises gene sequence encoding a EtfA3 polypeptide. In some embodiments, the nucleic acid comprises a etfA3 gene sequence. In certain embodiments, the nucleic acid comprising the etfA3 gene sequence has at least about 80% identity with SEQ ID NO: 3. In certain embodiments, the nucleic acid comprising the etfA3 gene sequence has at least about 90% identity with SEQ ID NO: 3. In certain embodiments, the nucleic acid comprising the etfA3 gene sequence has at least about 95% identity with SEQ ID NO: 3. In some embodiments, the nucleic acid comprising the etfA3 gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3. In some specific embodiments, the nucleic acid comprising the etfA3 gene sequence comprises SEQ ID NO: 3. In other specific embodiments the nucleic acid comprising the etfA3 gene sequence consists of SEQ ID NO: 3. 
     In some embodiments, the nucleic acid comprises gene sequence encoding a ThiA1 polypeptide. In some embodiments, the nucleic acid comprises a thiA1 gene sequence. In certain embodiments, the nucleic acid comprising the thiA1 gene sequence has at least about 80% identity with SEQ ID NO: 4. In certain embodiments, the nucleic acid comprising the thiA1 gene sequence has at least about 90% identity with SEQ ID NO: 4. In certain embodiments, the nucleic acid comprising the thiA1 gene sequence has at least about 95% identity with SEQ ID NO: 4. In some embodiments, the nucleic acid comprising the thiA1 gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. In some specific embodiments, the nucleic acid comprising the thiA1 gene sequence comprises SEQ ID NO: 4. In other specific embodiments the nucleic acid comprising the thiA1 gene sequence consists of SEQ ID NO: 4. 
     In some embodiments, the nucleic acid comprises gene sequence encoding a Hbd polypeptide. In some embodiments, the nucleic acid comprises a hbd gene sequence. In certain embodiments, the nucleic acid comprising the hbd gene sequence has at least about 80% identity with SEQ ID NO: 5. In certain embodiments, the nucleic acid comprising the hbdgene sequence has at least about 90% identity with SEQ ID NO: 5. In certain embodiments, the nucleic acid comprising the hbdgene sequence has at least about 95% identity with SEQ ID NO: 5. In some embodiments, the nucleic acid comprising the hbd gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5. In some specific embodiments, the nucleic acid comprising the hbd gene sequence comprises SEQ ID NO: 5. In other specific embodiments the nucleic acid comprising the hbd gene sequence consists of SEQ ID NO: 5. 
     In some embodiments, the nucleic acid comprises gene sequence encoding a Crt2 polypeptide. In some embodiments, the nucleic acid comprises a crt2 gene sequence. In certain embodiments, the nucleic acid comprising the crt2 gene sequence has at least about 80% identity with SEQ ID NO: 6. In certain embodiments, the nucleic acid comprising the crt2 gene sequence has at least about 90% identity with SEQ ID NO: 6. In certain embodiments, the nucleic acid comprising the crt2 gene sequence has at least about 95% identity with SEQ ID NO: 6. In some embodiments, the nucleic acid comprising the crt2 gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6. In some specific embodiments, the nucleic acid comprising the crt2 gene sequence comprises SEQ ID NO: 6. In other specific embodiments the nucleic acid comprising the crt2 gene sequence consists of SEQ ID NO: 6. 
     In some embodiments, the nucleic acid comprises gene sequence encoding a Pbt polypeptide. In some embodiments, the nucleic acid comprises a pbt gene sequence. In certain embodiments, the nucleic acid comprising the pbt gene sequence has at least about 80% identity with SEQ ID NO: 7. In certain embodiments, the nucleic acid comprising the pbt gene sequence has at least about 90% identity with SEQ ID NO: 7. In certain embodiments, the nucleic acid comprising the pbt gene sequence has at least about 95% identity with SEQ ID NO: 7. In some embodiments, the nucleic acid comprising the pbt gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 7. In some specific embodiments, the nucleic acid comprising the pbt gene sequence comprises SEQ ID NO: 7. In other specific embodiments the nucleic acid comprising the pbt gene sequence consists of SEQ ID NO: 7. 
     In some embodiments, the nucleic acid comprises gene sequence encoding a Buk polypeptide. In some embodiments, the nucleic acid comprises a buk gene sequence. In certain embodiments, the nucleic acid comprising the buk gene sequence has at least about 80% identity with SEQ ID NO: 8. In certain embodiments, the nucleic acid comprising the buk gene sequence has at least about 90% identity with SEQ ID NO: 8. In certain embodiments, the nucleic acid comprising the buk gene sequence has at least about 95% identity with SEQ ID NO: 8. In some embodiments, the nucleic acid comprising the buk gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 8. In some specific embodiments, the nucleic acid comprising the buk gene sequence comprises SEQ ID NO: 8. In other specific embodiments the nucleic acid comprising the buk gene sequence consists of SEQ ID NO: 8. 
     In some embodiments, the nucleic acid comprises gene sequence encoding a Ter polypeptide. In some embodiments, the nucleic acid comprises a ter gene sequence. In certain embodiments, the nucleic acid comprising the ter gene sequence has at least about 80% identity with SEQ ID NO: 9. In certain embodiments, the nucleic acid comprising the ter gene sequence has at least about 90% identity with SEQ ID NO: 9. In certain embodiments, the nucleic acid comprising the ter gene sequence has at least about 95% identity with SEQ ID NO: 9. In some embodiments, the nucleic acid comprising the ter gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9. In some specific embodiments, the nucleic acid comprising the ter gene sequence comprises SEQ ID NO: 9. In other specific embodiments the nucleic acid comprising the ter gene sequence consists of SEQ ID NO: 9. 
     In some embodiments, the nucleic acid comprises gene sequence encoding a TesB polypeptide. In some embodiments, the nucleic acid comprises a tesB gene sequence. In certain embodiments, the nucleic acid comprising the tesB gene sequence has at least about 80% identity with SEQ ID NO: 10. In certain embodiments, the nucleic acid comprising the tesB gene sequence has at least about 90% identity with SEQ ID NO: 10. In certain embodiments, the nucleic acid comprising the tesB gene sequence has at least about 95% identity with SEQ ID NO: 10. In some embodiments, the nucleic acid comprising the tesB gene sequence has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. In some specific embodiments, the nucleic acid comprising the tesB gene sequence comprises SEQ ID NO: 10. In other specific embodiments the nucleic acid comprising the tesB gene sequence consists of SEQ ID NO: 10. 
     In other embodiments, the disclosure provides novel nucleic acids for producing butyrate in which the nucleic acid comprises gene sequence encoding one or more butyrogenic gene cassette(s). In some embodiments, the nucleic acid comprises gene sequence encoding a butyrogenic gene cassette comprising Bcd2, EtfB3, EtfA3, ThiA1, Hbd, Crt2, Pbt, and Buk. In some embodiments, the nucleic acid comprises a butyrogenic gene cassette(s) cassette comprising bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, and buk gene sequence. In some embodiments, the nucleic acid comprises gene sequence encoding a butyrogenic gene cassette comprising ThiA1, Hbd, Crt2, Pbt, Buk, and Ter. In some embodiments, the nucleic acid comprises a butyrogenic gene cassette(s) cassette comprising thiA1, hbd, crt2, pbt, buk, and ter gene sequence. In some embodiments, the nucleic acid comprises gene sequence encoding a butyrogenic gene cassette comprising Ter, ThiA1, Hbd, Crt2, and TesB. In some embodiments, the nucleic acid comprises a butyrogenic gene cassette(s) cassette comprising ter, thiA1, hbd, crt2, and tesB gene sequence. 
     In any of the nucleic acid embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides that produce butyrate is operably linked to an inducible promoter. In said embodiments, the inducible promoter is directly or indirectly induced by exogenous environmental conditions. In any of the nucleic acid embodiments described above and elsewhere herein, the gene sequence encoding one or more polypeptides that produce butyrate is operably linked to an constitutive promoter. In some embodiments, the nucleic acid is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the nucleic acid is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the nucleic acid is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. Inducible promoters and constitutive promoters are described in more detail infra. 
     One or more of the nucleic acids encoding butyrate biosynthesis genes may be functionally replaced or modified, e.g., codon optimized. 
     Pharmaceutical Compositions and Formulations 
     Pharmaceutical compositions comprising the genetically engineered microorganisms of the invention may be used to inhibit inflammatory mechanisms in the gut, restore and tighten gut mucosal barrier function, and/or treat or prevent autoimmunedisorders. Pharmaceutical compositions comprising one or more genetically engineered bacteria, and/or one or more genetically engineered virus, alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers are provided. 
     In certain embodiments, the pharmaceutical composition comprises one species, strain, or subtype of bacteria that are engineered to comprise the genetic modifications described herein, e.g., to produce an anti-inflammation and/or gut barrier enhancer molecule. In alternate embodiments, the pharmaceutical composition comprises two or more species, strains, and/or subtypes of bacteria that are each engineered to comprise the genetic modifications described herein, e.g., to produce an anti-inflammation and/or gut barrier enhancer molecule. 
     In certain embodiments, a combination of two or more different genetically engineered microorganisms can be administered at the same time. In some embodiments, the method comprises administering the a subject a combination of two or more genetically engineered microorganisms, a first microorganism which expresses a first payload, and at least a second microorganism which expresses a second payload. In some embodiments, the method comprises compositions comprising a combination of two or more genetically engineered microorganisms, a first microorganisms which expresses a first payload, and at least a second microorganism which expresses a second payload. 
     The pharmaceutical compositions described herein may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., “Remington&#39;s Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.). In some embodiments, the pharmaceutical compositions are subjected to tabletting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. 
     Appropriate formulation depends on the route of administration. 
     The genetically engineered microorganisms may be formulated into pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g., oral, topical, injectable, intravenous, sub-cutaneous, immediate-release, pulsatile-release, delayed-release, or sustained release). Suitable dosage amounts for the genetically engineered bacteria may range from about 105 to 1012 bacteria, e.g., approximately 105 bacteria, approximately 106 bacteria, approximately 107 bacteria, approximately 108 bacteria, approximately 109 bacteria, approximately 1010 bacteria, approximately 1011 bacteria, or approximately 1011 bacteria. The composition may be administered once or more daily, weekly, or monthly. The composition may be administered before, during, or following a meal. In one embodiment, the pharmaceutical composition is administered before the subject eats a meal. In one embodiment, the pharmaceutical composition is administered currently with a meal. In on embodiment, the pharmaceutical composition is administered after the subject eats a meal 
     The genetically engineered bacteria or genetically engineered virus may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, buffering agents, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents. For example, the pharmaceutical composition may include, but is not limited to, the addition of calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20. In some embodiments, the genetically engineered bacteria of the invention may be formulated in a solution of sodium bicarbonate, e.g., 1 molar solution of sodium bicarbonate (to buffer an acidic cellular environment, such as the stomach, for example). The genetically engineered bacteria may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. 
     The genetically engineered microorganisms may be administered intravenously, e.g., by infusion or injection. 
     The genetically engineered microorganisms of the disclosure may be administered intrathecally. In some embodiments, the genetically engineered microorganisms of the invention may be administered orally. The genetically engineered microorganisms disclosed herein may be administered topically and formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well known to one of skill in the art. See, e.g., “Remington&#39;s Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa. In an embodiment, for non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are employed. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, e.g., osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art. In one embodiment, the pharmaceutical composition comprising the recombinant bacteria of the invention may be formulated as a hygiene product. For example, the hygiene product may be an antibacterial formulation, or a fermentation product such as a fermentation broth. Hygiene products may be, for example, shampoos, conditioners, creams, pastes, lotions, and lip balms. 
     The genetically engineered microorganisms disclosed herein may be administered orally and formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose compositions such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). Disintegrating agents may also be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate. 
     Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose, carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and tragacanth); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., calcium, aluminum, zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, L-leucine, magnesium stearate, talc, or silica); disintegrants (e.g., starch, potato starch, sodium starch glycolate, sugars, cellulose derivatives, silica powders); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. A coating shell may be present, and common membranes include, but are not limited to, polylactide, polyglycolic acid, polyanhydride, other biodegradable polymers, alginate-polylysine-alginate (APA), alginate-polymethylene-co-guanidine-alginate (A-PMCG-A), hydroymethylacrylate-methyl methacrylate (HEMA-MMA), multilayered HEMA-MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC), acrylonitrile/sodium methallylsulfonate (AN-69), polyethylene glycol/poly pentamethylcyclopentasiloxane/polydimethylsiloxane (PEG/PD5/PDMS), poly N,N-dimethyl acrylamide (PDMAAm), siliceous encapsulates, cellulose sulphate/sodium alginate/polymethylene-co-guanidine (CS/A/PMCG), cellulose acetate phthalate, calcium alginate, k-carrageenan-locust bean gum gel beads, gellan-xanthan beads, poly(lactide-co-glycolides), carrageenan, starch poly-anhydrides, starch polymethacrylates, polyamino acids, and enteric coating polymers. 
     In some embodiments, the genetically engineered microorganisms are enterically coated for release into the gut or a particular region of the gut, for example, the large intestine. The typical pH profile from the stomach to the colon is about 1-4 (stomach), 5.5-6 (duodenum), 7.3-8.0 (ileum), and 5.5-6.5 (colon). In some diseases, the pH profile may be modified. In some embodiments, the coating is degraded in specific pH environments in order to specify the site of release. In some embodiments, at least two coatings are used. In some embodiments, the outside coating and the inside coating are degraded at different pH levels. 
     Liquid preparations for oral administration may take the form of solutions, syrups, suspensions, or a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable agents such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of the genetically engineered microorganisms described herein. 
     In one embodiment, the genetically engineered microorganisms of the disclosure may be formulated in a composition suitable for administration to pediatric subjects. As is well known in the art, children differ from adults in many aspects, including different rates of gastric emptying, pH, gastrointestinal permeability, etc. (Ivanovska et al., Pediatrics, 134(2):361-372, 2014). Moreover, pediatric formulation acceptability and preferences, such as route of administration and taste attributes, are critical for achieving acceptable pediatric compliance. Thus, in one embodiment, the composition suitable for administration to pediatric subjects may include easy-to-swallow or dissolvable dosage forms, or more palatable compositions, such as compositions with added flavors, sweeteners, or taste blockers. In one embodiment, a composition suitable for administration to pediatric subjects may also be suitable for administration to adults. 
     In one embodiment, the composition suitable for administration to pediatric subjects may include a solution, syrup, suspension, elixir, powder for reconstitution as suspension or solution, dispersible/effervescent tablet, chewable tablet, gummy candy, lollipop, freezer pop, troche, chewing gum, oral thin strip, orally disintegrating tablet, sachet, soft gelatin capsule, sprinkle oral powder, or granules. In one embodiment, the composition is a gummy candy, which is made from a gelatin base, giving the candy elasticity, desired chewy consistency, and longer shelf-life. In some embodiments, the gummy candy may also comprise sweeteners or flavors. 
     In one embodiment, the composition suitable for administration to pediatric subjects may include a flavor. As used herein, “flavor” is a substance (liquid or solid) that provides a distinct taste and aroma to the formulation. Flavors also help to improve the palatability of the formulation. Flavors include, but are not limited to, strawberry, vanilla, lemon, grape, bubble gum, and cherry. 
     In certain embodiments, the genetically engineered microorganisms may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject&#39;s diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. 
     In another embodiment, the pharmaceutical composition comprising the recombinant bacteria of the invention may be a comestible product, for example, a food product. In one embodiment, the food product is milk, concentrated milk, fermented milk (yogurt, sour milk, frozen yogurt, lactic acid bacteria-fermented beverages), milk powder, ice cream, cream cheeses, dry cheeses, soybean milk, fermented soybean milk, vegetable-fruit juices, fruit juices, sports drinks, confectionery, candies, infant foods (such as infant cakes), nutritional food products, animal feeds, or dietary supplements. In one embodiment, the food product is a fermented food, such as a fermented dairy product. In one embodiment, the fermented dairy product is yogurt. In another embodiment, the fermented dairy product is cheese, milk, cream, ice cream, milk shake, or kefir. In another embodiment, the recombinant bacteria of the invention are combined in a preparation containing other live bacterial cells intended to serve as probiotics. In another embodiment, the food product is a beverage. In one embodiment, the beverage is a fruit juice-based beverage or a beverage containing plant or herbal extracts. In another embodiment, the food product is a jelly or a pudding. Other food products suitable for administration of the recombinant bacteria of the invention are well known in the art. For example, see U.S. 2015/0359894 and US 2015/0238545, the entire contents of each of which are expressly incorporated herein by reference. In yet another embodiment, the pharmaceutical composition of the invention is injected into, sprayed onto, or sprinkled onto a food product, such as bread, yogurt, or cheese. 
     In some embodiments, the composition is formulated for intraintestinal administration, intrajejunal administration, intraduodenal administration, intraileal administration, gastric shunt administration, or intracolic administration, via nanoparticles, nanocapsules, microcapsules, or microtablets, which are enterically coated or uncoated. The pharmaceutical compositions may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and/or dispersing agents. 
     The genetically engineered microorganisms described herein may be administered intranasally, formulated in an aerosol form, spray, mist, or in the form of drops, and conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Pressurized aerosol dosage units may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., of gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. 
     The genetically engineered microorganisms may be administered and formulated as depot preparations. Such long acting formulations may be administered by implantation or by injection, including intravenous injection, subcutaneous injection, local injection, direct injection, or infusion. For example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt). 
     In some embodiments, disclosed herein are pharmaceutically acceptable compositions in single dosage forms. Single dosage forms may be in a liquid or a solid form. Single dosage forms may be administered directly to a patient without modification or may be diluted or reconstituted prior to administration. In certain embodiments, a single dosage form may be administered in bolus form, e.g., single injection, single oral dose, including an oral dose that comprises multiple tablets, capsule, pills, etc. In alternate embodiments, a single dosage form may be administered over a period of time, e.g., by infusion. 
     Single dosage forms of the pharmaceutical composition may be prepared by portioning the pharmaceutical composition into smaller aliquots, single dose containers, single dose liquid forms, or single dose solid forms, such as tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. A single dose in a solid form may be reconstituted by adding liquid, typically sterile water or saline solution, prior to administration to a patient. 
     In other embodiments, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release. In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see e.g., U.S. Pat. No. 5,989,463). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used. 
     Dosage regimens may be adjusted to provide a therapeutic response. Dosing can depend on several factors, including severity and responsiveness of the disease, route of administration, time course of treatment (days to months to years), and time to amelioration of the disease. For example, a single bolus may be administered at one time, several divided doses may be administered over a predetermined period of time, or the dose may be reduced or increased as indicated by the therapeutic situation. The specification for the dosage is dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved. Dosage values may vary with the type and severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the treating clinician. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or animal models. For example, LD50, ED50, EC50, and IC50 may be determined, and the dose ratio between toxic and therapeutic effects (LD50/ED50) may be calculated as the therapeutic index. Compositions that exhibit toxic side effects may be used, with careful modifications to minimize potential damage to reduce side effects. Dosing may be estimated initially from cell culture assays and animal models. The data obtained from in vitro and in vivo assays and animal studies can be used in formulating a range of dosage for use in humans. 
     The ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. If the mode of administration is by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. 
     The pharmaceutical compositions may be packaged in a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent. In one embodiment, one or more of the pharmaceutical compositions is supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In an embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions is supplied as a dry sterile lyophilized powder in a hermetically sealed container stored between 2° C. and 8° C. and administered within 1 hour, within 3 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, or within one week after being reconstituted. Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Other suitable bulking agents include glycine and arginine, either of which can be included at a concentration of 0-0.05%, and polysorbate-80 (optimally included at a concentration of 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants. The pharmaceutical composition may be prepared as an injectable solution and can further comprise an agent useful as an adjuvant, such as those used to increase absorption or dispersion, e.g., hyaluronidase. 
     Methods of Treatment 
     Another aspect of the invention provides methods of treating autoimmune disorders, diarrheal diseases, IBD, related diseases, and other diseases that benefit from reduced gut inflammation and/or enhanced gut barrier function. In some embodiments, the invention provides for the use of at least one genetically engineered species, strain, or subtype of bacteria described herein for the manufacture of a medicament. In some embodiments, the invention provides for the use of at least one genetically engineered species, strain, or subtype of bacteria described herein for the manufacture of a medicament for treating autoimmune disorders, diarrheal diseases, IBD, related diseases, and other diseases that benefit from reduced gut inflammation and/or enhanced gut barrier function. In some embodiments, the invention provides at least one genetically engineered species, strain, or subtype of bacteria described herein for use in treating autoimmune disorders, diarrheal diseases, IBD, related diseases, and other diseases that benefit from reduced gut inflammation and/or enhanced gut barrier function. 
     In some embodiments, the diarrheal disease is selected from the group consisting of acute watery diarrhea, e.g., cholera, acute bloody diarrhea, e.g., dysentery, and persistent diarrhea. In some embodiments, the IBD or related disease is selected from the group consisting of Crohn&#39;s disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, diversion colitis, Behcet&#39;s disease, intermediate colitis, short bowel syndrome, ulcerative proctitis, proctosigmoiditis, left-sided colitis, pancolitis, and fulminant colitis. In some embodiments, the disease or condition is an autoimmune disorder selected from the group consisting of acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison&#39;s disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticarial, axonal &amp; neuronal neuropathies, Balo disease, Behcet&#39;s disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn&#39;s disease, Cogan&#39;s syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devic&#39;s disease (neuromyelitis optica), discoid lupus, Dressler&#39;s syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture&#39;s syndrome, granulomatosis with polyangiitis (GPA), Graves&#39; disease, Guillain-Barre syndrome, Hashimoto&#39;s encephalitis, Hashimoto&#39;s thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile idiopathic arthritis, juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus (systemic lupus erythematosus), chronic Lyme disease, Meniere&#39;s disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren&#39;s ulcer, Mucha-IIabennann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic&#39;s), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, &amp; III autoimmune polyglandular syndromes, polymyalgia rheumatic, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud&#39;s phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter&#39;s syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren&#39;s syndrome, sperm &amp; testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE), Susac&#39;s syndrome, sympathetic ophthalmia, Takayasu&#39;s arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, asthma, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener&#39;s granulomatosis. In some embodiments, the invention provides methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these diseases, including but not limited to diarrhea, bloody stool, mouth sores, perianal disease, abdominal pain, abdominal cramping, fever, fatigue, weight loss, iron deficiency, anemia, appetite loss, weight loss, anorexia, delayed growth, delayed pubertal development, and inflammation of the skin, eyes, joints, liver, and bile ducts. In some embodiments, the invention provides methods for reducing gut inflammation and/or enhancing gut barrier function, thereby ameliorating or preventing a systemic autoimmune disorder, e.g., asthma (Arrieta et al., 2015). 
     The method may comprise preparing a pharmaceutical composition with at least one genetically engineered species, strain, or subtype of bacteria described herein, and administering the pharmaceutical composition to a subject in a therapeutically effective amount. In some embodiments, the genetically engineered bacteria of the invention are administered orally in a liquid suspension. In some embodiments, the genetically engineered bacteria of the invention are lyophilized in a gel cap and administered orally. In some embodiments, the genetically engineered bacteria of the invention are administered via a feeding tube. In some embodiments, the genetically engineered bacteria of the invention are administered rectally, e.g., by enema. In some embodiments, the genetically engineered bacteria of the invention are administered topically, intraintestinally, intrajejunally, intraduodenally, intraileally, and/or intracolically. 
     In some embodiments, the genetically engineered viruses are prepared for delivery, taking into consideration the need for efficient delivery and for overcoming the host antiviral immune response. Approaches to evade antiviral response include the administration of different viral serotypes as par of the treatment regimen (serotype switching), formulation, such as polymer coating to mask the virus from antibody recognition and the use of cells as delivery vehicles. 
     In another embodiment, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release. In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see e.g., U.S. Pat. No. 5,989,463). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used. 
     The genetically engineered bacteria of the invention may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. 
     In certain embodiments, the pharmaceutical composition described herein is administered to reduce gut inflammation, enhance gut barrier function, and/or treat or prevent an autoimmune disorder in a subject. In some embodiments, the methods of the present disclosure may reduce gut inflammation in a subject by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels in an untreated or control subject. In some embodiments, the methods of the present disclosure may enhance gut barrier function in a subject by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels in an untreated or control subject. In some embodiments, changes in inflammation and/or gut barrier function are measured by comparing a subject before and after administration of the pharmaceutical composition. In some embodiments, the method of treating or ameliorating the autoimmune disorder and/or the disease or condition associated with gut inflammation and/or compromised gut barrier function allows one or more symptoms of the disease or condition to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. 
     In some embodiments, reduction is measured by comparing the levels of inflammation in a subject before and after administration of the pharmaceutical composition. In one embodiment, the levels of inflammation is reduced in the gut of the subject. In one embodiment, gut barrier function is enhanced in the gut of the subject. In another embodiment, levels of inflammation is reduced in the blood of the subject. In another embodiment, the levels of inflammation is reduced in the plasma of the subject. In another embodiment, levels of inflammation is reduced in the brain of the subject. 
     In one embodiment, the pharmaceutical composition described herein is administered to reduce levels of inflammation in a subject to normal levels. In another embodiment, the pharmaceutical composition described herein is administered to reduce levels of inflammation in a subject below normal. 
     In some embodiments, the method of treating the autoimmune disorder allows one or more symptoms of the condition or disorder to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of treating the disorder, allows one or more symptoms of the condition or disorder to improve by at least about two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, or ten-fold. 
     Before, during, and after the administration of the pharmaceutical composition, gut inflammation and/or barrier function in the subject may be measured in a biological sample, such as blood, serum, plasma, urine, fecal matter, peritoneal fluid, intestinal mucosal scrapings, a sample collected from a tissue, and/or a sample collected from the contents of one or more of the following: the stomach, duodenum, jejunum, ileum, cecum, colon, rectum, and anal canal. In some embodiments, the methods may include administration of the compositions of the invention to enhance gut barrier function and/or to reduce gut inflammation to baseline levels, e.g., levels comparable to those of a healthy control, in a subject. In some embodiments, the methods may include administration of the compositions of the invention to reduce gut inflammation to undetectable levels in a subject, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% of the subject&#39;s levels prior to treatment. In some embodiments, the methods may include administration of the compositions of the invention to enhance gut barrier function in a subject by about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100% or more of the subject&#39;s levels prior to treatment. 
     In certain embodiments, the recombinant bacteria are  E. coli  Nissle. The recombinant bacteria may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al., 2009) or by activation of a kill switch, several hours or days after administration. Thus, the pharmaceutical composition comprising the recombinant bacteria may be re-administered at a therapeutically effective dose and frequency. In alternate embodiments, the recombinant bacteria are not destroyed within hours or days after administration and may propagate and colonize the gut. 
     The pharmaceutical composition may be administered alone or in combination with one or more additional therapeutic agents, e.g., corticosteroids, aminosalicylates, anti-inflammatory agents. In some embodiments, the pharmaceutical composition is administered in conjunction with an anti-inflammatory drug (e.g., mesalazine, prednisolone, methylprednisolone, butesonide), an immunosuppressive drug (e.g., azathioprine, 6-mercaptopurine, methotrexate, cyclosporine, tacrolimus), an antibiotic (e.g., metronidazole, ornidazole, clarithromycin, rifaximin, ciprofloxacin, anti-TB), other probiotics, and/or biological agents (e.g., infliximab, adalimumab, certolizumab pegol) (Triantafillidis et al., 2011). An important consideration in the selection of the one or more additional therapeutic agents is that the agent(s) should be compatible with the genetically engineered bacteria of the invention, e.g., the agent(s) must not kill the bacteria In one embodiments, the bacterial cells disclosed herein are administered to a subject once daily. In another embodiment, the bacterial cells disclosed herein are administered to a subject twice daily. In another embodiment, the bacterial cells disclosed herein are administered to a subject in combination with a meal. In another embodiment, the bacterial cells disclosed herein are administered to a subject prior to a meal. In another embodiment, the bacterial cells disclosed herein are administered to a subject after a meal. The dosage of the pharmaceutical composition and the frequency of administration may be selected based on the severity of the symptoms and the progression of the disease. The appropriate therapeutically effective dose and/or frequency of administration can be selected by a treating clinician. 
     Treatment In Vivo 
     The genetically engineered bacteria of the invention may be evaluated in vivo, e.g., in an animal model. Any suitable animal model of a disease or condition associated with gut inflammation, compromised gut barrier function, and/or an autoimmune disorder may be used (see, e.g., Mizoguchi, 2012). The animal model may be a mouse model of IBD, e.g., a CD45RB Hi  T cell transfer model or a dextran sodium sulfate (DSS) model. The animal model may be a mouse model of type 1 diabetes (T1D), and T1D may be induced by treatment with streptozotocin. 
     Colitis is characterized by inflammation of the inner lining of the colon, and is one form of IBD. In mice, modeling colitis often involves the aberrant expression of T cells and/or cytokines. One exemplary mouse model of IBD can be generated by sorting CD4+ T cells according to their levels of CD45RB expression, and adoptively transferring CD4+ T cells with high CD45RB expression from normal donor mice into immunodeficient mice. Non-limiting examples of immunodeficient mice that may be used for transfer include severe combined immunodeficient (SCID) mice (Morrissey et al., 1993; Powrie et al., 1993), and recombination activating gene 2 (RAG2)-deficient mice (Corazza et al., 1999). The transfer of CD45RB Hi  T cells into immunodeficient mice, e.g., via intravenous or intraperitoneal injection, results in epithelial cell hyperplasia, tissue damage, and severe mononuclear cell infiltration within the colon (Byrne et al., 2005; Dohi et al., 2004; Wei et al., 2005). In some embodiments, the genetically engineered bacteria of the invention may be evaluated in a CD45RB Hi  T cell transfer mouse model of IBD. 
     Another exemplary animal model of IBD can be generated by supplementing the drinking water of mice with dextran sodium sulfate (DSS) (Martinez et al., 2006; Okayasu et al., 1990; Whittem et al., 2010). Treatment with DSS results in epithelial damage and robust inflammation in the colon lasting several days. Single treatments may be used to model acute injury, or acute injury followed by repair. Mice treated acutely show signs of acute colitis, including bloody stool, rectal bleeding, diarrhea, and weight loss (Okayasu et al., 1990). In contrast, repeat administration cycles of DSS may be used to model chronic inflammatory disease. Mice that develop chronic colitis exhibit signs of colonic mucosal regeneration, such as dysplasia, lymphoid follicle formation, and shortening of the large intestine (Okayasu et al., 1990). In some embodiments, the genetically engineered bacteria of the invention may be evaluated in a DSS mouse model of IBD. 
     In some embodiments, the genetically engineered bacteria of the invention is administered to the animal, e.g., by oral gavage, and treatment efficacy is determined, e.g., by endoscopy, colon translucency, fibrin attachment, mucosal and vascular pathology, and/or stool characteristics. In some embodiments, the animal is sacrificed, and tissue samples are collected and analyzed, e.g., colonic sections are fixed and scored for inflammation and ulceration, and/or homogenized and analyzed for myeloperoxidase activity and cytokine levels (e.g., IL-1β, TNF-α, IL-6, IFN-γ and IL-10). 
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     EXAMPLES 
     The following examples provide illustrative embodiments of the disclosure. One of ordinary skill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the disclosure. Such modifications and variations are encompassed within the scope of the disclosure. The Examples do not in any way limit the disclosure. 
     Example 1. Construction of Vectors for Producing Therapeutic Molecules 
     Butyrate 
     To facilitate inducible production of butyrate in  Escherichia coli  Nissle, the eight genes of the butyrate production pathway from  Peptoclostridium difficile  630 (bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, and buk; NCBI; Table 2 and Table 36), as well as transcriptional and translational elements, are synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322 to create pLogic031 (bcd2-etfB3-etfA3-thiA1-hbd-crt2-pbt buk butyrate cassette, also referred to as bcd2-etfB3-etfA3 butyrate cassette, SEQ ID NO: 162). 
     The gene products of the bcd2-etfA3-etfB3 genes form a complex that converts crotonyl-CoA to butyryl-CoA and may exhibit dependence on oxygen as a co-oxidant. Because the recombinant bacteria of the invention are designed to produce butyrate in an oxygen-limited environment (e.g. the mammalian gut), that dependence on oxygen could have a negative effect of butyrate production in the gut. It has been shown that a single gene from  Treponema denticola , trans-2-enoynl-CoA reductase (ter, Table 2 and Table 36), can functionally replace this three gene complex in an oxygen-independent manner. Therefore, a second butyrate gene cassette in which the ter gene replaces the bcd2-etfA3-etfB3 genes of the first butyrate cassette is synthesized (Genewiz, Cambridge, Mass.). The ter gene is codon-optimized for  E. coli  codon usage using Integrated DNA Technologies online codon optimization tool (https://www.idtdna.com/CodonOpt). The second butyrate gene cassette, as well as transcriptional and translational elements, is synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322 to create pLogic046 (ter-thiA1-hbd-crt2-pbt buk butyrate cassette, also referred to herein as ter butyrate cassette or pbt buk butyrate cassette, SEQ ID NO: 163). 
     In a third butyrate gene cassette, the pbt and buk genes are replaced with tesB (SEQ ID NO: 10). TesB is a thioesterase found in  E. coli  that cleaves off the butyrate from butyryl-coA, thus obviating the need for pbt-buk (see, e.g.,  FIG. 2  and Table 2 and Table 36). The third butyrate gene cassette, as well as transcriptional and translational elements, is synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322 to create pLOGIC046-delta pbt.buk/tesB+ (ter-thiA1-hbd-crt2-tesb butyrate cassette, also referred to herein as tesB butyrate cassette, SEQ ID NO: 164). Table 36 lists non-limiting examples for sequences of the three cassettes. 
     
       
         
           
               
             
               
                 TABLE 36 
               
             
            
               
                   
               
               
                 Butyrate Cassette Sequences 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 Description 
                 Sequence 
                 NO 
               
               
                   
               
               
                 bcd2-etfB3- 
                 atggatttaaattctaaaaaatatcagatgcttaaagagctatatgtaagcttcgctgaaaa 
                 SEQ ID 
               
               
                 etfA3-thiA 1 - 
                 tgaagttaaacctttagcaacagaacttgatgaagaagaaagatttccttatgaaacagt 
                 NO: 162 
               
               
                 hb- crt2-pbt- 
                 ggaaaaaatggcaaaagcaggaatgatgggtataccatatccaaaagaatatggtgg 
                   
               
               
                 buk butyrate 
                 agaaggtggagacactgtaggatatataatggcagttgaagaattgtctagagtttgtgg 
                   
               
               
                 cassette 
                 tactacaggagttatattatcagctcatacatctcttggctcatggcctatatatcaatatgg 
                   
               
               
                   
                 taatgaagaacaaaaacaaaaattcttaagaccactagcaagtggagaaaaattagga 
                   
               
               
                   
                 gcatttggtcttactgagcctaatgctggtacagatgcgtctggccaacaaacaactgct 
                   
               
               
                   
                 gttttagacggggatgaatacatacttaatggctcaaaaatatttataacaaacgcaatag 
                   
               
               
                   
                 ctggtgacatatatgtagtaatggcaatgactgataaatctaaggggaacaaaggaata 
                   
               
               
                   
                 tcagcatttatagttgaaaaaggaactcctgggtttagctttggagttaaagaaaagaaa 
                   
               
               
                   
                 atgggtataagaggttcagctacgagtgaattaatatttgaggattgcagaatacctaaa 
                   
               
               
                   
                 gaaaatttacttggaaaagaaggtcaaggatttaagatagcaatgtctactcttgatggtg 
                   
               
               
                   
                 gtagaattggtatagctgcacaagctttaggtttagcacaaggtgctcttgatgaaactgt 
                   
               
               
                   
                 taaatatgtaaaagaaagagtacaatttggtagaccattatcaaaattccaaaatacaca 
                   
               
               
                   
                 attccaattagctgatatggaagttaaggtacaagcggctagacaccttgtatatcaagc 
                   
               
               
                   
                 agctataaataaagacttaggaaaaccttatggagtagaagcagcaatggcaaaattat 
                   
               
               
                   
                 ttgcagctgaaacagctatggaagttactacaaaagctgtacaacttcatggaggatatg 
                   
               
               
                   
                 gatacactcgtgactatccagtagaaagaatgatgagagatgctaagataactgaaata 
                   
               
               
                   
                 tatgaaggaactagtgaagttcaaagaatggttatttcaggaaaactattaaaatagtaa 
                   
               
               
                   
                 gaaggagatatacatatggaggaaggatttatgaatatagtcgtttgtataaaacaagttc 
                   
               
               
                   
                 cagatacaacagaagttaaactagatcctaatacaggtactttaattagagatggagtac 
                   
               
               
                   
                 caagtataataaaccctgatgataaagcaggtttagaagaagctataaaattaaaagaa 
                   
               
               
                   
                 gaaatgggtgctcatgtaactgttataacaatgggacctcctcaagcagatatggcttta 
                   
               
               
                   
                 aaagaagctttagcaatgggtgcagatagaggtatattattaacagatagagcatttgcg 
                   
               
               
                   
                 ggtgctgatacttgggcaacttcatcagcattagcaggagcattaaaaaatatagattttg 
                   
               
               
                   
                 atattataatagctggaagacaggcgatagatggagatactgcacaagttggacctcaa 
                   
               
               
                   
                 atagctgaacatttaaatcttccatcaataacatatgctgaagaaataaaaactgaaggtg 
                   
               
               
                   
                 aatatgtattagtaaaaagacaatttgaagattgttgccatgacttaaaagttaaaatgcca 
                   
               
               
                   
                 tgccttataacaactcttaaagatatgaacacaccaagatacatgaaagttggaagaata 
                   
               
               
                   
                 tatgatgctttcgaaaatgatgtagtagaaacatggactgtaaaagatatagaagttgac 
                   
               
               
                   
                 ccttctaatttaggtcttaaaggttctccaactagtgtatttaaatcatttacaaaatcagtta 
                   
               
               
                   
                 aaccagaggtacaatatacaatgaagatgcgaaaacatcagctggaattatcatagat 
                   
               
               
                   
                 aaattaaaagagaagtatatcatataataagaaggagatatacatatgggtaacgttttag 
                   
               
               
                   
                 tagtaatagaacaaagagaaaatgtaattcaaactgtttctttagaattactaggaaaggc 
                   
               
               
                   
                 tacagaaatagcaaaagattatgatacaaaagtttctgcattacttttaggtagtaaggta 
                   
               
               
                   
                 gaaggtttaatagatacattagcacactatggtgcagatgaggtaatagtagtagatgat 
                   
               
               
                   
                 gaagctttagcagtgtatacaactgaaccatatacaaaagcagcttatgaagcaataaa 
                   
               
               
                   
                 agcagctgaccctatagttgtattatttggtgcaacttcaataggtagagatttagcgcct 
                   
               
               
                   
                 agagtttctgctagaatacatacaggtcttactgctgactgtacaggtcttgcagtagctg 
                   
               
               
                   
                 aagatacaaaattattattaatgacaagacctgcctttggtggaaatataatggcaacaat 
                   
               
               
                   
                 agtttgtaaagatttcagacctcaaatgtctacagttagaccaggggttatgaagaaaaa 
                   
               
               
                   
                 tgaacctgatgaaactaaagaagctgtaattaaccgtttcaaggtagaatttaatgatgct 
                   
               
               
                   
                 gataaattagttcaagttgtacaagtaataaaagaagctaaaaaacaagttaaaatagaa 
                   
               
               
                   
                 gatgctaagatattagtttctgctggacgtggaatgggtggaaaagaaaacttagacata 
                   
               
               
                   
                 ctttatgaattagctgaaattataggtggagaagtttctggttctcgtgccactatagatgc 
                   
               
               
                   
                 aggttggttagataaagcaagacaagttggtcaaactggtaaaactgtaagaccagac 
                   
               
               
                   
                 ctttatatagcatgtggtatataggagcaatacaacatatagctggtatggaagatgctg 
                   
               
               
                   
                 agtttatagttgctataaataaaaatccagaagctccaatatttaaatatgctgatgttggta 
                   
               
               
                   
                 tagttggagatgttcataaagtgcttccagaacttatcagtcagttaagtgttgcaaaaga 
                   
               
               
                   
                 aaaaggtgaagttttagctaactaataagaaggagatatacatatgagagaagtagtaat 
                   
               
               
                   
                 tgccagtgcagctagaacagcagtaggaagttttggaggagcatttaaatcagtttcag 
                   
               
               
                   
                 cggtagagttaggggtaacagcagctaaagaagctataaaaagagctaacataactcc 
                   
               
               
                   
                 agatatgatagatgaatctcttttagggggagtacttacagcaggtcttggacaaaatata 
                   
               
               
                   
                 gcaagacaaatagcattaggagcaggaataccagtagaaaaaccagctatgactataa 
                   
               
               
                   
                 atatagtttgtggttctggattaagatctgtttcaatggcatctcaacttatagcattaggtg 
                   
               
               
                   
                 atgctgatataatgttagttggtggagctgaaaacatgagtatgtctccttatttagtacca 
                   
               
               
                   
                 agtgcgagatatggtgcaagaatgggtgatgctgcttttgttgattcaatgataaaagat 
                   
               
               
                   
                 ggattatcagacatatttaataactatcacatgggtattactgctgaaaacatagcagagc 
                   
               
               
                   
                 aatggaatataactagagaagaacaagatgaattagctcttgcaagtcaaaataaagct 
                   
               
               
                   
                 gaaaaagctcaagctgaaggaaaatttgatgaagaaatagttcctgttgttataaaagga 
                   
               
               
                   
                 agaaaaggtgacactgtagtagataaagatgaatatattaagcctggcactacaatgga 
                   
               
               
                   
                 gaaacttgctaagttaagacctgcatttaaaaaagatggaacagttactgctggtaatgc 
                   
               
               
                   
                 atcaggaataaatgatggtgctgctatgttagtagtaatggctaaagaaaaagctgaag 
                   
               
               
                   
                 aactaggaatagagcctcttgcaactatagtttcttatggaacagctggtgttgaccctaa 
                   
               
               
                   
                 aataatgggatatggaccagttccagcaactaaaaaagctttagaagctgctaatatga 
                   
               
               
                   
                 ctattgaagatatagatttagttgaagctaatgaggcatttgctgcccaatctgtagctgta 
                   
               
               
                   
                 ataagagacttaaatatagatatgaataaagttaatgttaatggtggagcaatagctatag 
                   
               
               
                   
                 gacatccaataggatgctcaggagcaagaatacttactacacttttatatgaaatgaaga 
                   
               
               
                   
                 gaagagatgctaaaactggtcttgctacactttgtataggcggtggaatgggaactactt 
                   
               
               
                   
                 taatagttaagagatagtaagaaggagatatacatatgaaattagctgtaataggtagtg 
                   
               
               
                   
                 gaactatgggaagtggtattgtacaaacttttgcaagttgtggacatgatgtatgtttaaa 
                   
               
               
                   
                 gagtagaactcaaggtgctatagataaatgtttagctttattagataaaaatttaactaagtt 
                   
               
               
                   
                 agttactaagggaaaaatggatgaagctacaaaagcagaaatattaagtcatgttagttc 
                   
               
               
                   
                 aactactaattatgaagatttaaaagatatggatttaataatagaagcatctgtagaagac 
                   
               
               
                   
                 atgaatataaagaaagatgttttcaagttactagatgaattatgtaaagaagatactatctt 
                   
               
               
                   
                 ggcaacaaatacttcatcattatctataacagaaatagcacttctactaagcgcccagat 
                   
               
               
                   
                 aaagttataggaatgcatttctttaatccagttcctatgatgaaattagttgaagttataagt 
                   
               
               
                   
                 ggtcagttaacatcaaaagttacttttgatacagtatttgaattatctaagagtatcaataaa 
                   
               
               
                   
                 gtaccagtagatgtatctgaatctcctggatttgtagtaaatagaatacttatacctatgata 
                   
               
               
                   
                 aatgaagctgttggtatatatgcagatggtgttgcaagtaaagaagaaatagatgaagct 
                   
               
               
                   
                 atgaaattaggagcaaaccatccaatgggaccactagcattaggtgatttaatcggatta 
                   
               
               
                   
                 gatgttgttttagctataatgaacgttttatatactgaatttggagatactaaatatagacctc 
                   
               
               
                   
                 atccacttttagctaaaatggttagagctaatcaattaggaagaaaaactaagataggatt 
                   
               
               
                   
                 ctatgattataataaataataagaaggagatatacatatgagtacaagtgatgttaaagttt 
                   
               
               
                   
                 atgagaatgtagctgttgaagtagatggaaatatatgtacagtgaaaatgaatagaccta 
                   
               
               
                   
                 aagcccttaatgcaataaattcaaagactttagaagaactttatgaagtatttgtagatatt 
                   
               
               
                   
                 aataatgatgaaactattgatgttgtaatattgacaggggaaggaaaggcatttgtagct 
                   
               
               
                   
                 ggagcagatattgcatacatgaaagatttagatgctgtagctgctaaagattttagtatctt 
                   
               
               
                   
                 aggagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatagctgctgtaa 
                   
               
               
                   
                 acggatttgctttaggtggaggatgtgaacttgcaatggcatgtgatataagaattgcatc 
                   
               
               
                   
                 tgctaaagctaaatttggtcagccagaagtaactcttggaataactccaggatatggag 
                   
               
               
                   
                 gaactcaaaggcttacaagattggttggaatggcaaaagcaaaagaattaatctttaca 
                   
               
               
                   
                 ggtcaagttataaaagctgatgaagctgaaaaaatagggctagtaaatagagtcgttga 
                   
               
               
                   
                 gccagacattttaatagaagaagttgagaaattagctaagataatagctaaaaatgctca 
                   
               
               
                   
                 gcttgcagttagatactctaaagaagcaatacaacttggtgctcaaactgatataaatact 
                   
               
               
                   
                 ggaatagatatagaatctaatttatttggtctttgtttttcaactaaagaccaaaaagaagg 
                   
               
               
                   
                 aatgtcagctttcgttgaaaagagagaagctaactttataaaagggtaataagaaggag 
                   
               
               
                   
                 atatacatatgagaagttttgaagaagtaattaagtttgcaaaagaaagaggacctaaaa 
                   
               
               
                   
                 ctatatcagtagcatgttgccaagataaagaagttttaatggcagttgaaatggctagaa 
                   
               
               
                   
                 aagaaaaaatagcaaatgccattttagtaggagatatagaaaagactaaagaaattgca 
                   
               
               
                   
                 aaaagcatagacatggatatcgaaaattatgaactgatagatataaaagatttagcagaa 
                   
               
               
                   
                 gcatctctaaaatctgttgaattagtttcacaaggaaaagccgacatggtaatgaaaggc 
                   
               
               
                   
                 ttagtagacacatcaataatactaaaagcagttttaaataaagaagtaggtcttagaactg 
                   
               
               
                   
                 gaaatgtattaagtcacgtagcagtatttgatgtagagggatatgatagattatttttcgta 
                   
               
               
                   
                 actgacgcagctatgaacttagctcctgatacaaatactaaaaagcaaatcatagaaaat 
                   
               
               
                   
                 gcttgcacagtagcacattcattagatataagtgaaccaaaagttgctgcaatatgcgca 
                   
               
               
                   
                 aaagaaaaagtaaatccaaaaatgaaagatacagttgaagctaaagaactagaagaa 
                   
               
               
                   
                 atgtatgaaagaggagaaatcaaaggttgtatggttggtgggccttttgcaattgataat 
                   
               
               
                   
                 gcagtatctttagaagcagctaaacataaaggtataaatcatcctgtagcaggacgagc 
                   
               
               
                   
                 tgatatattattagccccagatattgaaggtggtaacatattatataaagctttggtattcttc 
                   
               
               
                   
                 tcaaaatcaaaaaatgcaggagttatagttggggctaaagcaccaataatattaacttct 
                   
               
               
                   
                 agagcagacagtgaagaaactaaactaaactcaatagctttaggtgttttaatggcagc 
                   
               
               
                   
                 aaaggcataataagaaggagatatacatatgagcaaaatatttaaaatcttaacaataaa 
                   
               
               
                   
                 tcctggttcgacatcaactaaaatagctgtatttgataatgaggatttagtatttgaaaaaa 
                   
               
               
                   
                 ctttaagacattcttcagaagaaataggaaaatatgagaaggtgtctgaccaatttgaatt 
                   
               
               
                   
                 tcgtaaacaagtaatagaagaagctctaaaagaaggtggagtaaaaacatctgaattag 
                   
               
               
                   
                 atgctgtagtaggtagaggaggacttcttaaacctataaaaggtggtacttattcagtaa 
                   
               
               
                   
                 gtgctgctatgattgaagatttaaaagtgggagttttaggagaacacgcttcaaacctag 
                   
               
               
                   
                 gtggaataatagcaaaacaaataggtgaagaagtaaatgttccttcatacatagtagac 
                   
               
               
                   
                 cctgttgttgtagatgaattagaagatgttgctagaatttctggtatgcctgaaataagtag 
                   
               
               
                   
                 agcaagtgtagtacatgctttaaatcaaaaggcaatagcaagaagatatgctagagaaa 
                   
               
               
                   
                 taaacaagaaatatgaagatataaatcttatagttgcacacatgggtggaggagtttctgt 
                   
               
               
                   
                 tggagctcataaaaatggtaaaatagtagatgttgcaaacgcattagatggagaaggac 
                   
               
               
                   
                 ctttctctccagaaagaagtggtggactaccagtaggtgcattagtaaaaatgtgctttag 
                   
               
               
                   
                 tggaaaatatactcaagatgaaattaaaaagaaaataaaaggtaatggcggactagttg 
                   
               
               
                   
                 catacttaaacactaatgatgctagagaagttgaagaaagaattgaagctggtgatgaa 
                   
               
               
                   
                 aaagctaaattagtatatgaagctatggcatatcaaatctctaaagaaataggagctagt 
                   
               
               
                   
                 gctgcagttcttaagggagatgtaaaagcaatattattaactggtggaatcgcatattcaa 
                   
               
               
                   
                 aaatgtttacagaaatgattgcagatagagttaaatttatagcagatgtaaaagtttatcca 
                   
               
               
                   
                 ggtgaagatgaaatgattgcattagctcaaggtggacttagagttttaactggtgaagaa 
                   
               
               
                   
                 gaggctcaagtttatgataactaataa 
                   
               
               
                   
               
               
                 ter-thiA 1 -hbd- 
                 atgatcgtaaaacctatggtacgcaacaatatctgcctgaacgcccatcctcagggctg 
                 SEQ ID 
               
               
                 crt2-pbt buk 
                 caagaagggagtggaagatcagattgaatataccaagaaacgcattaccgcagaagt 
                 NO: 163 
               
               
                 butyrate 
                 caaagctggcgcaaaagctccaaaaaacgttctggtgcttggctgctcaaatggttacg 
                   
               
               
                 cassette 
                 gcctggcgagccgcattactgctgcgttcggatacggggctgcgaccatcggcgtgtc 
                   
               
               
                   
                 ctttgaaaaagcgggttcagaaaccaaatatggtacaccgggatggtacaataatttgg 
                   
               
               
                   
                 catttgatgaagcggcaaaacgcgagggtctttatagcgtgacgatcgacggcgatgc 
                   
               
               
                   
                 gttttcagacgagatcaaggcccaggtaattgaggaagccaaaaaaaaaggtatcaaa 
                   
               
               
                   
                 tttgatctgatcgtatacagcttggccagcccagtacgtactgatcctgatacaggtatca 
                   
               
               
                   
                 tgcacaaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaacagtagatccg 
                   
               
               
                   
                 tttactggcgagctgaaggaaatctccgcggaaccagcaaatgacgaggaagcagcc 
                   
               
               
                   
                 gccactgttaaagttatggggggtgaagattgggaacgttggattaagcagctgtcgaa 
                   
               
               
                   
                 ggaaggcctcttagaagaaggctgtattaccttggcctatagttatattggccctgaagc 
                   
               
               
                   
                 tacccaagctttgtaccgtaaaggcacaatcggcaaggccaaagaacacctggaggc 
                   
               
               
                   
                 cacagcacaccgtctcaacaaagagaacccgtcaatccgtgccttcgtgagcgtgaat 
                   
               
               
                   
                 aaaggcctggtaacccgcgcaagcgccgtaatcccggtaatccctctgtatctcgcca 
                   
               
               
                   
                 gcttgttcaaagtaatgaaagagaagggcaatcatgaaggttgtattgaacagatcacg 
                   
               
               
                   
                 cgtctgtacgccgagcgcctgtaccgtaaagatggtacaattccagttgatgaggaaaa 
                   
               
               
                   
                 tcgcattcgcattgatgattgggagttagaagaagacgtccagaaagcggtatccgcgt 
                   
               
               
                   
                 tgatggagaaagtcacgggtgaaaacgcagaatctctcactgacttagcggggtaccg 
                   
               
               
                   
                 ccatgatttcttagctagtaacggctttgatgtagaaggtattaattatgaagcggaagttg 
                   
               
               
                   
                 aacgcttcgaccgtatctgataagaaggagatatacatatgagagaagtagtaattgcc 
                   
               
               
                   
                 agtgcagctagaacagcagtaggaagttttggaggagcatttaaatcagtttcagcggt 
                   
               
               
                   
                 agagttaggggtaacagcagctaaagaagctataaaaagagctaacataactccagat 
                   
               
               
                   
                 atgatagatgaatctcttttagggggagtacttacagcaggtcttggacaaaatatagca 
                   
               
               
                   
                 agacaaatagcattaggagcaggaataccagtagaaaaaccagctatgactataaata 
                   
               
               
                   
                 tagtttgtggttctggattaagatctgtttcaatggcatctcaacttatagcattaggtgatg 
                   
               
               
                   
                 ctgatataatgttagttggtggagctgaaaacatgagtatgtctccttatttagtaccaagt 
                   
               
               
                   
                 gcgagatatggtgcaagaatgggtgatgctgcttttgttgattcaatgataaaagatgga 
                   
               
               
                   
                 ttatcagacatatttaataactatcacatgggtattactgctgaaaacatagcagagcaat 
                   
               
               
                   
                 ggaatataactagagaagaacaagatgaattagctcttgcaagtcaaaataaagctgaa 
                   
               
               
                   
                 aaagctcaagctgaaggaaaatttgatgaagaaatagttcctgttgttataaaaggaaga 
                   
               
               
                   
                 aaaggtgacactgtagtagataaagatgaatatattaagcctggcactacaatggagaa 
                   
               
               
                   
                 acttgctaagttaagacctgcatttaaaaaagatggaacagttactgctggtaatgcatca 
                   
               
               
                   
                 ggaataaatgatggtgctgctatgttagtagtaatggctaaagaaaaagctgaagaact 
                   
               
               
                   
                 aggaatagagcctcttgcaactatagtttcttatggaacagctggtgttgaccctaaaata 
                   
               
               
                   
                 atgggatatggaccagttccagcaactaaaaaagctttagaagctgctaatatgactatt 
                   
               
               
                   
                 gaagatatagatttagttgaagctaatgaggcatttgctgcccaatctgtagctgtaataa 
                   
               
               
                   
                 gagacttaaatatagatatgaataaagttaatgttaatggtggagcaatagctataggac 
                   
               
               
                   
                 atccaataggatgctcaggagcaagaatacttactacacttttatatgaaatgaagagaa 
                   
               
               
                   
                 gagatgctaaaactggtcttgctacactttgtataggcggtggaatgggaactactttaat 
                   
               
               
                   
                 agttaagagatagtaagaaggagatatacatatgaaattagctgtaataggtagtggaa 
                   
               
               
                   
                 ctatgggaagtggtattgtacaaacttttgcaagttgtggacatgatgtatgtttaaagagt 
                   
               
               
                   
                 agaactcaaggtgctatagataaatgtttagctttattagataaaaatttaactaagttagtt 
                   
               
               
                   
                 actaagggaaaaatggatgaagctacaaaagcagaaatattaagtcatgttagttcaac 
                   
               
               
                   
                 tactaattatgaagatttaaaagatatggatttaataatagaagcatctgtagaagacatg 
                   
               
               
                   
                 aatataaagaaagatgttttcaagttactagatgaattatgtaaagaagatactatcttggc 
                   
               
               
                   
                 aacaaatacttcatcattatctataacagaaatagcttcttctactaagcgcccagataaa 
                   
               
               
                   
                 gttataggaatgcatttctttaatccagttcctatgatgaaattagttgaagttataagtggt 
                   
               
               
                   
                 cagttaacatcaaaagttacttttgatacagtatttgaattatctaagagtatcaataaagta 
                   
               
               
                   
                 ccagtagatgtatctgaatctcctggatttgtagtaaatagaatacttatacctatgataaat 
                   
               
               
                   
                 gaagctgttggtatatatgcagatggtgttgcaagtaaagaagaaatagatgaagctat 
                   
               
               
                   
                 gaaattaggagcaaaccatccaatgggaccactagcattaggtgatttaatcggattag 
                   
               
               
                   
                 atgttgttttagctataatgaacgttttatatactgaatttggagatactaaatatagacctca 
                   
               
               
                   
                 tccacttttagctaaaatggttagagctaatcaattaggaagaaaaactaagataggattc 
                   
               
               
                   
                 tatgattataataaataataagaaggagatatacatatgagtacaagtgatgttaaagttta 
                   
               
               
                   
                 tgagaatgtagctgttgaagtagatggaaatatatgtacagtgaaaatgaatagacctaa 
                   
               
               
                   
                 agcccttaatgcaataaattcaaagactttagaagaactttatgaagtatttgtagatatta 
                   
               
               
                   
                 ataatgatgaaactattgatgttgtaatattgacaggggaaggaaaggcatttgtagctg 
                   
               
               
                   
                 gagcagatattgcatacatgaaagatttagatgctgtagctgctaaagattttagtatctta 
                   
               
               
                   
                 ggagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatagctgctgtaaa 
                   
               
               
                   
                 cggatttgctttaggtggaggatgtgaacttgcaatggcatgtgatataagaattgcatct 
                   
               
               
                   
                 gctaaagctaaatttggtcagccagaagtaactcttggaataactccaggatatggagg 
                   
               
               
                   
                 aactcaaaggcttacaagattggttggaatggcaaaagcaaaagaattaatctttacag 
                   
               
               
                   
                 gtcaagttataaaagctgatgaagctgaaaaaatagggctagtaaatagagtcgttgag 
                   
               
               
                   
                 ccagacattttaatagaagaagttgagaaattagctaagataatagctaaaaatgctcag 
                   
               
               
                   
                 cttgcagttagatactCtaaagaagmatacaacttggtgCtcaaaCtgatataaatactg 
                   
               
               
                   
                 gaatagatatagaatctaatttatttggtctttgtttttcaactaaagaccaaaaagaagga 
                   
               
               
                   
                 atgtcagctttcgttgaaaagagagaagctaactttataaaagggtaataagaaggaga 
                   
               
               
                   
                 tatacatatgagaagttttgaagaagtaattaagtttgcaaaagaaagaggacctaaaac 
                   
               
               
                   
                 tatatcagtagcatgttgccaagataaagaagttttaatggcagttgaaatggctagaaa 
                   
               
               
                   
                 agaaaaaatagcaaatgccattttagtaggagatatagaaaagactaaagaaattgcaa 
                   
               
               
                   
                 aaagcatagacatggatatcgaaaattatgaactgatagatataaaagatttagcagaa 
                   
               
               
                   
                 gcatctctaaaatctgttgaattagtttcacaaggaaaagccgacatggtaatgaaaggc 
                   
               
               
                   
                 ttagtagacacatcaataatactaaaagcagttttaaataaagaagtaggtcttagaactg 
                   
               
               
                   
                 gaaatgtattaagtcacgtagcagtatttgatgtagagggatatgatagattatttttcgta 
                   
               
               
                   
                 actgacgcagctatgaacttagctcctgatacaaatactaaaaagcaaatcatagaaaat 
                   
               
               
                   
                 gcttgcacagtagacattcattagatataagtgaaccaaaagttgctgcaatatgcgca 
                   
               
               
                   
                 aaagaaaaagtaaatccaaaaatgaaagatacagttgaagctaaagaactagaagaa 
                   
               
               
                   
                 atgtatgaaagaggagaaatcaaaggttgtatggttggtgggccttttgcaattgataat 
                   
               
               
                   
                 gcagtatctttagaagcagctaaacataaaggtataaatcatcctgtagcaggacgagc 
                   
               
               
                   
                 tgatatattattagccccagatattgaaggtggtaacatattatataaagctttggtattcttc 
                   
               
               
                   
                 tcaaaatcaaaaaatgcaggagttatagttggggctaaagcaccaataatattaacttct 
                   
               
               
                   
                 agagcagacagtgaagaaactaaactaaactcaatagctttaggtgttttaatggcagc 
                   
               
               
                   
                 aaaggcataataagaaggagatatacatatgagcaaaatatttaaaatcttaacaataaa 
                   
               
               
                   
                 tcctggttcgacatcaactaaaatagctgtatttgataatgaggatttagtatttgaaaaaa 
                   
               
               
                   
                 ctttaagacattcttcagaagaaataggaaaatatgagaaggtgtctgaccaatttgaatt 
                   
               
               
                   
                 tcgtaaacaagtaatagaagaagctctaaaagaaggtggagtaaaaacatctgaattag 
                   
               
               
                   
                 atgctgtagtaggtagaggaggacttcttaaacctataaaaggtggtacttattcagtaa 
                   
               
               
                   
                 gtgctgctatgattgaagatttaaaagtgggagttttaggagaacacgcttcaaacctag 
                   
               
               
                   
                 gtggaataatagcaaaacaaataggtgaagaagtaaatgttccttcatacatagtagac 
                   
               
               
                   
                 cctgttgttgtagatgaattagaagatgttgctagaatttctggtatgcctgaaataagtag 
                   
               
               
                   
                 agcaagtgtagtacatgctttaaatcaaaaggcaatagcaagaagatatgctagagaaa 
                   
               
               
                   
                 taaacaagaaatatgaagatataaatcttatagttgcacacatgggtggaggagtttctgt 
                   
               
               
                   
                 tggagctcataaaaatggtaaaatagtagatgttgcaaacgcattagatggagaaggac 
                   
               
               
                   
                 ctttctctccagaaagaagtggtggactaccagtaggtgcattagtaaaaatgtgctttag 
                   
               
               
                   
                 tggaaaatatactcaagatgaaattaaaaagaaaataaaaggtaatggcggactagttg 
                   
               
               
                   
                 catacttaaacactaatgatgctagagaagttgaagaaagaattgaagctggtgatgaa 
                   
               
               
                   
                 aaagctaaattagtatatgaagctatggcatatcaaatctctaaagaaataggagctagt 
                   
               
               
                   
                 gctgcagttcttaagggagatgtaaaagcaatattattaactggtggaatcgcatattcaa 
                   
               
               
                   
                 aaatgtttacagaaatgattgcagatagagttaaatttatagcagatgtaaaagtttatcca 
                   
               
               
                   
                 ggtgaagatgaaatgattgcattagctcaaggtggacttagagttttaactggtgaagaa 
                   
               
               
                   
                 gaggctcaagtttatgataactaataa 
                   
               
               
                   
               
               
                 ter-thiA 1 -hbd- 
                 atgatcgtaaaacctatggtacgcaacaatatctgcctgaacgcccatcctcagggctg 
                 SEQ ID 
               
               
                 crt2-tesb 
                 caagaagggagtggaagatcagattgaatataccaagaaacgcattaccgcagaagt 
                 NO: 164 
               
               
                 butyrate 
                 caaagctggcgcaaaagctccaaaaaacgttctggtgcttggctgctcaaatggttacg 
                   
               
               
                 cassette 
                 gcctggcgagccgcattactgctgcgttcggatacggggctgcgaccatcggcgtgtc 
                   
               
               
                   
                 ctttgaaaaagcgggttcagaaaccaaatatggtacaccgggatggtacaataatttgg 
                   
               
               
                   
                 catttgatgaagcggcaaaacgcgagggtctttatagcgtgacgatcgacggcgatgc 
                   
               
               
                   
                 gttttcagacgagatcaaggcccaggtaattgaggaagccaaaaaaaaaggtatcaaa 
                   
               
               
                   
                 tttgatctgatcgtatacagcttggccagcccagtacgtactgatcctgatacaggtatca 
                   
               
               
                   
                 tgcacaaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaacagtagatccg 
                   
               
               
                   
                 tttactggcgagctgaaggaaatctccgcggaaccagcaaatgacgaggaagcagcc 
                   
               
               
                   
                 gccactgttaaagttatggggggtgaagattgggaacgttggattaagcagctgtcgaa 
                   
               
               
                   
                 ggaaggcctcttagaagaaggctgtattaccttggcctatagttatattggccctgaagc 
                   
               
               
                   
                 tacccaagctttgtaccgtaaaggcacaatcggcaaggccaaagaacacctggaggc 
                   
               
               
                   
                 cacagcacaccgtctcaacaaagagaacccgtcaatccgtgccttcgtgagcgtgaat 
                   
               
               
                   
                 aaaggcctggtaacccgcgcaagcgccgtaatcccggtaatccctctgtatctcgcca 
                   
               
               
                   
                 gcttgttcaaagtaatgaaagagaagggcaatcatgaaggttgtattgaacagatcacg 
                   
               
               
                   
                 cgtctgtacgccgagcgcctgtaccgtaaagatggtacaattccagttgatgaggaaaa 
                   
               
               
                   
                 tcgcattcgcattgatgattgggagttagaagaagacgtccagaaagcggtatccgcgt 
                   
               
               
                   
                 tgatggagaaagtcacgggtgaaaacgcagaatctctcactgacttagcggggtaccg 
                   
               
               
                   
                 ccatgatttcttagctagtaacggctttgatgtagaaggtattaattatgaagcggaagttg 
                   
               
               
                   
                 aacgcttcgaccgtatctgataagaaggagatatacatatgagagaagtagtaattgcc 
                   
               
               
                   
                 agtgcagctagaacagcagtaggaagttttggaggagcatttaaatcagtttcagcggt 
                   
               
               
                   
                 agagttaggggtaacagcagctaaagaagctataaaaagagctaacataactccagat 
                   
               
               
                   
                 atgatagatgaatctcttttagggggagtacttacagcaggtcttggacaaaatatagca 
                   
               
               
                   
                 agacaaatagcattaggagcaggaataccagtagaaaaaccagctatgactataaata 
                   
               
               
                   
                 tagtttgtggttctggattaagatctgtttcaatggcatctcaacttatagcattaggtgatg 
                   
               
               
                   
                 ctgatataatgttagttggtggagctgaaaacatgagtatgtctccttatttagtaccaagt 
                   
               
               
                   
                 gcgagatatggtgcaagaatgggtgatgctgcttttgttgattcaatgataaaagatgga 
                   
               
               
                   
                 ttatcagacatatttaataactatcacatgggtattactgctgaaaacatagcagagcaat 
                   
               
               
                   
                 ggaatataactagagaagaacaagatgaattagctcttgcaagtcaaaataaagctgaa 
                   
               
               
                   
                 aaagctcaagctgaaggaaaatttgatgaagaaatagttcctgttgttataaaaggaaga 
                   
               
               
                   
                 aaaggtgacactgtagtagataaagatgaatatattaagcctggcactacaatggagaa 
                   
               
               
                   
                 acttgctaagttaagacctgcatttaaaaaagatggaacagttactgctggtaatgcatca 
                   
               
               
                   
                 ggaataaatgatggtgctgctatgttagtagtaatggctaaagaaaaagctgaagaact 
                   
               
               
                   
                 aggaatagagcctcttgcaactatagtttcttatggaacagctggtgttgaccctaaaata 
                   
               
               
                   
                 atgggatatggaccagttccagcaactaaaaaagctttagaagctgctaatatgactatt 
                   
               
               
                   
                 gaagatatagatttagttgaagctaatgaggcatttgctgcccaatctgtagctgtaataa 
                   
               
               
                   
                 gagacttaaatatagatatgaataaagttaatgttaatggtggagcaatagctataggac 
                   
               
               
                   
                 atccaataggatgctcaggagcaagaatacttactacacttttatatgaaatgaagagaa 
                   
               
               
                   
                 gagatgctaaaactggtcttgctacactttgtataggcggtggaatgggaactactttaat 
                   
               
               
                   
                 agttaagagatagtaagaaggagatatacatatgaaattagctgtaataggtagtggaa 
                   
               
               
                   
                 ctatgggaagtggtattgtacaaacttttgcaagttgtggacatgatgtatgtttaaagagt 
                   
               
               
                   
                 agaactcaaggtgctatagataaatgtttagctttattagataaaaatttaactaagttagtt 
                   
               
               
                   
                 actaagggaaaaatggatgaagctacaaaagcagaaatattaagtcatgttagttcaac 
                   
               
               
                   
                 tactaattatgaagatttaaaagatatggatttaataatagaagcatctgtagaagacatg 
                   
               
               
                   
                 aatataaagaaagatgttttcaagttactagatgaattatgtaaagaagatactatcttggc 
                   
               
               
                   
                 aacaaatacttcatcattatctataacagaaatagcttcttctactaagcgcccagataaa 
                   
               
               
                   
                 gttataggaatgcatttctttaatccagttcctatgatgaaattagttgaagttataagtggt 
                   
               
               
                   
                 cagttaacatcaaaagttacttttgatacagtatttgaattatctaagagtatcaataaagta 
                   
               
               
                   
                 ccagtagatgtatctgaatctcctggatttgtagtaaatagaatacttatacctatgataaat 
                   
               
               
                   
                 gaagctgttggtatatatgcagatggtgttgcaagtaaagaagaaatagatgaagctat 
                   
               
               
                   
                 gaaattaggagcaaaccatccaatgggaccactagcattaggtgatttaatcggattag 
                   
               
               
                   
                 atgttgttttagctataatgaacgttttatatactgaatttggagatactaaatatagacctca 
                   
               
               
                   
                 tccacttttagctaaaatggttagagctaatcaattaggaagaaaaactaagataggattc 
                   
               
               
                   
                 tatgattataataaataataagaaggagatatacatatgagtacaagtgatgttaaagttta 
                   
               
               
                   
                 tgagaatgtagctgttgaagtagatggaaatatatgtacagtgaaaatgaatagacctaa 
                   
               
               
                   
                 agcccttaatgcaataaattcaaagactttagaagaactttatgaagtatttgtagatatta 
                   
               
               
                   
                 ataatgatgaaactattgatgttgtaatattgacaggggaaggaaaggcatttgtagctg 
                   
               
               
                   
                 gagcagatattgcatacatgaaagatttagatgctgtagctgctaaagattttagtatctta 
                   
               
               
                   
                 ggagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatagctgctgtaaa 
                   
               
               
                   
                 cggatttgctttaggtggaggatgtgaacttgcaatggcatgtgatataagaattgcatct 
                   
               
               
                   
                 gctaaagctaaatttggtcagccagaagtaactcttggaataactccaggatatggagg 
                   
               
               
                   
                 aactcaaaggcttacaagattggttggaatggcaaaagcaaaagaattaatctttacag 
                   
               
               
                   
                 gtcaagttataaaagctgatgaagctgaaaaaatagggctagtaaatagagtcgttgag 
                   
               
               
                   
                 ccagacattttaatagaagaagttgagaaattagctaagataatagctaaaaatgctcag 
                   
               
               
                   
                 cttgcagttagatactctaaagaagcaatacaacttggtgctcaaactgatataaatactg 
                   
               
               
                   
                 gaatagatatagaatctaatttatttggtctttgtttttcaactaaagaccaaaaagaagga 
                   
               
               
                   
                 atgtcagctttcgttgaaaagagagaagctaactttataaaagggtaataagaaggaga 
                   
               
               
                   
                 tatacatatgAGTCAGGCGCTAAAAAATTTACTGACATTGT 
                   
               
               
                   
                 TAAATCTGGAAAAAATTGAGGAAGGACTCTTTCGCG 
                   
               
               
                   
                 GCCAGAGTGAAGATTTAGGTTTACGCCAGGTGTTTG 
                   
               
               
                   
                 GCGGCCAGGTCGTGGGTCAGGCCTTGTATGCTGCAA 
                   
               
               
                   
                 AAGAGACCGTCCCTGAAGAGCGGCTGGTACATTCGT 
                   
               
               
                   
                 TTCACAGCTACTTTCTTCGCCCTGGCGATAGTAAGAA 
                   
               
               
                   
                 GCCGATTATTTATGATGTCGAAACGCTGCGTGACGG 
                   
               
               
                   
                 TAACAGCTTCAGCGCCCGCCGGGTTGCTGCTATTCA 
                   
               
               
                   
                 AAACGGCAAACCGATTTTTTATATGACTGCCTCTTTC 
                   
               
               
                   
                 CAGGCACCAGAAGCGGGTTTCGAACATCAAAAAAC 
                   
               
               
                   
                 AATGCCGTCCGCGCCAGCGCCTGATGGCCTCCCTTC 
                   
               
               
                   
                 GGAAACGCAAATCGCCCAATCGCTGGCGCACCTGCT 
                   
               
               
                   
                 GCCGCCAGTGCTGAAAGATAAATTCATCTGCGATCG 
                   
               
               
                   
                 TCCGCTGGAAGTCCGTCCGGTGGAGTTTCATAACCC 
                   
               
               
                   
                 ACTGAAAGGTCACGTCGCAGAACCACATCGTCAGGT 
                   
               
               
                   
                 GTGGATCCGCGCAAATGGTAGCGTGCCGGATGACCT 
                   
               
               
                   
                 GCGCGTTCATCAGTATCTGCTCGGTTACGCTTCTGAT 
                   
               
               
                   
                 CTTAACTTCCTGCCGGTAGCTCTACAGCCGCACGGC 
                   
               
               
                   
                 ATCGGTTTTCTCGAACCGGGGATTCAGATTGCCACC 
                   
               
               
                   
                 ATTGACCATTCCATGTGGTTCCATCGCCCGTTTAATT 
                   
               
               
                   
                 TGAATGAATGGCTGCTGTATAGCGTGGAGAGCACCT 
                   
               
               
                   
                 CGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGAGT 
                   
               
               
                   
                 TTTATACCCAAGACGGCGTACTGGTTGCCTCGACCG 
                   
               
               
                   
                 TTCAGGAAGGGGTGATGCGTAATCACAATtaa 
               
               
                   
               
            
           
         
       
     
     In certain constructs, the butyrate gene cassette (e.g., bcd2-etfB3-etfA3-thiA1-hbd-crt2-pbt buk butyrate cassette (pLogic031), and/or ter-thiA1-hbd-crt2-pbt buk butyrate cassette (pLogic046) and/or ter-thiA1-hbd-crt2-tesb butyrate cassette (pLOGIC046-delta pbt.buk/tesB+)) is placed under the control of an RNS-responsive regulatory region, e.g., norB. In some embodiments, the butyrate gene cassette is placed under the control of an RNS-responsive regulatory region, e.g., norB. and the bacteria further comprises a gene encoding a corresponding RNS-responsive transcription factor, e.g., nsrR (see, e.g., Table 37 and Table 38 and SEQ ID NO: 167). 
     Table 37 depicts the nucleic acid sequence of an exemplary RNS-regulated construct comprising a gene encoding nsrR, a regulatory region of norB, and a butyrogenic gene cassette (pLogic031-nsrR-norB-butyrate construct; SEQ ID NO: 165). The sequence encoding NsrR is underlined and bolded, and the NsrR binding site, i.e., a regulatory region of norB is   Table 38 depicts the nucleic acid sequence of an exemplary RNS-regulated construct comprising a gene encoding nsrR, a regulatory region of norB, and a butyrogenic gene cassette (pLogic046-nsrR-norB-butyrate construct; SEQ ID NO: 166). The sequence encoding NsrR is underlined and bolded, and the NsrR binding site, i.e., a regulatory region of norB is  . 
     Table 39 (SEQ ID NO: 167) depicts the nucleic acid sequence of an exemplary RNS-regulated construct comprising a gene encoding nsrR, a regulatory region of norB, and a butyrogenic gene cassette (pLOGIC046-delta pbt.buk/tesB+-nsrR-norB-butyrate construct (SEQ ID NO: 167). 
     In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 165, 166, 167, or a functional fragment thereof. 
     
       
         
           
               
             
               
                 TABLE 37 
               
               
                   
               
               
                 Nucleotide sequences of pLogic031-nsrR-norB-butyrate construct 
               
               
                 Nucleotide sequences of pLogic031-nsrR-norB-butyrate construct 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 165) 
               
               
                 ttatta   tcgcaccgcaatcgggattttcgattcataaagcaggtcgtaggtcggcttgtt     
               
               
                   
               
               
                 
                   
                     gagcaggtcttgcagcgtgaaaccgtccagatacgtgaaaaacgacttcattgcaccgcc 
                   
                 
               
               
                   
               
               
                 
                   
                     gagtatgcccgtcagccggcaggacggcgtaatcaggcattcgttgttcgggcccataca 
                   
                 
               
               
                   
               
               
                 
                   
                     ctcgaccagctgcatcggttcgaggtggcggacgaccgcgccgatattgatgcgttcggg 
                   
                 
               
               
                   
               
               
                 
                   
                     cggcgcggccagcctcagcccgccgcctttcccgcgtacgctgtgcaagaacccgccttt 
                   
                 
               
               
                   
               
               
                 
                   
                     gaccagcgcggtaaccactttcatcaaatggcttttggaaatgccgtaggtcgaggcgat 
                   
                 
               
               
                   
               
               
                 
                   
                     ggtggcgatattgaccagcgcgtcgtcgttgacggcggtgtagatgaggacgcgcagccc 
                   
                 
               
               
                   
               
               
                     gtagtcggtatgttgggtcagatacat   acaacctccttagtacatgcaaaattatttcta 
               
               
                   
               
               
                 gagcaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagttgagtt 
               
               
                   
               
               
                 gaggaattataacaggaagaaatattcctcatacgcttgtaattcctctatggttgttga 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 aaataattttgtttaactttaagaaggagatatacatatggatttaaattctaaaaaata 
               
               
                   
               
               
                 tcagatgcttaaagagctatatgtaagcttcgctgaaaatgaagttaaacctttagcaac 
               
               
                   
               
               
                 agaacttgatgaagaagaaagatttccttatgaaacagtggaaaaaatggcaaaagcagg 
               
               
                   
               
               
                 aatgatgggtataccatatccaaaagaatatggtggagaaggtggagacactgtaggata 
               
               
                   
               
               
                 tataatggcagttgaagaattgtctagagtttgtggtactacaggagttatattatcagc 
               
               
                   
               
               
                 tcatacatctcttggctcatggcctatatatcaatatggtaatgaagaacaaaaacaaaa 
               
               
                   
               
               
                 attcttaagaccactagcaagtggagaaaaattaggagcatttggtcttactgagcctaa 
               
               
                   
               
               
                 tgctggtacagatgcgtctggccaacaaacaactgctgttttagacggggatgaatacat 
               
               
                   
               
               
                 acttaatggctcaaaaatatttataacaaacgcaatagctggtgacatatatgtagtaat 
               
               
                   
               
               
                 ggcaatgactgataaatctaaggggaacaaaggaatatcagcatttatagttgaaaaagg 
               
               
                   
               
               
                 aactcctgggtttagctttggagttaaagaaaagaaaatgggtataagaggttcagctac 
               
               
                   
               
               
                 gagtgaattaatatttgaggattgcagaatacctaaagaaaatttacttggaaaagaagg 
               
               
                   
               
               
                 tcaaggatttaagatagcaatgtctactcttgatggtggtagaattggtatagctgcaca 
               
               
                   
               
               
                 agctttaggtttagcacaaggtgctcttgatgaaactgttaaatatgtaaaagaaagagt 
               
               
                   
               
               
                 acaatttggtagaccattatcaaaattccaaaatacacaattccaattagctgatatgga 
               
               
                   
               
               
                 agttaaggtacaagcggctagacaccttgtatatcaagcagctataaataaagacttagg 
               
               
                   
               
               
                 aaaaccttatggagtagaagcagcaatggcaaaattatttgcagctgaaacagctatgga 
               
               
                   
               
               
                 agttactacaaaagctgtacaacttcatggaggatatggatacactcgtgactatccagt 
               
               
                   
               
               
                 agaaagaatgatgagagatgctaagataactgaaatatatgaaggaactagtgaagttca 
               
               
                   
               
               
                 aagaatggttatttcaggaaaactattaaaatagtaagaaggagatatacatatggagga 
               
               
                   
               
               
                 aggatttatgaatatagtcgtttgtataaaacaagttccagatacaacagaagttaaact 
               
               
                   
               
               
                 agatcctaatacaggtactttaattagagatggagtaccaagtataataaaccctgatga 
               
               
                   
               
               
                 taaagcaggtttagaagaagctataaaattaaaagaagaaatgggtgctcatgtaactgt 
               
               
                   
               
               
                 tataacaatgggacctcctcaagcagatatggctttaaaagaagctttagcaatgggtgc 
               
               
                   
               
               
                 agatagaggtatattattaacagatagagcatttgcgggtgctgatacttgggcaacttc 
               
               
                   
               
               
                 atcagcattagcaggagcattaaaaaatatagattttgatattataatagctggaagaca 
               
               
                   
               
               
                 ggcgatagatggagatactgcacaagttggacctcaaatagctgaacatttaaatcttcc 
               
               
                   
               
               
                 atcaataacatatgctgaagaaataaaaactgaaggtgaatatgtattagtaaaaagaca 
               
               
                   
               
               
                 atttgaagattgttgccatgacttaaaagttaaaatgccatgccttataacaactcttaa 
               
               
                   
               
               
                 agatatgaacacaccaagatacatgaaagttggaagaatatatgatgctttcgaaaatga 
               
               
                   
               
               
                 tgtagtagaaacatggactgtaaaagatatagaagttgacccttctaatttaggtcttaa 
               
               
                   
               
               
                 aggttctccaactagtgtatttaaatcatttacaaaatcagttaaaccagctggtacaat 
               
               
                   
               
               
                 atacaatgaagatgcgaaaacatcagctggaattatcatagataaattaaaagagaagta 
               
               
                   
               
               
                 tatcatataataagaaggagatatacatatgggtaacgttttagtagtaatagaacaaag 
               
               
                   
               
               
                 agaaaatgtaattcaaactgtttctttagaattactaggaaaggctacagaaatagcaaa 
               
               
                   
               
               
                 agattatgatacaaaagtttctgcattacttttaggtagtaaggtagaaggtttaataga 
               
               
                   
               
               
                 tacattagcacactatggtgcagatgaggtaatagtagtagatgatgaagctttagcagt 
               
               
                   
               
               
                 gtatacaactgaaccatatacaaaagcagcttatgaagcaataaaagcagctgaccctat 
               
               
                   
               
               
                 agttgtattatttggtgcaacttcaataggtagagatttagcgcctagagtttctgctag 
               
               
                   
               
               
                 aatacatacaggtcttactgctgactgtacaggtcttgcagtagctgaagatacaaaatt 
               
               
                   
               
               
                 attattaatgacaagacctgcctttggtggaaatataatggcaacaatagtttgtaaaga 
               
               
                   
               
               
                 tttcagacctcaaatgtctacagttagaccaggggttatgaagaaaaatgaacctgatga 
               
               
                   
               
               
                 aactaaagaagctgtaattaaccgtttcaaggtagaatttaatgatgctgataaattagt 
               
               
                   
               
               
                 tcaagttgtacaagtaataaaagaagctaaaaaacaagttaaaatagaagatgctaagat 
               
               
                   
               
               
                 attagtttctgctggacgtggaatgggtggaaaagaaaacttagacatactttatgaatt 
               
               
                   
               
               
                 agctgaaattataggtggagaagtttctggttctcgtgccactatagatgcaggttggtt 
               
               
                   
               
               
                 agataaagcaagacaagttggtcaaactggtaaaactgtaagaccagacctttatatagc 
               
               
                   
               
               
                 atgtggtatatctggagcaatacaacatatagctggtatggaagatgctgagtttatagt 
               
               
                   
               
               
                 tgctataaataaaaatccagaagctccaatatttaaatatgctgatgttggtatagttgg 
               
               
                   
               
               
                 agatgttcataaagtgcttccagaacttatcagtcagttaagtgttgcaaaagaaaaagg 
               
               
                   
               
               
                 tgaagttttagctaactaataagaaggagatatacatatgagagaagtagtaattgccag 
               
               
                   
               
               
                 tgcagctagaacagcagtaggaagttttggaggagcatttaaatcagtttcagcggtaga 
               
               
                   
               
               
                 gttaggggtaacagcagctaaagaagctataaaaagagctaacataactccagatatgat 
               
               
                   
               
               
                 agatgaatctcttttagggggagtacttacagcaggtcttggacaaaatatagcaagaca 
               
               
                   
               
               
                 aatagcattaggagcaggaataccagtagaaaaaccagctatgactataaatatagtttg 
               
               
                   
               
               
                 tggttctggattaagatctgtttcaatggcatctcaacttatagcattaggtgatgctga 
               
               
                   
               
               
                 tataatgttagttggtggagctgaaaacatgagtatgtctccttatttagtaccaagtgc 
               
               
                   
               
               
                 gagatatggtgcaagaatgggtgatgctgcttttgttgattcaatgataaaagatggatt 
               
               
                   
               
               
                 atcagacatatttaataactatcacatgggtattactgctgaaaacatagcagagcaatg 
               
               
                   
               
               
                 gaatataactagagaagaacaagatgaattagctcttgcaagtcaaaataaagctgaaaa 
               
               
                   
               
               
                 agctcaagctgaaggaaaatttgatgaagaaatagttcctgttgttataaaaggaagaaa 
               
               
                   
               
               
                 aggtgacactgtagtagataaagatgaatatattaagcctggcactacaatggagaaact 
               
               
                   
               
               
                 tgctaagttaagacctgcatttaaaaaagatggaacagttactgctggtaatgcatcagg 
               
               
                   
               
               
                 aataaatgatggtgctgctatgttagtagtaatggctaaagaaaaagctgaagaactagg 
               
               
                   
               
               
                 aatagagcctcttgcaactatagtttcttatggaacagctggtgttgaccctaaaataat 
               
               
                   
               
               
                 gggatatggaccagttccagcaactaaaaaagctttagaagctgctaatatgactattga 
               
               
                   
               
               
                 agatatagatttagttgaagctaatgaggcatttgctgcccaatctgtagctgtaataag 
               
               
                   
               
               
                 agacttaaatatagatatgaataaagttaatgttaatggtggagcaatagctataggaca 
               
               
                   
               
               
                 tccaataggatgctcaggagcaagaatacttactacacttttatatgaaatgaagagaag 
               
               
                   
               
               
                 agatgctaaaactggtcttgctacactttgtataggcggtggaatgggaactactttaat 
               
               
                   
               
               
                 agttaagagatagtaagaaggagatatacatatgaaattagctgtaataggtagtggaac 
               
               
                   
               
               
                 tatgggaagtggtattgtacaaacttttgcaagttgtggacatgatgtatgtttaaagag 
               
               
                   
               
               
                 tagaactcaaggtgctatagataaatgtttagctttattagataaaaatttaactaagtt 
               
               
                   
               
               
                 agttactaagggaaaaatggatgaagctacaaaagcagaaatattaagtcatgttagttc 
               
               
                   
               
               
                 aactactaattatgaagatttaaaagatatggatttaataatagaagcatctgtagaaga 
               
               
                   
               
               
                 catgaatataaagaaagatgttttcaagttactagatgaattatgtaaagaagatactat 
               
               
                   
               
               
                 cttggcaacaaatacttcatcattatctataacagaaatagcttcttctactaagcgccc 
               
               
                   
               
               
                 agataaagttataggaatgcatttctttaatccagttcctatgatgaaattagttgaagt 
               
               
                   
               
               
                 tataagtggtcagttaacatcaaaagttacttttgatacagtatttgaattatctaagag 
               
               
                   
               
               
                 tatcaataaagtaccagtagatgtatctgaatctcctggatttgtagtaaatagaatact 
               
               
                   
               
               
                 tatacctatgataaatgaagctgttggtatatatgcagatggtgttgcaagtaaagaaga 
               
               
                   
               
               
                 aatagatgaagctatgaaattaggagcaaaccatccaatgggaccactagcattaggtga 
               
               
                   
               
               
                 tttaatcggattagatgttgttttagctataatgaacgttttatatactgaatttggaga 
               
               
                   
               
               
                 tactaaatatagacctcatccacttttagctaaaatggttagagctaatcaattaggaag 
               
               
                   
               
               
                 aaaaactaagataggattctatgattataataaataataagaaggagatatacatatgag 
               
               
                   
               
               
                 tacaagtgatgttaaagtttatgagaatgtagctgttgaagtagatggaaatatatgtac 
               
               
                   
               
               
                 agtgaaaatgaatagacctaaagcccttaatgcaataaattcaaagactttagaagaact 
               
               
                   
               
               
                 ttatgaagtatttgtagatattaataatgatgaaactattgatgttgtaatattgacagg 
               
               
                   
               
               
                 ggaaggaaaggcatttgtagctggagcagatattgcatacatgaaagatttagatgctgt 
               
               
                   
               
               
                 agctgctaaagattttagtatcttaggagcaaaagcttttggagaaatagaaaatagtaa 
               
               
                   
               
               
                 aaaagtagtgatagctgctgtaaacggatttgctttaggtggaggatgtgaacttgcaat 
               
               
                   
               
               
                 ggcatgtgatataagaattgcatctgctaaagctaaatttggtcagccagaagtaactct 
               
               
                   
               
               
                 tggaataactccaggatatggaggaactcaaaggcttacaagattggttggaatggcaaa 
               
               
                   
               
               
                 agcaaaagaattaatctttacaggtcaagttataaaagctgatgaagctgaaaaaatagg 
               
               
                   
               
               
                 gctagtaaatagagtcgttgagccagacattttaatagaagaagttgagaaattagctaa 
               
               
                   
               
               
                 gataatagctaaaaatgctcagcttgcagttagatactctaaagaagcaatacaacttgg 
               
               
                   
               
               
                 tgctcaaactgatataaatactggaatagatatagaatctaatttatttggtctttgttt 
               
               
                   
               
               
                 ttcaactaaagaccaaaaagaaggaatgtcagctttcgttgaaaagagagaagctaactt 
               
               
                   
               
               
                 tataaaagggtaataagaaggagatatacatatgagaagttttgaagaagtaattaagtt 
               
               
                   
               
               
                 tgcaaaagaaagaggacctaaaactatatcagtagcatgttgccaagataaagaagtttt 
               
               
                   
               
               
                 aatggcagttgaaatggctagaaaagaaaaaatagcaaatgccattttagtaggagatat 
               
               
                   
               
               
                 agaaaagactaaagaaattgcaaaaagcatagacatggatatcgaaaattatgaactgat 
               
               
                   
               
               
                 agatataaaagatttagcagaagcatctctaaaatctgttgaattagtttcacaaggaaa 
               
               
                   
               
               
                 agccgacatggtaatgaaaggcttagtagacacatcaataatactaaaagcagttttaaa 
               
               
                   
               
               
                 taaagaagtaggtcttagaactggaaatgtattaagtcacgtagcagtatttgatgtaga 
               
               
                   
               
               
                 gggatatgatagattatttttcgtaactgacgcagctatgaacttagctcctgatacaaa 
               
               
                   
               
               
                 tactaaaaagcaaatcatagaaaatgcttgcacagtagcacattcattagatataagtga 
               
               
                   
               
               
                 accaaaagttgctgcaatatgcgcaaaagaaaaagtaaatccaaaaatgaaagatacagt 
               
               
                   
               
               
                 tgaagctaaagaactagaagaaatgtatgaaagaggagaaatcaaaggttgtatggttgg 
               
               
                   
               
               
                 tgggccttttgcaattgataatgcagtatctttagaagcagctaaacataaaggtataaa 
               
               
                   
               
               
                 tcatcctgtagcaggacgagctgatatattattagccccagatattgaaggtggtaacat 
               
               
                   
               
               
                 attatataaagctttggtattcttctcaaaatcaaaaaatgcaggagttatagttggggc 
               
               
                   
               
               
                 taaagcaccaataatattaacttctagagcagacagtgaagaaactaaactaaactcaat 
               
               
                   
               
               
                 agctttaggtgttttaatggcagcaaaggcataataagaaggagatatacatatgagcaa 
               
               
                   
               
               
                 aatatttaaaatcttaacaataaatcctggttcgacatcaactaaaatagctgtatttga 
               
               
                   
               
               
                 taatgaggatttagtatttgaaaaaactttaagacattcttcagaagaaataggaaaata 
               
               
                   
               
               
                 tgagaaggtgtctgaccaatttgaatttcgtaaacaagtaatagaagaagctctaaaaga 
               
               
                   
               
               
                 aggtggagtaaaaacatctgaattagatgctgtagtaggtagaggaggacttcttaaacc 
               
               
                   
               
               
                 tataaaaggtggtacttattcagtaagtgctgctatgattgaagatttaaaagtgggagt 
               
               
                   
               
               
                 tttaggagaacacgcttcaaacctaggtggaataatagcaaaacaaataggtgaagaagt 
               
               
                   
               
               
                 aaatgttccttcatacatagtagaccctgttgttgtagatgaattagaagatgttgctag 
               
               
                   
               
               
                 aatttctggtatgcctgaaataagtagagcaagtgtagtacatgctttaaatcaaaaggc 
               
               
                   
               
               
                 aatagcaagaagatatgctagagaaataaacaagaaatatgaagatataaatcttatagt 
               
               
                   
               
               
                 tgcacacatgggtggaggagtttctgttggagctcataaaaatggtaaaatagtagatgt 
               
               
                   
               
               
                 tgcaaacgcattagatggagaaggacctttctctccagaaagaagtggtggactaccagt 
               
               
                   
               
               
                 aggtgcattagtaaaaatgtgctttagtggaaaatatactcaagatgaaattaaaaagaa 
               
               
                   
               
               
                 aataaaaggtaatggcggactagttgcatacttaaacactaatgatgctagagaagttga 
               
               
                   
               
               
                 agaaagaattgaagctggtgatgaaaaagctaaattagtatatgaagctatggcatatca 
               
               
                   
               
               
                 aatctctaaagaaataggagctagtgctgcagttcttaagggagatgtaaaagcaatatt 
               
               
                   
               
               
                 attaactggtggaatcgcatattcaaaaatgtttacagaaatgattgcagatagagttaa 
               
               
                   
               
               
                 atttatagcagatgtaaaagtttatccaggtgaagatgaaatgattgcattagctcaagg 
               
               
                   
               
               
                 tggacttagagttttaactggtgaagaagaggctcaagtttatgataactaataa 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 38 
               
               
                   
               
               
                 pLogic046-nsrR-norB-butyrate construct 
               
               
                 Nucleotide sequences of pLogic046-nsrR-norB-butyrate construct 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 166) 
               
               
                 ttatta   tcgcaccgcaatcgggattttcgattcataaagcaggtcgtaggtcggcttgtt     
               
               
                   
               
               
                 
                   
                     gagcaggtcttgcagcgtgaaaccgtccagatacgtgaaaaacgacttcattgcaccgcc 
                   
                 
               
               
                   
               
               
                 
                   
                     gagtatgcccgtcagccggcaggacggcgtaatcaggcattcgttgttcgggcccataca 
                   
                 
               
               
                   
               
               
                 
                   
                     ctcgaccagctgcatcggttcgaggtggcggacgaccgcgccgatattgatgcgttcggg 
                   
                 
               
               
                   
               
               
                 
                   
                     cggcgcggccagcctcagcccgccgcctttcccgcgtacgctgtgcaagaacccgccttt 
                   
                 
               
               
                   
               
               
                 
                   
                     gaccagcgcggtaaccactttcatcaaatggcttttggaaatgccgtaggtcgaggcgat 
                   
                 
               
               
                   
               
               
                 
                   
                     ggtggcgatattgaccagcgcgtcgtcgttgacggcggtgtagatgaggacgcgcagccc 
                   
                 
               
               
                   
               
               
                     gtagtcggtatgttgggtcagatacat   acaacctccttagtacatgcaaaattatttcta 
               
               
                   
               
               
                 gagcaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagttgagtt 
               
               
                   
               
               
                 gaggaattataacaggaagaaatattcctcatacgcttgtaattcctctatggttgttga 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 aaataattttgtttaactttaagaaggagatatacatatgatcgtaaaacctatggtacg 
               
               
                   
               
               
                 caacaatatctgcctgaacgcccatcctcagggctgcaagaagggagtggaagatcagat 
               
               
                   
               
               
                 tgaatataccaagaaacgcattaccgcagaagtcaaagctggcgcaaaagctccaaaaaa 
               
               
                   
               
               
                 cgttctggtgcttggctgctcaaatggttacggcctggcgagccgcattactgctgcgtt 
               
               
                   
               
               
                 cggatacggggctgcgaccatcggcgtgtcctttgaaaaagcgggttcagaaaccaaata 
               
               
                   
               
               
                 tggtacaccgggatggtacaataatttggcatttgatgaagcggcaaaacgcgagggtct 
               
               
                   
               
               
                 ttatagcgtgacgatcgacggcgatgcgttttcagacgagatcaaggcccaggtaattga 
               
               
                   
               
               
                 ggaagccaaaaaaaaaggtatcaaatttgatctgatcgtatacagcttggccagcccagt 
               
               
                   
               
               
                 acgtactgatcctgatacaggtatcatgcacaaaagcgttttgaaaccctttggaaaaac 
               
               
                   
               
               
                 gttcacaggcaaaacagtagatccgtttactggcgagctgaaggaaatctccgcggaacc 
               
               
                   
               
               
                 agcaaatgacgaggaagcagccgccactgttaaagttatggggggtgaagattgggaacg 
               
               
                   
               
               
                 ttggattaagcagctgtcgaaggaaggcctcttagaagaaggctgtattaccttggccta 
               
               
                   
               
               
                 tagttatattggccctgaagctacccaagctttgtaccgtaaaggcacaatcggcaaggc 
               
               
                   
               
               
                 caaagaacacctggaggccacagcacaccgtctcaacaaagagaacccgtcaatccgtgc 
               
               
                   
               
               
                 cttcgtgagcgtgaataaaggcctggtaacccgcgcaagcgccgtaatcccggtaatccc 
               
               
                   
               
               
                 tctgtatctcgccagcttgttcaaagtaatgaaagagaagggcaatcatgaaggttgtat 
               
               
                   
               
               
                 tgaacagatcacgcgtctgtacgccgagcgcctgtaccgtaaagatggtacaattccagt 
               
               
                   
               
               
                 tgatgaggaaaatcgcattcgcattgatgattgggagttagaagaagacgtccagaaagc 
               
               
                   
               
               
                 ggtatccgcgttgatggagaaagtcacgggtgaaaacgcagaatctctcactgacttagc 
               
               
                   
               
               
                 ggggtaccgccatgatttcttagctagtaacggctttgatgtagaaggtattaattatga 
               
               
                   
               
               
                 agcggaagttgaacgcttcgaccgtatctgataagaaggagatatacatatgagagaagt 
               
               
                   
               
               
                 agtaattgccagtgcagctagaacagcagtaggaagttttggaggagcatttaaatcagt 
               
               
                   
               
               
                 ttcagcggtagagttaggggtaacagcagctaaagaagctataaaaagagctaacataac 
               
               
                   
               
               
                 tccagatatgatagatgaatctcttttagggggagtacttacagcaggtcttggacaaaa 
               
               
                   
               
               
                 tatagcaagacaaatagcattaggagcaggaataccagtagaaaaaccagctatgactat 
               
               
                   
               
               
                 aaatatagtttgtggttctggattaagatctgtttcaatggcatctcaacttatagcatt 
               
               
                   
               
               
                 aggtgatgctgatataatgttagttggtggagctgaaaacatgagtatgtctccttattt 
               
               
                   
               
               
                 agtaccaagtgcgagatatggtgcaagaatgggtgatgctgcttttgttgattcaatgat 
               
               
                   
               
               
                 aaaagatggattatcagacatatttaataactatcacatgggtattactgctgaaaacat 
               
               
                   
               
               
                 agcagagcaatggaatataactagagaagaacaagatgaattagctcttgcaagtcaaaa 
               
               
                   
               
               
                 taaagctgaaaaagctcaagctgaaggaaaatttgatgaagaaatagttcctgttgttat 
               
               
                   
               
               
                 aaaaggaagaaaaggtgacactgtagtagataaagatgaatatattaagcctggcactac 
               
               
                   
               
               
                 aatggagaaacttgctaagttaagacctgcatttaaaaaagatggaacagttactgctgg 
               
               
                   
               
               
                 taatgcatcaggaataaatgatggtgctgctatgttagtagtaatggctaaagaaaaagc 
               
               
                   
               
               
                 tgaagaactaggaatagagcctcttgcaactatagtttcttatggaacagctggtgttga 
               
               
                   
               
               
                 ccctaaaataatgggatatggaccagttccagcaactaaaaaagctttagaagctgctaa 
               
               
                   
               
               
                 tatgactattgaagatatagatttagttgaagctaatgaggcatttgctgcccaatctgt 
               
               
                   
               
               
                 agctgtaataagagacttaaatatagatatgaataaagttaatgttaatggtggagcaat 
               
               
                   
               
               
                 agctataggacatccaataggatgctcaggagcaagaatacttactacacttttatatga 
               
               
                   
               
               
                 aatgaagagaagagatgctaaaactggtcttgctacactttgtataggcggtggaatggg 
               
               
                   
               
               
                 aactactttaatagttaagagatagtaagaaggagatatacatatgaaattagctgtaat 
               
               
                   
               
               
                 aggtagtggaactatgggaagtggtattgtacaaacttttgcaagttgtggacatgatgt 
               
               
                   
               
               
                 atgtttaaagagtagaactcaaggtgctatagataaatgtttagctttattagataaaaa 
               
               
                   
               
               
                 tttaactaagttagttactaagggaaaaatggatgaagctacaaaagcagaaatattaag 
               
               
                   
               
               
                 tcatgttagttcaactactaattatgaagatttaaaagatatggatttaataatagaagc 
               
               
                   
               
               
                 atctgtagaagacatgaatataaagaaagatgttttcaagttactagatgaattatgtaa 
               
               
                   
               
               
                 agaagatactatcttggcaacaaatacttcatcattatctataacagaaatagcttcttc 
               
               
                   
               
               
                 tactaagcgcccagataaagttataggaatgcatttctttaatccagttcctatgatgaa 
               
               
                   
               
               
                 attagttgaagttataagtggtcagttaacatcaaaagttacttttgatacagtatttga 
               
               
                   
               
               
                 attatctaagagtatcaataaagtaccagtagatgtatctgaatctcctggatttgtagt 
               
               
                   
               
               
                 aaatagaatacttatacctatgataaatgaagctgttggtatatatgcagatggtgttgc 
               
               
                   
               
               
                 aagtaaagaagaaatagatgaagctatgaaattaggagcaaaccatccaatgggaccact 
               
               
                   
               
               
                 agcattaggtgatttaatcggattagatgttgttttagctataatgaacgttttatatac 
               
               
                   
               
               
                 tgaatttggagatactaaatatagacctcatccacttttagctaaaatggttagagctaa 
               
               
                   
               
               
                 tcaattaggaagaaaaactaagataggattctatgattataataaataataagaaggaga 
               
               
                   
               
               
                 tatacatatgagtacaagtgatgttaaagtttatgagaatgtagctgttgaagtagatgg 
               
               
                   
               
               
                 aaatatatgtacagtgaaaatgaatagacctaaagcccttaatgcaataaattcaaagac 
               
               
                   
               
               
                 tttagaagaactttatgaagtatttgtagatattaataatgatgaaactattgatgttgt 
               
               
                   
               
               
                 aatattgacaggggaaggaaaggcatttgtagctggagcagatattgcatacatgaaaga 
               
               
                   
               
               
                 tttagatgctgtagctgctaaagattttagtatcttaggagcaaaagcttttggagaaat 
               
               
                   
               
               
                 agaaaatagtaaaaaagtagtgatagctgctgtaaacggatttgctttaggtggaggatg 
               
               
                   
               
               
                 tgaacttgcaatggcatgtgatataagaattgcatctgctaaagctaaatttggtcagcc 
               
               
                   
               
               
                 agaagtaactcttggaataactccaggatatggaggaactcaaaggcttacaagattggt 
               
               
                   
               
               
                 tggaatggcaaaagcaaaagaattaatctttacaggtcaagttataaaagctgatgaagc 
               
               
                   
               
               
                 tgaaaaaatagggctagtaaatagagtcgttgagccagacattttaatagaagaagttga 
               
               
                   
               
               
                 gaaattagctaagataatagctaaaaatgctcagcttgcagttagatactctaaagaagc 
               
               
                   
               
               
                 aatacaacttggtgctcaaactgatataaatactggaatagatatagaatctaatttatt 
               
               
                   
               
               
                 tggtctttgtttttcaactaaagaccaaaaagaaggaatgtcagctttcgttgaaaagag 
               
               
                   
               
               
                 agaagctaactttataaaagggtaataagaaggagatatacatatgagaagttttgaaga 
               
               
                   
               
               
                 agtaattaagtttgcaaaagaaagaggacctaaaactatatcagtagcatgttgccaaga 
               
               
                   
               
               
                 taaagaagttttaatggcagttgaaatggctagaaaagaaaaaatagcaaatgccatttt 
               
               
                   
               
               
                 agtaggagatatagaaaagactaaagaaattgcaaaaagcatagacatggatatcgaaaa 
               
               
                   
               
               
                 ttatgaactgatagatataaaagatttagcagaagcatctctaaaatctgttgaattagt 
               
               
                   
               
               
                 ttcacaaggaaaagccgacatggtaatgaaaggcttagtagacacatcaataatactaaa 
               
               
                   
               
               
                 agcagttttaaataaagaagtaggtcttagaactggaaatgtattaagtcacgtagcagt 
               
               
                   
               
               
                 atttgatgtagagggatatgatagattatttttcgtaactgacgcagctatgaacttagc 
               
               
                   
               
               
                 tcctgatacaaatactaaaaagcaaatcatagaaaatgcttgcacagtagcacattcatt 
               
               
                   
               
               
                 agatataagtgaaccaaaagttgctgcaatatgcgcaaaagaaaaagtaaatccaaaaat 
               
               
                   
               
               
                 gaaagatacagttgaagctaaagaactagaagaaatgtatgaaagaggagaaatcaaagg 
               
               
                   
               
               
                 ttgtatggttggtgggccttttgcaattgataatgcagtatctttagaagcagctaaaca 
               
               
                   
               
               
                 taaaggtataaatcatcctgtagcaggacgagctgatatattattagccccagatattga 
               
               
                   
               
               
                 aggtggtaacatattatataaagctttggtattcttctcaaaatcaaaaaatgcaggagt 
               
               
                   
               
               
                 tatagttggggctaaagcaccaataatattaacttctagagcagacagtgaagaaactaa 
               
               
                   
               
               
                 actaaactcaatagctttaggtgttttaatggcagcaaaggcataataagaaggagatat 
               
               
                   
               
               
                 acatatgagcaaaatatttaaaatcttaacaataaatcctggttcgacatcaactaaaat 
               
               
                   
               
               
                 agctgtatttgataatgaggatttagtatttgaaaaaactttaagacattcttcagaaga 
               
               
                   
               
               
                 aataggaaaatatgagaaggtgtctgaccaatttgaatttcgtaaacaagtaatagaaga 
               
               
                   
               
               
                 agctctaaaagaaggtggagtaaaaacatctgaattagatgctgtagtaggtagaggagg 
               
               
                   
               
               
                 acttcttaaacctataaaaggtggtacttattcagtaagtgctgctatgattgaagattt 
               
               
                   
               
               
                 aaaagtgggagttttaggagaacacgcttcaaacctaggtggaataatagcaaaacaaat 
               
               
                   
               
               
                 aggtgaagaagtaaatgttccttcatacatagtagaccctgttgttgtagatgaattaga 
               
               
                   
               
               
                 agatgttgctagaatttctggtatgcctgaaataagtagagcaagtgtagtacatgcttt 
               
               
                   
               
               
                 aaatcaaaaggcaatagcaagaagatatgctagagaaataaacaagaaatatgaagatat 
               
               
                   
               
               
                 aaatcttatagttgcacacatgggtggaggagtttctgttggagctcataaaaatggtaa 
               
               
                   
               
               
                 aatagtagatgttgcaaacgcattagatggagaaggacctttctctccagaaagaagtgg 
               
               
                   
               
               
                 tggactaccagtaggtgcattagtaaaaatgtgctttagtggaaaatatactcaagatga 
               
               
                   
               
               
                 aattaaaaagaaaataaaaggtaatggcggactagttgcatacttaaacactaatgatgc 
               
               
                   
               
               
                 tagagaagttgaagaaagaattgaagctggtgatgaaaaagctaaattagtatatgaagc 
               
               
                   
               
               
                 tatggcatatcaaatctctaaagaaataggagctagtgctgcagttcttaagggagatgt 
               
               
                   
               
               
                 aaaagcaatattattaactggtggaatcgcatattcaaaaatgtttacagaaatgattgc 
               
               
                   
               
               
                 agatagagttaaatttatagcagatgtaaaagtttatccaggtgaagatgaaatgattgc 
               
               
                   
               
               
                 attagctcaaggtggacttagagttttaactggtgaagaagaggctcaagtttatgataa 
               
               
                   
               
               
                 ctaataa 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 39 
               
               
                   
               
               
                 pLOGIC046-delta pbt.buk/tesB+  
               
               
                 -nsrR-norB-butyrate construct 
               
               
                 pLOGIC046-delta pbt.buk/tesB+30-nsrR-norB- 
               
               
                 butyrate construct SEQ ID NO: 167 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 ttatta tcgcaccgcaatcgggattttcgattcataaagcaggtcgtag   
               
               
                   
               
               
                 
                   gtcggcttgttgagcaggtcttgcagcgtgaaaccgtccagatacgtga 
                 
               
               
                   
               
               
                 
                   aaaacgacttcattgcaccgccgagtatgcccgtcagccggcaggacgg 
                 
               
               
                   
               
               
                 
                   cgtaatcaggcattcgttgttcgggcccatacactcgaccagctgcatc 
                 
               
               
                   
               
               
                 
                   ggttcgaggtggcggacgaccgcgccgatattgatgcgttcgggcggcg 
                 
               
               
                   
               
               
                 
                   cggccagcctcagcccgccgcctttcccgcgtacgctgtgcaagaaccc 
                 
               
               
                   
               
               
                 
                   gcctttgaccagcgcggtaaccactttcatcaaatggcttttggaaatg 
                 
               
               
                   
               
               
                 
                   ccgtaggtcgaggcgatggtggcgatattgaccagcgcgtcgtcgttga 
                 
               
               
                   
               
               
                 
                   cggcggtgtagatgaggacgcgcagcccgtagtcggtatgttgggtcag 
                 
               
               
                   
               
               
                 
                   atacat 
                   acaacctccttagtacatgcaaaattatttctagagcaacata 
                 
               
               
                   
               
               
                 
                   cgagccggaagcataaagtgtaaagcctggggtgcctaatgagttgagt 
                 
               
               
                   
               
               
                 
                   tgaggaattataacaggaagaaatattcctcatacgcttgtaattcctc 
                 
               
               
                   
               
               
                 
                   tatggttgttgacaattaatcatcggctcgtataatgtataacattcat 
                 
               
               
                   
               
               
                 
                   attttgtgaattttaaactctagaaataattttgtttaactttaagaag 
                 
               
               
                   
               
               
                   gagatatacat atgatcgtaaaacctatggtacgcaacaatatctgcct 
               
               
                   
               
               
                 gaacgcccatcctcagggctgcaagaagggagtggaagatcagattgaa 
               
               
                   
               
               
                 tataccaagaaacgcattaccgcagaagtcaaagctggcgcaaaagctc 
               
               
                   
               
               
                 caaaaaacgttctggtgcttggctgctcaaatggttacggcctggcgag 
               
               
                   
               
               
                 ccgcattactgctgcgttcggatacggggctgcgaccatcggcgtgtcc 
               
               
                   
               
               
                 tttgaaaaagcgggttcagaaaccaaatatggtacaccgggatggtaca 
               
               
                   
               
               
                 ataatttggcatttgatgaagcggcaaaacgcgagggtctttatagcgt 
               
               
                   
               
               
                 gacgatcgacggcgatgcgttttcagacgagatcaaggcccaggtaatt 
               
               
                   
               
               
                 gaggaagccaaaaaaaaaggtatcaaatttgatctgatcgtatacagct 
               
               
                   
               
               
                 tggccagcccagtacgtactgatcctgatacaggtatcatgcacaaaag 
               
               
                   
               
               
                 cgttttgaaaccctttggaaaaacgttcacaggcaaaacagtagatccg 
               
               
                   
               
               
                 tttactggcgagctgaaggaaatctccgcggaaccagcaaatgacgagg 
               
               
                   
               
               
                 aagcagccgccactgttaaagttatggggggtgaagattgggaacgttg 
               
               
                   
               
               
                 gattaagcagctgtcgaaggaaggcctcttagaagaaggctgtattacc 
               
               
                   
               
               
                 ttggcctatagttatattggccctgaagctacccaagctttgtaccgta 
               
               
                   
               
               
                 aaggcacaatcggcaaggccaaagaacacctggaggccacagcacaccg 
               
               
                   
               
               
                 tctcaacaaagagaacccgtcaatccgtgccttcgtgagcgtgaataaa 
               
               
                   
               
               
                 ggcctggtaacccgcgcaagcgccgtaatcccggtaatccctctgtatc 
               
               
                   
               
               
                 tcgccagcttgttcaaagtaatgaaagagaagggcaatcatgaaggttg 
               
               
                   
               
               
                 tattgaacagatcacgcgtctgtacgccgagcgcctgtaccgtaaagat 
               
               
                   
               
               
                 ggtacaattccagttgatgaggaaaatcgcattcgcattgatgattggg 
               
               
                   
               
               
                 agttagaagaagacgtccagaaagcggtatccgcgttgatggagaaagt 
               
               
                   
               
               
                 cacgggtgaaaacgcagaatctctcactgacttagcggggtaccgccat 
               
               
                   
               
               
                 gatttcttagctagtaacggctttgatgtagaaggtattaattatgaag 
               
               
                   
               
               
                 cggaagttgaacgcttcgaccgtatctgataagaaggagatatacatat 
               
               
                   
               
               
                 gagagaagtagtaattgccagtgcagctagaacagcagtaggaagtttt 
               
               
                   
               
               
                 ggaggagcatttaaatcagtttcagcggtagagttaggggtaacagcag 
               
               
                   
               
               
                 ctaaagaagctataaaaagagctaacataactccagatatgatagatga 
               
               
                   
               
               
                 atctcttttagggggagtacttacagcaggtcttggacaaaatatagca 
               
               
                   
               
               
                 agacaaatagcattaggagcaggaataccagtagaaaaaccagctatga 
               
               
                   
               
               
                 ctataaatatagtttgtggttctggattaagatctgtttcaatggcatc 
               
               
                   
               
               
                 tcaacttatagcattaggtgatgctgatataatgttagttggtggagct 
               
               
                   
               
               
                 gaaaacatgagtatgtctccttatttagtaccaagtgcgagatatggtg 
               
               
                   
               
               
                 caagaatgggtgatgctgcttttgttgattcaatgataaaagatggatt 
               
               
                   
               
               
                 atcagacatatttaataactatcacatgggtattactgctgaaaacata 
               
               
                   
               
               
                 gcagagcaatggaatataactagagaagaacaagatgaattagctcttg 
               
               
                   
               
               
                 caagtcaaaataaagctgaaaaagctcaagctgaaggaaaatttgatga 
               
               
                   
               
               
                 agaaatagttcctgttgttataaaaggaagaaaaggtgacactgtagta 
               
               
                   
               
               
                 gataaagatgaatatattaagcctggcactacaatggagaaacttgcta 
               
               
                   
               
               
                 agttaagacctgcatttaaaaaagatggaacagttactgctggtaatgc 
               
               
                   
               
               
                 atcaggaataaatgatggtgctgctatgttagtagtaatggctaaagaa 
               
               
                   
               
               
                 aaagctgaagaactaggaatagagcctcttgcaactatagtttcttatg 
               
               
                   
               
               
                 gaacagctggtgttgaccctaaaataatgggatatggaccagttccagc 
               
               
                   
               
               
                 aactaaaaaagctttagaagctgctaatatgactattgaagatatagat 
               
               
                   
               
               
                 ttagttgaagctaatgaggcatttgctgcccaatctgtagctgtaataa 
               
               
                   
               
               
                 gagacttaaatatagatatgaataaagttaatgttaatggtggagcaat 
               
               
                   
               
               
                 agctataggacatccaataggatgctcaggagcaagaatacttactaca 
               
               
                   
               
               
                 cttttatatgaaatgaagagaagagatgctaaaactggtcttgctacac 
               
               
                   
               
               
                 tttgtataggcggtggaatgggaactactttaatagttaagagatagta 
               
               
                   
               
               
                 agaaggagatatacatatgaaattagctgtaataggtagtggaactatg 
               
               
                   
               
               
                 ggaagtggtattgtacaaacttttgcaagttgtggacatgatgtatgtt 
               
               
                   
               
               
                 taaagagtagaactcaaggtgctatagataaatgtttagctttattaga 
               
               
                   
               
               
                 taaaaatttaactaagttagttactaagggaaaaatggatgaagctaca 
               
               
                   
               
               
                 aaagcagaaatattaagtcatgttagttcaactactaattatgaagatt 
               
               
                   
               
               
                 taaaagatatggatttaataatagaagcatctgtagaagacatgaatat 
               
               
                   
               
               
                 aaagaaagatgttttcaagttactagatgaattatgtaaagaagatact 
               
               
                   
               
               
                 atcttggcaacaaatacttcatcattatctataacagaaatagcttctt 
               
               
                   
               
               
                 ctactaagcgcccagataaagttataggaatgcatttctttaatccagt 
               
               
                   
               
               
                 tcctatgatgaaattagttgaagttataagtggtcagttaacatcaaaa 
               
               
                   
               
               
                 gttacttttgatacagtatttgaattatctaagagtatcaataaagtac 
               
               
                   
               
               
                 cagtagatgtatctgaatctcctggatttgtagtaaatagaatacttat 
               
               
                   
               
               
                 acctatgataaatgaagctgttggtatatatgcagatggtgttgcaagt 
               
               
                   
               
               
                 aaagaagaaatagatgaagctatgaaattaggagcaaaccatccaatgg 
               
               
                   
               
               
                 gaccactagcattaggtgatttaatcggattagatgttgttttagctat 
               
               
                   
               
               
                 aatgaacgttttatatactgaatttggagatactaaatatagacctcat 
               
               
                   
               
               
                 ccacttttagctaaaatggttagagctaatcaattaggaagaaaaacta 
               
               
                   
               
               
                 agataggattctatgattataataaataataagaaggagatatacatat 
               
               
                   
               
               
                 gagtacaagtgatgttaaagtttatgagaatgtagctgttgaagtagat 
               
               
                   
               
               
                 ggaaatatatgtacagtgaaaatgaatagacctaaagcccttaatgcaa 
               
               
                   
               
               
                 taaattcaaagactttagaagaactttatgaagtatttgtagatattaa 
               
               
                   
               
               
                 taatgatgaaactattgatgttgtaatattgacaggggaaggaaaggca 
               
               
                   
               
               
                 tttgtagctggagcagatattgcatacatgaaagatttagatgctgtag 
               
               
                   
               
               
                 ctgctaaagattttagtatcttaggagcaaaagcttttggagaaataga 
               
               
                   
               
               
                 aaatagtaaaaaagtagtgatagctgctgtaaacggatttgctttaggt 
               
               
                   
               
               
                 ggaggatgtgaacttgcaatggcatgtgatataagaattgcatctgcta 
               
               
                   
               
               
                 aagctaaatttggtcagccagaagtaactcttggaataactccaggata 
               
               
                   
               
               
                 tggaggaactcaaaggcttacaagattggttggaatggcaaaagcaaaa 
               
               
                   
               
               
                 gaattaatctttacaggtcaagttataaaagctgatgaagctgaaaaaa 
               
               
                   
               
               
                 tagggctagtaaatagagtcgttgagccagacattttaatagaagaagt 
               
               
                   
               
               
                 tgagaaattagctaagataatagctaaaaatgctcagcttgcagttaga 
               
               
                   
               
               
                 tactctaaagaagcaatacaacttggtgctcaaactgatataaatactg 
               
               
                   
               
               
                 gaatagatatagaatctaatttatttggtctttgtttttcaactaaaga 
               
               
                   
               
               
                 ccaaaaagaaggaatgtcagctttcgttgaaaagagagaagctaacttt 
               
               
                   
               
               
                 ataaaagggtaataagaaggagatatacatatgAGTCAGGCGCTAAAAA 
               
               
                   
               
               
                 ATTTACTGACATTGTTAAATCTGGAAAAAATTGAGGAAGGACTCTTTCG 
               
               
                   
               
               
                 CGGCCAGAGTGAAGATTTAGGTTTACGCCAGGTGTTTGGCGGCCAGGTC 
               
               
                   
               
               
                 GTGGGTCAGGCCTTGTATGCTGCAAAAGAGACCGTCCCTGAAGAGCGGC 
               
               
                   
               
               
                 TGGTACATTCGTTTCACAGCTACTTTCTTCGCCCTGGCGATAGTAAGAA 
               
               
                   
               
               
                 GCCGATTATTTATGATGTCGAAACGCTGCGTGACGGTAACAGCTTCAGC 
               
               
                   
               
               
                 GCCCGCCGGGTTGCTGCTATTCAAAACGGCAAACCGATTTTTTATATGA 
               
               
                   
               
               
                 CTGCCTCTTTCCAGGCACCAGAAGCGGGTTTCGAACATCAAAAAACAAT 
               
               
                   
               
               
                 GCCGTCCGCGCCAGCGCCTGATGGCCTCCCTTCGGAAACGCAAATCGCC 
               
               
                   
               
               
                 CAATCGCTGGCGCACCTGCTGCCGCCAGTGCTGAAAGATAAATTCATCT 
               
               
                   
               
               
                 GCGATCGTCCGCTGGAAGTCCGTCCGGTGGAGTTTCATAACCCACTGAA 
               
               
                   
               
               
                 AGGTCACGTCGCAGAACCACATCGTCAGGTGTGGATCCGCGCAAATGGT 
               
               
                   
               
               
                 AGCGTGCCGGATGACCTGCGCGTTCATCAGTATCTGCTCGGTTACGCTT 
               
               
                   
               
               
                 CTGATCTTAACTTCCTGCCGGTAGCTCTACAGCCGCACGGCATCGGTTT 
               
               
                   
               
               
                 TCTCGAACCGGGGATTCAGATTGCCACCATTGACCATTCCATGTGGTTC 
               
               
                   
               
               
                 CATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATAGCGTGGAGAGCA 
               
               
                   
               
               
                 CCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGAGTTTTATACCCA 
               
               
                   
               
               
                 AGACGGCGTACTGGTTGCCTCGACCGTTCAGGAAGGGGTGATGCGTAAT 
               
               
                   
               
               
                 CACAATtaa 
               
               
                   
               
            
           
         
       
     
     In certain constructs, the butyrate gene cassette (e.g., bcd2-etfB3-etfA3-thiA1-hbd-crt2-pbt buk butyrate cassette (pLogic031), and/or ter-thiA1-hbd-crt2-pbt buk butyrate cassette (pLogic046) and/or ter-thiA1-hbd-crt2-tesb butyrate cassette (pLOGIC046-delta pbt.buk/tesB+)) is placed under the control of an ROS-responsive regulatory region, e.g., oxyS. In certain constructs, the butyrate gene cassette (e.g., bcd2-etfB3-etfA3-thiA1-hbd-crt2-pbt buk butyrate cassette (pLogic031), and/or ter-thiA1-hbd-crt2-pbt buk butyrate cassette (pLogic046) and/or ter-thiA1-hbd-crt2-tesb butyrate cassette (pLOGIC046-delta pbt.buk/tesB+)) is placed under the control of an ROS-responsive regulatory region, e.g., oxyS, and the bacteria further comprises a gene encoding a corresponding ROS-responsive transcription factor, e.g., oxyR (see, e.g., the tables and elsewhere herein). 
     Nucleic acid sequences of exemplary ROS-regulated constructs comprising an oxyS promoter are shown in Table 40 and Table 41 and Table 43. The nucleic acid sequence of an exemplary construct encoding OxyR is shown in Table 42. Table 40 depicts the nucleic acid sequence of an exemplary ROS-regulated construct comprising an oxyS promoter and a butyrogenic gene cassette (pLogic031-oxyS-butyrate construct; SEQ ID NO: 168). Table 41 depicts the nucleic acid sequence of an exemplary ROS-regulated construct comprising an oxyS promoter and a butyrogenic gene cassette (pLogic046-oxyS-butyrate construct; SEQ ID NO: 169). Table 42 depicts the nucleic acid sequence of an exemplary construct encoding OxyR (pZA22-oxyR construct; SEQ ID NO: 170). Table 43 depicts the nucleic acid sequence of an exemplary ROS-regulated construct comprising an oxyS promoter and a butyrogenic gene cassette (pLOGIC046-delta pbt.buk/tesB+-oxyS-butyrate construct; SEQ ID NO: 171). 
     In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 168, 169, 170, or 171, or a functional fragment thereof. 
     
       
         
           
               
             
               
                 TABLE 40 
               
               
                   
               
               
                 pLogic031-oxyS-butyrate  
               
               
                 construct (SEQ ID NO: 168) 
               
               
                 Nucleotide sequences of pLogic031-oxyS- 
               
               
                 butyrate construct (SEQ ID NO: 168) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 ctcgagttcattatccatcctccatcgccacgatagttcatggcgatag 
               
               
                   
               
               
                 gtagaatagcaatgaacgattatccctatcaagcattctgactgataat 
               
               
                   
               
               
                 tgctcacacgaattcattaaagaggagaaaggtaccatggatttaaatt 
               
               
                   
               
               
                 ctaaaaaatatcagatgcttaaagagctatatgtaagcttcgctgaaaa 
               
               
                   
               
               
                 tgaagttaaacctttagcaacagaacttgatgaagaagaaagatttcct 
               
               
                   
               
               
                 tatgaaacagtggaaaaaatggcaaaagcaggaatgatgggtataccat 
               
               
                   
               
               
                 atccaaaagaatatggtggagaaggtggagacactgtaggatatataat 
               
               
                   
               
               
                 ggcagttgaagaattgtctagagtttgtggtactacaggagttatatta 
               
               
                   
               
               
                 tcagctcatacatctcttggctcatggcctatatatcaatatggtaatg 
               
               
                   
               
               
                 aagaacaaaaacaaaaattcttaagaccactagcaagtggagaaaaatt 
               
               
                   
               
               
                 aggagcatttggtcttactgagcctaatgctggtacagatgcgtctggc 
               
               
                   
               
               
                 caacaaacaactgctgttttagacggggatgaatacatacttaatggct 
               
               
                   
               
               
                 caaaaatatttataacaaacgcaatagctggtgacatatatgtagtaat 
               
               
                   
               
               
                 ggcaatgactgataaatctaaggggaacaaaggaatatcagcatttata 
               
               
                   
               
               
                 gttgaaaaaggaactcctgggtttagctttggagttaaagaaaagaaaa 
               
               
                   
               
               
                 tgggtataagaggttcagctacgagtgaattaatatttgaggattgcag 
               
               
                   
               
               
                 aatacctaaagaaaatttacttggaaaagaaggtcaaggatttaagata 
               
               
                   
               
               
                 gcaatgtctactcttgatggtggtagaattggtatagctgcacaagctt 
               
               
                   
               
               
                 taggtttagcacaaggtgctcttgatgaaactgttaaatatgtaaaaga 
               
               
                   
               
               
                 aagagtacaatttggtagaccattatcaaaattccaaaatacacaattc 
               
               
                   
               
               
                 caattagctgatatggaagttaaggtacaagcggctagacaccttgtat 
               
               
                   
               
               
                 atcaagcagctataaataaagacttaggaaaaccttatggagtagaagc 
               
               
                   
               
               
                 agcaatggcaaaattatttgcagctgaaacagctatggaagttactaca 
               
               
                   
               
               
                 aaagctgtacaacttcatggaggatatggatacactcgtgactatccag 
               
               
                   
               
               
                 tagaaagaatgatgagagatgctaagataactgaaatatatgaaggaac 
               
               
                   
               
               
                 tagtgaagttcaaagaatggttatttcaggaaaactattaaaatagtaa 
               
               
                   
               
               
                 gaaggagatatacatatggaggaaggatttatgaatatagtcgtttgta 
               
               
                   
               
               
                 taaaacaagttccagatacaacagaagttaaactagatcctaatacagg 
               
               
                   
               
               
                 tactttaattagagatggagtaccaagtataataaaccctgatgataaa 
               
               
                   
               
               
                 gcaggtttagaagaagctataaaattaaaagaagaaatgggtgctcatg 
               
               
                   
               
               
                 taactgttataacaatgggacctcctcaagcagatatggctttaaaaga 
               
               
                   
               
               
                 agctttagcaatgggtgcagatagaggtatattattaacagatagagca 
               
               
                   
               
               
                 tttgcgggtgctgatacttgggcaacttcatcagcattagcaggagcat 
               
               
                   
               
               
                 taaaaaatatagattttgatattataatagctggaagacaggcgataga 
               
               
                   
               
               
                 tggagatactgcacaagttggacctcaaatagctgaacatttaaatctt 
               
               
                   
               
               
                 ccatcaataacatatgctgaagaaataaaaactgaaggtgaatatgtat 
               
               
                   
               
               
                 tagtaaaaagacaatttgaagattgttgccatgacttaaaagttaaaat 
               
               
                   
               
               
                 gccatgccttataacaactcttaaagatatgaacacaccaagatacatg 
               
               
                   
               
               
                 aaagttggaagaatatatgatgctttcgaaaatgatgtagtagaaacat 
               
               
                   
               
               
                 ggactgtaaaagatatagaagttgacccttctaatttaggtcttaaagg 
               
               
                   
               
               
                 ttctccaactagtgtatttaaatcatttacaaaatcagttaaaccagct 
               
               
                   
               
               
                 ggtacaatatacaatgaagatgcgaaaacatcagctggaattatcatag 
               
               
                   
               
               
                 ataaattaaaagagaagtatatcatataataagaaggagatatacatat 
               
               
                   
               
               
                 gggtaacgttttagtagtaatagaacaaagagaaaatgtaattcaaact 
               
               
                   
               
               
                 gtttctttagaattactaggaaaggctacagaaatagcaaaagattatg 
               
               
                   
               
               
                 atacaaaagtttctgcattacttttaggtagtaaggtagaaggtttaat 
               
               
                   
               
               
                 agatacattagcacactatggtgcagatgaggtaatagtagtagatgat 
               
               
                   
               
               
                 gaagctttagcagtgtatacaactgaaccatatacaaaagcagcttatg 
               
               
                   
               
               
                 aagcaataaaagcagctgaccctatagttgtattatttggtgcaacttc 
               
               
                   
               
               
                 aataggtagagatttagcgcctagagtttctgctagaatacatacaggt 
               
               
                   
               
               
                 cttactgctgactgtacaggtcttgcagtagctgaagatacaaaattat 
               
               
                   
               
               
                 tattaatgacaagacctgcctttggtggaaatataatggcaacaatagt 
               
               
                   
               
               
                 ttgtaaagatttcagacctcaaatgtctacagttagaccaggggttatg 
               
               
                   
               
               
                 aagaaaaatgaacctgatgaaactaaagaagctgtaattaaccgtttca 
               
               
                   
               
               
                 aggtagaatttaatgatgctgataaattagttcaagttgtacaagtaat 
               
               
                   
               
               
                 aaaagaagctaaaaaacaagttaaaatagaagatgctaagatattagtt 
               
               
                   
               
               
                 tctgctggacgtggaatgggtggaaaagaaaacttagacatactttatg 
               
               
                   
               
               
                 aattagctgaaattataggtggagaagtttctggttctcgtgccactat 
               
               
                   
               
               
                 agatgcaggttggttagataaagcaagacaagttggtcaaactggtaaa 
               
               
                   
               
               
                 actgtaagaccagacctttatatagcatgtggtatatctggagcaatac 
               
               
                   
               
               
                 aacatatagctggtatggaagatgctgagtttatagttgctataaataa 
               
               
                   
               
               
                 aaatccagaagctccaatatttaaatatgctgatgttggtatagttgga 
               
               
                   
               
               
                 gatgttcataaagtgcttccagaacttatcagtcagttaagtgttgcaa 
               
               
                   
               
               
                 aagaaaaaggtgaagttttagctaactaataagaaggagatatacatat 
               
               
                   
               
               
                 gagagaagtagtaattgccagtgcagctagaacagcagtaggaagtttt 
               
               
                   
               
               
                 ggaggagcatttaaatcagtttcagcggtagagttaggggtaacagcag 
               
               
                   
               
               
                 ctaaagaagctataaaaagagctaacataactccagatatgatagatga 
               
               
                   
               
               
                 atctcttttagggggagtacttacaggaggtcttggacaaaatatagca 
               
               
                   
               
               
                 agacaaatagcattaggaggaggaataccagtagaaaaaccagctatga 
               
               
                   
               
               
                 ctataaatatagtttgtggttctggattaagatctgtttcaatggcatc 
               
               
                   
               
               
                 tcaacttatagcattaggtgatgctgatataatgttagttggtggagct 
               
               
                   
               
               
                 gaaaacatgagtatgtctccttatttagtaccaagtgcgagatatggtg 
               
               
                   
               
               
                 caagaatgggtgatgctgcttttgttgattcaatgataaaagatggatt 
               
               
                   
               
               
                 atcagacatatttaataactatcacatgggtattactgctgaaaacata 
               
               
                   
               
               
                 gcagagcaatggaatataactagagaagaacaagatgaattagctcttg 
               
               
                   
               
               
                 caagtcaaaataaagctgaaaaagctcaagctgaaggaaaatttgatga 
               
               
                   
               
               
                 agaaatagttcctgttgttataaaaggaagaaaaggtgacactgtagta 
               
               
                   
               
               
                 gataaagatgaatatattaagcctggcactacaatggagaaacttgcta 
               
               
                   
               
               
                 agttaagacctgcatttaaaaaagatggaacagttactgctggtaatgc 
               
               
                   
               
               
                 atcaggaataaatgatggtgctgctatgttagtagtaatggctaaagaa 
               
               
                   
               
               
                 aaagctgaagaactaggaatagagcctcttgcaactatagtttcttatg 
               
               
                   
               
               
                 gaacagctggtgttgaccctaaaataatgggatatggaccagttccagc 
               
               
                   
               
               
                 aactaaaaaagctttagaagctgctaatatgactattgaagatatagat 
               
               
                   
               
               
                 ttagttgaagctaatgaggcatttgctgcccaatctgtagctgtaataa 
               
               
                   
               
               
                 gagacttaaatatagatatgaataaagttaatgttaatggtggagcaat 
               
               
                   
               
               
                 agctataggacatccaataggatgctcaggagcaagaatacttactaca 
               
               
                   
               
               
                 cttttatatgaaatgaagagaagagatgctaaaactggtcttgctacac 
               
               
                   
               
               
                 tttgtataggcggtggaatgggaactactttaatagttaagagatagta 
               
               
                   
               
               
                 agaaggagatatacatatgaaattagctgtaataggtagtggaactatg 
               
               
                   
               
               
                 ggaagtggtattgtacaaacttttgcaagttgtggacatgatgtatgtt 
               
               
                   
               
               
                 taaagagtagaactcaaggtgctatagataaatgtttagctttattaga 
               
               
                   
               
               
                 taaaaatttaactaagttagttactaagggaaaaatggatgaagctaca 
               
               
                   
               
               
                 aaagcagaaatattaagtcatgttagttcaactactaattatgaagatt 
               
               
                   
               
               
                 taaaagatatggatttaataatagaagcatctgtagaagacatgaatat 
               
               
                   
               
               
                 aaagaaagatgttttcaagttactagatgaattatgtaaagaagatact 
               
               
                   
               
               
                 atcttggcaacaaatacttcatcattatctataacagaaatagcttctt 
               
               
                   
               
               
                 ctactaagcgcccagataaagttataggaatgcatttctttaatccagt 
               
               
                   
               
               
                 tcctatgatgaaattagttgaagttataagtggtcagttaacatcaaaa 
               
               
                   
               
               
                 gttacttttgatacagtatttgaattatctaagagtatcaataaagtac 
               
               
                   
               
               
                 cagtagatgtatctgaatctcctggatttgtagtaaatagaatacttat 
               
               
                   
               
               
                 acctatgataaatgaagctgttggtatatatgcagatggtgttgcaagt 
               
               
                   
               
               
                 aaagaagaaatagatgaagctatgaaattaggagcaaaccatccaatgg 
               
               
                   
               
               
                 gaccactagcattaggtgatttaatcggattagatgttgttttagctat 
               
               
                   
               
               
                 aatgaacgttttatatactgaatttggagatactaaatatagacctcat 
               
               
                   
               
               
                 ccacttttagctaaaatggttagagctaatcaattaggaagaaaaacta 
               
               
                   
               
               
                 agataggattctatgattataataaataataagaaggagatatacatat 
               
               
                   
               
               
                 gagtacaagtgatgttaaagtttatgagaatgtagctgttgaagtagat 
               
               
                   
               
               
                 ggaaatatatgtacagtgaaaatgaatagacctaaagcccttaatgcaa 
               
               
                   
               
               
                 taaattcaaagactttagaagaactttatgaagtatttgtagatattaa 
               
               
                   
               
               
                 taatgatgaaactattgatgttgtaatattgacaggggaaggaaaggca 
               
               
                   
               
               
                 tttgtagctggaggagatattgcatacatgaaagatttagatgctgtag 
               
               
                   
               
               
                 ctgctaaagattttagtatcttaggagcaaaagcttttggagaaataga 
               
               
                   
               
               
                 aaatagtaaaaaagtagtgatagctgctgtaaacggatttgctttaggt 
               
               
                   
               
               
                 ggaggatgtgaacttgcaatggcatgtgatataagaattgcatctgcta 
               
               
                   
               
               
                 aagctaaatttggtcagccagaagtaactcttggaataactccaggata 
               
               
                   
               
               
                 tggaggaactcaaaggcttacaagattggttggaatggcaaaagcaaaa 
               
               
                   
               
               
                 gaattaatctttacaggtcaagttataaaagctgatgaagctgaaaaaa 
               
               
                   
               
               
                 tagggctagtaaatagagtcgttgagccagacattttaatagaagaagt 
               
               
                   
               
               
                 tgagaaattagctaagataatagctaaaaatgctcagcttgcagttaga 
               
               
                   
               
               
                 tactctaaagaagcaatacaacttggtgctcaaactgatataaatactg 
               
               
                   
               
               
                 gaatagatatagaatctaatttatttggtctttgtttttcaactaaaga 
               
               
                   
               
               
                 ccaaaaagaaggaatgtcagctttcgttgaaaagagagaagctaacttt 
               
               
                   
               
               
                 ataaaagggtaataagaaggagatatacatatgagaagttttgaagaag 
               
               
                   
               
               
                 taattaagtttgcaaaagaaagaggacctaaaactatatcagtagcatg 
               
               
                   
               
               
                 ttgccaagataaagaagttttaatggcagttgaaatggctagaaaagaa 
               
               
                   
               
               
                 aaaatagcaaatgccattttagtaggagatatagaaaagactaaagaaa 
               
               
                   
               
               
                 ttgcaaaaagcatagacatggatatcgaaaattatgaactgatagatat 
               
               
                   
               
               
                 aaaagatttagcagaagcatctctaaaatctgttgaattagtttcacaa 
               
               
                   
               
               
                 ggaaaagccgacatggtaatgaaaggcttagtagacacatcaataatac 
               
               
                   
               
               
                 taaaagcagttttaaataaagaagtaggtcttagaactggaaatgtatt 
               
               
                   
               
               
                 aagtcacgtagcagtatttgatgtagagggatatgatagattatttttc 
               
               
                   
               
               
                 gtaactgacgcagctatgaacttagctcctgatacaaatactaaaaagc 
               
               
                   
               
               
                 aaatcatagaaaatgcttgcacagtagcacattcattagatataagtga 
               
               
                   
               
               
                 accaaaagttgctgcaatatgcgcaaaagaaaaagtaaatccaaaaatg 
               
               
                   
               
               
                 aaagatacagttgaagctaaagaactagaagaaatgtatgaaagaggag 
               
               
                   
               
               
                 aaatcaaaggttgtatggttggtgggccttttgcaattgataatgcagt 
               
               
                   
               
               
                 atctttagaagcagctaaacataaaggtataaatcatcctgtagcagga 
               
               
                   
               
               
                 cgagctgatatattattagccccagatattgaaggtggtaacatattat 
               
               
                   
               
               
                 ataaagctttggtattcttctcaaaatcaaaaaatgcaggagttatagt 
               
               
                   
               
               
                 tggggctaaagcaccaataatattaacttctagagcagacagtgaagaa 
               
               
                   
               
               
                 actaaactaaactcaatagctttaggtgttttaatggcagcaaaggcat 
               
               
                   
               
               
                 aataagaaggagatatacatatgagcaaaatatttaaaatcttaacaat 
               
               
                   
               
               
                 aaatcctggttcgacatcaactaaaatagctgtatttgataatgaggat 
               
               
                   
               
               
                 ttagtatttgaaaaaactttaagacattcttcagaagaaataggaaaat 
               
               
                   
               
               
                 atgagaaggtgtctgaccaatttgaatttcgtaaacaagtaatagaaga 
               
               
                   
               
               
                 agctctaaaagaaggtggagtaaaaacatctgaattagatgctgtagta 
               
               
                   
               
               
                 ggtagaggaggacttcttaaacctataaaaggtggtacttattcagtaa 
               
               
                   
               
               
                 gtgctgctatgattgaagatttaaaagtgggagttttaggagaacacgc 
               
               
                   
               
               
                 ttcaaacctaggtggaataatagcaaaacaaataggtgaagaagtaaat 
               
               
                   
               
               
                 gttccttcatacatagtagaccctgttgttgtagatgaattagaagatg 
               
               
                   
               
               
                 ttgctagaatttctggtatgcctgaaataagtagagcaagtgtagtaca 
               
               
                   
               
               
                 tgctttaaatcaaaaggcaatagcaagaagatatgctagagaaataaac 
               
               
                   
               
               
                 aagaaatatgaagatataaatcttatagttgcacacatgggtggaggag 
               
               
                   
               
               
                 tttctgttggagctcataaaaatggtaaaatagtagatgttgcaaacgc 
               
               
                   
               
               
                 attagatggagaaggacctttctctccagaaagaagtggtggactacca 
               
               
                   
               
               
                 gtaggtgcattagtaaaaatgtgctttagtggaaaatatactcaagatg 
               
               
                   
               
               
                 aaattaaaaagaaaataaaaggtaatggcggactagttgcatacttaaa 
               
               
                   
               
               
                 cactaatgatgctagagaagttgaagaaagaattgaagctggtgatgaa 
               
               
                   
               
               
                 aaagctaaattagtatatgaagctatggcatatcaaatctctaaagaaa 
               
               
                   
               
               
                 taggagctagtgctgcagttcttaagggagatgtaaaagcaatattatt 
               
               
                   
               
               
                 aactggtggaatcgcatattcaaaaatgtttacagaaatgattgcagat 
               
               
                   
               
               
                 agagttaaatttatagcagatgtaaaagtttatccaggtgaagatgaaa 
               
               
                   
               
               
                 tgattgcattagctcaaggtggacttagagttttaactggtgaagaaga 
               
               
                   
               
               
                 ggctcaagtttatgataactaataa 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 41 
               
               
                   
               
               
                 pLogic046-oxyS-butyrate  
               
               
                 construct (SEQ ID NO: 169) 
               
               
                 Nucleotide sequences of pLogic046-oxyS-butyrate  
               
               
                 construct (SEQ ID NO: 169) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 ctcgagttcattatccatcctccatcgccacgatagttcatggcgatag 
               
               
                   
               
               
                 gtagaatagcaatgaacgattatccctatcaagcattctgactgataat 
               
               
                   
               
               
                 tgctcacacgaattcattaaagaggagaaaggtaccatgatcgtaaaac 
               
               
                   
               
               
                 ctatggtacgcaacaatatctgcctgaacgcccatcctcagggctgcaa 
               
               
                   
               
               
                 gaagggagtggaagatcagattgaatataccaagaaacgcattaccgca 
               
               
                   
               
               
                 gaagtcaaagctggcgcaaaagctccaaaaaacgttctggtgcttggct 
               
               
                   
               
               
                 gctcaaatggttacggcctggcgagccgcattactgctgcgttcggata 
               
               
                   
               
               
                 cggggctgcgaccatcggcgtgtcctttgaaaaagcgggttcagaaacc 
               
               
                   
               
               
                 aaatatggtacaccgggatggtacaataatttggcatttgatgaagcgg 
               
               
                   
               
               
                 caaaacgcgagggtctttatagcgtgacgatcgacggcgatgcgttttc 
               
               
                   
               
               
                 agacgagatcaaggcccaggtaattgaggaagccaaaaaaaaaggtatc 
               
               
                   
               
               
                 aaatttgatctgatcgtatacagcttggccagcccagtacgtactgatc 
               
               
                   
               
               
                 ctgatacaggtatcatgcacaaaagcgttttgaaaccctttggaaaaac 
               
               
                   
               
               
                 gttcacaggcaaaacagtagatccgtttactggcgagctgaaggaaate 
               
               
                   
               
               
                 tccgcggaaccagcaaatgacgaggaagcagccgccactgttaaagtta 
               
               
                   
               
               
                 tggggggtgaagattgggaacgttggattaagcagctgtcgaaggaagg 
               
               
                   
               
               
                 cctcttagaagaaggctgtattaccttggcctatagttatattggccct 
               
               
                   
               
               
                 gaagctacccaagctttgtaccgtaaaggcacaatcggcaaggccaaag 
               
               
                   
               
               
                 aacacctggaggccacagcacaccgtctcaacaaagagaacccgtcaat 
               
               
                   
               
               
                 ccgtgccttcgtgagcgtgaataaaggcctggtaacccgcgcaagcgcc 
               
               
                   
               
               
                 gtaatcccggtaatccctctgtatctcgccagcttgttcaaagtaatga 
               
               
                   
               
               
                 aagagaagggcaatcatgaaggttgtattgaacagatcacgcgtctgta 
               
               
                   
               
               
                 cgccgagcgcctgtaccgtaaagatggtacaattccagttgatgaggaa 
               
               
                   
               
               
                 aatcgcattcgcattgatgattgggagttagaagaagacgtccagaaag 
               
               
                   
               
               
                 cggtatccgcgttgatggagaaagtcacgggtgaaaacgcagaatctct 
               
               
                   
               
               
                 cactgacttagcggggtaccgccatgatttcttagctagtaacggcttt 
               
               
                   
               
               
                 gatgtagaaggtattaattatgaagcggaagttgaacgcttcgaccgta 
               
               
                   
               
               
                 tctgataagaaggagatatacatatgagagaagtagtaattgccagtgc 
               
               
                   
               
               
                 agctagaacagcagtaggaagttttggaggagcatttaaatcagtttca 
               
               
                   
               
               
                 gcggtagagttaggggtaacagcagctaaagaagctataaaaagagcta 
               
               
                   
               
               
                 acataactccagatatgatagatgaatctcttttagggggagtacttac 
               
               
                   
               
               
                 aggaggtcttggacaaaatatagcaagacaaatagcattaggagcagga 
               
               
                   
               
               
                 ataccagtagaaaaaccagctatgactataaatatagtttgtggttctg 
               
               
                   
               
               
                 gattaagatctgtttcaatggcatctcaacttatagcattaggtgatgc 
               
               
                   
               
               
                 tgatataatgttagttggtggagctgaaaacatgagtatgtctccttat 
               
               
                   
               
               
                 ttagtaccaagtgcgagatatggtgcaagaatgggtgatgctgcttttg 
               
               
                   
               
               
                 ttgattcaatgataaaagatggattatcagacatatttaataactatca 
               
               
                   
               
               
                 catgggtattactgctgaaaacatagcagagcaatggaatataactaga 
               
               
                   
               
               
                 gaagaacaagatgaattagetcttgcaagtcaaaataaagctgaaaaag 
               
               
                   
               
               
                 ctcaagctgaaggaaaatttgatgaagaaatagttcctgttgttataaa 
               
               
                   
               
               
                 aggaagaaaaggtgacactgtagtagataaagatgaatatattaagcct 
               
               
                   
               
               
                 ggcactacaatggagaaacttgctaagttaagacctgcatttaaaaaag 
               
               
                   
               
               
                 atggaacagttactgctggtaatgcatcaggaataaatgatggtgctgc 
               
               
                   
               
               
                 tatgttagtagtaatggctaaagaaaaagctgaagaactaggaatagag 
               
               
                   
               
               
                 cctcttgcaactatagtttcttatggaacagctggtgttgaccctaaaa 
               
               
                   
               
               
                 taatgggatatggaccagttccagcaactaaaaaagctttagaagctgc 
               
               
                   
               
               
                 taatatgactattgaagatatagatttagttgaagctaatgaggcattt 
               
               
                   
               
               
                 gctgcccaatctgtagctgtaataagagacttaaatatagatatgaata 
               
               
                   
               
               
                 aagttaatgttaatggtggagcaatagctataggacatccaataggatg 
               
               
                   
               
               
                 ctcaggagcaagaatacttactacacttttatatgaaatgaagagaaga 
               
               
                   
               
               
                 gatgctaaaactggtcttgctacactttgtataggcggtggaatgggaa 
               
               
                   
               
               
                 ctactttaatagttaagagatagtaagaaggagatatacatatgaaatt 
               
               
                   
               
               
                 agctgtaataggtagtggaactatgggaagtggtattgtacaaactttt 
               
               
                   
               
               
                 gcaagttgtggacatgatgtatgtttaaagagtagaactcaaggtgcta 
               
               
                   
               
               
                 tagataaatgtttagctttattagataaaaatttaactaagttagttac 
               
               
                   
               
               
                 taagggaaaaatggatgaagctacaaaagcagaaatattaagtcatgtt 
               
               
                   
               
               
                 agttcaactactaattatgaagatttaaaagatatggatttaataatag 
               
               
                   
               
               
                 aagcatctgtagaagacatgaatataaagaaagatgttttcaagttact 
               
               
                   
               
               
                 agatgaattatgtaaagaagatactatcttggcaacaaatacttcatca 
               
               
                   
               
               
                 ttatctataacagaaatagcttcttctactaagcgcccagataaagtta 
               
               
                   
               
               
                 taggaatgcatttctttaatccagttcctatgatgaaattagttgaagt 
               
               
                   
               
               
                 tataagtggtcagttaacatcaaaagttacttttgatacagtatttgaa 
               
               
                   
               
               
                 ttatctaagagtatcaataaagtaccagtagatgtatctgaatctcctg 
               
               
                   
               
               
                 gatttgtagtaaatagaatacttatacctatgataaatgaagctgttgg 
               
               
                   
               
               
                 tatatatgcagatggtgttgcaagtaaagaagaaatagatgaagctatg 
               
               
                   
               
               
                 aaattaggagcaaaccatccaatgggaccactagcattaggtgatttaa 
               
               
                   
               
               
                 tcggattagatgttgttttagctataatgaacgttttatatactgaatt 
               
               
                   
               
               
                 tggagatactaaatatagacctcatccacttttagctaaaatggttaga 
               
               
                   
               
               
                 gctaatcaattaggaagaaaaactaagataggattctatgattataata 
               
               
                   
               
               
                 aataataagaaggagatatacatatgagtacaagtgatgttaaagttta 
               
               
                   
               
               
                 tgagaatgtagctgttgaagtagatggaaatatatgtacagtgaaaatg 
               
               
                   
               
               
                 aatagacctaaagcccttaatgcaataaattcaaagactttagaagaac 
               
               
                   
               
               
                 tttatgaagtatttgtagatattaataatgatgaaactattgatgttgt 
               
               
                   
               
               
                 aatattgacaggggaaggaaaggcatttgtagctggagcagatattgca 
               
               
                   
               
               
                 tacatgaaagatttagatgctgtagctgctaaagattttagtatcttag 
               
               
                   
               
               
                 gagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatagc 
               
               
                   
               
               
                 tgctgtaaacggatttgctttaggtggaggatgtgaacttgcaatggca 
               
               
                   
               
               
                 tgtgatataagaattgcatctgctaaagctaaatttggtcagccagaag 
               
               
                   
               
               
                 taactcttggaataactccaggatatggaggaactcaaaggcttacaag 
               
               
                   
               
               
                 attggttggaatggcaaaagcaaaagaattaatctttacaggtcaagtt 
               
               
                   
               
               
                 ataaaagctgatgaagctgaaaaaatagggctagtaaatagagtcgttg 
               
               
                   
               
               
                 agccagacattttaatagaagaagttgagaaattagctaagataatagc 
               
               
                   
               
               
                 taaaaatgctcagcttgcagttagatactctaaagaagcaatacaactt 
               
               
                   
               
               
                 ggtgctcaaactgatataaatactggaatagatatagaatctaatttat 
               
               
                   
               
               
                 ttggtctttgtttttcaactaaagaccaaaaagaaggaatgtcagcttt 
               
               
                   
               
               
                 cgttgaaaagagagaagctaactttataaaagggtaataagaaggagat 
               
               
                   
               
               
                 atacatatgagaagttttgaagaagtaattaagtttgcaaaagaaagag 
               
               
                   
               
               
                 gacctaaaactatatcagtagcatgttgccaagataaagaagttttaat 
               
               
                   
               
               
                 ggcagttgaaatggctagaaaagaaaaaatagcaaatgccattttagta 
               
               
                   
               
               
                 ggagatatagaaaagactaaagaaattgcaaaaagcatagacatggata 
               
               
                   
               
               
                 tcgaaaattatgaactgatagatataaaagatttagcagaagcatctct 
               
               
                   
               
               
                 aaaatctgttgaattagtttcacaaggaaaagccgacatggtaatgaaa 
               
               
                   
               
               
                 ggcttagtagacacatcaataatactaaaagcagttttaaataaagaag 
               
               
                   
               
               
                 taggtcttagaactggaaatgtattaagtcacgtagcagtatttgatgt 
               
               
                   
               
               
                 agagggatatgatagattatttttcgtaactgacgcagctatgaactta 
               
               
                   
               
               
                 gctcctgatacaaatactaaaaagcaaatcatagaaaatgcttgcacag 
               
               
                   
               
               
                 tagcacattcattagatataagtgaaccaaaagttgctgcaatatgcgc 
               
               
                   
               
               
                 aaaagaaaaagtaaatccaaaaatgaaagatacagttgaagctaaagaa 
               
               
                   
               
               
                 ctagaagaaatgtatgaaagaggagaaatcaaaggttgtatggttggtg 
               
               
                   
               
               
                 ggccttttgcaattgataatgcagtatctttagaagcagctaaacataa 
               
               
                   
               
               
                 aggtataaatcatcctgtagcaggacgagctgatatattattagcccca 
               
               
                   
               
               
                 gatattgaaggtggtaacatattatataaagctttggtattcttctcaa 
               
               
                   
               
               
                 aatcaaaaaatgcaggagttatagttggggctaaagcaccaataatatt 
               
               
                   
               
               
                 aacttctagagcagacagtgaagaaactaaactaaactcaatagcttta 
               
               
                   
               
               
                 ggtgttttaatggcagcaaaggcataataagaaggagatatacatatga 
               
               
                   
               
               
                 gcaaaatatttaaaatcttaacaataaatcctggttcgacatcaactaa 
               
               
                   
               
               
                 aatagctgtatttgataatgaggatttagtatttgaaaaaactttaaga 
               
               
                   
               
               
                 cattcttcagaagaaataggaaaatatgagaaggtgtctgaccaatttg 
               
               
                   
               
               
                 aatttcgtaaacaagtaatagaagaagctctaaaagaaggtggagtaaa 
               
               
                   
               
               
                 aacatctgaattagatgctgtagtaggtagaggaggacttcttaaacct 
               
               
                   
               
               
                 ataaaaggtggtacttattcagtaagtgctgctatgattgaagatttaa 
               
               
                   
               
               
                 aagtgggagttttaggagaacacgcttcaaacctaggtggaataatagc 
               
               
                   
               
               
                 aaaacaaataggtgaagaagtaaatgttccttcatacatagtagaccct 
               
               
                   
               
               
                 gttgttgtagatgaattagaagatgttgctagaatttctggtatgcctg 
               
               
                   
               
               
                 aaataagtagagcaagtgtagtacatgctttaaatcaaaaggcaatagc 
               
               
                   
               
               
                 aagaagatatgctagagaaataaacaagaaatatgaagatataaatctt 
               
               
                   
               
               
                 atagttgcacacatgggtggaggagtttctgttggagctcataaaaatg 
               
               
                   
               
               
                 gtaaaatagtagatgttgcaaacgcattagatggagaaggacctttctc 
               
               
                   
               
               
                 tccagaaagaagtggtggactaccagtaggtgcattagtaaaaatgtgc 
               
               
                   
               
               
                 tttagtggaaaatatactcaagatgaaattaaaaagaaaataaaaggta 
               
               
                   
               
               
                 atggcggactagttgcatacttaaacactaatgatgctagagaagttga 
               
               
                   
               
               
                 agaaagaattgaagctggtgatgaaaaagctaaattagtatatgaagct 
               
               
                   
               
               
                 atggcatatcaaatctctaaagaaataggagctagtgctgcagttctta 
               
               
                   
               
               
                 agggagatgtaaaagcaatattattaactggtggaatcgcatattcaaa 
               
               
                   
               
               
                 aatgtttacagaaatgattgcagatagagttaaatttatagcagatgta 
               
               
                   
               
               
                 aaagtttatccaggtgaagatgaaatgattgcattagctcaaggtggac 
               
               
                   
               
               
                 ttagagttttaactggtgaagaagaggctcaagtttatgataactaata 
               
               
                   
               
               
                 a 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 42 
               
               
                   
               
               
                 pZA22-oxyR construct (SEQ ID NO: 170) 
               
               
                 Nucleotide sequences of pZA22-oxyR  
               
               
                 construct (SEQ ID NO: 170) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 ctcgagatgctagcaattgtgagcggataacaattgacattgtgagcgg 
               
               
                   
               
               
                 ataacaagatactgageacatcagcaggacgcactgaccttaattaaaa 
               
               
                   
               
               
                 gaattcattaaagaggagaaaggtaccatgaatattcgtgatcttgagt 
               
               
                   
               
               
                 acctggtggcattggctgaacaccgccattttcggcgtgcggcagattc 
               
               
                   
               
               
                 ctgccacgttagccagccgacgcttagcgggcaaattcgtaagctggaa 
               
               
                   
               
               
                 gatgagctgggcgtgatgttgctggagcggaccagccgtaaagtgttgt 
               
               
                   
               
               
                 tcacccaggcgggaatgctgctggtggatcaggcgcgtaccgtgctgcg 
               
               
                   
               
               
                 tgaggtgaaagtccttaaagagatggcaagccagcagggcgagacgatg 
               
               
                   
               
               
                 tccggaccgctgcacattggtttgattcccacagttggaccgtacctgc 
               
               
                   
               
               
                 taccgcatattatccctatgctgcaccagacctttccaaagctggaaat 
               
               
                   
               
               
                 gtatctgcatgaagcacagacccaccagttactggcgcaactggacagc 
               
               
                   
               
               
                 ggcaaactcgattgcgtgatcctcgcgctggtgaaagagagcgaagcat 
               
               
                   
               
               
                 tcattgaagtgccgttgtttgatgagccaatgttgctggctatctatga 
               
               
                   
               
               
                 agatcacccgtgggcgaaccgcgaatgcgtaccgatggccgatctggca 
               
               
                   
               
               
                 ggggaaaaactgctgatgctggaagatggtcactgtttgcgcgatcagg 
               
               
                   
               
               
                 caatgggtttctgttttgaagccggggcggatgaagatacacacttccg 
               
               
                   
               
               
                 cgcgaccagcctggaaactctgcgcaacatggtggcggcaggtagcggg 
               
               
                   
               
               
                 atcactttactgccagcgctggctgtgccgccggagcgcaaacgcgatg 
               
               
                   
               
               
                 gggttgtttatctgccgtgcattaagccggaaccacgccgcactattgg 
               
               
                   
               
               
                 cctggtttatcgtcctggctcaccgctgcgcagccgctatgagcagctg 
               
               
                   
               
               
                 gcagaggccatccgcgcaagaatggatggccatttcgataaagttttaa 
               
               
                   
               
               
                 aacaggcggtttaaggatcccatggtacgcgtgctagaggcatcaaata 
               
               
                   
               
               
                 aaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtt 
               
               
                   
               
               
                 tgtcggtgaacgctctcctgagtaggacaaatccgccgccctagaccta 
               
               
                   
               
               
                 ggggatatattccgcttcctcgctcactgactcgctacgctcggtcgtt 
               
               
                   
               
               
                 cgactgcggcgagcggaaatggcttacgaacggggcggagatttcctgg 
               
               
                   
               
               
                 aagatgccaggaagatacttaacagggaagtgagagggccgcggcaaag 
               
               
                   
               
               
                 ccgtttttccataggctccgcccccctgacaagcatcacgaaatctgac 
               
               
                   
               
               
                 gctcaaatcagtggtggcgaaacccgacaggactataaagataccaggc 
               
               
                   
               
               
                 gtttccccctggcggctccctcgtgcgctctcctgttcctgcctttcgg 
               
               
                   
               
               
                 tttaccggtgtcattccgctgttatggccgcgtttgtctcattccacgc 
               
               
                   
               
               
                 ctgacactcagttccgggtaggcagttcgctccaagctggactgtatgc 
               
               
                   
               
               
                 acgaaccccccgttcagtccgaccgctgcgccttatccggtaactatcg 
               
               
                   
               
               
                 tcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagcc 
               
               
                   
               
               
                 actggtaattgatttagaggagttagtcttgaagtcatgcgccggttaa 
               
               
                   
               
               
                 ggctaaactgaaaggacaagttttggtgactgcgctcctccaagccagt 
               
               
                   
               
               
                 tacctcggttcaaagagttggtagctcagagaaccttcgaaaaaccgcc 
               
               
                   
               
               
                 ctgcaaggcggttttttcgttttcagagcaagagattacgcgcagacca 
               
               
                   
               
               
                 aaacgatctcaagaagatcatcttattaatcagataaaatatttctaga 
               
               
                   
               
               
                 tttcagtgcaatttatctcttcaaatgtagcacctgaagtcagccccat 
               
               
                   
               
               
                 acgatataagttgttactagtgcttggattctcaccaataaaaaacgcc 
               
               
                   
               
               
                 cggcggcaaccgagcgttctgaacaaatccagatggagttctgaggtca 
               
               
                   
               
               
                 ttactggatctatcaacaggagtccaagcgagctctcgaaccccagagt 
               
               
                   
               
               
                 cccgctcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcg 
               
               
                   
               
               
                 aatcgggagcggcgataccgtaaagcacgaggaagcggtcagcccattc 
               
               
                   
               
               
                 gccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctga 
               
               
                   
               
               
                 tagcggtccgccacacccagccggccacagtcgatgaatccagaaaagc 
               
               
                   
               
               
                 ggccattttccaccatgatattcggcaagcaggcatcgccatgggtcac 
               
               
                   
               
               
                 gacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaacagt 
               
               
                   
               
               
                 tcggctggcgcgagcccctgatgctcttcgtccagatcatcctgatcga 
               
               
                   
               
               
                 caagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgc 
               
               
                   
               
               
                 ttggtggtcgaatgggcaggtagccggatcaagcgtatgcagccgccgc 
               
               
                   
               
               
                 attgcatcagccatgatggatactttctcggcaggagcaaggtgagatg 
               
               
                   
               
               
                 acaggagatcctgccccggcacttcgcccaatagcagccagtcccttcc 
               
               
                   
               
               
                 cgcttcagtgacaacgtcgagcacagctgcgcaaggaacgcccgtcgtg 
               
               
                   
               
               
                 gccagccacgatagccgcgctgcctcgtcctgcagttcattcagggcac 
               
               
                   
               
               
                 cggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacag 
               
               
                   
               
               
                 ccggaacacggcggcatcagagcagccgattgtctgttgtgcccagtca 
               
               
                   
               
               
                 tagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatc 
               
               
                   
               
               
                 catcttgttcaatcatgcgaaacgatcctcatcctgtctcttgatcaga 
               
               
                   
               
               
                 tcttgatcccctgcgccatcagatccttggcggcaagaaagccatccag 
               
               
                   
               
               
                 tttactttgcagggcttcccaaccttaccagagggcgccccagctggca 
               
               
                   
               
               
                 attccgacgtctaagaaaccattattatcatgacattaacctataaaaa 
               
               
                   
               
               
                 taggcgtatcacgaggccctttcgtcttcac 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 43 
               
               
                   
               
               
                 pLOGIC046-delta pbt.buk/tesB+-oxyS-butyrate  
               
               
                 construct 
               
               
                 Nucleotide sequences of pLOGIC046-delta  
               
               
                 pbt.buk/tesB+-oxyS-butyrate  
               
               
                 construct(SEQ ID NO: 171) 
               
               
                   
               
             
            
               
                 Ctcgagttcattatccatcctccatcgccacgatagttcatggcgatag 
               
               
                   
               
               
                 gtagaatagcaatgaacgattatccctatcaagcattctgactgataat 
               
               
                   
               
               
                 tgctcacacgaattcattaaagaggagaaaggtaccatgatcgtaaaac 
               
               
                   
               
               
                 ctatggtacgcaacaatatctgcctgaacgcccatcctcagggctgcaa 
               
               
                   
               
               
                 gaagggagtggaagatcagattgaatataccaagaaacgcattaccgca 
               
               
                   
               
               
                 gaagtcaaagctggcgcaaaagctccaaaaaacgttctggtgcttggct 
               
               
                   
               
               
                 gctcaaatggttacggcctggcgagccgcattactgctgcgttcggata 
               
               
                   
               
               
                 cggggctgcgaccatcggcgtgtcctttgaaaaagcgggttcagaaacc 
               
               
                   
               
               
                 aaatatggtacaccgggatggtacaataatttggcatttgatgaagcgg 
               
               
                   
               
               
                 caaaacgcgagggtctttatagcgtgacgatcgacggcgatgcgttttc 
               
               
                   
               
               
                 agacgagatcaaggcccaggtaattgaggaagccaaaaaaaaaggtatc 
               
               
                   
               
               
                 aaatttgatctgatcgtatacagcttggccagcccagtacgtactgatc 
               
               
                   
               
               
                 ctgatacaggtatcatgcacaaaagcgttttgaaaccctttggaaaaac 
               
               
                   
               
               
                 gttcacaggcaaaacagtagatccgtttactggcgagctgaaggaaatc 
               
               
                   
               
               
                 tccgcggaaccagcaaatgacgaggaagcagccgccactgttaaagtta 
               
               
                   
               
               
                 tggggggtgaagattgggaacgttggattaagcagctgtcgaaggaagg 
               
               
                   
               
               
                 cctcttagaagaaggctgtattaccttggcctatagttatattggccct 
               
               
                   
               
               
                 gaagctacccaagctttgtaccgtaaaggcacaatcggcaaggccaaag 
               
               
                   
               
               
                 aacacctggaggccacagcacaccgtctcaacaaagagaacccgtcaat 
               
               
                   
               
               
                 ccgtgccttcgtgagcgtgaataaaggcctggtaacccgcgcaagcgcc 
               
               
                   
               
               
                 gtaatcccggtaatccctctgtatctcgccagcttgttcaaagtaatga 
               
               
                   
               
               
                 aagagaagggcaatcatgaaggttgtattgaacagatcacgcgtctgta 
               
               
                   
               
               
                 cgccgagcgcctgtaccgtaaagatggtacaattccagttgatgaggaa 
               
               
                   
               
               
                 aatcgcattcgcattgatgattgggagttagaagaagacgtccagaaag 
               
               
                   
               
               
                 cggtatccgcgttgatggagaaagtcacgggtgaaaacgcagaatctct 
               
               
                   
               
               
                 cactgacttagcggggtaccgccatgatttcttagctagtaacggcttt 
               
               
                   
               
               
                 gatgtagaaggtattaattatgaagcggaagttgaacgcttcgaccgta 
               
               
                   
               
               
                 tctgataagaaggagatatacatatgagagaagtagtaattgccagtgc 
               
               
                   
               
               
                 agctagaacagcagtaggaagttttggaggagcatttaaatcagtttca 
               
               
                   
               
               
                 gcggtagagttaggggtaacagcagctaaagaagctataaaaagagcta 
               
               
                   
               
               
                 acataactccagatatgatagatgaatctcttttagggggagtacttac 
               
               
                   
               
               
                 agcaggtcttggacaaaatatagcaagacaaatagcattaggagcagga 
               
               
                   
               
               
                 ataccagtagaaaaaccagctatgactataaatatagtttgtggttctg 
               
               
                   
               
               
                 gattaagatctgtttcaatggcatctcaacttatagcattaggtgatgc 
               
               
                   
               
               
                 tgatataatgttagttggtggagctgaaaacatgagtatgtctccttat 
               
               
                   
               
               
                 ttagtaccaagtgcgagatatggtgcaagaatgggtgatgctgcttttg 
               
               
                   
               
               
                 ttgattcaatgataaaagatggattatcagacatatttaataactatca 
               
               
                   
               
               
                 catgggtattactgctgaaaacatagcagagcaatggaatataactaga 
               
               
                   
               
               
                 gaagaacaagatgaattagctcttgcaagtcaaaataaagctgaaaaag 
               
               
                   
               
               
                 ctcaagctgaaggaaaatttgatgaagaaatagttcctgttgttataaa 
               
               
                   
               
               
                 aggaagaaaaggtgacactgtagtagataaagatgaatatattaagcct 
               
               
                   
               
               
                 ggcactacaatggagaaacttgctaagttaagacctgcatttaaaaaag 
               
               
                   
               
               
                 atggaacagttactgctggtaatgcatcaggaataaatgatggtgctgc 
               
               
                   
               
               
                 tatgttagtagtaatggctaaagaaaaagctgaagaactaggaatagag 
               
               
                   
               
               
                 cctcttgcaactatagtttcttatggaacagctggtgttgaccctaaaa 
               
               
                   
               
               
                 taatgggatatggaccagttccagcaactaaaaaagctttagaagctgc 
               
               
                   
               
               
                 taatatgactattgaagatatagatttagttgaagctaatgaggcattt 
               
               
                   
               
               
                 gctgcccaatctgtagctgtaataagagacttaaatatagatatgaata 
               
               
                   
               
               
                 aagttaatgttaatggtggagcaatagctataggacatccaataggatg 
               
               
                   
               
               
                 ctcaggagcaagaatacttactacacttttatatgaaatgaagagaaga 
               
               
                   
               
               
                 gatgctaaaactggtcttgctacactttgtataggcggtggaatgggaa 
               
               
                   
               
               
                 ctactttaatagttaagagatagtaagaaggagatatacatatgaaatt 
               
               
                   
               
               
                 agctgtaataggtagtggaactatgggaagtggtattgtacaaactttt 
               
               
                   
               
               
                 gcaagttgtggacatgatgtatgtttaaagagtagaactcaaggtgcta 
               
               
                   
               
               
                 tagataaatgtttagctttattagataaaaatttaactaagttagttac 
               
               
                   
               
               
                 taagggaaaaatggatgaagctacaaaagcagaaatattaagtcatgtt 
               
               
                   
               
               
                 agttcaactactaattatgaagatttaaaagatatggatttaataatag 
               
               
                   
               
               
                 aagcatctgtagaagacatgaatataaagaaagatgttttcaagttact 
               
               
                   
               
               
                 agatgaattatgtaaagaagatactatcttggcaacaaatacttcatca 
               
               
                   
               
               
                 ttatctataacagaaatagcttcttctactaagcgcccagataaagtta 
               
               
                   
               
               
                 taggaatgcatttctttaatccagttcctatgatgaaattagttgaagt 
               
               
                   
               
               
                 tataagtggtcagttaacatcaaaagttacttttgatacagtatttgaa 
               
               
                   
               
               
                 ttatctaagagtatcaataaagtaccagtagatgtatctgaatctcctg 
               
               
                   
               
               
                 gatttgtagtaaatagaatacttatacctatgataaatgaagctgttgg 
               
               
                   
               
               
                 tatatatgcagatggtgttgcaagtaaagaagaaatagatgaagctatg 
               
               
                   
               
               
                 aaattaggagcaaaccatccaatgggaccactagcattaggtgatttaa 
               
               
                   
               
               
                 tcggattagatgttgttttagctataatgaacgttttatatactgaatt 
               
               
                   
               
               
                 tggagatactaaatatagacctcatccacttttagctaaaatggttaga 
               
               
                   
               
               
                 gctaatcaattaggaagaaaaactaagataggattctatgattataata 
               
               
                   
               
               
                 aataataagaaggagatatacatatgagtacaagtgatgttaaagttta 
               
               
                   
               
               
                 tgagaatgtagctgttgaagtagatggaaatatatgtacagtgaaaatg 
               
               
                   
               
               
                 aatagacctaaagcccttaatgcaataaattcaaagactttagaagaac 
               
               
                   
               
               
                 tttatgaagtatttgtagatattaataatgatgaaactattgatgttgt 
               
               
                   
               
               
                 aatattgacaggggaaggaaaggcatttgtagctggagcagatattgca 
               
               
                   
               
               
                 tacatgaaagatttagatgctgtagctgctaaagattttagtatcttag 
               
               
                   
               
               
                 gagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatagc 
               
               
                   
               
               
                 tgctgtaaacggatttgctttaggtggaggatgtgaacttgcaatggca 
               
               
                   
               
               
                 tgtgatataagaattgcatctgctaaagctaaatttggtcagccagaag 
               
               
                   
               
               
                 taactcttggaataactccaggatatggaggaactcaaaggcttacaag 
               
               
                   
               
               
                 attggttggaatggcaaaagcaaaagaattaatctttacaggtcaagtt 
               
               
                   
               
               
                 ataaaagctgatgaagctgaaaaaatagggetagtaaatagagtcgttg 
               
               
                   
               
               
                 agccagacattttaatagaagaagttgagaaattagctaagataatagc 
               
               
                   
               
               
                 taaaaatgctcagcttgcagttagatactctaaagaagcaatacaactt 
               
               
                   
               
               
                 ggtgctcaaactgatataaatactggaatagatatagaatctaatttat 
               
               
                   
               
               
                 ttggtctttgtttttcaactaaagaccaaaaagaaggaatgtcagcttt 
               
               
                   
               
               
                 cgttgaaaagagagaagctaactttataaaagggtaataagaaggagat 
               
               
                   
               
               
                 atacatatgAGTCAGGCGCTAAAAAATTTACTGACATTGTTAAATCTGG 
               
               
                   
               
               
                 AAAAAATTGAGGAAGGACTCTTTCGCGGCCAGAGTGAAGATTTAGGTTT 
               
               
                   
               
               
                 ACGCCAGGTGTTTGGCGGCCAGGTCGTGGGTCAGGCCTTGTATGCTGCA 
               
               
                   
               
               
                 AAAGAGACCGTCCCTGAAGAGCGGCTGGTACATTCGTTTCACAGCTACT 
               
               
                   
               
               
                 TTCTTCGCCCTGGCGATAGTAAGAAGCCGATTATTTATGATGTCGAAAC 
               
               
                   
               
               
                 GCTGCGTGACGGTAACAGCTTCAGCGCCCGCCGGGTTGCTGCTATTCAA 
               
               
                   
               
               
                 AACGGCAAACCGATTTTTTATATGACTGCCTCTTTCCAGGCACCAGAAG 
               
               
                   
               
               
                 CGGGTTTCGAACATCAAAAAACAATGCCGTCCGCGCCAGCGCCTGATGG 
               
               
                   
               
               
                 CCTCCCTTCGGAAACGCAAATCGCCCAATCGCTGGCGCACCTGCTGCCG 
               
               
                   
               
               
                 CCAGTGCTGAAAGATAAATTCATCTGCGATCGTCCGCTGGAAGTCCGTC 
               
               
                   
               
               
                 CGGTGGAGTTTCATAACCCACTGAAAGGTCACGTCGCAGAACCACATCG 
               
               
                   
               
               
                 TCAGGTGTGGATCCGCGCAAATGGTAGCGTGCCGGATGACCTGCGCGTT 
               
               
                   
               
               
                 CATCAGTATCTGCTCGGTTACGCTTCTGATCTTAACTTCCTGCCGGTAG 
               
               
                   
               
               
                 CTCTACAGCCGCACGGCATCGGTTTTCTCGAACCGGGGATTCAGATTGC 
               
               
                   
               
               
                 CACCATTGACCATTCCATGTGGTTCCATCGCCCGTTTAATTTGAATGAA 
               
               
                   
               
               
                 TGGCTGCTGTATAGCGTGGAGAGCACCTCGGCGTCCAGCGCACGTGGCT 
               
               
                   
               
               
                 TTGTGCGCGGTGAGTTTTATACCCAAGACGGCGTACTGGTTGCCTCGAC 
               
               
                   
               
               
                 CGTTCAGGAAGGGGTGATGCGTAATCACAATtaa 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the butyrate gene cassette (e.g., bcd2-etfB3-etfA3-thiA1-hbd-crt2-pbt buk butyrate cassette (pLogic031), and/or ter-thiA1-hbd-crt2-pbt buk butyrate cassette (pLogic046) and/or ter-thiA1-hbd-crt2-tesb butyrate cassette (pLOGIC046-delta pbt.buk/tesB+)) is placed under the control of a FNR regulatory region selected from Table 21 or 22 and SEQ ID NOs: 141-157. In certain constructs, the FNR-responsive promoter is further fused to a strong ribosome binding site sequence. For efficient translation of butyrate genes, each synthetic gene in the operon was separated by a 15 base pair ribosome binding site derived from the T7 promoter/translational start site. 
     Example 2. Construction of Vectors for Overproducing Butyrate Using an Inducible Tet Promoter-Butyrate Circuit 
     To facilitate inducible production of butyrate in  Escherichia coli  Nissle, the eight genes of the butyrate production pathway from  Peptoclostridium difficile  630 (bcd2, etfB3, etfA3, thiA1, hbd, crt2, bpt, and buk; NCBI), as well as transcriptional and translational elements, were synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322 to create pLogic031. For efficient translation of butyrate genes, each synthetic gene in the operon was separated by a 15 base pair ribosome binding site derived from the T7 promoter. 
     The gene products of bcd2-etfA3-etfB3 form a complex that convert crotonyl-CoA to butyryl-CoA, and may show some dependence on oxygen as a co-oxidant. For reasons described in Example 1, a second plasmid was generated, in which bcd2-etfA3-etfB3 was replaced with (trans-2-enoynl-CoA reductase; ter from  Treponema denticola  capable of butyrate production in  E. coli . Inverse PCR was used to amplify the entire sequence of pLogic031 outside of the bcd-etfA3-etfB3 region. The ter gene was codon optimized for  E. coli  codon usage using Integrated DNA technologies online codon optimization tool, synthesized (Genewiz, Cambridge, Mass.), and cloned into this inverse PCR fragment using Gibson assembly to create pLogic046. 
     A third butyrate gene cassette was further generated, in which the pbt and buk genes were replaced with tesB (SEQ ID NO: 10). TesB is a thioesterase found in  E. coli  that cleaves off the butyrate from butyryl-coA, thus obviating the need for pbt-buk (see  FIG. 2 ). The third butyrate gene cassette, as well as transcriptional and translational elements, is synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322 to create pLOGIC046-delta pbt.buk/tesB+(ter-thiA1-hbd-crt2-tesb butyrate cassette, also referred to herein as tesB butyrate cassette). 
     As synthesized, the all three butyrate gene cassettes were placed under control of a tetracycline-inducible promoter, with the let repressor (tetR) expressed constitutively, divergent from the tet-inducible synthetic butyrate operon. 
     Nucleic acid sequences of tetracycline-regulated constructs comprising a let promoter are shown in Table 44 and Table 45 and Table 46. Table 44 depicts the nucleic acid sequence of an exemplary tetracycline-regulated construct comprising a tet promoter and a butyrogenic gene cassette (pLogic031-tet-butyrate construct; SEQ ID NO: 78). The sequence encoding TetR is underlined, and the overlapping tetR/tetA promoters are  . Table 45 depicts the nucleic acid sequence of an exemplary tetracycline-regulated construct comprising a let promoter and a butyrogenic gene cassette (pLogic046-tet-butyrate construct; SEQ ID NO: 79). The sequence encoding TetR is underlined, and the overlapping tetR/tetA promoters are  . 
     Table 46 depicts the nucleic acid sequence of an exemplary tetracycline-regulated construct (pLOGIC046-delta pbt.buk/tesB+-tet-butyrate construct) comprising a reverse complement of the tetR repressor (underlined), an intergenic region containing divergent promoters controlling tetR and the butyrate operon and their respective RBS (bold), and the butyrate genes separated by RBS. 
     In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 172, 173, or 174 or a functional fragment thereof. 
     
       
         
           
               
             
               
                 TABLE 44 
               
               
                   
               
               
                 pLogic031-tet-butyrate construct (SEQ ID NO: 172) 
               
               
                 Nucleotide sequences of pLogic031-tet-butyrate construct 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 172) 
               
               
                 gtaaaacgacggccagtgaattcg ttaagacccactttcacatttaagttgtttttctaatccgcatatg   
               
               
                   
               
               
                 
                   atcaattcaaggccgaataagaaggctggctctgcaccttggtgatcaaataattcgatagcttgtcgta 
                 
               
               
                   
               
               
                 ataatggcggcatactatcagtagtaggtgtttccctttcttctttagcgacttgatgctcttgatcttc 
               
               
                   
               
               
                 
                   caatacgcaacctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctagtgaaaaacct 
                 
               
               
                   
               
               
                 
                   tgttggcataaaaaggctaattgattttcgagagtttcatactgtttttctgtaggccgtgtacctaaat 
                 
               
               
                   
               
               
                 
                   gtacttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatc 
                 
               
               
                   
               
               
                 
                   ttgccagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcg 
                 
               
               
                   
               
               
                 
                   tcgagcaaagcccgcttattttttacatgccaatacaatgtaggctgctctacacctagcttctgggcga 
                 
               
               
                   
               
               
                 
                   gtttacgggttgttaaaccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttact 
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 atatggatttaaattctaaaaaatatcagatgcttaaagagctatatgtaagcttcgctgaaaatgaagt 
               
               
                   
               
               
                 taaacctttagcaacagaacttgatgaagaagaaagatttccttatgaaacagtggaaaaaatggcaaaa 
               
               
                   
               
               
                 gcaggaatgatgggtataccatatccaaaagaatatggtggagaaggtggagacactgtaggatatataa 
               
               
                   
               
               
                 tggcagttgaagaattgtctagagtttgtggtactacaggagttatattatcagctcatacatctcttgg 
               
               
                   
               
               
                 ctcatggcctatatatcaatatggtaatgaagaacaaaaacaaaaattcttaagaccactagcaagtgga 
               
               
                   
               
               
                 gaaaaattaggagcatttggtcttactgagcctaatgctggtacagatgcgtctggccaacaaacaactg 
               
               
                   
               
               
                 ctgttttagacggggatgaatacatacttaatggctcaaaaatatttataacaaacgcaatagctggtga 
               
               
                   
               
               
                 catatatgtagtaatggcaatgactgataaatctaaggggaacaaaggaatatcagcatttatagttgaa 
               
               
                   
               
               
                 aaaggaactcctgggtttagctttggagttaaagaaaagaaaatgggtataagaggttcagctacgagtg 
               
               
                   
               
               
                 aattaatatttgaggattgcagaatacctaaagaaaatttacttggaaaagaaggtcaaggatttaagat 
               
               
                   
               
               
                 agcaatgtctactcttgatggtggtagaattggtatagctgcacaagctttaggtttagcacaaggtgct 
               
               
                   
               
               
                 cttgatgaaactgttaaatatgtaaaagaaagagtacaatttggtagaccattatcaaaattccaaaata 
               
               
                   
               
               
                 cacaattccaattagctgatatggaagttaaggtacaagcggctagacaccttgtatatcaagcagctat 
               
               
                   
               
               
                 aaataaagacttaggaaaaccttatggagtagaagcagcaatggcaaaattatttgcagctgaaacagct 
               
               
                   
               
               
                 atggaagttactacaaaagctgtacaacttcatggaggatatggatacactcgtgactatccagtagaaa 
               
               
                   
               
               
                 gaatgatgagagatgctaagataactgaaatatatgaaggaactagtgaagttcaaagaatggttatttc 
               
               
                   
               
               
                 aggaaaactattaaaatagtaagaaggagatatacatatggaggaaggatttatgaatatagtcgtttgt 
               
               
                   
               
               
                 ataaaacaagttccagatacaacagaagttaaactagatcctaatacaggtactttaattagagatggag 
               
               
                   
               
               
                 taccaagtataataaaccctgatgataaagcaggtttagaagaagctataaaattaaaagaagaaatggg 
               
               
                   
               
               
                 tgctcatgtaactgttataacaatgggacctcctcaagcagatatggctttaaaagaagctttagcaatg 
               
               
                   
               
               
                 ggtgcagatagaggtatattattaacagatagagcatttgcgggtgctgatacttgggcaacttcatcag 
               
               
                   
               
               
                 cattagcaggagcattaaaaaatatagattttgatattataatagctggaagacaggcgatagatggaga 
               
               
                   
               
               
                 tactgcacaagttggacctcaaatagctgaacatttaaatcttccatcaataacatatgctgaagaaata 
               
               
                   
               
               
                 aaaactgaaggtgaatatgtattagtaaaaagacaatttgaagattgttgccatgacttaaaagttaaaa 
               
               
                   
               
               
                 tgccatgccttataacaactcttaaagatatgaacacaccaagatacatgaaagttggaagaatatatga 
               
               
                   
               
               
                 tgctttcgaaaatgatgtagtagaaacatggactgtaaaagatatagaagttgacccttctaatttaggt 
               
               
                   
               
               
                 cttaaaggttctccaactagtgtatttaaatcatttacaaaatcagttaaaccagctggtacaatataca 
               
               
                   
               
               
                 atgaagatgcgaaaacatcagctggaattatcatagataaattaaaagagaagtatatcatataataaga 
               
               
                   
               
               
                 aggagatatacatatgggtaacgttttagtagtaatagaacaaagagaaaatgtaattcaaactgtttct 
               
               
                   
               
               
                 ttagaattactaggaaaggctacagaaatagcaaaagattatgatacaaaagtttctgcattacttttag 
               
               
                   
               
               
                 gtagtaaggtagaaggtttaatagatacattagcacactatggtgcagatgaggtaatagtagtagatga 
               
               
                   
               
               
                 tgaagctttagcagtgtatacaactgaaccatatacaaaagcagcttatgaagcaataaaagcagctgac 
               
               
                   
               
               
                 cctatagttgtattatttggtgcaacttcaataggtagagatttagcgcctagagtttctgctagaatac 
               
               
                   
               
               
                 atacaggtcttactgctgactgtacaggtcttgcagtagctgaagatacaaaattattattaatgacaag 
               
               
                   
               
               
                 acctgcctttggtggaaatataatggcaacaatagtttgtaaagatttcagacctcaaatgtctacagtt 
               
               
                   
               
               
                 agaccaggggttatgaagaaaaatgaacctgatgaaactaaagaagctgtaattaaccgtttcaaggtag 
               
               
                   
               
               
                 aatttaatgatgctgataaattagttcaagttgtacaagtaataaaagaagctaaaaaacaagttaaaat 
               
               
                   
               
               
                 agaagatgctaagatattagtttctgctggacgtggaatgggtggaaaagaaaacttagacatactttat 
               
               
                   
               
               
                 gaattagctgaaattataggtggagaagtttctggttctcgtgccactatagatgcaggttggttagata 
               
               
                   
               
               
                 aagcaagacaagttggtcaaactggtaaaactgtaagaccagacctttatatagcatgtggtatatctgg 
               
               
                   
               
               
                 agcaatacaacatatagctggtatggaagatgctgagtttatagttgctataaataaaaatccagaagct 
               
               
                   
               
               
                 ccaatatttaaatatgctgatgttggtatagttggagatgttcataaagtgcttccagaacttatcagtc 
               
               
                   
               
               
                 agttaagtgttgcaaaagaaaaaggtgaagttttagctaactaataagaaggagatatacatatgagaga 
               
               
                   
               
               
                 agtagtaattgccagtgcagctagaacagcagtaggaagttttggaggagcatttaaatcagtttcagcg 
               
               
                   
               
               
                 gtagagttaggggtaacagcagctaaagaagctataaaaagagctaacataactccagatatgatagatg 
               
               
                   
               
               
                 aatctcttttagggggagtacttacagcaggtcttggacaaaatatagcaagacaaatagcattaggagc 
               
               
                   
               
               
                 aggaataccagtagaaaaaccagctatgactataaatatagtttgtggttctggattaagatctgtttca 
               
               
                   
               
               
                 atggcatctcaacttatagcattaggtgatgctgatataatgttagttggtggagctgaaaacatgagta 
               
               
                   
               
               
                 tgtctccttatttagtaccaagtgcgagatatggtgcaagaatgggtgatgctgcttttgttgattcaat 
               
               
                   
               
               
                 gataaaagatggattatcagacatatttaataactatcacatgggtattactgctgaaaacatagcagag 
               
               
                   
               
               
                 caatggaatataactagagaagaacaagatgaattagctcttgcaagtcaaaataaagctgaaaaagctc 
               
               
                   
               
               
                 aagctgaaggaaaatttgatgaagaaatagttcctgttgttataaaaggaagaaaaggtgacactgtagt 
               
               
                   
               
               
                 agataaagatgaatatattaagcctggcactacaatggagaaacttgctaagttaagacctgcatttaaa 
               
               
                   
               
               
                 aaagatggaacagttactgctggtaatgcatcaggaataaatgatggtgctgctatgttagtagtaatgg 
               
               
                   
               
               
                 ctaaagaaaaagctgaagaactaggaatagagcctcttgcaactatagtttcttatggaacagctggtgt 
               
               
                   
               
               
                 tgaccctaaaataatgggatatggaccagttccagcaactaaaaaagctttagaagctgctaatatgact 
               
               
                   
               
               
                 attgaagatatagatttagttgaagctaatgaggcatttgctgcccaatctgtagctgtaataagagact 
               
               
                   
               
               
                 taaatatagatatgaataaagttaatgttaatggtggagcaatagctataggacatccaataggatgctc 
               
               
                   
               
               
                 aggagcaagaatacttactacacttttatatgaaatgaagagaagagatgctaaaactggtcttgctaca 
               
               
                   
               
               
                 ctttgtataggcggtggaatgggaactactttaatagttaagagatagtaagaaggagatatacatatga 
               
               
                   
               
               
                 aattagctgtaataggtagtggaactatgggaagtggtattgtacaaacttttgcaagttgtggacatga 
               
               
                   
               
               
                 tgtatgtttaaagagtagaactcaaggtgctatagataaatgtttagctttattagataaaaatttaact 
               
               
                   
               
               
                 aagttagttactaagggaaaaatggatgaagctacaaaagcagaaatattaagtcatgttagttcaacta 
               
               
                   
               
               
                 ctaattatgaagatttaaaagatatggatttaataatagaagcatctgtagaagacatgaatataaagaa 
               
               
                   
               
               
                 agatgttttcaagttactagatgaattatgtaaagaagatactatcttggcaacaaatacttcatcatta 
               
               
                   
               
               
                 tctataacagaaatagcttcttctactaagcgcccagataaagttataggaatgcatttctttaatccag 
               
               
                   
               
               
                 ttcctatgatgaaattagttgaagttataagtggtcagttaacatcaaaagttacttttgatacagtatt 
               
               
                   
               
               
                 tgaattatctaagagtatcaataaagtaccagtagatgtatctgaatctcctggatttgtagtaaataga 
               
               
                   
               
               
                 atacttatacctatgataaatgaagctgttggtatatatgcagatggtgttgcaagtaaagaagaaatag 
               
               
                   
               
               
                 atgaagctatgaaattaggagcaaaccatccaatgggaccactagcattaggtgatttaatcggattaga 
               
               
                   
               
               
                 tgttgttttagctataatgaacgttttatatactgaatttggagatactaaatatagacctcatccactt 
               
               
                   
               
               
                 ttagctaaaatggttagagctaatcaattaggaagaaaaactaagataggattctatgattataataaat 
               
               
                   
               
               
                 aataagaaggagatatacatatgagtacaagtgatgttaaagtttatgagaatgtagctgttgaagtaga 
               
               
                   
               
               
                 tggaaatatatgtacagtgaaaatgaatagacctaaagcccttaatgcaataaattcaaagactttagaa 
               
               
                   
               
               
                 gaactttatgaagtatttgtagatattaataatgatgaaactattgatgttgtaatattgacaggggaag 
               
               
                   
               
               
                 gaaaggcatttgtagctggagcagatattgcatacatgaaagatttagatgctgtagctgctaaagattt 
               
               
                   
               
               
                 tagtatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatagctgctgtaaac 
               
               
                   
               
               
                 ggatttgctttaggtggaggatgtgaacttgcaatggcatgtgatataagaattgcatctgctaaagcta 
               
               
                   
               
               
                 aatttggtcagccagaagtaactcttggaataactccaggatatggaggaactcaaaggcttacaagatt 
               
               
                   
               
               
                 ggttggaatggcaaaagcaaaagaattaatctttacaggtcaagttataaaagctgatgaagctgaaaaa 
               
               
                   
               
               
                 atagggctagtaaatagagtcgttgagccagacattttaatagaagaagttgagaaattagctaagataa 
               
               
                   
               
               
                 tagctaaaaatgctcagcttgcagttagatactctaaagaagcaatacaacttggtgctcaaactgatat 
               
               
                   
               
               
                 aaatactggaatagatatagaatctaatttatttggtctttgtttttcaactaaagaccaaaaagaagga 
               
               
                   
               
               
                 atgtcagctttcgttgaaaagagagaagctaactttataaaagggtaataagaaggagatatacatatga 
               
               
                   
               
               
                 gaagttttgaagaagtaattaagtttgcaaaagaaagaggacctaaaactatatcagtagcatgttgcca 
               
               
                   
               
               
                 agataaagaagttttaatggcagttgaaatggctagaaaagaaaaaatagcaaatgccattttagtagga 
               
               
                   
               
               
                 gatatagaaaagactaaagaaattgcaaaaagcatagacatggatatcgaaaattatgaactgatagata 
               
               
                   
               
               
                 taaaagatttagcagaagcatctctaaaatctgttgaattagtttcacaaggaaaagccgacatggtaat 
               
               
                   
               
               
                 gaaaggcttagtagacacatcaataatactaaaagcagttttaaataaagaagtaggtcttagaactgga 
               
               
                   
               
               
                 aatgtattaagtcacgtagcagtatttgatgtagagggatatgatagattatttttcgtaactgacgcag 
               
               
                   
               
               
                 ctatgaacttagctcctgatacaaatactaaaaagcaaatcatagaaaatgcttgcacagtagcacattc 
               
               
                   
               
               
                 attagatataagtgaaccaaaagttgctgcaatatgcgcaaaagaaaaagtaaatccaaaaatgaaagat 
               
               
                   
               
               
                 acagttgaagctaaagaactagaagaaatgtatgaaagaggagaaatcaaaggttgtatggttggtgggc 
               
               
                   
               
               
                 cttttgcaattgataatgcagtatctttagaagcagctaaacataaaggtataaatcatcctgtagcagg 
               
               
                   
               
               
                 acgagctgatatattattagccccagatattgaaggtggtaacatattatataaagctttggtattcttc 
               
               
                   
               
               
                 tcaaaatcaaaaaatgcaggagttatagttggggctaaagcaccaataatattaacttctagagcagaca 
               
               
                   
               
               
                 gtgaagaaactaaactaaactcaatagctttaggtgttttaatggcagcaaaggcataataagaaggaga 
               
               
                   
               
               
                 tatacatatgagcaaaatatttaaaatcttaacaataaatcctggttcgacatcaactaaaatagctgta 
               
               
                   
               
               
                 tttgataatgaggatttagtatttgaaaaaactttaagacattcttcagaagaaataggaaaatatgaga 
               
               
                   
               
               
                 aggtgtctgaccaatttgaatttcgtaaacaagtaatagaagaagctctaaaagaaggtggagtaaaaac 
               
               
                   
               
               
                 atctgaattagatgctgtagtaggtagaggaggacttcttaaacctataaaaggtggtacttattcagta 
               
               
                   
               
               
                 agtgctgctatgattgaagatttaaaagtgggagttttaggagaacacgcttcaaacctaggtggaataa 
               
               
                   
               
               
                 tagcaaaacaaataggtgaagaagtaaatgttccttcatacatagtagaccctgttgttgtagatgaatt 
               
               
                   
               
               
                 agaagatgttgctagaatttctggtatgcctgaaataagtagagcaagtgtagtacatgctttaaatcaa 
               
               
                   
               
               
                 aaggcaatagcaagaagatatgctagagaaataaacaagaaatatgaagatataaatcttatagttgcac 
               
               
                   
               
               
                 acatgggtggaggagtttctgttggagctcataaaaatggtaaaatagtagatgttgcaaacgcattaga 
               
               
                   
               
               
                 tggagaaggacctttctctccagaaagaagtggtggactaccagtaggtgcattagtaaaaatgtgcttt 
               
               
                   
               
               
                 agtggaaaatatactcaagatgaaattaaaaagaaaataaaaggtaatggcggactagttgcatacttaa 
               
               
                   
               
               
                 acactaatgatgctagagaagttgaagaaagaattgaagctggtgatgaaaaagctaaattagtatatga 
               
               
                   
               
               
                 agctatggcatatcaaatctctaaagaaataggagctagtgctgcagttcttaagggagatgtaaaagca 
               
               
                   
               
               
                 atattattaactggtggaatcgcatattcaaaaatgtttacagaaatgattgcagatagagttaaattta 
               
               
                   
               
               
                 tagcagatgtaaaagtttatccaggtgaagatgaaatgattgcattagctcaaggtggacttagagtttt 
               
               
                   
               
               
                 aactggtgaagaagaggctcaagtttatgataactaataa 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 45 
               
               
                   
               
               
                 pLogic046-tet-butyrate construct (SEQ ID NO: 173) 
               
               
                 Nucleotide sequences of pLogic046-tet-butyrate construct 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 173) 
               
               
                 gtaaaacgacggccagtgaattcg ttaagacccactttcacatttaagttgtttttctaatccgcatatg   
               
               
                   
               
               
                 
                   atcaattcaaggccgaataagaaggctggctctgcaccttggtgatcaaataattcgatagcttgtcgta 
                 
               
               
                   
               
               
                 
                   ataatggcggcatactatcagtagtaggtgtttccctttcttctttagcgacttgatgctcttgatcttc 
                 
               
               
                   
               
               
                 
                   caatacgcaacctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctagtgaaaaacct 
                 
               
               
                   
               
               
                 
                   tgttggcataaaaaggctaattgattttcgagagtttcatactgtttttctgtaggccgtgtacctaaat 
                 
               
               
                   
               
               
                 
                   gtactttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaaaaaaatc 
                 
               
               
                   
               
               
                 
                   ttgccagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcg 
                 
               
               
                   
               
               
                 
                   tcgagcaaagcccgcttattttttacatgccaatacaatgtaggctgctctacacctagcttctgggcga 
                 
               
               
                   
               
               
                 gtttacgggttgttaaaccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttact 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 atatgatcgtaaaacctatggtacgcaacaatatctgcctgaacgcccatcctcagggctgcaagaaggg 
               
               
                   
               
               
                 agtggaagatcagattgaatataccaagaaacgcattaccgcagaagtcaaagctggcgcaaaagctcca 
               
               
                   
               
               
                 aaaaacgttctggtgcttggctgctcaaatggttacggcctggcgagccgcattactgctgcgttcggat 
               
               
                   
               
               
                 acggggctgcgaccatcggcgtgtcctttgaaaaagcgggttcagaaaccaaatatggtacaccgggatg 
               
               
                   
               
               
                 gtacaataatttggcatttgatgaagcggcaaaacgcgagggtctttatagcgtgacgatcgacggcgat 
               
               
                   
               
               
                 gcgttttcagacgagatcaaggcccaggtaattgaggaagccaaaaaaaaaggtatcaaatttgatctga 
               
               
                   
               
               
                 tcgtatacagcttggccagcccagtacgtactgatcctgatacaggtatcatgcacaaaagcgttttgaa 
               
               
                   
               
               
                 accctttggaaaaacgttcacaggcaaaacagtagatccgtttactggcgagctgaaggaaatctccgcg 
               
               
                   
               
               
                 gaaccagcaaatgacgaggaagcagccgccactgttaaagttatggggggtgaagattgggaacgttgga 
               
               
                   
               
               
                 ttaagcagctgtcgaaggaaggcctcttagaagaaggctgtattaccttggcctatagttatattggccc 
               
               
                   
               
               
                 tgaagctacccaagctttgtaccgtaaaggcacaatcggcaaggccaaagaacacctggaggccacagca 
               
               
                   
               
               
                 caccgtctcaacaaagagaacccgtcaatccgtgccttcgtgagcgtgaataaaggcctggtaacccgcg 
               
               
                   
               
               
                 caagcgccgtaatcccggtaatccctctgtatctcgccagcttgttcaaagtaatgaaagagaagggcaa 
               
               
                   
               
               
                 tcatgaaggttgtattgaacagatcacgcgtctgtacgccgagcgcctgtaccgtaaagatggtacaatt 
               
               
                   
               
               
                 ccagttgatgaggaaaatcgcattcgcattgatgattgggagttagaagaagacgtccagaaagcggtat 
               
               
                   
               
               
                 ccgcgttgatggagaaagtcacgggtgaaaacgcagaatctctcactgacttagcggggtaccgccatga 
               
               
                   
               
               
                 tttcttagctagtaacggctttgatgtagaaggtattaattatgaagcggaagttgaacgcttcgaccgt 
               
               
                   
               
               
                 atctgataagaaggagatatacatatgagagaagtagtaattgccagtgcagctagaacagcagtaggaa 
               
               
                   
               
               
                 gttttggaggagcatttaaatcagtttcagcggtagagttaggggtaacagcagctaaagaagctataaa 
               
               
                   
               
               
                 aagagctaacataactccagatatgatagatgaatctcttttagggggagtacttacagcaggtcttgga 
               
               
                   
               
               
                 caaaatatagcaagacaaatagcattaggagcaggaataccagtagaaaaaccagctatgactataaata 
               
               
                   
               
               
                 tagtttgtggttctggattaagatctgtttcaatggcatctcaacttatagcattaggtgatgctgatat 
               
               
                   
               
               
                 aatgttagttggtggagctgaaaacatgagtatgtctccttatttagtaccaagtgcgagatatggtgca 
               
               
                   
               
               
                 agaatgggtgatgctgcttttgttgattcaatgataaaagatggattatcagacatatttaataactatc 
               
               
                   
               
               
                 acatgggtattactgctgaaaacatagcagagcaatggaatataactagagaagaacaagatgaattagc 
               
               
                   
               
               
                 tcttgcaagtcaaaataaagctgaaaaagctcaagctgaaggaaaatttgatgaagaaatagttcctgtt 
               
               
                   
               
               
                 gttataaaaggaagaaaaggtgacactgtagtagataaagatgaatatattaagcctggcactacaatgg 
               
               
                   
               
               
                 agaaacttgctaagttaagacctgcatttaaaaaagatggaacagttactgctggtaatgcatcaggaat 
               
               
                   
               
               
                 aaatgatggtgctgctatgttagtagtaatggctaaagaaaaagctgaagaactaggaatagagcctctt 
               
               
                   
               
               
                 gcaactatagtttcttatggaacagctggtgttgaccctaaaataatgggatatggaccagttccagcaa 
               
               
                   
               
               
                 ctaaaaaagctttagaagctgctaatatgactattgaagatatagatttagttgaagctaatgaggcatt 
               
               
                   
               
               
                 tgctgcccaatctgtagctgtaataagagacttaaatatagatatgaataaagttaatgttaatggtgga 
               
               
                   
               
               
                 gcaatagctataggacatccaataggatgctcaggagcaagaatacttactacacttttatatgaaatga 
               
               
                   
               
               
                 agagaagagatgctaaaactggtcttgctacactttgtataggcggtggaatgggaactactttaatagt 
               
               
                   
               
               
                 taagagatagtaagaaggagatatacatatgaaattagctgtaataggtagtggaactatgggaagtggt 
               
               
                   
               
               
                 attgtacaaacttttgcaagttgtggacatgatgtatgtttaaagagtagaactcaaggtgctatagata 
               
               
                   
               
               
                 aatgtttagctttattagataaaaatttaactaagttagttactaagggaaaaatggatgaagctacaaa 
               
               
                   
               
               
                 agcagaaatattaagtcatgttagttcaactactaattatgaagatttaaaagatatggatttaataata 
               
               
                   
               
               
                 gaagcatctgtagaagacatgaatataaagaaagatgttttcaagttactagatgaattatgtaaagaag 
               
               
                   
               
               
                 atactatcttggcaacaaatacttcatcattatctataacagaaatagcttcttctactaagcgcccaga 
               
               
                   
               
               
                 taaagttataggaatgcatttctttaatccagttcctatgatgaaattagttgaagttataagtggtcag 
               
               
                   
               
               
                 ttaacatcaaaagttacttttgatacagtatttgaattatctaagagtatcaataaagtaccagtagatg 
               
               
                   
               
               
                 tatctgaatctcctggatttgtagtaaatagaatacttatacctatgataaatgaagctgttggtatata 
               
               
                   
               
               
                 tgcagatggtgttgcaagtaaagaagaaatagatgaagctatgaaattaggagcaaaccatccaatggga 
               
               
                   
               
               
                 ccactagcattaggtgatttaatcggattagatgttgttttagctataatgaacgttttatatactgaat 
               
               
                   
               
               
                 ttggagatactaaatatagacctcatccacttttagctaaaatggttagagctaatcaattaggaagaaa 
               
               
                   
               
               
                 aactaagataggattctatgattataataaataataagaaggagatatacatatgagtacaagtgatgtt 
               
               
                   
               
               
                 aaagtttatgagaatgtagctgttgaagtagatggaaatatatgtacagtgaaaatgaatagacctaaag 
               
               
                   
               
               
                 cccttaatgcaataaattcaaagactttagaagaactttatgaagtatttgtagatattaataatgatga 
               
               
                   
               
               
                 aactattgatgttgtaatattgacaggggaaggaaaggcatttgtagctggagcagatattgcatacatg 
               
               
                   
               
               
                 aaagatttagatgctgtagctgctaaagattttagtatcttaggagcaaaagcttttggagaaatagaaa 
               
               
                   
               
               
                 atagtaaaaaagtagtgatagctgctgtaaacggatttgctttaggtggaggatgtgaacttgcaatggc 
               
               
                   
               
               
                 atgtgatataagaattgcatctgctaaagctaaatttggtcagccagaagtaactcttggaataactcca 
               
               
                   
               
               
                 ggatatggaggaactcaaaggcttacaagattggttggaatggcaaaagcaaaagaattaatctttacag 
               
               
                   
               
               
                 gtcaagttataaaagctgatgaagctgaaaaaatagggctagtaaatagagtcgttgagccagacatttt 
               
               
                   
               
               
                 aatagaagaagttgagaaattagctaagataatagctaaaaatgctcagcttgcagttagatactctaaa 
               
               
                   
               
               
                 gaagcaatacaacttggtgctcaaactgatataaatactggaatagatatagaatctaatttatttggtc 
               
               
                   
               
               
                 tttgtttttcaactaaagaccaaaaagaaggaatgtcagctttcgttgaaaagagagaagctaactttat 
               
               
                   
               
               
                 aaaagggtaataagaaggagatatacatatgagaagttttgaagaagtaattaagtttgcaaaagaaaga 
               
               
                   
               
               
                 ggacctaaaactatatcagtagcatgttgccaagataaagaagttttaatggcagttgaaatggctagaa 
               
               
                   
               
               
                 aagaaaaaatagcaaatgccattttagtaggagatatagaaaagactaaagaaattgcaaaaagcataga 
               
               
                   
               
               
                 catggatatcgaaaattatgaactgatagatataaaagatttagcagaagcatctctaaaatctgttgaa 
               
               
                   
               
               
                 ttagtttcacaaggaaaagccgacatggtaatgaaaggcttagtagacacatcaataatactaaaagcag 
               
               
                   
               
               
                 ttttaaataaagaagtaggtcttagaactggaaatgtattaagtcacgtagcagtatttgatgtagaggg 
               
               
                   
               
               
                 atatgatagattatttttcgtaactgacgcagctatgaacttagctcctgatacaaatactaaaaagcaa 
               
               
                   
               
               
                 atcatagaaaatgcttgcacagtagcacattcattagatataagtgaaccaaaagttgctgcaatatgcg 
               
               
                   
               
               
                 caaaagaaaaagtaaatccaaaaatgaaagatacagttgaagctaaagaactagaagaaatgtatgaaag 
               
               
                   
               
               
                 aggagaaatcaaaggttgtatggttggtgggccttttgcaattgataatgcagtatctttagaagcagct 
               
               
                   
               
               
                 aaacataaaggtataaatcatcctgtagcaggacgagctgatatattattagccccagatattgaaggtg 
               
               
                   
               
               
                 gtaacatattatataaagctttggtattcttctcaaaatcaaaaaatgcaggagttatagttggggctaa 
               
               
                   
               
               
                 agcaccaataatattaacttctagagcagacagtgaagaaactaaactaaactcaatagctttaggtgtt 
               
               
                   
               
               
                 ttaatggcagcaaaggcataataagaaggagatatacatatgagcaaaatatttaaaatcttaacaataa 
               
               
                   
               
               
                 atcctggttcgacatcaactaaaatagctgtatttgataatgaggatttagtatttgaaaaaactttaag 
               
               
                   
               
               
                 acattcttcagaagaaataggaaaatatgagaaggtgtctgaccaatttgaatttcgtaaacaagtaata 
               
               
                   
               
               
                 gaagaagctctaaaagaaggtggagtaaaaacatctgaattagatgctgtagtaggtagaggaggacttc 
               
               
                   
               
               
                 ttaaacctataaaaggtggtacttattcagtaagtgctgctatgattgaagatttaaaagtgggagtttt 
               
               
                   
               
               
                 aggagaacacgcttcaaacctaggtggaataatagcaaaacaaataggtgaagaagtaaatgttccttca 
               
               
                   
               
               
                 tacatagtagaccctgttgttgtagatgaattagaagatgttgctagaatttctggtatgcctgaaataa 
               
               
                   
               
               
                 gtagagcaagtgtagtacatgctttaaatcaaaaggcaatagcaagaagatatgctagagaaataaacaa 
               
               
                   
               
               
                 gaaatatgaagatataaatcttatagttgcacacatgggtggaggagtttctgttggagctcataaaaat 
               
               
                   
               
               
                 ggtaaaatagtagatgttgcaaacgcattagatggagaaggacctttctctccagaaagaagtggtggac 
               
               
                   
               
               
                 taccagtaggtgcattagtaaaaatgtgctttagtggaaaatatactcaagatgaaattaaaaagaaaat 
               
               
                   
               
               
                 aaaaggtaatggcggactagttgcatacttaaacactaatgatgctagagaagttgaagaaagaattgaa 
               
               
                   
               
               
                 gctggtgatgaaaaagctaaattagtatatgaagctatggcatatcaaatctctaaagaaataggagcta 
               
               
                   
               
               
                 gtgctgcagttcttaagggagatgtaaaagcaatattattaactggtggaatcgcatattcaaaaatgtt 
               
               
                   
               
               
                 tacagaaatgattgcagatagagttaaatttatagcagatgtaaaagtttatccaggtgaagatgaaatg 
               
               
                   
               
               
                 attgcattagctcaaggtggacttagagttttaactggtgaagaagaggctcaagtttatgataactaat 
               
               
                   
               
               
                 aa 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 46 
               
               
                   
               
               
                 pLOGIC046-delta pbt.buk/tesB+-tet-butyrate  
               
               
                 construct (SEQ ID NO: 174) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 SEQ ID NO: 174 
               
               
                 gtaaaacgacggccagtgaattcg ttaagacccactttcacatttaagt   
               
               
                   
               
               
                 
                   tgtttttctaatccgcatatgatcaattcaaggccgaataagaaggctg 
                 
               
               
                   
               
               
                 
                   gctctgcaccttggtgatcaaataattcgatagcttgtcgtaataatgg 
                 
               
               
                   
               
               
                 
                   cggcatactatcagtagtaggtgtttccctttcttctttagcgacttga 
                 
               
               
                   
               
               
                 
                   tgctcttgatcttccaatacgcaacctaaagtaaaatgccccacagcgc 
                 
               
               
                   
               
               
                 
                   tgagtgcatataatgcattctctagtgaaaaaccttgttggcataaaaa 
                 
               
               
                   
               
               
                 
                   ggctaattgattttcgagagtttcatactgtttttctgtaggccgtgta 
                 
               
               
                   
               
               
                 
                   cctaaatgtacttttgctccatcgcgatgacttagtaaagcacatctaa 
                 
               
               
                   
               
               
                 
                   aacttttagcgttattacgtaaaaaatcttgccagctttccccttctaa 
                 
               
               
                   
               
               
                 
                   agggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcg 
                 
               
               
                   
               
               
                 
                   tcgagcaaagcccgcttattttttacatgccaatacaatgtaggctgct 
                 
               
               
                   
               
               
                 
                   ctacacctagcttctgggcgagtttacgggttgttaaaccttcgattcc 
                 
               
               
                   
               
               
                 
                   gacctcattaagcagctctaatgcgctgttaatcactttacttttatct 
                 
               
               
                   
               
               
                 
                   aatctagacat 
                   cattaattcctaatttttgttgacactctatcattgat 
                 
               
               
                   
               
               
                 
                   agagttattttaccactccctatcagtgatagagaaaagtgaactctag 
                 
               
               
                   
               
               
                   aaataattttgtttaactttaagaaggagatatacat atgatcgtaaaa 
               
               
                   
               
               
                 cctatggtacgcaacaatatctgcctgaacgcccatcctcagggctgca 
               
               
                   
               
               
                 agaagggagtggaagatcagattgaatataccaagaaacgcattaccgc 
               
               
                   
               
               
                 agaagtcaaagctggcgcaaaagctccaaaaaacgttctggtgcttggc 
               
               
                   
               
               
                 tgctcaaatggttacggcctggcgagccgcattactgctgcgttcggat 
               
               
                   
               
               
                 acggggctgcgaccatcggcgtgtcctttgaaaaagcgggttcagaaac 
               
               
                   
               
               
                 caaatatggtacaccgggatggtacaataatttggcatttgatgaagcg 
               
               
                   
               
               
                 gcaaaacgcgagggtctttatagcgtgacgatcgacggcgatgcgtttt 
               
               
                   
               
               
                 cagacgagatcaaggcccaggtaattgaggaagccaaaaaaaaaggtat 
               
               
                   
               
               
                 caaatttgatctgatcgtatacagcttggccagcccagtacgtactgat 
               
               
                   
               
               
                 cctgatacaggtatcatgcacaaaagcgttttgaaaccctttggaaaaa 
               
               
                   
               
               
                 cgttcacaggcaaaacagtagatccgtttactggcgagctgaaggaaat 
               
               
                   
               
               
                 ctccgcggaaccagcaaatgacgaggaagcagccgccactgttaaagtt 
               
               
                   
               
               
                 atggggggtgaagattgggaacgttggattaagcagctgtcgaaggaag 
               
               
                   
               
               
                 gcctcttagaagaaggctgtattaccttggcctatagttatattggccc 
               
               
                   
               
               
                 tgaagctacccaagctttgtaccgtaaaggcacaatcggcaaggccaaa 
               
               
                   
               
               
                 gaacacctggaggccacagcacaccgtctcaacaaagagaacccgtcaa 
               
               
                   
               
               
                 tccgtgccttcgtgagcgtgaataaaggcctggtaacccgcgcaagcgc 
               
               
                   
               
               
                 cgtaatcccggtaatccctctgtatctcgccagcttgttcaaagtaatg 
               
               
                   
               
               
                 aaagagaagggcaatcatgaaggttgtattgaacagatcacgcgtctgt 
               
               
                   
               
               
                 acgccgagcgcctgtaccgtaaagatggtacaattccagttgatgagga 
               
               
                   
               
               
                 aaatcgcattcgcattgatgattgggagttagaagaagacgtccagaaa 
               
               
                   
               
               
                 gcggtatccgcgttgatggagaaagtcacgggtgaaaacgcagaatctc 
               
               
                   
               
               
                 tcactgacttagcggggtaccgccatgatttcttagctagtaacggctt 
               
               
                   
               
               
                 tgatgtagaaggtattaattatgaagcggaagttgaacgcttcgaccgt 
               
               
                   
               
               
                 atctgataagaaggagatatacatatgagagaagtagtaattgccagtg 
               
               
                   
               
               
                 cagctagaacagcagtaggaagttttggaggagcatttaaatcagtttc 
               
               
                   
               
               
                 agcggtagagttaggggtaacagcagctaaagaagctataaaaagagct 
               
               
                   
               
               
                 aacataactccagatatgatagatgaatctcttttagggggagtactta 
               
               
                   
               
               
                 cagcaggtcttggacaaaatatagcaagacaaatagcattaggagcagg 
               
               
                   
               
               
                 aataccagtagaaaaaccagctatgactataaatatagtttgtggttct 
               
               
                   
               
               
                 ggattaagatctgtttcaatggcatctcaacttatagcattaggtgatg 
               
               
                   
               
               
                 ctgatataatgttagttggtggagctgaaaacatgagtatgtctcctta 
               
               
                   
               
               
                 tttagtaccaagtgcgagatatggtgcaagaatggatgatgctgctttt 
               
               
                   
               
               
                 gttgattcaatgataaaagatggattatcagacatatttaataactatc 
               
               
                   
               
               
                 acatgggtattactgctgaaaacatagcagagcaatggaatataactag 
               
               
                   
               
               
                 agaagaacaagatgaattagctcttgcaagtcaaaataaagctgaaaaa 
               
               
                   
               
               
                 gctcaagctgaaggaaaatttgatgaagaaatagttcctgttgttataa 
               
               
                   
               
               
                 aaggaagaaaaggtgacactgtagtagataaagatgaatatattaagcc 
               
               
                   
               
               
                 tggcactacaatggagaaacttgctaagttaagacctgcatttaaaaaa 
               
               
                   
               
               
                 gatggaacagttactgctggtaatgcatcaggaataaatgatggtgctg 
               
               
                   
               
               
                 ctatgttagtagtaatggctaaagaaaaagctgaagaactaggaataga 
               
               
                   
               
               
                 gcctcttgcaactatagtttcttatggaacagctggtgttgaccctaaa 
               
               
                   
               
               
                 ataatgggatatggaccagttccagcaactaaaaaagctttagaagctg 
               
               
                   
               
               
                 ctaatatgactattgaagatatagatttagttgaagctaatgaggcatt 
               
               
                   
               
               
                 tgctgcccaatctgtagctgtaataagagacttaaatatagatatgaat 
               
               
                   
               
               
                 aaagttaatgttaatggtggagcaatagctataggacatccaataggat 
               
               
                   
               
               
                 gctcaggagcaagaatacttactacacttttatatgaaatgaagagaag 
               
               
                   
               
               
                 agatgctaaaactggtcttgctacactttgtataggcggtggaatggga 
               
               
                   
               
               
                 actactttaatagttaagagatagtaagaaggagatatacatatgaaat 
               
               
                   
               
               
                 tagctgtaataggtagtggaactatgggaagtggtattgtacaaacttt 
               
               
                   
               
               
                 tgcaagttgtggacatgatgtatgtttaaagagtagaactcaaggtgct 
               
               
                   
               
               
                 atagataaatgtttagctttattagataaaaatttaactaagttagtta 
               
               
                   
               
               
                 ctaagggaaaaatggatgaagctacaaaagcagaaatattaagtcatgt 
               
               
                   
               
               
                 tagttcaactactaattatgaagatttaaaagatatggatttaataata 
               
               
                   
               
               
                 gaagcatctgtagaagacatgaatataaagaaagatgttttcaagttac 
               
               
                   
               
               
                 tagatgaattatgtaaagaagatactatcttggcaacaaatacttcatc 
               
               
                   
               
               
                 attatctataacagaaatagcttcttctactaagcgcccagataaagtt 
               
               
                   
               
               
                 ataggaatgcatttctttaatccagttcctatgatgaaattagttgaag 
               
               
                   
               
               
                 ttataagtggtcagttaacatcaaaagttacttttgatacagtatttga 
               
               
                   
               
               
                 attatctaagagtatcaataaagtaccagtagatgtatctgaatctcct 
               
               
                   
               
               
                 ggatttgtagtaaatagaatacttatacctatgataaatgaagctgttg 
               
               
                   
               
               
                 gtatatatgcagatggtgttgcaagtaaagaagaaatagatgaagctat 
               
               
                   
               
               
                 gaaattaggagcaaaccatccaatgggaccactagcattaggtgattta 
               
               
                   
               
               
                 atcggattagatgttgttttagctataatgaacgttttatatactgaat 
               
               
                   
               
               
                 ttggagatactaaatatagacctcatccacttttagctaaaatggttag 
               
               
                   
               
               
                 agctaatcaattaggaagaaaaactaagataggattctatgattataat 
               
               
                   
               
               
                 aaataataagaaggagatatacatatgagtacaagtgatgttaaagttt 
               
               
                   
               
               
                 atgagaatgtagctgttgaagtagatggaaatatatgtacagtgaaaat 
               
               
                   
               
               
                 gaatagacctaaagcccttaatgcaataaattcaaagactttagaagaa 
               
               
                   
               
               
                 ctttatgaagtatttgtagatattaataatgatgaaactattgatgttg 
               
               
                   
               
               
                 taatattgacaggggaaggaaaggcatttgtagctggagcagatattgc 
               
               
                   
               
               
                 atacatgaaagatttagatgctgtagctgctaaagattttagtatctta 
               
               
                   
               
               
                 ggagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatag 
               
               
                   
               
               
                 ctgctgtaaacggatttgctttaggtggaggatgtgaacttgcaatggc 
               
               
                   
               
               
                 atgtgatataagaattgcatctgctaaagctaaatttggtcagccagaa 
               
               
                   
               
               
                 gtaactcttggaataactccaggatatggaggaactcaaaggcttacaa 
               
               
                   
               
               
                 gattggttggaatggcaaaagcaaaagaattaatctttacaggtcaagt 
               
               
                   
               
               
                 tataaaagctgatgaagctgaaaaaatagggctagtaaatagagtcgtt 
               
               
                   
               
               
                 gagccagacattttaatagaagaagttgagaaattagctaagataatag 
               
               
                   
               
               
                 ctaaaaatgctcagcttgcagttagatactctaaagaagcaatacaact 
               
               
                   
               
               
                 tggtgctcaaactgatataaatactggaatagatatagaatctaattta 
               
               
                   
               
               
                 tttggtctttgtttttcaactaaagaccaaaaagaaggaatgtcagctt 
               
               
                   
               
               
                 tcgttgaaaagagagaagctaactttataaaagggtaataagaaggaga 
               
               
                   
               
               
                 tatacatatgAGTCAGGCGCTAAAAAATTTACTGACATTGTTAAATCTG 
               
               
                   
               
               
                 GAAAAAATTGAGGAAGGACTCTTTCGCGGCCAGAGTGAAGATTTAGGTT 
               
               
                   
               
               
                 TACGCCAGGTGTTTGGCGGCCAGGTCGTGGGTCAGGCCTTGTATGCTGC 
               
               
                   
               
               
                 AAAAGAGACCGTCCCTGAAGAGCGGCTGGTACATTCGTTTCACAGCTAC 
               
               
                   
               
               
                 TTTCTTCGCCCTGGCGATAGTAAGAAGCCGATTATTTATGATGTCGAAA 
               
               
                   
               
               
                 CGCTGCGTGACGGTAACAGCTTCAGCGCCCGCCGGGTTGCTGCTATTCA 
               
               
                   
               
               
                 AAACGGCAAACCGATTTTTTATATGACTGCCTCTTTCCAGGCACCAGAA 
               
               
                   
               
               
                 GCGGGTTTCGAACATCAAAAAACAATGCCGTCCGCGCCAGCGCCTGATG 
               
               
                   
               
               
                 GCCTCCCTTCGGAAACGCAAATCGCCCAATCGCTGGCGCACCTGCTGCC 
               
               
                   
               
               
                 GCCAGTGCTGAAAGATAAATTCATCTGCGATCGTCCGCTGGAAGTCCGT 
               
               
                   
               
               
                 CCGGTGGAGTTTCATAACCCACTGAAAGGTCACGTCGCAGAACCACATC 
               
               
                   
               
               
                 GTCAGGTGTGGATCCGCGCAAATGGTAGCGTGCCGGATGACCTGCGCGT 
               
               
                   
               
               
                 TCATCAGTATCTGCTCGGTTACGCTTCTGATCTTAACTTCCTGCCGGTA 
               
               
                   
               
               
                 GCTCTACAGCCGCACGGCATCGGTTTTCTCGAACCGGGGATTCAGATTG 
               
               
                   
               
               
                 CCACCATTGACCATTCCATGTGGTTCCATCGCCCGTTTAATTTGAATGA 
               
               
                   
               
               
                 ATGGCTGCTGTATAGCGTGGAGAGCACCTCGGCGTCCAGCGCACGTGGC 
               
               
                   
               
               
                 TTTGTGCGCGGTGAGTTTTATACCCAAGACGGCGTACTGGTTGCCTCGA 
               
               
                   
               
               
                 CCGTTCAGGAAGGGGTGATGCGTAATCACAATtaa 
               
               
                   
               
            
           
         
       
     
     Butyrate, IL-10, IL-22, GLP-2 
     In certain constructs, in addition to the butyrate production pathways described above, the  Escherichia coli  Nissle are further engineered to produce one or more molecules selected from IL-10, IL-2, IL-22, IL-27, SOD, kyurenine, kyurenic acid, and GLP-2 using the methods described above. In some embodiments, the bacteria comprise a gene cassette for producing butyrate as described above, and a gene encoding IL-10 (see, e.g., SEQ ID NO: 134, SEQ ID NO: 193, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 194). In some embodiments, the bacteria comprise a gene cassette for producing butyrate as described above, and a gene encoding IL-2 (see, e.g., SEQ ID NO: 135). In some embodiments, the bacteria comprise a gene cassette for producing butyrate as described above, and a gene encoding IL-22 (see, e.g., SEQ ID NO: 136, SEQ ID NO: 195, SEQ ID NO: 196). In some embodiments, the bacteria comprise a gene cassette for producing butyrate as described above, and a gene encoding IL-27 (see, e.g., SEQ ID NO: 137). In some embodiments, the bacteria comprise a gene cassette for producing butyrate as described above, and a gene encoding SOD (see, e.g., SEQ ID NO: 138). In some embodiments, the bacteria comprise a gene cassette for producing butyrate as described above, and a gene encoding GLP-2 (see, e.g., SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 136189, SEQ ID NO: 190, SEQ ID NO: 192). In some embodiments, the bacteria comprise a gene cassette for producing butyrate as described above, and a gene or gene cassette for producing kyurenine or kyurenic acid. In some embodiments, the bacteria comprise a gene cassette for producing butyrate as described above, and a gene encoding IL-10, IL-22, and GLP-2. In one embodiment, each of the genes or gene cassettes is placed under the control of a FNR regulatory region selected from SEQ ID NO: 141 through SEQ ID NO: 157 (Table 21 and Table 22). In an alternate embodiment, each of the genes or gene cassettes is placed under the control of an RNS-responsive regulatory region, e.g., norB, and the bacteria further comprises a gene encoding a corresponding RNS-responsive transcription factor, e.g., nsrR (see, e.g., Table 27 and elsewhere herein). In yet another embodiment, each of the genes or gene cassettes is placed under the control of an ROS-responsive regulatory region, e.g., oxyS, and the bacteria further comprises a gene encoding a corresponding ROS-responsive transcription factor, e.g., oxyR (see, e.g., Table 28 and Table 29 and elsewhere herein). In certain constructs, one or more of the genes is placed under the control of a tetracycline-inducible or constitutive promoter. 
     Butyrate, Propionate, IL-10, IL-22, IL-2, IL-27 
     In certain constructs, in addition to the butyrate production pathways described above, the  Escherichia coli  Nissle are further engineered to produce propionate, and one or more molecules selected from IL-10, IL-2, IL-22, IL-27, SOD, kyurenine, kyurenic acid, and GLP-2 using the methods described above. In certain constructs, in addition to the butyrate production pathways described above, the  Escherichia coli  Nissle are further engineered to produce propionate, and one or more molecules selected from IL-10, IL-2, and IL-22. In certain constructs, in addition to the butyrate production pathways described above, the  Escherichia coli  Nissle are further engineered to produce propionate, and one or more molecules selected from IL-10, IL-2, and IL-27. In some embodiments, the genetically engineered bacteria further comprise acrylate pathway genes for propionate biosynthesis, pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC. In an alternate embodiment, the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, thrA fbr , thrB, thrC, ilvA fbr , aceE, aceF, and lpd. In another alternate embodiment, the genetically engineered bacteria comprise thrA fbr , thrB, thrC, ilvA fbr , aceE, aceF, lpd, and tesB. 
     The bacteria comprise a gene cassette for producing butyrate as described above, a gene cassette for producing propionate as described above, a gene encoding IL-10 (see, e.g., 49), a gene encoding IL-27 (see, e.g., SEQ ID NO: 137), a gene encoding IL-22 (see, e.g., SEQ ID NO: 136, SEQ ID NO: 195, SEQ ID NO: 196), and a gene encoding IL-2 (see, e.g., SEQ ID NO: 135). In one embodiment, each of the genes or gene cassettes is placed under the control of a FNR regulatory region selected from SEQ ID NOs: 141-157 (Table 21 and 22). In an alternate embodiment, each of the genes or gene cassettes is placed under the control of an RNS-responsive regulatory region, e.g., norB, and the bacteria further comprises a gene encoding a corresponding RNS-responsive transcription factor, e.g., nsrR (see, e.g., Table 23). In yet another embodiment, each of the genes or gene cassettes is placed under the control of an ROS-responsive regulatory region, e.g., oxyS, and the bacteria further comprises a gene encoding a corresponding ROS-responsive transcription factor, e.g., oxyR (see, e.g., Table 24 and elsewhere herein). In certain constructs, one or more of the genes is placed under the control of a tetracycline-inducible or constitutive promoter. 
     Butyrate, Propionate, IL-10, L-22, SOD, GLP-2, Kynurenine 
     In certain constructs, in addition to the butyrate production pathways described above, the  Escherichia coli  Nissle are further engineered to produce one or more molecules selected from IL-10, IL-22, SOD, GLP-2, and kynurenine using the methods described above. In certain constructs, in addition to the butyrate production pathways described above, the  Escherichia coli  Nissle are further engineered to produce propionate, and one or more molecules selected from IL-10, IL-22, SOD, GLP-2, and kynurenine using the methods described above. In certain constructs, in addition to the butyrate production pathways described above, the  Escherichia coli  Nissle are further engineered to produce IL-10, IL-27, IL-22, SOD, GLP-2, and kynurenine using the methods described above. In certain constructs, in addition to the butyrate production pathways described above, the  Escherichia coli  Nissle are further engineered to produce propionate, IL-10, IL-27, IL-22, SOD, GLP-2, and kynurenine using the methods described above. In some embodiments, the genetically engineered bacteria further comprise acrylate pathway genes for propionate biosynthesis, pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC. In an alternate embodiment, the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, thrA fbr , thrB, thrC, ilvA fbr , aceE, aceF, and lpd. In another alternate embodiment, the genetically engineered bacteria comprise thrA fbr , thrB, thrC, ilvA fbr , aceE, aceF, lpd, and tesB. 
     The bacteria comprise a gene cassette for producing butyrate as described above, a gene cassette for producing propionate as described above, a gene encoding IL-10 (see, e.g., SEQ ID NO: 134), a gene encoding IL-22 (see, e.g., SEQ ID NO: 136, SEQ ID NO: 195, SEQ ID NO: 196), a gene encoding SOD (see, e.g., SEQ ID NO: 138), a gene encoding GLP-2 or a GLP-2 analog or GLP-2 polypeptide (see, e.g., SEQ ID NO: 139, SEQ ID NO:140, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO: 192), and a gene or gene cassette for producing kynurenine. In one embodiment, each of the genes or gene cassettes is placed under the control of a FNR regulatory region selected from SEQ ID NO: 141 though SEQ ID NO: 157 (Table 21 and Table 22). In an alternate embodiment, each of the genes or gene cassettes is placed under the control of an RNS-responsive regulatory region, e.g., norB, and the bacteria further comprises a gene encoding a corresponding RNS-responsive transcription factor, e.g., nsrR (see, e.g., Table 23 and elsewhere herein). In yet another embodiment, each of the genes or gene cassettes is placed under the control of an ROS-responsive regulatory region, e.g., oxyS, and the bacteria further comprises a gene encoding a corresponding ROS-responsive transcription factor, e.g., oxyR (see, e.g., Table 24 and Table 25 and elsewhere herein). In certain constructs, one or more of the genes is placed under the control of a tetracycline-inducible or constitutive promoter. 
     Butyrate, Propionate, IL-10, IL-27, IL-22, IL-2, SOD, GLP-2, Kynurenine 
     In certain constructs, in addition to the butyrate production pathways described above, the  Escherichia coli  Nissle are further engineered to produce one or more molecules selected from IL-10, IL-27, IL-22, IL-2, SOD, GLP-2, and kynurenine using the methods described above. In certain constructs, in addition to the butyrate production pathways described above, the  Escherichia coli  Nissle are further engineered to produce propionate and one or more molecules selected from IL-10, IL-27, IL-22, IL-2, SOD, GLP-2, and kynurenine using the methods described above. In certain constructs, in addition to the butyrate production pathways described above, the  Escherichia coli  Nissle are further engineered to produce IL-10, IL-27, IL-22, SOD, GLP-2, and kynurenine using the methods described above. In some embodiments, the genetically engineered bacteria further comprise acrylate pathway genes for propionate biosynthesis, pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC. In an alternate embodiment, the genetically engineered bacteria comprise pyruvate pathway genes for propionate biosynthesis, thrA fbr , thrB, thrC, ilvA fbr , aceE, aceF, and lpd. In another alternate embodiment, the genetically engineered bacteria comprise thrA fbr , thrB, thrC, ilvA fbr , aceE, aceF, lpd, and tesB. 
     The bacteria comprise a gene cassette for producing butyrate as described above, a gene cassette for producing propionate as described above, a gene encoding IL-10 (see, e.g., SEQ ID NO: 134, SEQ ID NO: 193, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 194), a gene encoding IL-27 (see, e.g., SEQ ID NO: 137), a gene encoding IL-22 (see, e.g., SEQ ID NO: 51), a gene encoding IL-2 (see, e.g., SEQ ID NO: 50), a gene encoding SOD (see, e.g., SEQ ID NO: 53), a gene encoding GLP-2 (see, e.g., SEQ ID NO: 54), and a gene or gene cassette for producing kynurenine. In one embodiment, each of the genes or gene cassettes is placed under the control of a FNR regulatory region selected from SEQ ID NO: 141 through SEQ ID NO: 157 (Table 21 and Table 22). In an alternate embodiment, each of the genes or gene cassettes is placed under the control of an RNS-responsive regulatory region, e.g., norB, and the bacteria further comprises a gene encoding a corresponding RNS-responsive transcription factor, e.g., nsrR (see, e.g., Table 23 and Table 24 and elsewhere herein). In yet another embodiment, each of the genes or gene cassettes is placed under the control of an ROS-responsive regulatory region, e.g., oxyS, and the bacteria further comprises a gene encoding a corresponding ROS-responsive transcription factor, e.g., oxyR (see, e.g., Table 24 and Table 25 and elsewhere herein). In certain constructs, one or more of the genes is placed under the control of a tetracycline-inducible or constitutive promoter. 
     In some embodiments, bacterial genes may be disrupted or deleted to produce an auxotrophic strain. These include, but are not limited to, genes required for oligonucleotide synthesis, amino acid synthesis, and cell wall synthesis, as shown in Table 33. 
     Example 3. Transforming  E. coli    
     Each plasmid is transformed into  E. coli  Nissle or  E. coli  DH5a. All tubes, solutions, and cuvettes are pre-chilled to 4° C. An overnight culture of  E. coli  Nissle or  E. coli  DH5a is diluted 1:100 in 5 mL of lysogeny broth (LB) and grown until it reached an OD 600  of 0.4-0.6. The cell culture medium contains a selection marker, e.g., ampicillin, that is suitable for the plasmid. The  E. coli  cells are then centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 1 mL of 4° C. water. The  E. coli  are again centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 0.5 mL of 4° C. water. The  E. coli  are again centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are finally resuspended in 0.1 mL of 4° C. water. The electroporator is set to 2.5 kV. 0.5 μg of one of the above plasmids is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. One mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 37° C. for 1 hr. The cells are spread out on an LB plate containing ampicillin and incubated overnight. 
     In alternate embodiments, the butyrate cassette can be inserted into the Nissle genome through homologous recombination (Genewiz, Cambridge, Mass.). Organization of the constructs and nucleotide sequences are provided herein. Organization of the constructs and nucleotide sequences are shown in  FIG. 2 . To create a vector capable of integrating the synthesized butyrate cassette construct into the chromosome, Gibson assembly was first used to add 1000 bp sequences of DNA homologous to the Nissle lacZ locus into the R6K origin plasmid pKD3. This targets DNA cloned between these homology arms to be integrated into the lacZ locus in the Nissle genome. Gibson assembly was used to clone the fragment between these arms. PCR was used to amplify the region from this plasmid containing the entire sequence of the homology arms, as well as the butyrate cassette between them. This PCR fragment was used to transform electrocompetent Nissle-pKD46, a strain that contains a temperature-sensitive plasmid encoding the lambda red recombinase genes. After transformation, cells were grown out for 2 hours before plating on chloramphenicol at 20 ug/mL at 37 degrees C. Growth at 37 degrees C. also cures the pKD46 plasmid. Transformants containing cassette were chloramphenicol resistant and lac-minus (lac-). 
     Example 4. Production of Butyrate in Recombinant  E. coli  Using Tet-Inducible Promoter 
     Production of butyrate was assessed in  E. coli  Nissle strains containing butyrate cassettes described above in order to determine the effect of oxygen on butyrate production. The tet-inducible cassettes tested include (1) tet-butyrate cassette comprising all eight genes (pLOGIC031); (2) tet-butyrate cassette in which the ter is substituted (pLOGIC046) and (3) tet-butyrate cassette in which tesB is substituted in place of pbt and buk genes. 
     All incubations are performed at 37° C. Cultures of  E. coli  strains DH5a and Nissle transformed with the butyrate cassettes are grown overnight in LB and then diluted 1:200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells were grown with shaking (250 rpm) for 4-6 h and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N 2 , 5% CO 2 , 5% H2). One mL culture aliquots were prepared in 1.5 mL capped tubes and incubated in a stationary incubator to limit culture aeration. One tube is removed at each time point (0, 1, 2, 4, and 20 hours) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment. 
       FIG. 11  depicts bar graphs of butyrate production using the different butyrate-producing circuits shown in  FIG. 2 . 
       FIG. 11A  shows butyrate production in strains pLOGIC031 and pLOGIC046 in the presence and absence of oxygen, in which there is no significant difference in butyrate production. Enhanced butyrate production was shown in Nissle in low copy plasmid expressing pLOGIC046 which contain a deletion of the final two genes (ptb-buk) and their replacement with the endogenous  E. coli  tesB gene (a thioesterase that cleaves off the butyrate portion from butyryl CoA). 
     Example 5. Tet-Driven and RNS Driven In Vitro Butyrate Production in Recombinant  E. coli    
     All incubations were performed at 37 C. Lysogeny broth (LB)-grown overnight cultures of  E. coli  Nissle transformed with pLogic031 or pLogic046 were subcultured 1:100 into 10 mL of M9 minimal medium containing 0.5% glucose and grown shaking (200 rpm) for 2 h, at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100 ng/mL to induce expression the butyrate operon from pLogic031 or pLogic046. After 2 hours of induction, cells were spun down, supernatant was discarded, and the cells were resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant was then analyzed at indicated time points ((0 up to 24 hours, as shown in  FIG. 21 ) to assess levels of butyrate production by LC-MS. As seen in  FIG. 21  butyrate production is greater in the strain comprising the pLogic046 construct than the strain comprising the pLogic03 construct. 
     Production of butyrate was also assessed in  E. coli  Nissle strains containing the butyrate cassettes driven by an RNS promoter described above (pLogic031-nsrR-norB-butyrate operon construct and pLogic046-nsrR-norB-butyrate operon construct) in order to determine the effect of nitrogen on butyrate production. Overnight bacterial cultures were diluted 1:100 into fresh LB and grown for 1.5 hrs to allow entry into early log phase. At this point, long half-life nitric oxide donor (DETA-NO; diethylenetriamine-nitric oxide adduct) was added to cultures at a final concentration of 0.3 mM to induce expression from plasmid. After 2 hours of induction, cells were spun down, supernatant was discarded, and the cells were resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant was then analyzed at indicated time points (0 up to 24 hours, as shown in  FIG. 22 ) to assess levels of butyrate production. As seen in  FIG. 22 , genetically engineered Nissle comprising pLogic031-nsrR-norB-butyrate operon construct) or (pLogic046-nsrR-norB-butyrate operon construct) produced significantly more butyrate as compared to wild-type Nissle. 
     Example 6. In Vitro Production of Butyrate in Recombinant  E. coli  Using an Inducible Tet Promoter Butyrate Circuit 
     NuoB is a protein complex involved in the oxidation of NADH during respiratory growth (form of growth requiring electron transport). Preventing the coupling of NADH oxidation to electron transport allows an increase in the amount of NADH being used to support butyrate production. To test whether Preventing the coupling of NADH oxidation to electron transport would allow increased butyrate production, NuoB mutants having NuoB deletion were obtained. 
     All incubations were performed at 37° C. Lysogeny broth (LB)-grown overnight cultures of  E. coli  strains DH5a and Nissle containing pLogic031 or pLogic046 were subcultured 1:100 into 10 mL of M9 minimal medium containing 0.2% glucose and grown shaking (200 rpm) for 2 h, at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100 ng/mL to induce expression the butyrate operon from pLogic031 or pLogic046. Cultures were incubated either shaking in flasks (+O 2 ) or in the anaerobic chamber (−O 2 ) and samples were removed, and butyrate was quantitated at 2, 4, and 24 hr via LC-MS. See  FIG. 13 , which depicts a graph of butyrate production using different butyrate-producing circuits comprising a nuoB gene deletion.  FIG. 13  shows the BW25113 strain of  E. coli , which is a common cloning strain and the background of the KEIO collection of  E. coli  mutants.  FIG. 13  shows that compared with wild-type Nissle, deletion of NuoB results in greater production of butyrate. 
     Example 7. Production of Butyrate in Recombinant  E. coli    
     In vitro production of butyrate under the control of a tetracycline promoter was compared between (1) Butyrate gene cassette (pLOGIC046-ter-thiA1-hbd-crt2-pbt buk butyrate) and (2) butyrate cassette in which the pbt and buk genes were placed with tesB (pLOGIC046-deltapbt-buk/tesB+-butyrate; SEQ ID NO: 56). 
     Overnight bacterial cultures were diluted 1:100 into fresh LB and grown for 1.5 hrs to allow entry into early log phase. At this point, anhydrous tetracycline (ATC) was added to cultures at a final concentration of 100 ng/mL to induce expression of butyrate genes from plasmid. After 2 hours of induction, cells were spun down, supernatant was discarded, and the cells were resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant was then analyzed at indicated time points to assess levels of butyrate production. As shown in  FIG. 11B , replacement of pbt and buk with tesB leads to greater levels of butyrate production. 
     Example 8. Construction of Vectors for Overproducing Butyrate (FNR Driven) 
     The three butyrate cassettes described in Example 1 (see, e.g., Table 36, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165) are placed under the control of a FNR regulatory region selected from (SEQ ID NO: 141 through SEQ ID NO: 157) (Table 21 and Table 22) In certain constructs, the FNR-responsive promoter is further fused to a strong ribosome binding site sequence. For efficient translation of butyrate genes, each synthetic gene in the operon was separated by a 15 base pair ribosome binding site derived from the T7 promoter/translational start site. In certain embodiments, a ydfZ promoter was used. In other embodiments, a FNRS promoter is used. 
     Example 9. FNR and RNS Driven In Vitro Production of Butyrate in Recombinant  E. coli    
     Production of butyrate is assessed in  E. coli  Nissle strains containing the butyrate cassettes described above driven by an FNR promoter in order to determine the effect of oxygen on butyrate production. All incubations are performed at 37° C. Cultures of  E. coli  strains DH5a and Nissle transformed with the butyrate cassettes are grown overnight in LB and then diluted 1:200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells are grown with shaking (250 rpm) for 4-6 h and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N 2 , 5% CO 2 , 5% H 2 ). One mL culture aliquots are prepared in 1.5 mL capped tubes and incubated in a stationary incubator to limit culture aeration. One tube is removed at each time point (0, 1, 2, 4, and 20 hours) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment. 
     In an alternate embodiment, production of butyrate is assessed in  E. coli  Nissle strains containing the butyrate cassettes described above driven by an RNS promoter in order to determine the effect of nitrogen on butyrate production. Overnight bacterial cultures are diluted 1:100 into fresh LB and grown for 1.5 hrs to allow entry into early log phase. At this point, long half-life nitric oxide donor (DETA-NO; diethylenetriamine-nitric oxide adduct) is added to cultures at a final concentration of 0.3 mM to induce expression from plasmid. After 2 hours of induction, cells are spun down, supernatant is discarded, and the cells are resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant is then analyzed at indicated time points to assess levels of butyrate production. 
     Example 10. Production of Butyrate in Recombinant  E. coli    
     The effect of oxygen and glucose on FNR promoter driven butyrate production was compared between  E. coli  Nissle strains SYN501 (comprises pSC101 PydfZ-ter butyrate plasmid, i.e., (ter-thiA1-hbd-crt2-pbt-buk genes under the control of a ydfZ promoter) SYN-UCD500 (comprises pSC101 PydfZ-bcd butyrate plasmid, i.e, bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, and buk under control of the ydfZ promoter) and SYN-UCD506 (comprises pSC101 nirB-bcd butyrate plasmid, i.e., i.e, bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, and buk under control of the nirB promoter. 
     All incubations were performed at 37° C. Cultures of  E. coli  Nissle strains transformed with the butyrate cassettes were grown overnight in LB and then diluted 1:200 into 4 mL of M9 minimal medium containing 0.5% glucose. The cells were grown with shaking (250 rpm) for 4-6 h and incubated anaerobically in a Coy anaerobic chamber (supplying 90% N 2 , 5% CO 2 , 5% H 2 ) for 4 hours. Cells were washed and resuspended in minimal media w/0.5% glucose and incubated microaerobically to monitor butyrate production over time. One aliquot was removed at each time point (2, 8, and 24 hours) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment. As seen in  FIG. 14B , SYN-501 led to significant butyrate production under anaerobic conditions. 
     In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 175, 176, 177, or 178, or a functional fragment thereof. 
     
       
         
           
               
             
               
                 TABLE 47 
               
             
            
               
                   
               
               
                 ydfZ-butyrate cassettes 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 Description 
                 Sequence 
                 NO 
               
               
                   
               
               
                 YdfZ 
                 CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTT 
                 SEQ ID 
               
               
                 promoter 
                 CCCCCGACTTATGGCTCATGCATGCATCAAAAAAG 
                 NO: 175 
               
               
                   
                 ATGTGAGCTTGATCAAAAACAAAAAATATTTCACTC 
                   
               
               
                   
                 GACAGGAGTATTTATATTGCGCCCGGATCCCTCTAG 
                   
               
               
                   
                 AAATAATTTTGTTTAACTTTAAGAAGGAGATATACA 
                   
               
               
                   
                 T 
                   
               
               
                   
               
               
                 YdfZ-bcd2- 
                 CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTT 
                 SEQ ID 
               
               
                 etfB3-etfA3- 
                 CCCCCGACTTATGGCTCATGCATGCATCAAAAAAG 
                 NO: 176 
               
               
                 thiA1-hb- 
                 ATGTGAGCTTGATCAAAAACAAAAAATATTTCACTC 
                   
               
               
                 crt2-pbt-buk 
                 GACAGGAGTATTTATATTGCGCCCGGATCCCTCTAG 
                   
               
               
                 butyrate 
                 AAATAATTTTGTTTAACTTTAAGAAGGAGATATACA 
                   
               
               
                 cassette 
                 T 
                   
               
               
                   
                 atggatttaaattctaaaaaatatcagatgcttaaagagctatatgtaagcttcgctgaaa 
                   
               
               
                   
                 atgaagttaaacctttagcaacagaacttgatgaagaagaaagatttccttatgaaaca 
                   
               
               
                   
                 gtggaaaaaatggcaaaagcaggaatgatgggtataccatatccaaaagaatatggt 
                   
               
               
                   
                 ggagaaggtggagacactgtaggatatataatggcagttgaagaattgtctagagttt 
                   
               
               
                   
                 gtggtactacaggagttatattatcagctcatacatctcttggctcatggcctatatatca 
                   
               
               
                   
                 atatggtaatgaagaacaaaaacaaaaattcttaagaccactagcaagtggagaaaa 
                   
               
               
                   
                 attaggagcatttggtcttactgagcctaatgctggtacagatgcgtctggccaacaaa 
                   
               
               
                   
                 caactgctgttttagacggggatgaatacatacttaatggctcaaaaatatttataacaa 
                   
               
               
                   
                 acgcaatagctggtgacatatatgtagtaatggcaatgactgataaatctaaggggaa 
                   
               
               
                   
                 caaaggaatatcagcatttatagttgaaaaaggaactcctgggtttagctttggagttaa 
                   
               
               
                   
                 agaaaagaaaatgggtataagaggttcagctacgagtgaattaatatttgaggattgca 
                   
               
               
                   
                 gaatacctaaagaaaatttacttggaaaagaaggtcaaggatttaagatagcaatgtct 
                   
               
               
                   
                 actcttgatggtggtagaattggtatagctgcacaagctttaggtttagcacaaggtgct 
                   
               
               
                   
                 cttgatgaaactgttaaatatgtaaaagaaagagtacaatttggtagaccattatcaaaa 
                   
               
               
                   
                 ttccaaaatacacaattccaattagctgatatggaagttaaggtacaagcggctagaca 
                   
               
               
                   
                 ccttgtatatcaagcagctataaataaagacttaggaaaaccttatggagtagaagcag 
                   
               
               
                   
                 caatggcaaaattatttgcagctgaaacagctatggaagttactacaaaagctgtacaa 
                   
               
               
                   
                 cttcatggaggatatggatacactcgtgactatccagtagaaagaatgatgagagatg 
                   
               
               
                   
                 ctaagataactgaaatatatgaaggaactagtgaagttcaaagaatggttatttcagga 
                   
               
               
                   
                 aaactattaaaatagtaagaaggagatatacatatggaggaaggatttatgaatatagt 
                   
               
               
                   
                 cgtttgtataaaacaagttccagatacaacagaagttaaactagatcctaatacaggta 
                   
               
               
                   
                 ctttaattagagatggagtaccaagtataataaaccctgatgataaagcaggatagaa 
                   
               
               
                   
                 gaagctataaaattaaaagaagaaatgggtgctcatgtaactgttataacaatgggacc 
                   
               
               
                   
                 tcctcaagcagatatggctttaaaagaagctttagcaatgggtgcagatagaggtatat 
                   
               
               
                   
                 tattaacagatagagcatttgcgggtgctgatacttgggcaacttcatcagcattagca 
                   
               
               
                   
                 ggagcattaaaaaatatagattttgatattataatagctggaagacaggcgatagatgg 
                   
               
               
                   
                 agatactgcacaagttggacctcaaatagctgaacatttaaatcttccatcaataacata 
                   
               
               
                   
                 tgctgaagaaataaaaactgaaggtgaatatgtattagtaaaaagacaatttgaagatt 
                   
               
               
                   
                 gttgccatgacttaaaagttaaaatgccatgccttataacaactcttaaagatatgaaca 
                   
               
               
                   
                 caccaagatacatgaaagttggaagaatatatgatgctttcgaaaatgatgtagtagaa 
                   
               
               
                   
                 acatggactgtaaaagatatagaagttgacccttctaatttaggtcttaaaggttctccaa 
                   
               
               
                   
                 ctagtgtatttaaatcatttacaaaatcagttaaaccagctggtacaatatacaatgaaga 
                   
               
               
                   
                 tgcgaaaacatcagctggaattatcatagataaattaaaagagaagtatatcatataata 
                   
               
               
                   
                 agaaggagatatacatatgggtaacgttttagtagtaatagaacaaagagaaaatgta 
                   
               
               
                   
                 attcaaactgtttctttagaattactaggaaaggctacagaaatagcaaaagattatgat 
                   
               
               
                   
                 acaaaagtttctgcattacttttaggtagtaaggtagaaggtttaatagatacattagcac 
                   
               
               
                   
                 actatggtgcagatgaggtaatagtagtagatgatgaagctttagcagtgtatacaact 
                   
               
               
                   
                 gaaccatatacaaaagcagcttatgaagcaataaaagcagctgaccctatagttgtatt 
                   
               
               
                   
                 atttggtgcaacttcaataggtagagatttagcgcctagagtactgctagaatacatac 
                   
               
               
                   
                 aggtcttactgctgactgtacaggtcttgcagtagctgaagatacaaaattattattaatg 
                   
               
               
                   
                 acaagacctgcctttggtggaaatataatggcaacaatagtttgtaaagatttcagacct 
                   
               
               
                   
                 caaatgtctacagttagaccaggggttatgaagaaaaatgaacctgatgaaactaaag 
                   
               
               
                   
                 aagctgtaattaaccgtttcaaggtagaatttaatgatgctgataaattagttcaagttgta 
                   
               
               
                   
                 caagtaataaaagaagctaaaaaacaagttaaaatagaagatgctaagatattagtttc 
                   
               
               
                   
                 tgctggacgtggaatgggtggaaaagaaaacttagacatactttatgaattagctgaaa 
                   
               
               
                   
                 ttataggtggagaagtttctggttctcgtgccactatagatgcaggttggttagataaag 
                   
               
               
                   
                 caagacaagttggtcaaactggtaaaactgtaagaccagacattatatagcatgtggt 
                   
               
               
                   
                 atatctggagcaatacaacatatagctggtatggaagatgctgagtttatagttgctata 
                   
               
               
                   
                 aataaaaatccagaagctccaatatttaaatatgctgatgttggtatagttggagatgttc 
                   
               
               
                   
                 ataaagtgcttccagaacttatcagtcagttaagtgttgcaaaagaaaaaggtgaagttt 
                   
               
               
                   
                 tagctaactaataagaaggagatatacatatgagagaagtagtaattgccagtgcagc 
                   
               
               
                   
                 tagaacagcagtaggaagttttggaggagcatttaaatcagtttcagcggtagagttag 
                   
               
               
                   
                 gggtaacagcagctaaagaagctataaaaagagctaacataactccagatatgatag 
                   
               
               
                   
                 atgaatctcttttagggggagtacttacagcaggtcttggacaaaatatagcaagacaa 
                   
               
               
                   
                 atagcattaggagcaggaataccagtagaaaaaccagctatgactataaatatagtttg 
                   
               
               
                   
                 tggttctggattaagatctgtttcaatggcatctcaacttatagcattaggtgatgctgata 
                   
               
               
                   
                 taatgttagttggtggagctgaaaacatgagtatgtctccttatttagtaccaagtgcga 
                   
               
               
                   
                 gatatggtgcaagaatgggtgatgctgcttttgttgattcaatgataaaagatggattatc 
                   
               
               
                   
                 agacatatttaataactatcacatgggtattactgctgaaaacatagcagagcaatgga 
                   
               
               
                   
                 atataactagagaagaacaagatgaattagctcttgcaagtcaaaataaagctgaaaa 
                   
               
               
                   
                 agctcaagctgaaggaaaatttgatgaagaaatagttcctgttgttataaaaggaagaa 
                   
               
               
                   
                 aaggtgacactgtagtagataaagatgaatatattaagcctggcactacaatggagaa 
                   
               
               
                   
                 acttgctaagttaagacctgcatttaaaaaagatggaacagttactgctggtaatgcatc 
                   
               
               
                   
                 aggaataaatgatggtgctgctatgttagtagtaatggctaaagaaaaagctgaagaa 
                   
               
               
                   
                 ctaggaatagagcctcttgcaactatagtttcttatggaacagctggtgttgaccctaaa 
                   
               
               
                   
                 ataatgggatatggaccagttccagcaactaaaaaagctttagaagctgctaatatgac 
                   
               
               
                   
                 tattgaagatatagatttagttgaagctaatgaggcatttgctgcccaatctgtagctgta 
                   
               
               
                   
                 ataagagacttaaatatagatatgaataaagttaatgttaatggtggagcaatagctata 
                   
               
               
                   
                 ggacatccaataggatgctcaggagcaagaatacttactacacttttatatgaaatgaa 
                   
               
               
                   
                 gagaagagatgctaaaactggtcttgctacactttgtataggcggtggaatgggaact 
                   
               
               
                   
                 actttaatagttaagagatagtaagaaggagatatacatatgaaattagctgtaataggt 
                   
               
               
                   
                 agtggaactatgggaagtggtattgtacaaacttttgcaagttgtggacatgatgtatgtt 
                   
               
               
                   
                 taaagagtagaactcaaggtgctatagataaatgtttagctttattagataaaaatttaact 
                   
               
               
                   
                 aagttagttactaagggaaaaatggatgaagctacaaaagcagaaatattaagtcatgt 
                   
               
               
                   
                 tagttcaactactaattatgaagatttaaaagatatggatttaataatagaagcatctgtag 
                   
               
               
                   
                 aagacatgaatataaagaaagatgttttcaagttactagatgaattatgtaaagaagata 
                   
               
               
                   
                 ctatcttggcaacaaatacttcatcattatctataacagaaatagcttcttctactaagcgc 
                   
               
               
                   
                 ccagataaagttataggaatgcatttctttaatccagttcctatgatgaaattagttgaagt 
                   
               
               
                   
                 tataagtggtcagttaacatcaaaagttacttttgatacagtatttgaattatctaagagtat 
                   
               
               
                   
                 caataaagtaccagtagatgtatctgaatctcctggatttgtagtaaatagaatacttata 
                   
               
               
                   
                 cctatgataaatgaagctgttggtatatatgcagatggtgttgcaagtaaagaagaaat 
                   
               
               
                   
                 agatgaagctatgaaattaggagcaaaccatccaatgggaccactagcattaggtgat 
                   
               
               
                   
                 ttaatcggattagatgttgttttagctataatgaacgttttatatactgaatttggagatacta 
                   
               
               
                   
                 aatatagacctcatccacttttagctaaaatggttagagctaatcaattaggaagaaaaa 
                   
               
               
                   
                 ctaagataggattctatgattataataaataataagaaggagatatacatatgagtacaa 
                   
               
               
                   
                 gtgatgttaaagtttatgagaatgtagctgttgaagtagatggaaatatatgtacagtga 
                   
               
               
                   
                 aaatgaatagacctaaagcccttaatgcaataaattcaaagactttagaagaactttatg 
                   
               
               
                   
                 aagtatttgtagatattaataatgatgaaactattgatgttgtaatattgacaggggaagg 
                   
               
               
                   
                 aaaggcatttgtagctggagcagatattgcatacatgaaagatttagatgctgtagctg 
                   
               
               
                   
                 ctaaagattttagtatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaaa 
                   
               
               
                   
                 gtagtgatagctgctgtaaacggatttgctttaggtggaggatgtgaacttgcaatggc 
                   
               
               
                   
                 atgtgatataagaattgcatctgctaaagctaaatttggtcagccagaagtaactcttgg 
                   
               
               
                   
                 aataactccaggatatggaggaactcaaaggcttacaagattggttggaatggcaaaa 
                   
               
               
                   
                 gcaaaagaattaatctttacaggtcaagttataaaagctgatgaagctgaaaaaatagg 
                   
               
               
                   
                 gctagtaaatagagtcgttgagccagacattttaatagaagaagttgagaaattagcta 
                   
               
               
                   
                 agataatagctaaaaatgctcagcttgcagttagatactctaaagaagcaatacaactt 
                   
               
               
                   
                 ggtgctcaaactgatataaatactggaatagatatagaatctaatttatttggtctttgttttt 
                   
               
               
                   
                 caactaaagaccaaaaagaaggaatgtcagctttcgttgaaaagagagaagctaactt 
                   
               
               
                   
                 tataaaagggtaataagaaggagatatacatatgagaagttttgaagaagtaattaagtt 
                   
               
               
                   
                 tgcaaaagaaagaggacctaaaactatatcagtagcatgttgccaagataaagaagtt 
                   
               
               
                   
                 ttaatggcagttgaaatggctagaaaagaaaaaatagcaaatgccattttagtaggag 
                   
               
               
                   
                 atatagaaaagactaaagaaattgcaaaaagcatagacatggatatcgaaaattatga 
                   
               
               
                   
                 actgatagatataaaagatttagcagaagcatctctaaaatctgttgaattagtttcacaa 
                   
               
               
                   
                 ggaaaagccgacatggtaatgaaaggcttagtagacacatcaataatactaaaagca 
                   
               
               
                   
                 gttttaaataaagaagtaggtcttagaactggaaatgtattaagtcacgtagcagtatttg 
                   
               
               
                   
                 atgtagagggatatgatagattatttttcgtaactgacgcagctatgaacttagctcctga 
                   
               
               
                   
                 tacaaatactaaaaagcaaatcatagaaaatgcttgcacagtagcacattcattagatat 
                   
               
               
                   
                 aagtgaaccaaaagttgctgcaatatgcgcaaaagaaaaagtaaatccaaaaatgaa 
                   
               
               
                   
                 agatacagttgaagctaaagaactagaagaaatgtatgaaagaggagaaatcaaag 
                   
               
               
                   
                 gttgtatggttggtgggccttttgcaattgataatgcagtatctttagaagcagctaaaca 
                   
               
               
                   
                 taaaggtataaatcatcctgtagcaggacgagctgatatattattagccccagatattga 
                   
               
               
                   
                 aggtggtaacatattatataaagctttggtattcttctcaaaatcaaaaaatgcaggagtt 
                   
               
               
                   
                 atagttggggctaaagcaccaataatattaacttctagagcagacagtgaagaaacta 
                   
               
               
                   
                 aactaaactcaatagattaggtgttttaatggcagcaaaggcataataagaaggagat 
                   
               
               
                   
                 atacatatgagcaaaatatttaaaatcttaacaataaatcctggttcgacatcaactaaaa 
                   
               
               
                   
                 tagctgtatttgataatgaggatttagtatttgaaaaaactttaagacattcttcagaagaa 
                   
               
               
                   
                 ataggaaaatatgagaaggtgtctgaccaatttgaatttcgtaaacaagtaatagaaga 
                   
               
               
                   
                 agctctaaaagaaggtggagtaaaaacatctgaattagatgctgtagtaggtagagga 
                   
               
               
                   
                 ggacttcttaaacctataaaaggtggtacttattcagtaagtgctgctatgattgaagattt 
                   
               
            
           
           
               
               
               
               
            
               
                   
                 acgcttcaaacctaggtggaataatagcaaaaca 
                   
                   
               
               
                   
                 aataggtgaagaagtaaatgttccttcatacatagtagaccctgttgttgtagatgaatta 
                   
                   
               
               
                   
                 gaagatgttgctagaatttctggtatgcctgaaataagtagagcaagtgtagtacatgct 
                   
                   
               
               
                   
                 ttaaatcaaaaggcaatagcaagaagatatgctagagaaataaacaagaaatatgaa 
                   
                   
               
               
                   
                 gatataaatcttatagttgcacacatgggtggaggagtttctgttggagctcataaaaat 
                   
                   
               
               
                   
                 ggtaaaatagtagatgttgcaaacgcattagatggagaaggacctttctctccagaaa 
                   
                   
               
               
                   
                 gaagtggtggactaccagtaggtgcattagtaaaaatgtgctttagtggaaaatatact 
                   
                   
               
               
                   
                 caagatgaaattaaaaagaaaataaaaggtaatggcggactagttgcatacttaaaca 
                   
                   
               
               
                   
                 ctaatgatgctagagaagttgaagaaagaattgaagctggtgatgaaaaagctaaatt 
                   
                   
               
               
                   
                 agtatatgaagctatggcatatcaaatctctaaagaaataggagctagtgctgcagttct 
                   
                   
               
               
                   
                 taagggagatgtaaaagcaatattattaactggtggaatcgcatattcaaaaatgtttac 
                   
                   
               
               
                   
                 agaaatgattgcagatagagttaaatttatagcagatgtaaaagtttatccaggtgaaga 
                   
                   
               
               
                   
                 tgaaatgattgcattagctcaaggtggacttagagttttaactggtgaagaagaggctc 
                   
                   
               
               
                   
                 aagtttatgataactaataa 
                   
                   
               
               
                   
               
               
                 YdfZ-ter- 
                 CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTT 
                 SEQ ID 
                   
               
               
                 thiA1-hbd- 
                 CCCCCGACTTATGGCTCATGCATGCATCAAAAAAG 
                 NO: 177 
                   
               
               
                 crt2-pbt-buk 
                 ATGTGAGCTTGATCAAAAACAAAAAATATTTCACTC 
                   
                   
               
               
                   
                 GACAGGAGTATTTATATTGCGCCCGGATCCCTCTAG 
                   
                   
               
               
                   
                 AAATAATTTTGTTTAACTTTAAGAAGGAGATATACA 
                   
                   
               
               
                   
                 Tatgatcgtaaaacctatggtacgcaacaatatctgcctgaacgcccatcctcagggc 
                   
                   
               
               
                   
                 tgcaagaagggagtggaagatcagattgaatataccaagaaacgcattaccgcaga 
                   
                   
               
               
                   
                 agtcaaagctggcgcaaaagctccaaaaaacgttctggtgcttggctgctcaaatggt 
                   
                   
               
               
                   
                 tacggcctggcgagccgcattactgctgcgttcggatacggggctgc gaccatcggc 
                   
                   
               
               
                   
                 gtgtcctttgaaaaagcgggttcagaaaccaaatatggtacaccgggatggtacaata 
                   
                   
               
               
                   
                 atttggcatttgatgaagcggcaaaacgcgagggtattatagcgtgacgatcgacgg 
                   
                   
               
               
                   
                 cgatgcgttttcagacgagatcaaggcccaggtaattgaggaagccaaaaaaaaag 
                   
                   
               
               
                   
                 gtatcaaatttgatctgatcgtatacagcttggccagcccagtacgtactgatcctgata 
                   
                   
               
               
                   
                 caggtatcatgcacaaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaac 
                   
                   
               
               
                   
                 agtagatccgtttactggcgagctgaaggaaatctccgcggaaccagcaaatgacga 
                   
                   
               
               
                   
                 ggaagcagccgccactgttaaagttatggggggtgaagattgggaacgttggattaa 
                   
                   
               
               
                   
                 gcagctgtcgaaggaaggcctcttagaagaaggctgtattaccttggcctatagttata 
                   
                   
               
               
                   
                 ttggccctgaagctacccaagctttgtaccgtaaaggcacaatcggcaaggccaaag 
                   
                   
               
               
                   
                 aacacctggaggccacagcacaccgtctcaacaaagagaacccgtcaatccgtgcc 
                   
                   
               
               
                   
                 ttcgtgagcgtgaataaaggcctggtaacccgcgcaagcgccgtaatcccggtaatc 
                   
                   
               
               
                   
                 cctctgtatctcgccagcttgttcaaagtaatgaaagagaagggcaatcatgaaggttg 
                   
                   
               
               
                   
                 tattgaacagatcacgcgtctgtacgccgagcgcctgtaccgtaaagatggtacaatt 
                   
                   
               
               
                   
                 ccagttgatgaggaaaatcgcattcgcattgatgattgggagttagaagaagacgtcc 
                   
                   
               
               
                   
                 agaaagcggtatccgcgttgatggagaaagtcacgggtgaaaacgcagaatctctca 
                   
                   
               
               
                   
                 ctgacttagcggggtaccgccatgatttcttagctagtaacggattgatgtagaaggta 
                   
                   
               
               
                   
                 ttaattatgaagcggaagttgaacgcttcgaccgtatctgataagaaggagatatacat 
                   
                   
               
               
                   
                 atgagagaagtagtaattgccagtgcagctagaacagcagtaggaagttttggagga 
                   
                   
               
               
                   
                 gcatttaaatcagtttcagcggtagagttaggggtaacagcagctaaagaagctataa 
                   
                   
               
               
                   
                 aaagagctaacataactccagatatgatagatgaatctcttttagggggagtacttaca 
                   
                   
               
               
                   
                 gcaggtcttggacaaaatatagcaagacaaatagcattaggagcaggaataccagta 
                   
                   
               
               
                   
                 gaaaaaccagctatgactataaatatagtttgtggttctggattaagatctgtttcaatgg 
                   
                   
               
               
                   
                 catctcaacttatagcattaggtgatgctgatataatgttagttggtggagctgaaaacat 
                   
                   
               
               
                   
                 gagtatgtctccttatttagtaccaagtgcgagatatggtgcaagaatgggtgatgctg 
                   
                   
               
               
                   
                 cttttgttgattcaatgataaaagatggattatcagacatatttaataactatcacatgggt 
                   
                   
               
               
                   
                 attactgctgaaaacatagcagagcaatggaatataactagagaagaacaagatgaat 
                   
                   
               
               
                   
                 tagctcttgcaagtcaaaataaagctgaaaaagctcaagctgaaggaaaatttgatga 
                   
                   
               
               
                   
                 agaaatagttcctgttgttataaaaggaagaaaaggtgacactgtagtagataaagatg 
                   
                   
               
               
                   
                 aatatattaagcctggcactacaatggagaaacttgctaagttaagacctgcatttaaaa 
                   
                   
               
               
                   
                 aagatggaacagttactgctggtaatgcatcaggaataaatgatggtgctgctatgtta 
                   
                   
               
               
                   
                 gtagtaatggctaaagaaaaagctgaagaactaggaatagagcctcttgcaactatag 
                   
                   
               
               
                   
                 tttcttatggaacagaggtgttgaccctaaaataatgggatatggaccagttccagcaa 
                   
                   
               
               
                   
                 ctaaaaaagctttagaagctgctaatatgactattgaagatatagatttagttgaagctaa 
                   
                   
               
               
                   
                 tgaggcatttgctgcccaatctgtagctgtaataagagacttaaatatagatatgaataa 
                   
                   
               
               
                   
                 agttaatgttaatggtggagcaatagctataggacatccaataggatgctcaggagca 
                   
                   
               
               
                   
                 agaatacttactacacttttatatgaaatgaagagaagagatgctaaaactggtcttgct 
                   
                   
               
               
                   
                 acactttgtataggcggtggaatgggaactactttaatagttaagagatagtaagaagg 
                   
                   
               
               
                   
                 agatatacatatgaaattagctgtaataggtagtggaactatgggaagtggtattgtaca 
                   
                   
               
               
                   
                 aacttttgcaagttgtggacatgatgtatgtttaaagagtagaactcaaggtgctatagat 
                   
                   
               
               
                   
                 aaatgtttagctttattagataaaaatttaactaagttagttactaagggaaaaatggatg 
                   
                   
               
               
                   
                 aagctacaaaagcagaaatattaagtcatgttagttcaactactaattatgaagatttaaa 
                   
                   
               
               
                   
                 agatatggatttaataatagaagcatctgtagaagacatgaatataaagaaagatgtttt 
                   
                   
               
               
                   
                 caagttactagatgaattatgtaaagaagatactatcttggcaacaaatacttcatcatta 
                   
                   
               
               
                   
                 tctataacagaaatagcttcttctactaagcgcccagataaagttataggaatgcatttct 
                   
                   
               
               
                   
                 ttaatccagttcctatgatgaaattagttgaagttataagtggtcagttaacatcaaaagtt 
                   
                   
               
               
                   
                 acttttgatacagtatttgaattatctaagagtatcaataaagtaccagtagatgtatctga 
                   
                   
               
               
                   
                 atctcaggatttgtagtaaatagaatacttatacctatgataaatgaagctgttggtatat 
                   
                   
               
               
                   
                 atgcagatggtgttgcaagtaaagaagaaatagatgaagctatgaaattaggagcaa 
                   
                   
               
               
                   
                 accatccaatgggaccactagcattaggtgatttaatcggattagatgttgttttagctat 
                   
                   
               
               
                   
                 aatgaacgttttatatactgaatttggagatactaaatatagacctcatccacttttagcta 
                   
                   
               
               
                   
                 aaatggttagagctaatcaattaggaagaaaaactaagataggattctatgattataata 
                   
                   
               
               
                   
                 aataataagaaggagatatacatatgagtacaagtgatgttaaagtttatgagaatgtag 
                   
                   
               
               
                   
                 ctgttgaagtagatggaaatatatgtacagtgaaaatgaatagacctaaagcccttaat 
                   
                   
               
               
                   
                 gcaataaattcaaagactttagaagaactttatgaagtatttgtagatattaataatgatga 
                   
                   
               
               
                   
                 aactattgatgttgtaatattgacaggggaaggaaaggcatttgtagctggagcagata 
                   
                   
               
               
                   
                 ttgcatacatgaaagatttagatgctgtagctgctaaagattttagtatcttaggagcaaa 
                   
                   
               
               
                   
                 agcttaggagaaatagaaaatagtaaaaaagtagtgatagctgctgtaaacggatttg 
                   
                   
               
               
                   
                 ctttaggtggaggatgtgaacttgcaatggcatgtgatataagaattgcatctgctaaag 
                   
                   
               
               
                   
                 ctaaatttggtcagccagaagtaactcttggaataactccaggatatggaggaactcaa 
                   
                   
               
               
                   
                 aggcttacaagattggttggaatggcaaaagcaaaagaattaatctttacaggtcaagt 
                   
                   
               
               
                   
                 tataaaagctgatgaagctgaaaaaatagggctagtaaatagagtcgttgagccagac 
                   
                   
               
               
                   
                 attttaatagaagaagttgagaaattagctaagataatagctaaaaatgctcagcttgca 
                   
                   
               
               
                   
                 gttagatactctaaagaagcaatacaacttggtgctcaaactgatataaatactggaata 
                   
                   
               
               
                   
                 gatatagaatctaatttatttggtctttgtttttcaactaaagaccaaaaagaaggaatgtc 
                   
                   
               
               
                   
                 agctttcgttgaaaagagagaagctaactttataaaagggtaataagaaggagatata 
                   
                   
               
               
                   
                 catatgagaagttttgaagaagtaattaagtttgcaaaagaaagaggacctaaaactat 
                   
                   
               
               
                   
                 atcagtagcatgttgccaagataaagaagttttaatggcagttgaaatggctagaaaag 
                   
                   
               
               
                   
                 aaaaaatagcaaatgccattttagtaggagatatagaaaagactaaagaaattgcaaa 
                   
                   
               
               
                   
                 aagcatagacatggatatcgaaaattatgaactgatagatataaaagatttagcagaag 
                   
                   
               
               
                   
                 catctctaaaatctgttgaattagtttcacaaggaaaagccgacatggtaatgaaaggc 
                   
                   
               
               
                   
                 ttagtagacacatcaataatactaaaagcagttttaaataaagaagtaggtcttagaact 
                   
                   
               
               
                   
                 ggaaatgtattaagtcacgtagcagtatttgatgtagagggatatgatagattatttttcgt 
                   
                   
               
               
                   
                 aactgacgcagctatgaacttagctcctgatacaaatactaaaaagcaaatcatagaa 
                   
                   
               
               
                   
                 aatgcttgcacagtagcacattcattagatataagtgaaccaaaagttgctgcaatatgc 
                   
                   
               
               
                   
                 gcaaaagaaaaagtaaatccaaaaatgaaagatacagttgaagctaaagaactagaa 
                   
                   
               
               
                   
                 gaaatgtatgaaagaggagaaatcaaaggttgtatggttggtgggccttttgcaattga 
                   
                   
               
               
                   
                 taatgcagtatctttagaagcagctaaacataaaggtataaatcatcctgtagcaggac 
                   
                   
               
               
                   
                 gagctgatatattattagccccagatattgaaggtggtaacatattatataaagctttggt 
                   
                   
               
               
                   
                 attcttctcaaaatcaaaaaatgcaggagttatagaggggctaaagcaccaataatatt 
                   
                   
               
               
                   
                 aacttctagagcagacagtgaagaaactaaactaaactcaatagctttaggtgttttaat 
                   
                   
               
               
                   
                 ggcagcaaaggcataataagaaggagatatacatatgagcaaaatatttaaaatctta 
                   
                   
               
               
                   
                 acaataaatcctggttcgacatcaactaaaatagctgtatttgataatgaggatttagtatt 
                   
                   
               
               
                   
                 tgaaaaaactttaagacattatcagaagaaataggaaaatatgagaaggtgtctgacc 
                   
                   
               
               
                   
                 aatttgaatttcgtaaacaagtaatagaagaagctctaaaagaaggtggagtaaaaac 
                   
                   
               
               
                   
                 atctgaattagatgctgtagtaggtagaggaggacttcttaaacctataaaaggtggta 
                   
                   
               
               
                   
                 cttattcagtaagtgctgctatgattgaagatttaaaagtgggagttttaggagaacacg 
                   
                   
               
               
                   
                 cttcaaacctaggtggaataatagcaaaacaaataggtgaagaagtaaatgttccttca 
                   
                   
               
               
                   
                 tacatagtagaccctgttgttgtagatgaattagaagatgttgctagaatttctggtatgc 
                   
                   
               
               
                   
                 ctgaaataagtagagcaagtgtagtacatgctttaaatcaaaaggcaatagcaagaag 
                   
                   
               
               
                   
                 atatgctagagaaataaacaagaaatatgaagatataaatcttatagttgcacacatgg 
                   
                   
               
               
                   
                 gtggaggagtttctgttggagctcataaaaatggtaaaatagtagatgttgcaaacgca 
                   
                   
               
               
                   
                 ttagatggagaaggacctttctctccagaaagaagtggtggactaccagtaggtgcat 
                   
                   
               
               
                   
                 tagtaaaaatgtgctttagtggaaaatatactcaagatgaaattaaaaagaaaataaaa 
                   
                   
               
               
                   
                 ggtaatggcggactagttgcatacttaaacactaatgatgctagagaagttgaagaaa 
                   
                   
               
               
                   
                 gaattgaagctggtgatgaaaaagctaaattagtatatgaagctatggcatatcaaatct 
                   
                   
               
               
                   
                 ctaaagaaataggagctagtgctgcagttcttaagggagatgtaaaagcaatattatta 
                   
                   
               
               
                   
                 actggtggaatcgcatattcaaaaatgtttacagaaatgattgcagatagagttaaattta 
                   
                   
               
               
                   
                 tagcagatgtaaaagtttatccaggtgaagatgaaatgattgcattagctcaaggtgga 
                   
                   
               
               
                   
                 cttagagttttaactggtgaagaagaggctcaagtttatgataactaataa 
                   
                   
               
               
                   
               
               
                 Ydfz- ter- 
                 CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTT 
                 SEQ ID 
                   
               
               
                 thiA1-hbd- 
                 CCCCCGACTTATGGCTCATGCATGCATCAAAAAAG 
                 NO: 178 
                   
               
               
                 crt2-tesb 
                 ATGTGAGCTTGATCAAAAACAAAAAATATTTCACTC 
                   
                   
               
               
                 butyrate 
                 GACAGGAGTATTTATATTGCGCCCGGATCCCTCTAG 
                   
                   
               
               
                 cassette 
                 AAATAATTTTGTTTAACTTTAAGAAGGAGATATACA 
                   
                   
               
               
                   
                 T 
                   
                   
               
               
                   
                 atgatcgtaaaacctatggtacgcaacaatatctgcctgaacgcccatcctcagggct 
                   
                   
               
               
                   
                 gcaagaagggagtggaagatcagattgaatataccaagaaacgcattaccgcagaa 
                   
                   
               
               
                   
                 gtcaaagctggcgcaaaagctccaaaaaacgttctggtgcttggctgctcaaatggtt 
                   
                   
               
               
                   
                 acggcctggcgagccgcattactgctgcgttcggatacggggctgcgaccatcggc 
                   
                   
               
               
                   
                 gtgtcctttgaaaaagcgggttcagaaaccaaatatggtacaccgggatggtacaata 
                   
                   
               
               
                   
                 atttggcatttgatgaagcggcaaaacgcgagggtctttatagcgtgacgatcgacgg 
                   
                   
               
               
                   
                 cgatgcgttttcagacgagatcaaggcccaggtaattgaggaagccaaaaaaaaag 
                   
                   
               
               
                   
                 gtatcaaatttgatctgatcgtatacagcttggccagcccagtacgtactgatcctgata 
                   
                   
               
               
                   
                 caggtatcatgcacaaaagcgttttgaaacccatttggaaaaacgttcacaggcaaaac 
                   
                   
               
               
                   
                 agtagatccgtttactggcgagctgaaggaaatctccgcggaaccagcaaatgacga 
                   
                   
               
               
                   
                 ggaagcagccgccactgttaaagttatggggggtgaagattgggaacgttggattaa 
                   
                   
               
               
                   
                 gcagctgtcgaaggaaggcctcttagaagaaggctgtattaccttggcctatagttata 
                   
                   
               
               
                   
                 ttggccctgaagctacccaagctttgtaccgtaaaggcacaatcggcaaggccaaag 
                   
                   
               
               
                   
                 aacacctggaggccacagcacaccgtctcaacaaagagaacccgtcaatccgtgcc 
                   
                   
               
               
                   
                 ttcgtgagcgtgaataaaggcctggtaacccgcgcaagcgccgtaatcccggtaatc 
                   
                   
               
               
                   
                 cctctgtatctcgccagcttgttcaaagtaatgaaagagaagggcaatcatgaaggttg 
                   
                   
               
               
                   
                 tattgaacagatcacgcgtctgtacgccgagcgcctgtaccgtaaagatggtacaatt 
                   
                   
               
               
                   
                 ccagttgatgaggaaaatcgcattcgcattgatgattgggagttagaagaagacgtcc 
                   
                   
               
               
                   
                 agaaagcggtatccgcgttgatggagaaagtcacgggtgaaaacgcagaatctctca 
                   
                   
               
               
                   
                 ctgacctagcggggtaccgccatgatttcttagctagtaacggctttgatgtagaaggta 
                   
                   
               
               
                   
                 ttaattatgaagcggaagttgaacgcttcgaccgtatctgataagaaggagatatacat 
                   
                   
               
               
                   
                 atgagagaagtagtaattgccagtgcagctagaacagcagtaggaagttttggagga 
                   
                   
               
               
                   
                 gcatttaaatcagtttcagcggtagagttaggggtaacagcagctaaagaagctataa 
                   
                   
               
               
                   
                 aaagagctaacataactccagatatgatagatgaatctcttttagggggagtacttaca 
                   
                   
               
               
                   
                 gcaggtcttggacaaaatatagcaagacaaatagcattaggagcaggaataccagta 
                   
                   
               
               
                   
                 gaaaaaccagctatgactataaatatagtttgtggttctggattaagatctgtttcaatgg 
                   
                   
               
               
                   
                 catctcaacttatagcattaggtgatgctgatataatgttagttggtggagctgaaaacat 
                   
                   
               
               
                   
                 gagtatgtctccttatttagtaccaagtgcgagatatggtgcaagaatgggtgatgctg 
                   
                   
               
               
                   
                 cttttgttgattcaatgataaaagatggattatcagacatatttaataactatcacatgggt 
                   
                   
               
               
                   
                 attactgctgaaaacatagcagagcaatggaatataactagagaagaacaagatgaat 
                   
                   
               
               
                   
                 tagctcttgcaagtcaaaataaagctgaaaaagctcaagctgaaggaaaatttgatga 
                   
                   
               
               
                   
                 agaaatagttcctgttgttataaaaggaagaaaaggtgacactgtagtagataaagatg 
                   
                   
               
               
                   
                 aatatattaagcctggcactacaatggagaaacttgctaagttaagacctgcatttaaaa 
                   
                   
               
               
                   
                 aagatggaacagttactgctggtaatgcatcaggaataaatgatggtgctgctatgtta 
                   
                   
               
               
                   
                 gtagtaatggctaaagaaaaagctgaagaactaggaatagagcctcttgcaactatag 
                   
                   
               
               
                   
                 tttcttatggaacagctggtgttgaccctaaaataatgggatatggaccagttccagcaa 
                   
                   
               
               
                   
                 ctaaaaaagctttagaagctgctaatatgactattgaagatatagatttagttgaagctaa 
                   
                   
               
               
                   
                 tgaggcatttgctgcccaatctgtagctgtaataagagacttaaatatagatatgaataa 
                   
                   
               
               
                   
                 agttaatgttaatggtggagcaatagctataggacatccaataggatgctcaggagca 
                   
                   
               
               
                   
                 agaatacttactacacttttatatgaaatgaagagaagagatgctaaaactggtcttgct 
                   
                   
               
               
                   
                 acactttgtataggcggtggaatgggaactactttaatagttaagagatagtaagaagg 
                   
                   
               
               
                   
                 agatatacatatgaaattagctgtaataggtagtggaactatgggaagtggtattgtaca 
                   
                   
               
               
                   
                 aacttttgcaagttgtggacatgatgtatgtttaaagagtagaactcaaggtgctatagat 
                   
                   
               
               
                   
                 aaatgtttagctttattagataaaaatttaactaagttagttactaagggaaaaatggatg 
                   
                   
               
               
                   
                 aagctacaaaagcagaaatattaagtcatgttagttcaactactaattatgaagatttaaa 
                   
                   
               
               
                   
                 agatatggatttaataatagaagcatctgtagaagacatgaatataaagaaagatgtttt 
                   
                   
               
               
                   
                 caagttactagatgaattatgtaaagaagatactatcttggcaacaaatacttcatcatta 
                   
                   
               
               
                   
                 tctataacagaaatagcttcttctactaagcgcccagataaagttataggaatgcatttct 
                   
                   
               
               
                   
                 ttaatccagttcctatgatgaaattagttgaagttataagtggtcagttaacatcaaaagtt 
                   
                   
               
               
                   
                 acttttgatacagtatttgaattatctaagagtatcaataaagtaccagtagatgtatctga 
                   
                   
               
               
                   
                 atctcctggatttgtagtaaatagaatacttatacctatgataaatgaagctgttggtatat 
                   
                   
               
               
                   
                 atgcagatggtgttgcaagtaaagaagaaatagatgaagctatgaaattaggagcaa 
                   
                   
               
               
                   
                 accatccaatgggaccactagcattaggtgatttaatcggattagatgttgttttagctat 
                   
                   
               
               
                   
                 aatgaacgttttatatactgaatttggagatactaaatatagacctcatccacttttagcta 
                   
                   
               
               
                   
                 aaatggttagagctaatcaattaggaagaaaaactaagataggattctatgattataata 
                   
                   
               
               
                   
                 aataataagaaggagatatacatatgagtacaagtgatgttaaagtttatgagaatgtag 
                   
                   
               
               
                   
                 ctgttgaagtagatggaaatatatgtacagtgaaaatgaatagacctaaagcccttaat 
                   
                   
               
               
                   
                 gcaataaattcaaagactttagaagaactttatgaagtatttgtagatattaataatgatga 
                   
                   
               
               
                   
                 aactattgatgttgtaatattgacaggggaaggaaaggcatttgtagctggagcagata 
                   
                   
               
               
                   
                 ttgcatacatgaaagatttagatgctgtagctgctaaagattttagtatcttaggagcaaa 
                   
                   
               
               
                   
                 agcttttggagaaatagaaaatagtaaaaaagtagtgatagctgctgtaaacggatttg 
                   
                   
               
               
                   
                 ctttaggtggaggatgtgaacttgcaatggcatgtgatataagaattgcatctgctaaag 
                   
                   
               
               
                   
                 ctaaatttggtcagccagaagtaactcttggaataactccaggatatggaggaactcaa 
                   
                   
               
               
                   
                 aggcttacaagattggttggaatggcaaaagcaaaagaattaatctttacaggtcaagt 
                   
                   
               
               
                   
                 tataaaagctgatgaagctgaaaaaatagggctagtaaatagagtcgttgagccagac 
                   
                   
               
               
                   
                 attttaatagaagaagttgagaaattagctaagataatagctaaaaatgctcagcttgca 
                   
                   
               
               
                   
                 gttagatactctaaagaagcaatacaacttggtgctcaaactgatataaatactggaata 
                   
                   
               
               
                   
                 gatatagaatctaatttatttggtctttgtttttcaactaaagaccaaaaagaaggaatgtc 
                   
                   
               
               
                   
                 agctttcgttgaaaagagagaagctaactttataaaagggtaataagaaggagatata 
                   
                   
               
               
                   
                 catatgAGTCAGGCGCTAAAAAATTTACTGACATTGTT 
                   
                   
               
               
                   
                 AAATCTGGAAAAAATTGAGGAAGGACTCTTTCGCG 
                   
                   
               
               
                   
                 GCCAGAGTGAAGATTTAGGTTTACGCCAGGTGTTTG 
                   
                   
               
               
                   
                 GCGGCCAGGTCGTGGGTCAGGCCTTGTATGCTGCA 
                   
                   
               
               
                   
                 AAAGAGACCGTCCCTGAAGAGCGGCTGGTACATTC 
                   
                   
               
               
                   
                 GTTTCACAGCTACTTTCTTCGCCCTGGCGATAGTAA 
                   
                   
               
               
                   
                 GAAGCCGATTATTTATGATGTCGAAACGCTGCGTGA 
                   
                   
               
               
                   
                 CGGTAACAGCTTCAGCGCCCGCCGGGTTGCTGCTAT 
                   
                   
               
               
                   
                 TCAAAACGGCAAACCGATTTTTTATATGACTGCCTC 
                   
                   
               
               
                   
                 TTTCCAGGCACCAGAAGCGGGTTTCGAACATCAAA 
                   
                   
               
               
                   
                 AAACAATGCCGTCCGCGCCAGCGCCTGATGGCCTC 
                   
                   
               
               
                   
                 CCTTCGGAAACGCAAATCGCCCAATCGCTGGCGCA 
                   
                   
               
               
                   
                 CCTGCTGCCGCCAGTGCTGAAAGATAAATTCATCTG 
                   
                   
               
               
                   
                 CGATCGTCCGCTGGAAGTCCGTCCGGTGGAGTTTCA 
                   
                   
               
               
                   
                 TAACCCACTGAAAGGTCACGTCGCAGAACCACATC 
                   
                   
               
               
                   
                 GTCAGGTGTGGATCCGCGCAAATGGTAGCGTGCCG 
                   
                   
               
               
                   
                 GATGACCTGCGCGTTCATCAGTATCTGCTCGGTTAC 
                   
                   
               
               
                   
                 GCTTCTGATCTTAACTTCCTGCCGGTAGCTCTACAG 
                   
                   
               
               
                   
                 CCGCACGGCATCGGTTTTCTCGAACCGGGGATTCAG 
                   
                   
               
               
                   
                 ATTGCCACCATTGACCATTCCATGTGGTTCCATCGC 
                   
                   
               
               
                   
                 CCGTTTAATTTGAATGAATGGCTGCTGTATAGCGTG 
                   
                   
               
               
                   
                 GAGAGCACCTCGGCGTCCAGCGCACGTGGCTTTGT 
                   
                   
               
               
                   
                 GCGCGGTGAGTTTTATACCCAAGACGGCGTACTGGT 
                   
                   
               
               
                   
                 TGCCTCGACCGTTCAGGAAGGGGTGATGCGTAATC 
                   
                   
               
               
                   
                 ACAATtaa 
               
               
                   
               
            
           
         
       
     
     Example 11. Production of Butyrate in Recombinant  E. coli    
     The effect of oxygen and glucose on butyrate production was assessed in  E. coli  Nissle strains using a butyrate cassette driven by a FNR promoter (ter-thiA1-hbd-crt2-pbt-buk genes under the control of a ydfZ promoter). 
     All incubations were performed at 37° C. Cultures of  E. coli  strains DH5a and Nissle transformed with the butyrate cassettes were grown overnight in LB and then diluted 1:200 into 4 mL of LB containing no glucose or RCM medium containing 0.5% glucose. The cells were grown with shaking (250 rpm) for 4-6 h and incubated aerobically or anaerobically in a Coy anaerobic chamber (supplying 90% N 2 , 5% CO 2 , 5% H2). One mL culture aliquots were prepared in 1.5 mL capped tubes and incubated in a stationary incubator to limit culture aeration. One tube was removed at each time point (0, 1, 2, 4, and 20 hours) and analyzed for butyrate concentration by LC-MS to confirm that butyrate production in these recombinant strains can be achieved in a low-oxygen environment. 
       FIG. 14C  depicts butyrate production in strains comprising an FNR-butyrate cassette (having the ter substitution) in the presence/absence of glucose and oxygen and shows that bacteria need both glucose and anaerobic conditions for butyrate production from the FNR promoter. Cells were grown aerobically or anaerobically in media containing no glucose (LB) or in media containing glucose at 0.5% (RMC). Culture samples were taken at indicated time pints and supernatant fractions were assessed for butyrate concentration using LC-MS. These data show that SYN501 requires glucose for butyrate production and that in the presence of glucose butyrate production can be enhanced under anaerobic conditions when under the control of the anaerobic FNR-regulated ydfZ promoter. 
     Example 12. Optimization of a Low-Dose DSS-Induced Colitis Model for the Detection of Compromised Barrier Function 
     To Determine the optimal DDS concentration to administer to mice to be able to investigate compromised barrier function, as study was conducted in mice using various concentrations of DSS. 
     Briefly, C57BL6 mice (12 weeks, N=25) were treated with 0.25%, 0.5%, 1% and 1.5% DSS and FITC-dextran (4 kD). 
     On day 0 of the study, animals were weighed, and randomized mice into 5 treatment groups (n=5/group) according to weight as follows: Group 1-H2O Control, n=5; Group 2-0.25% DSS n=5; Group 3-0.5% DSS, n=5; Group 4-1% DSS, n=5; Group 5-1.5% DSS, n=5. Fecal pellets were collected and water was changed to DSS-containing water. Animals were again weighed on day one and three. On day two, blood samples were collected for spectrophotometric analysis of FITC. On day four, mice were fasted for 4 h and gavaged all mice with 0.6 mg/g FITC-dextran (4 kD). At 3 h post FITC-dex administration, animals were weighed and bled. Fecal pellets were collected and colon samples were harvested. Blood samples were processed for spectrophotometric analysis of FITC, and serum was prepared from whole blood. 
     Fecal pellets are analyzed for levels of mouse lipocalin2 and calprotectin by ELISA (RnD systems), as seen in  FIG. 14D . CRP levels are also analyzed by ELSA (R&amp;D Systems). Colon tissue is analyzed for increased levels of IL-1a/b, -6, -13, -18, CCL1, CXCL1, TNFα, IFNg EpCAM, MPO and G-CSF by qPCR. Serum was analyzed for FITC-dextran levels by spectrophotometry, and results are shown in  FIG. 15 . As seen in  FIG. 15 , 0.5% DSS is the lowest dose at which an increase in FITC dextran was observed. 
     Example 13. Comparison of Low-Dose DSS Concentrations and Different FITC MW for the Detection of Compromised Barrier Function 
     A study was conducted to determine the optimal DSS concentration (0.75 or 1.5%) and molecular weight FITC-Dextran (4 or 40 kDA) to administer to mice to be able to investigate compromised barrier function. 
     C57BL6 (9 weeks, n=18), were treated with DSS as follows DSS-0.75 and 1.5%; FITC-dextran (4 and 40 kD) and effects on molecular markers of colitis (as assessed by Spectrophotometry and ELISA) assessed, and body weight and overall animal health were monitored. 
     On day 0, mice were weighed and randomized mice into 3 treatment groups (n=6/group) according to weight as follows: Group 1—H2O Control, n=6; Group 2—0.75% DSS, n=6; Group 3—1.5% DSS, n=6. Water was changed to DSS-containing water. 
     Mice were again weighed on days 1-3. ON day 4, mice were fasted for 4 hours, and 3 mice from each group were gavaged with 0.6 mg/g of either 4 kDa or 40 kDa FITC-dextran. Mice 1-3 and 4-6 (as designated by tail marks) from each group were used for 4 kDa and 40 kDa FITC-dex administration respectively. At 3 h post FITC-dex administration, mice were weighed and bled, and fecal pellets were collected. Blood samples were processed for spectrophotometric analysis of FITC, and serum from whole blood was prepared. 
     Analysis of serum for FITC-dextran levels by spectrophotometry is shown in  FIG. 15 . 
     Example 14. Butyrate-Producing Bacterial Strain Reduces Gut Inflammation in a Low-Dose DSS-Induced Mouse Model of IBD 
     At Day 0, 40 C57BL6 mice (8 weeks of age) were weighed and randomized into the following five treatment groups (n=8 per group): H 2 O control (group 1); 0.5% DSS control (group 2); 0.5% DSS+100 mM butyrate (group 3); 0.5% DSS+SYN94 (group 4); and 0.5% DSS+SYN363 (group 5). After randomization, the cage water for group 3 was changed to water supplemented with butyrate (100 mM), and groups 4 and 5 were administered 100 μL of SYN94 and SYN363 by oral gavage, respectively. At Day 1, groups 4 and 5 were gavaged with bacteria in the morning, weighed, and gavaged again in the evening. Groups 4 and 5 were also gavaged once per day for Day 2 and Day 3. 
     At Day 4, groups 4 and 5 were gavaged with bacteria, and then all mice were weighed. Cage water was changed to either H 2 O+0.5% DSS (groups 2, 4, and 5), or H 2 O+0.5% DSS supplemented with 100 mM butyrate (group 3). Mice from groups 4 and 5 were gavaged again in the evening. On Days 5-7, groups 4 and 5 were gavaged with bacteria in the morning, weighed, and gavaged again in the evening. 
     At Day 8, all mice were fasted for 4 hours, and groups 4 and 5 were gavaged with bacteria immediately following the removal of food. All mice were then weighed, and gavaged with a single dose of FITC-dextran tracer (4 kDa, 0.6 mg/g body weight). Fecal pellets were collected; however, if colitis was severe enough to prevent feces collection, feces were harvested after euthanization. All mice were euthanized at exactly 3 hours following FITC-dextran administration. Animals were then cardiac bled and blood samples were processed to obtain serum. Levels of mouse lipocalin 2, calprotectin, and CRP-1 were quantified by ELISA, and serum levels of FITC-dextran were analyzed by spectrophotometry (see also Example 8). 
       FIG. 14D  shows lipocalin 2 (LCN2) levels in all treatment groups, as demonstrated by ELISA, on Day 8 of the study. Since LCN2 is a biomarker of inflammatory disease activity, these data suggest that SYN-501 produces enough butyrate to significantly reduce LCN2 concentrations, as well as gut inflammation, in a low-dose DSS-induced mouse model of IBD. 
     Example 15. Comparison of In Vitro Butyrate Production Efficacy of Chromosomal Insertion and Plasmid-Bearing Engineered Bacterial Strains 
     The in vitro butyrate production efficacy of engineered bacterial strains harboring a chromosomal insertion of a butyrate cassette was compared to a strain bearing a butyrate cassette on a plasmid. SYN1001 and SYN1002 harbor a chromosomal insertion between the agaI/rsmI locus of a butyrate cassette (either ter→tesB or ter→pbt-buk, respectively) driven by an fnr inducible promoter. These strains were compared side by side with the low copy plasmid strain SYN501 (Logic156 (pSC101 PydfZ-ter-&gt;pbt-buk butyrate plasmid) also driven by an fnr inducible promoter. Butyrate levels in the media were measured at 4 and 24 hours post anaerobic induction. 
     Briefly, 3 ml LB was inoculated with bacteria from frozen glycerol stocks. Bacteria were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 dilution into 10 ml LB (containing antibiotics) in a 125 ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5 h, and then transferred to the anaerobic chamber at 37 C for 4 h. Bacteria (2×10 8  CFU) were added to 1 ml M9 media containing 50 mM MOPS with 0.5% glucose in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At indicated times (4 and 24 h), 120 ul cells were removed and pelleted at 14,000 rpm for Imin, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at −80 C until analysis by LC-MS for butyrate concentrations (as described in Example 22). Results are depicted in  FIG. 19A , and show that SYN1001 and SYN1002 give comparable butyrate production to the plasmid strain SYN501. 
     In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 179, 180, 181, or 182, or a functional fragment thereof. 
     
       
         
           
               
             
               
                 TABLE 48 
               
             
            
               
                   
               
               
                 FRNRs Butyrate Cassette Sequences 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 Pfnrs-ter-thiA1-hbd-ctr2- 
                 GGTACCAGTTGTTCTTATTGGTGGTGTTGCTTTATGGTT 
               
               
                 tesB 
                 GCATCGTAGTAAATGGTTGTAACAAAAGCAATTTTTCC 
               
               
                 SEQ ID NO:179, e.g. 
                 GGCTGTCTGTATACAAAAACGCCGCAAAGTTTGAGCGA 
               
               
                 integrated into the 
                 AGTCAATAAACTCTCTACCCATTCAGGGCAATATCTCTC 
               
               
                 chromosome in SYN1001 
                 TTGGATCCAAAGTGAACTCTAGAAATAATTTTGTTTAAC 
               
               
                 Pfnrs: uppercase; butyrate 
                 TTTAAGAAGGAGATATACATatgatcgtaaaacctatggtacgcaacaat 
               
               
                 cassette: lower case 
                 atctgcctgaacgcccatcctcagggctgcaagaagggagtggaagatcagattgaatata 
               
               
                   
                 ccaagaaacgcattaccgcagaagtcaaagctggcgcaaaagctccaaaaaacgttctggt 
               
               
                   
                 gcttggctgctcaaatggttacggcctggcgagccgcattactgctgcgttcggatacgggg 
               
               
                   
                 ctgcgaccatcggcgtgtcctttgaaaaagcgggttcagaaaccaaatatggtacaccggg 
               
               
                   
                 atggtacaataatttggcatttgatgaagcggcaaaacgcgagggtctttatagcgtgacgat 
               
               
                   
                 cgacggcgatgcgttttcagacgagatcaaggcccaggtaattgaggaagccaaaaaaaa 
               
               
                   
                 aggtatcaaatttgatctgatcgtatacagcttggccagcccagtacgtactgatcctgataca 
               
               
                   
                 ggtatcatgcacaaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaacagtagat 
               
               
                   
                 ccgtttactggcgagctgaaggaaatctccgcggaaccagcaaatgacgaggaagcagcc 
               
               
                   
                 gccactgttaaagttatggggggtgaagattgggaacgttggattaagcagctgtcgaagga 
               
               
                   
                 aggcctcttagaagaaggctgtattaccttggcctatagttatattggccctgaagctacccaa 
               
               
                   
                 gctttgtaccgtaaaggcacaatcggcaaggccaaagaacacctggaggccacagcacac 
               
               
                   
                 cgtctcaacaaagagaacccgtcaatccgtgccttcgtgagcgtgaataaaggcctggtaac 
               
               
                   
                 ccgcgcaagcgccgtaatcccggtaatccctctgtatctcgccagcttgttcaaagtaatgaa 
               
               
                   
                 agagaagggcaatcatgaaggttgtattgaacagatcacgcgtctgtacgccgagcgcctgt 
               
               
                   
                 accgtaaagatggtacaattccagttgatgaggaaaatcgcattcgcattgatgattgggagtt 
               
               
                   
                 agaagaagacgtccagaaagcggtatccgcgttgatggagaaagtcacgggtgaaaacgc 
               
               
                   
                 agaatctctcactgacttagcggggtaccgccatgatttcttagctagtaacggctttgatgtag 
               
               
                   
                 aaggtattaattatgaagcggaagttgaacgcttcgaccgtatctgataagaaggagatatac 
               
               
                   
                 atatgagagaagtagtaattgccagtgcagctagaacagcagtaggaagttttggaggagc 
               
               
                   
                 atttaaatcagtttcagcggtagagttaggggtaacagcagctaaagaagctataaaaagag 
               
               
                   
                 ctaacataactccagatatgatagatgaatctcttttagggggagtacttacagcaggtcttgg 
               
               
                   
                 acaaaatatagcaagacaaatagcattaggagcaggaataccagtagaaaaaccagctatg 
               
               
                   
                 actataaatatagtttgtggttctggattaagatctgtttcaatggcatctcaacttatagcattag 
               
               
                   
                 gtgatgctgatataatgttagttggtggagctgaaaacatgagtatgtctccttatttagtaccaa 
               
               
                   
                 gtgcgagatatggtgcaagaatgggtgatgctgcttttgttgattcaatgataaaagatggatt 
               
               
                   
                 atcagacatatttaataactatcacatgggtattactgctgaaaacatagcagagcaatggaat 
               
               
                   
                 ataactagagaagaacaagatgaattagctcttgcaagtcaaaataaagctgaaaaagctca 
               
               
                   
                 agctgaaggaaaatttgatgaagaaatagttcctgttgttataaaaggaagaaaaggtgacac 
               
               
                   
                 tgtagtagataaagatgaatatattaagcctggcactacaatggagaaacttgctaagttaaga 
               
               
                   
                 cctgcatttaaaaaagatggaacagttactgctggtaatgcatcaggaataaatgatggtgct 
               
               
                   
                 gctatgttagtagtaatggctaaagaaaaagctgaagaactaggaatagagcctcttgcaact 
               
               
                   
                 atagtttcttatggaacagctggtgttgaccctaaaataatgggatatggaccagttccagcaa 
               
               
                   
                 ctaaaaaagctttagaagctgctaatatgactattgaagatatagatttagttgaagctaatgag 
               
               
                   
                 gcatttgctgcccaatctgtagctgtaataagagacttaaatatagatatgaataaagttaatgtt 
               
               
                   
                 aatggtggagcaatagctataggacatccaataggatgctcaggagcaagaatacttactac 
               
               
                   
                 acttttatatgaaatgaagagaagagatgctaaaactggtcttgctacactttgtataggcggtg 
               
               
                   
                 gaatgggaactactttaatagttaagagatagtaagaaggagatatacatatgaaattagctgt 
               
               
                   
                 aataggtagtggaactatgggaagtggtattgtacaaacttttgcaagttgtggacatgatgtat 
               
               
                   
                 gtttaaagagtagaactcaaggtgctatagataaatgtttagctttattagataaaaatttaacta 
               
               
                   
                 agttagttactaagggaaaaatggatgaagctacaaaagcagaaatattaagtcatgttagttc 
               
               
                   
                 aactactaattatgaagatttaaaagatatggatttaataatagaagcatctgtagaagacatga 
               
               
                   
                 atataaagaaagatgttttcaagttactagatgaattatgtaaagaagatactatcttggcaaca 
               
               
                   
                 aatacttcatcattatctataacagaaatagcttcttctactaagcgcccagataaagttatagga 
               
               
                   
                 atgcatttctttaatccagttcctatgatgaaattagttgaagttataagtggtcagttaacatcaa 
               
               
                   
                 aagttacttttgatacagtatttgaattatctaagagtatcaataaagtaccagtagatgtatctga 
               
               
                   
                 atctcctggatttgtagtaaatagaatacttatacctatgataaatgaagctgttggtatatatgca 
               
               
                   
                 gatggtgttgcaagtaaagaagaaatagatgaagctatgaaattaggagcaaaccatccaat 
               
               
                   
                 gggaccactagcattaggtgatttaatcggattagatgttgttttagctataatgaacgttttatat 
               
               
                   
                 actgaatttggagatactaaatatagacctcatccacttttagctaaaatggttagagctaatca 
               
               
                   
                 attaggaagaaaaactaagataggattctatgattataataaataataagaaggagatatacat 
               
               
                   
                 atgagtacaagtgatgttaaagtttatgagaatgtagctgttgaagtagatggaaatatatgtac 
               
               
                   
                 agtgaaaatgaatagacctaaagcccttaatgcaataaattcaaagactttagaagaactttat 
               
               
                   
                 gaagtatttgtagatattaataatgatgaaactattgatgttgtaatattgacaggggaaggaaa 
               
               
                   
                 ggcatttgtagctggagcagatattgcatacatgaaagatttagatgctgtagctgctaaagat 
               
               
                   
                 tttagtatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatagctg 
               
               
                   
                 ctgtaaacggatttgctttaggtggaggatgtgaacttgcaatggcatgtgatataagaattgc 
               
               
                   
                 atctgctaaagctaaatttggtcagccagaagtaactcttggaataactccaggatatggagg 
               
               
                   
                 aactcaaaggcttacaagattggttggaatggcaaaagcaaaagaattaatctttacaggtca 
               
               
                   
                 agttataaaagctgatgaagctgaaaaaatagggctagtaaatagagtcgttgagccagaca 
               
               
                   
                 ttttaatagaagaagttgagaaattagctaagataatagctaaaaatgctcagcttgcagttaga 
               
               
                   
                 tactctaaagaagcaatacaacttggtgctcaaactgatataaatactggaatagatatagaat 
               
               
                   
                 ctaatttatttggtctttgtttttcaactaaagaccaaaaagaaggaatgtcagctttcgttgaaaa 
               
               
                   
                 gagagaagctaactttataaaagggtaataagaaggagatatacatatgagtcaggcgctaa 
               
               
                   
                 aaaatttactgacattgttaaatctggaaaaaattgaggaaggactctttcgcggccagagtga 
               
               
                   
                 agatttaggtttacgccaggtgtttggcggccaggtcgtgggtcaggccttgtatgctgcaaa 
               
               
                   
                 agagaccgtccctgaagagcggctggtacattcgtttcacagctactttcttcgccctggcga 
               
               
                   
                 tagtaagaagccgattatttatgatgtcgaaacgctgcgtgacggtaacagcttcagcgcccg 
               
               
                   
                 ccgggttgctgctattcaaaacggcaaaccgattttttatatgactgcctctttccaggcaccag 
               
               
                   
                 aagcgggtttcgaacatcaaaaaacaatgccgtccgcgccagcgcctgatggcctcccttc 
               
               
                   
                 ggaaacgcaaatcgcccaatcgctggcgcacctgctgccgccagtgctgaaagataaattc 
               
               
                   
                 atctgcgatcgtccgctggaagtccgtccggtggagtttcataacccactgaaaggtcacgtc 
               
               
                   
                 gcagaaccacatcgtcaggtgtggatccgcgcaaatggtagcgtgccggatgacctgcgc 
               
               
                   
                 gttcatcagtatctgctcggttacgcttctgatcttaacttcctgccggtagctctacagccgca 
               
               
                   
                 cggcatcggttttctcgaaccggggattcagattgccaccattgaccattccatgtggttccat 
               
               
                   
                 cgcccgtttaatttgaatgaatggctgctgtatagcgtggagagcacctcggcgtccagcgc 
               
               
                   
                 acgtggctttgtgcgcggtgagttttatacccaagacggcgtactggttgcctcgaccgttca 
               
               
                   
                 ggaaggggtgatgcgtaatcacaattaa 
               
               
                   
               
               
                 Pfnrs-ter-thiA1-hbd-crt2- 
                 GGTACCAGTTGTTCTTATTGGTGGTGTTGCTTTATGGTT 
               
               
                 pbt-buk 
                 GCATCGTAGTAAATGGTTGTAACAAAAGCAATTTTTCC 
               
               
                 (SEQ ID NO: 180), e.g. 
                 GGCTGTCTGTATACAAAAACGCCGCAAAGTTTGAGCGA 
               
               
                 integrated into the 
                 AGTCAATAAACTCTCTACCCATTCAGGGCAATATCTCTC 
               
               
                 chromosome in SYN1002 
                 TTGGATCCAAAGTGAACTCTAGAAATAATTTTGTTTAAC 
               
               
                 Pfnrs: uppercase; butyrate 
                 TTTAAGAAGGAGATATACATatgatcgtaaaacctatggtacgcaacaat 
               
               
                 cassette: lower case 
                 atctgcctgaacgcccatcctcagggctgcaagaagggagtggaagatcagattgaatata 
               
               
                   
                 ccaagaaacgcattaccgcagaagtcaaagctggcgcaaaagctccaaaaaacgttctggt 
               
               
                   
                 gcttggctgctcaaatggttacggcctggcgagccgcattactgctgcgttcggatacgggg 
               
               
                   
                 ctgcgaccatcggcgtgtcctttgaaaaagcgggttcagaaaccaaatatggtacaccggg 
               
               
                   
                 atggtacaataatttggcatttgatgaagcggcaaaacgcgagggtctttatagcgtgacgat 
               
               
                   
                 cgacggcgatgcgttttcagacgagatcaaggcccaggtaattgaggaagccaaaaaaaa 
               
               
                   
                 aggtatcaaatttgatctgatcgtatacagcttggccagcccagtacgtactgatcctgataca 
               
               
                   
                 ggtatcatgcacaaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaacagtagat 
               
               
                   
                 ccgtttactggcgagctgaaggaaatctccgcggaaccagcaaatgacgaggaagcagcc 
               
               
                   
                 gccactgttaaagttatggggggtgaagattgggaacgttggattaagcagctgtcgaagga 
               
               
                   
                 aggcctcttagaagaaggctgtattaccttggcctatagttatattggccctgaagctacccaa 
               
               
                   
                 gctttgtaccgtaaaggcacaatcggcaaggccaaagaacacctggaggccacagcacac 
               
               
                   
                 cgtctcaacaaagagaacccgtcaatccgtgccttcgtgagcgtgaataaaggcctggtaac 
               
               
                   
                 ccgcgcaagcgccgtaatcccggtaatccctctgtatctcgccagcttgttcaaagtaatgaa 
               
               
                   
                 agagaagggcaatcatgaaggngtattgaacagatcacgcgtctgtacgccgagcgcctgt 
               
               
                   
                 accgtaaagatggtacaattccagttgatgaggaaaatcgcattcgcattgatgattgggagtt 
               
               
                   
                 agaagaagacgtccagaaagcggtatccgcgttgatggagaaagtcacgggtgaaaacgc 
               
               
                   
                 agaatctctcactgacttagcggggtaccgccatgatttcttagctagtaacggattgatgtag 
               
               
                   
                 aaggtattaattatgaagcggaagttgaacgcttcgaccgtatctgataagaaggagatatac 
               
               
                   
                 atatgagagaagtagtaattgccagtgcagctagaacagcagtaggaagttttggaggagc 
               
               
                   
                 atttaaatcagtttcagcggtagagttaggggtaacagcagctaaagaagctataaaaagag 
               
               
                   
                 ctaacataactccagatatgatagatgaatctcttttagggggagtacttacagcaggtcttgg 
               
               
                   
                 acaaaatatagcaagacaaatagcattaggagcaggaataccagtagaaaaaccagctatg 
               
               
                   
                 actataaatatagtttgtggttctggattaagatctgtttcaatggcatctcaacttatagcattag 
               
               
                   
                 gtgatgctgatataatgttagttggtggagctgaaaacatgagtatgtctccttatttagtaccaa 
               
               
                   
                 gtgcgagatatggtgcaagaatgggtgatgctgcttttgttgattcaatgataaaagatggatt 
               
               
                   
                 atcagacatatttaataactatcacatgggtattactgctgaaaacatagcagagcaatggaat 
               
               
                   
                 ataactagagaagaacaagatgaattagctcttgcaagtcaaaataaagctgaaaaagctca 
               
               
                   
                 agctgaaggaaaatttgatgaagaaatagttcctgttgttataaaaggaagaaaaggtgacac 
               
               
                   
                 tgtagtagataaagatgaatatattaagcctggcactacaatggagaaacttgctaagttaaga 
               
               
                   
                 cctgcatttaaaaaagatggaacagttactgctggtaatgcatcaggaataaatgatggtgct 
               
               
                   
                 gctatgttagtagtaatggctaaagaaaaagctgaagaactaggaatagagcctcttgcaact 
               
               
                   
                 atagtttcttatggaacagctggtgttgaccctaaaataatgggatatggaccagttccagcaa 
               
               
                   
                 ctaaaaaagctttagaagctgctaatatgactattgaagatatagatttagttgaagctaatgag 
               
               
                   
                 gcatttgctgcccaatctgtagctgtaataagagacttaaatatagatatgaataaagttaatgtt 
               
               
                   
                 aatggtggagcaatagctataggacatccaataggatgctcaggagcaagaatacttactac 
               
               
                   
                 acttttatatgaaatgaagagaagagatgctaaaactggtcttgctacactttgtataggcggtg 
               
               
                   
                 gaatgggaactactttaatagttaagagatagtaagaaggagatatacatatgaaattagctgt 
               
               
                   
                 aataggtagtggaactatgggaagtggtattgtacaaacttttgcaagttgtggacatgatgtat 
               
               
                   
                 gtttaaagagtagaactcaaggtgctatagataaatgtttagctttattagataaaaatttaacta 
               
               
                   
                 agttagttactaagggaaaaatggatgaagctacaaaagcagaaatattaagtcatgttagttc 
               
               
                   
                 aactactaattatgaagatttaaaagatatggatttaataatagaagcatctgtagaagacatga 
               
               
                   
                 atataaagaaagatgttttcaagttactagatgaattatgtaaagaagatactatcttggcaaca 
               
               
                   
                 aatacttcatcattatctataacagaaatagcttcttctactaagcgcccagataaagttatagga 
               
               
                   
                 atgcatttctttaatccagttcctatgatgaaattagttgaagttataagtggtcagttaacatcaa 
               
               
                   
                 aagttacttttgatacagtatttgaattatctaagagtatcaataaagtaccagtagatgtatctga 
               
               
                   
                 atctcctggatttgtagtaaatagaatacttatacctatgataaatgaagctgttggtatatatgca 
               
               
                   
                 gatggtgttgcaagtaaagaagaaatagatgaagctatgaaattaggagcaaaccatccaat 
               
               
                   
                 gggaccactagcattaggtgatttaatcggattagatgttgttttagctataatgaacgttttatat 
               
               
                   
                 actgaatttggagatactaaatatagacctcatccacttttagctaaaatggttagagctaatca 
               
               
                   
                 attaggaagaaaaactaagataggattctatgattataataaataataagaaggagatatacat 
               
               
                   
                 atgagtacaagtgatgttaaagtttatgagaatgtagctgttgaagtagatggaaatatatgtac 
               
               
                   
                 agtgaaaatgaatagacctaaagcccttaatgcaataaattcaaagactttagaagaactttat 
               
               
                   
                 gaagtatttgtagatattaataatgatgaaactattgatgttgtaatattgacaggggaaggaaa 
               
               
                   
                 ggcatttgtagctggagcagatattgcatacatgaaagatttagatgctgtagctgctaaagat 
               
               
                   
                 tttagtatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatagctg 
               
               
                   
                 ctgtaaacggatttgctttaggtggaggatgtgaacttgcaatggcatgtgatataagaattgc 
               
               
                   
                 atctgctaaagctaaatttggtcagccagaagtaactcttggaataactccaggatatggagg 
               
               
                   
                 aactcaaaggcttacaagattggttggaatggcaaaagcaaaagaattaatctttacaggtca 
               
               
                   
                 agttataaaagctgatgaagctgaaaaaatagggctagtaaatagagtcgttgagccagaca 
               
               
                   
                 ttttaatagaagaagttgagaaattagctaagataatagctaaaaatgctcagcttgcagttaga 
               
               
                   
                 tactctaaagaagcaatacaacttggtgctcaaactgatataaatactggaatagatatagaat 
               
               
                   
                 ctaatttatttggtctttgtttttcaactaaagaccaaaaagaaggaatgtcagctttcgttgaaaa 
               
               
                   
                 gagagaagctaactttataaaagggtaataagaaggagatatacatatgagaagttttgaaga 
               
               
                   
                 agtaattaagtttgcaaaagaaagaggacctaaaactatatcagtagcatgttgccaagataa 
               
               
                   
                 agaagttttaatggcagttgaaatggctagaaaagaaaaaatagcaaatgccattttagtagg 
               
               
                   
                 agatatagaaaagactaaagaaattgcaaaaagcatagacatggatatcgaaaattatgaact 
               
               
                   
                 gatagatataaaagatttagcagaagcatctctaaaatctgttgaattagtttcacaaggaaaa 
               
               
                   
                 gccgacatggtaatgaaaggcttagtagacacatcaataatactaaaagcagttttaaataaa 
               
               
                   
                 gaagtaggtcttagaactggaaatgtattaagtcacgtagcagtatttgatgtagagggatatg 
               
               
                   
                 atagattatttttcgtaactgacgcagctatgaacttagctcctgatacaaatactaaaaagcaa 
               
               
                   
                 atcatagaaaatgcttgcacagtagcacattcattagatataagtgaaccaaaagttgctgcaa 
               
               
                   
                 tatgcgcaaaagaaaaagtaaatccaaaaatgaaagatacagttgaagctaaagaactaga 
               
               
                   
                 agaaatgtatgaaagaggagaaatcaaaggttgtatggttggtgggccttttgcaattgataat 
               
               
                   
                 gcagtatctttagaagcagctaaacataaaggtataaatcatcctgtagcaggacgagctgat 
               
               
                   
                 atattattagccccagatattgaaggtggtaacatattatataaagctttggtattcttctcaaaat 
               
               
                   
                 caaaaaatgcaggagttatagttggggctaaagcaccaataatattaacttctagagcagaca 
               
               
                   
                 gtgaagaaactaaactaaactcaatagctttaggtgttttaatggcagcaaaggcataataag 
               
               
                   
                 aaggagatatacatatgagcaaaatatttaaaatcttaacaataaatcctggttcgacatcaact 
               
               
                   
                 aaaatagctgtatttgataatgaggatttagtatttgaaaaaactttaagacattcttcagaagaa 
               
               
                   
                 ataggaaaatatgagaaggtgtctgaccaatttgaatttcgtaaacaagtaatagaagaagct 
               
               
                   
                 ctaaaagaaggtggagtaaaaacatctgaattagatgctgtagtaggtagaggaggacttctt 
               
               
                   
                 aaacctataaaaggtggtacttattcagtaagtgctgctatgattgaagatttaaaagtgggagt 
               
               
                   
                 tttaggagaacacgcttcaaacctaggtggaataatagcaaaacaaataggtgaagaagtaa 
               
               
                   
                 atgttccttcatacatagtagaccctgttgttgtagatgaattagaagatgttgctagaatttctgg 
               
               
                   
                 tatgcctgaaataagtagagcaagtgtagtacatgctttaaatcaaaaggcaatagcaagaag 
               
               
                   
                 atatgctagagaaataaacaagaaatatgaagatataaatcttatagttgcacacatgggtgg 
               
               
                   
                 aggagtttctgttggagctcataaaaatggtaaaatagtagatgttgcaaacgcattagatgga 
               
               
                   
                 gaaggacctttctctccagaaagaagtggtggactaccagtaggtgcattagtaaaaatgtgc 
               
               
                   
                 tttagtggaaaatatactcaagatgaaattaaaaagaaaataaaaggtaatggcggactagtt 
               
               
                   
                 gcatacttaaacactaatgatgctagagaagttgaagaaagaattgaagctggtgatgaaaa 
               
               
                   
                 agctaaattagtatatgaagctatggcatatcaaatctctaaagaaataggagctagtgctgca 
               
               
                   
                 gttcttaagggagatgtaaaagcaatattattaactggtggaatcgcatattcaaaaatgtttac 
               
               
                   
                 agaaatgattgcagatagagttaaatttatagcagatgtaaaagtttatccaggtgaagatgaa 
               
               
                   
                 atgattgcattagctcaaggtggacttagagttttaactggtgaagaagaggctcaagtttatg 
               
               
                   
                 ataactaa 
               
               
                   
               
               
                 PfNRS (ribosome binding 
                 GGTACCAGTTGTTCTTATTGGTGGTGTTGCTTTATGGTT 
               
               
                 site is underlined) 
                 GCATCGTAGTAAATGGTTGTAACAAAAGCAATTTTTCC 
               
               
                 (SEQ ID NO: 181) 
                 GGCTGTCTGTATACAAAAACGCCGCAAAGTTTGAGCGA 
               
               
                   
                 AGTCAATAAACTCTCTACCCATTCAGGGCAATATCTCTC 
               
               
                   
                 TTGGATCCAAAGTGAACTCTAGAAATAATTTTGTTTAAC 
               
               
                   
                 TTTAAGAAGGAGATATACAT 
               
               
                   
               
               
                 Ribosome binding site and 
                 CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATAT 
               
               
                 leader region (SEQ ID 
                 ACAT 
               
               
                 NO:182) 
               
               
                   
               
            
           
         
       
     
     Example 16. Assessment of Intestinal Butyrate Levels in Response to SYN501 Administration in Mice 
     To determine efficacy of butyrate production by the genetically engineered bacteria in vivo, the levels of butyrate upon administration of SYN501 (Logic156 (pSC101 PydfZ-ter-&gt;pbt-buk butyrate plasmid)) to C57BL6 mice was first assessed in the feces. Water containing 100 mM butyrate was used as a control. 
     On day 1, C57BL6 mice (24 total animals) were weighed and randomized into 4 groups; Group 1: H20 control (n=6); Group 2-100 mM butyrate (n=6); Group 3-streptomycin resistant Nissle (n=6); Group 4-SYN501 (n=6). Mice were either gavaged with 100 ul streptomycin resistant Nissle or SYN501, and group 2 was changed to H20(+)100 mM butyrate at a dose of 10e10 cells/100 ul. On days 2-4, mice were weighted and Groups 3 and 4 were gavaged in the AM and the PM with streptomycin resistant Nissle or SYN501. On day 5, mice were weighed and Groups 3 and 4 were gavaged in the am with streptomycin resistant Nissle or SYN501, and feces was collected and butyrate concentrations determined as described in Example 23. Results are depicted in  FIG. 28 . Significantly greater levels of butyrate were detected in the feces of the mice gavaged with SYN501 as compared mice gavaged with the Nissle control or those given water only. Levels are close to 2 mM and higher than the levels seen in the mice fed with H20 (+) 200 mM butyrate. 
     Next the effects of SYN501 on levels of butyrate in the cecum, cecal effluent, large intestine, and large intestine effluent are assessed. Because baseline concentrations of butyrate are high in these compartments, an antibiotic treatment is administered in advance to clear out the bacteria responsible for butyrate production in the intestine. As a result, smaller differences in butyrate levels can be more accurately observed and measured. Water containing 100 mM butyrate is used as a control. 
     During week 1 of the study, animals are treated with an antibiotic cocktail in the drinking water to reduce the baseline levels of resident microflora. The antibiotic cocktail is composed of ABX-ampicillin, vancomycin, neomycin, and metronidazole. During week 2 animals are orally administered 100 ul of streptomycin resistant Nissle or engineered strain SYN501 twice a day for five days (at a dose of 10e10 cells/100 ul). 
     On day 1, C57BL6 (Female, 8 weeks) are separated into four groups as follows: Group 1: H2O control (n=10); Group 2: 100 mM butyrate (n=10); Group 3: streptomycin resistant Nissle (n=10); Group 4: SYN501 (n=10). Animals are weighed and feces is collected from the animals (T=0-time point). Animals are changed to H2O (+) antibiotic cocktail. On day 5, animals are weighed and feces is collected (time point T=5d). The H2O (+) antibiotic cocktail bottles are changed. On day 8, the mice are weighed and feces is collected. Mice of Group 3 and Group 4 are gavaged in the AM and PM with streptomycin resistant Nissle or SYN501. The water in all cages is changed to water without antibiotic. Group 2 is provided with 100 mM butyrate in H2O. On days 9-11, mice are weighed, and mice of Group 3 and Group 4 are gavaged in the AM and PM with streptomycin resistant Nissle or SYN501. On day 12, mice are gavaged with streptomycin resistant Nissle or SYN501 in the AM, and 4 hours post dose, blood is harvested, and cecal and large intestinal contents, and tissue, and feces are collected and processed for analysis. 
     Example 17. Comparison of Butyrate Production Levels Between the Genetically Engineered Bacteria Encoding a Butyrate Cassette and Selected Clostridia Strains 
     The efficacy of pbutyrate production in SYN501 (pSC101 PydfZ-ter-&gt;pbt-buk butyrate plasmid) was compared to CBM588 (Clostridia butyricum MIYARISAN, a Japanese probiotic strain),  Clostridium tyrobutyricum  VPI 5392 (Type Strain), and  Clostridium butyricum  NCTC 7423 (Type Strain). 
     Briefly, overnight cultures of SYN501 were diluted 1:100 were grown in RCM (Reinforced Clostridial Media, which is similar to LB but contains 0.5% glucose) at 37 C with shaking for 2 hours, then either moved into the anaerobic chamber or left aerobically shaking. Clostridial strains were only grown anaerobically. At indicated times (2, 8, 24, and 48 h), 120 ul cells were removed and pelleted at 14,000 rpm for Imin, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at −80 C until analysis by LC-MS for butyrate concentrations (as described in Example 18). Results are depicted in  FIG. 18 , and show that SYN501 produces butyrate levels comparable to  Clostridium  spp. in RCM media 
     Example 18. Quantification of Butyrate by LC-MS/MS 
     To obtain the butyrate measurements in Example 37 a LC-MS/MS protocol for butyrate quantification was used. 
     Sample Preparation 
     First, fresh 1000, 500, 250, 100, 20, 4 and 0.8 g/mL sodium butyrate standards were prepared in water. Then, 10 μL of sample (bacterial supernatants and standards) were pipetted into a V-bottom polypropylene 96-well plate, and 90 μL of 67% ACN (60 uL ACN+30 uL water per reaction) with 4 ug/mL of butyrate-d7 (CDN isotope) internal standard in final solution were added to each sample. The plate was heat-sealed, mixed well, and centrifuged at 4000 rpm for 5 minutes. In a round-bottom 96-well polypropylene plate, 20 μL of diluted samples were added to 180 μL of a buffer containing 10 mM MES pH4.5, 20 mM EDC (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide), and 20 mM TFEA (2,2,2-trifluroethylamine). The plate was again heat-sealed and mixed well, and samples were incubated at room temperature for 1 hour. 
     LC-MS/MS Method 
     Butyrate was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. HPLC Details are listed in Table 49 and Table 50. Tandem Mass Spectrometry details are found in Table 51. 
     
       
         
           
               
             
               
                 TABLE 49 
               
               
                   
               
               
                 HPLC Details 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Column 
                 Thermo Aquasil C18 column, 5 μm (50 × 2.1 mm) 
               
               
                 Mobile Phase A 
                 100% H2O, 0.1% Formic Acid 
               
               
                 Mobile Phase B 
                 100% ACN, 0.1% Formic Acid 
               
               
                 Injection volume 
                 10 uL 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 50 
               
             
            
               
                   
               
               
                 HPLC Method 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Total Time (min) 
                 Flow Rate (μL/min) 
                 A % 
                 B % 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0 
                 0.5 
                 100 
                 0 
               
               
                   
                 1 
                 0.5 
                 100 
                 0 
               
               
                   
                 2 
                 0.5 
                 10 
                 90 
               
               
                   
                 4 
                 0.5 
                 10 
                 90 
               
               
                   
                 4.01 
                 0.5 
                 100 
                 0 
               
               
                   
                 4.25 
                 0.5 
                 100 
                 0 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 51 
               
               
                   
               
               
                 Tandem Mass Spectrometry Details 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Ion Source 
                 HESI-II 
               
               
                   
                 Polarity 
                 Positive 
               
               
                   
                 SRM 
                 Butyrate 170.0/71.1, 
               
               
                   
                 transitions 
                 Butyrate d7 177.1/78.3 
               
               
                   
                   
               
            
           
         
       
     
     Example 19. Quantification of Butyrate in Feces by LC-MS/MS Sample Preparation 
     Fresh 1000, 500, 250, 100, 20, 4 and 0.8 μg/mL sodium butyrate standards were prepared in water. Single fecal pellets were ground in 100 uL water and centrifuged at 15,000 rpm for 5 min at 4° C. 10 μL of the sample (fecal supernatant and standards) were pipetted into a V-bottom polypropylene 96-well plate, and 901VL of the derivatizing solution containing 50 mM of 2-Hydrazinoquinoline (2-HQ), dipyridyl disulfide, and triphenylphospine in acetonitrile with 5 ug/mL of butyrate-d7 were added to each sample. The plate was heat-sealed and incubated at 60° C. for 1 hr. The plate was then centrifuged at 4,000 rpm for 5 min and 20 μL of the derivatized samples mixed to 180 μL of 22% acetonitrile with 0.1% formic acid. 
     LC-MS/MS Method 
     Butyrate was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. HPLC Details are listed in Table 52 and Table 53. Tandem Mass Spectrometry details are found in Table 54. 
     
       
         
           
               
             
               
                 TABLE 52 
               
               
                   
               
               
                 HPLC Details 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Column 
                 Luna phenomenex C18 column, 5 μm (100 × 2.1 mm) 
               
               
                 Mobile Phase A 
                 100% H2O, 0.1% Formic Acid 
               
               
                 Mobile Phase B 
                 100% ACN, 0.1% Formic Acid 
               
               
                 Injection volume 
                 10 uL 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 53 
               
             
            
               
                   
               
               
                 HPLC Method 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Total Time (min) 
                 Flow Rate (μL/min) 
                 A % 
                 B % 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0 
                 0.5 
                 95 
                 5 
               
               
                   
                 0.5 
                 0.5 
                 95 
                 5 
               
               
                   
                 1.5 
                 0.5 
                 10 
                 90 
               
               
                   
                 3.5 
                 0.5 
                 10 
                 90 
               
               
                   
                 3.51 
                 0.5 
                 95 
                 5 
               
               
                   
                 3.75 
                 0.5 
                 95 
                 5 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 54A 
               
               
                   
               
               
                 Tandem Mass Spectrometry Details 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Ion Source 
                 HESI-II 
               
               
                   
                 Polarity 
                 Positive 
               
               
                   
                 SRM 
                 Butyrate 230.1/143.1, 
               
               
                   
                 transitions 
                 Butyrate d7 237.1/143.1 
               
               
                   
                   
               
            
           
         
       
     
     Example 20. Generation of Butyrate and Acetate Producing Strains 
     A. Generation of an Acetate Overproducing Strain 
       E. coli  generates high levels of acetate as an end product of fermentation. In order generate enhanced acetate production, strain SYN2001 was generated, which harbors a deletion in the endogenous ldh (lactate dehydrogenase) gene, with the intention to prevent or reduce flux through the metabolic arm generating lactate, and thereby enhancing the flux through the metabolic arm generating acetate (see, e.g.,  FIG. 25 ). 
     Briefly, We deleted the gene encoding L-lactate dehydrogenase A (ldhA) to block carbon flux from pyruvate to lactate and improve acetate biosynthetic yield in  E. coli  Nissle. Knockout primers were synthesized (IDT) and a chloramphenicol-resistance antibiotic marker was inserted in place of the ldhA coding region to ensure the removal of the targeted gene. The ldhA gene on the  E. coli  Nissle genome was knocked out and replaced with the chloramphenicol resistance gene through allelic exchange, which was facilitated by the lambda red recombinase system. Proper knockout of the target gene in the Nissle genome was validated by the ability of the resulting Nissle strain to grow on chloramphenicol-containing LB plates or medium and further confirmed by PCR. This strain was designated SYN2001. 
     For this study, media M9 media containing 50 mM MOPS with 0.5% glucose was compared to media containing 0.5% glucuronic acid, as glucuronic acid better mimics available carbon sources in the gut. 
     SYN2001 and streptomycin resistant  E. coli  Nissle (SYN094) were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 into 10 ml LB (containing antibiotics) in a 125 ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5 h, and then transferred to the anaerobic chamber at 37 C for 4 h. Bacteria (2×10 8  CFU) were added to 1 ml M9 media containing 50 mM MOPS with 0.5% glucose or 0.5% glucuronic acid in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At 1, 2, 3, 4, 5, and 6 hours, cells were removed and pelleted at 14,000 rpm for 1 min, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at −80 C until analysis by LC-MS for acetate concentrations as described herein, e.g., in Example 21. 
     
       
         
           
               
             
               
                 TABLE 54B 
               
             
            
               
                   
               
               
                 Acetate production by SYN2001 from three different manufacturing experiments 
               
            
           
           
               
               
               
               
            
               
                   
                 Run1 
                 Run 2 
                 Run 3 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 [Acetate] 
                   
                 [Acetate] 
                   
                 [Acetate] 
                   
               
               
                 Strain 
                 in mM 
                 SD 
                 in mM 
                 SD 
                 in mM 
                 SD 
               
               
                   
               
               
                 SYN2001 
                 24.43737 
                 2.970942327 
                 21.26342667 
                 1.719791084 
                 31.58750134 
                 6.68461575 
               
               
                   
               
            
           
         
       
     
     Culture supernatants of SYN2001 produced between 21.2 and 31.5 mM acetate and an undetectable amount of butyrate (data not shown) under the above conditions in 3 independent production runs. Culture supernatant from run 3 was then used to generate the bioactivity results from cell based assays presented below in Example 63. 
     As seen in  FIG. 26A  and  FIG. 26B , the ldhA knockout  E. coli  Nissle strain SYN2001 has improved acetate productivity during over a 6 hour time course using either glucose or glucuronic acid as the main carbon source. 
     B. Generation of Strains which Produces Butyrate and Acetate 
     a. Knock Out of the Endogenous adhE and ldhA Genes 
     In order to improve acetate production while also producing high levels butyrate production, deletions in endogenous adhE (Aldehyde-alcohol dehydrogenase) and ldh (lactate dehydrogenase) were generated to prevent or reduce metabolic flux through pathways which do not result in acetate or butyrate production (see, e.g.,  FIG. 25 ). Aldehyde-alcohol dehydrogenase converts acetylCoA into acetaldehyde, which is then converted to ethanol. As a result, a mutation or deletion of adhE is expected to prevent the metabolic flux towards ethanol production and consequently allow for additional acetylCoA to be used for butyrate production. For this study, Nissle strains with either integrated FNRS ter-tesB or FNRS-ter-pbt-buk butyrate cassettes were used. Additionally, media M9 media containing 50 mM MOPS with 0.5% glucose was compared to media containing 0.5% glucuronic acid, as glucuronic acid better mimics available carbon sources in the gut. 
     Briefly, bacteria were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 into 10 ml LB (containing antibiotics) in a 125 ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5 h, and then transferred to the anaerobic chamber at 37 C for 4 h. Bacteria (2×10 8  CFU) were added to 1 ml M9 media containing 50 mM MOPS with 0.5% glucose or 0.5% glucuronic acid in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At 18 hours, cells were removed and pelleted at 14,000 rpm for 1 min, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at −80 C until analysis by LC-MS for butyrate and acetate concentrations as described herein, e.g., in Example 21. 
     As seen in  FIG. 26C  and  FIG. 26D , both integrated strains made similar amounts of acetate, and FNRS-ter-pbt-buk butyrate cassettes produced slightly more butyrate. Deletions in adhE and ldhA have similar effects on butyrate and acetate production. Acetate production was much greater in media containing 0.5% glucuronic acid. 
     b. Knock Out of the Endogenous frdA Gene 
     FrdA is one of two catalytic subunits in the four subunit fumarate reductase complex. Fumarate reductase converts fumarate (derived from phosphoenolpyruvate) to succinate along one arm of anaerobic metabolism. In a second study, the effect of a deletion in the endogenous frdA gene, which prevents metabolic flux through the phosphoenolpyruvate-&gt;succinate pathway, on acetate and butyrate production was assessed. For this study, SYN2005 (comprising FNRS-ter-tesB butyrate cassette integrated at the HA1/2 site and a deletion in the endogenous frd gene) was compared to SYN1004 (comprising the FNRS-ter-tesB butyrate cassette integrated at the HA1/2 site). 
     Bacteria were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 into 10 ml LB (containing antibiotics) in a 125 ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5 h, and then transferred to the anaerobic chamber at 37 C for 4 h. Bacteria (2×10 8  CFU) were added to 1 ml M9 media containing 50 mM MOPS with 0.5% glucose in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At 18 hours, cells were removed and pelleted at 14,000 rpm for 1 min, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at −80 C until analysis by LC-MS for butyrate and acetate concentrations as described herein, e.g., in Example 21. 
     Results are depicted in  FIG. 26E  and indicate that the frdA mutation in SYN2005 allowed increased acetate production relative to SYN1173. SYN1173 produces greater levels of butyrate than acetate, while SYN2005 produces similar levels of both acetate and butyrate. 
     In other studies, strains are generated with combinations of deletions in two or more of the aldE, ldhA, and frd genes and the effect of the deletions on acetate and butyrate production are assessed. 
     C. Butyrate Only Producing Strains 
     In order to generate a strain which can produce butyrate, but has a reduced ability to produce acetate, a deletion in the pta gene was introduced into a strain that contains an integrated butyrate cassette (Ter/TesB cassette) under the control of an FNR promoter (SYN2002). Phosphate acetyltransferase (Pta) catalyzes the conversion between acetyl-CoA and acetylphosphate, the first step in the metabolic arm leading to the generation of acetate (see., e.g.,  FIG. 25 ). As such inhibition of this step was assumed to help prevent accumulation of acetate. Additionally, a mutation in the adhE (aldehyde-alcohol dehydrogenase) gene was introduced. 
     Acetate and butyrate production in both strains was compared to a third strain which contains both the FNR-driven ter-pbt-buk butyrate cassette and the deletion in the endogenous ldhA gene (e.g., as described above). 
     For this study, bacteria from all three strains were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 into 10 ml LB (containing antibiotics) in a 125 ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5 h, and then transferred to the anaerobic chamber at 37 C for 4 h. Bacteria (2×10 8  CFU) were added to 1 ml M9 media containing 50 mM MOPS with 0.5% glucose in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At 18 hours, cells were removed and pelleted at 14,000 rpm for 1 min, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at −80 C until analysis by LC-MS for butyrate and acetate concentrations as described herein, e.g., in Example 21. 
     Results are depicted in  FIG. 26F , and show that the strain comprising the deletion in the endogenous ldhA gene produced acetate but no butyrate, the strain comprising the FNR-ter-tesB butyrate cassette and the aldhE deletion produced butyrate, but very low levels of acetate. The third strain, comprising the FNRter-tesB butyrate cassette and the deletions in the adhE and pta genes, made equal amounts of acetate and butyrate. 
     Example 21. Acetate and Butyrate Quantification in Bacterial Supernatant by LC-MS/MS 
     Sample Preparation 
     Ammonium acetate and Sodium butyrate stock (10 mg/mL) was prepared in water and aliquoted in 1.5 mL microcentrifuge tubes (100 μL) and stored at −20° C. Standards (1000, 500, 250, 100, 20, 4, 0.8 μg/mL) were prepared in water. Sample and standards (10 μL) were pipetted in a V-bottom polypropylene 96-well plate on ice. Derivatizing solution (90 μL) containing 50 mM of 2-Hydrazinoquinoline (2-HQ), dipyridyl disulfide, and triphenylphosphine in acetonitrile with 2 ug/mL of Sodium butyrate-d7 was added into the final solution. The plate was then heat-sealed with a ThermASeal foil and mixed well, and the samples were incubated at 60° C. for 1 hr for derivatization and centrifuged at 4000 rpm for 5 min. The derivatized samples (20 μL) were added to 180 μL of 0.1% formic acid in water/ACN (140:40) in a round-bottom 96-well plate. The plate was then heat-sealed with a ClearASeal sheet and mixed well. 
     LC-MS/MS Method 
     Derivatized metabolites were measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. Table 55 and Table 56 provides the summary of the LC-MS/MS method. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 55 
               
               
                   
                   
               
             
            
               
                   
                 Column: 
                 C18 column, 3 μm (100 × 2 mm) 
               
               
                   
                 Mobile Phase A: 
                 100% H20, 0.1% Formic Acid 
               
               
                   
                 Mobile Phase B: 
                 100% ACN, 0.1% Formic Acid 
               
               
                   
                 Injection volume: 
                 10 uL 
               
               
                   
                   
               
            
           
         
       
     
                     TABLE 56                  HPLC Method:                                     Time (min)   Flow Rale (μL/min)   A %   B %                                                 0   500   95   5           0.5   500   95   5           2.0   500   10   90           3.0   500   10   90           3.01   500   95   5           3.25   500   95   5                        
Table 57 summarizes Tandem Mass Spectrometry.
 
     
       
         
           
               
             
               
                 TABLE 57 
               
               
                   
               
               
                 Tandem Mass Spectrometry: 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Ion Source: 
                 HESI-II 
               
               
                   
                 Polarity: 
                 Positive 
               
            
           
           
               
            
               
                 SRM transitions: 
               
            
           
           
               
               
               
            
               
                   
                 Acetate: 
                 202.1/143.1 
               
               
                   
                 Butyrate: 
                 230.1/160.2 
               
               
                   
                 Butyrate-d7: 
                 237.1/160.2 
               
               
                   
                   
               
            
           
         
       
     
     Example 22. Production of Propionate Through the Sleeping Beauty Mutase Pathway in Genetically Engineered  E. coli  BW25113 and Nissle 
     In  E. coli , a four gene operon, sbm-ygfD-ygfD-ygfH (sleeping beauty mutase pathway) has been shown to encode a putative cobalamin-dependent pathway with the ability to produce propionate from succinate in vitro. While the sleeping beauty mutase pathway is present in  E. coli , it is not under the control of a strong promoter and has shown low activity in vivo. 
     The utility of this operon for the production of propionate was assessed. Because  E. coli  Nissle does not have the complete operon, initial experiments were conducted in  E. coli  K12 (BW25113). 
     First, the native promoter for the sleeping beauty mutase operon on the chromosome in the BW25113 strain was replaced with a fnr promoter (BW25113 ldhA::frt; PfnrS-SBM-cam). The sequence for this construct is provided in Table 58. Mutation of the lactate dehydrogenase gene (ldhA) reportedly increases propionate production, and this mutation is therefore also added in certain embodiments. 
     In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 184, or 184, or a functional fragment thereof. 
     
       
         
           
               
             
               
                 TABLE 58 
               
             
            
               
                   
               
               
                 SBM Construct Sequences 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 BW25113 fnrS SBM construct 
                 
                   
                   
                   
                 
               
               
                 (BW25113 frt-cam-frt-PfnrS- sbm, 
                 
                   
                   
                   
                 
               
               
                 ygfD, ygfG, ygfH), comprising rmB 
                      CCGCCGGGAGCG 
               
               
                 terminator 1, rrnB terminator 2 (both 
                 GATTTGAACGTTGCGAAGCAACGGCCCGGA 
               
               
                 italic, uppercase), cat promoter and cam 
                 GGGTGGCGGGCAGGACGCCCGCCATAAACT 
               
               
                 resistance gene (encoded on the 
                 GCCAGGCATCAAATTAAGC   
               
               
                 lagging strand underlined 
                      TGCGTGGCCAGTGCCAA 
               
               
                 uppercase), frt sites (italic underlined), 
                 GCTTGCATGCAGATTGCAGCATTACACGTCT 
               
               
                 FNRS promoter bold lowercase, with 
                 TGAGCGATTGTGTAGGCTGGAGCTGCTTC   
               
               
                 RBS and leader region bold and 
                 
                   
                   
                   
                 
               
               
                 underlined and FNR binding site in bold 
                    ATTTAAATGGCGCGCCTTAC 
               
               
                 and italics); sleeping beauty operon 
                 GCCCCGCCCTGCCA CTCATCGCAGTACTGTT   
               
               
                 (sbm, ygfD, ygfG, ygfH) bold and 
                 
                   GTATTCATTAAGCATCTGCCGACATGGAAGC 
                 
               
               
                 uppercase 
                 
                   CATCACAAACGGCATGATGAACCTGAATCGC 
                 
               
            
           
           
               
               
               
               
            
               
                 (SEQ ID NO: 183 
                 
                   CAGCGGCATCAGCACCTTGTCGCCTTGCGTA 
                 
                   
                   
               
               
                   
                 
                   TAATATTTGCCCATGGTGAAAACGGGGGCGA 
                 
                   
                   
               
               
                   
                 
                   AGAAGTTGTCCATATTGGCCACGTTTAAATC 
                 
                   
                   
               
               
                   
                 
                   AAAACTGGTGAAACTCACCCAGGGATTGGCT 
                 
                   
                   
               
               
                   
                 
                   GAGACGAAAAACATATTCTCAATAAACCCTT 
                 
                   
                   
               
               
                   
                 
                   TAGGGAAATAGGCCAGGTTTTCACCGTAACA 
                 
                   
                   
               
               
                   
                 
                   CGCCACATCTTGCGAATATATGTGTAGAAAC 
                 
                   
                   
               
               
                   
                 
                   TGCCGGAAATCGTCGTGGTATTCACTCCAGA 
                 
                   
                   
               
               
                   
                 
                   GCGATGAAAACGTTTCAGTTTGCTCATGGAA 
                 
                   
                   
               
               
                   
                 
                   AACGGTGTAACAAGGGTGAACACTATCCCAT 
                 
                   
                   
               
               
                   
                 
                   ATCACCAGCTCACCGTCTTTCATTGCCATAC 
                 
                   
                   
               
               
                   
                 
                   GTAATTCCGGATGAGCATTCATCAGGCGGGC 
                 
                   
                   
               
               
                   
                 
                   AAGAATGTGAATAAAGGCCGGATAAAACTTG 
                 
                   
                   
               
               
                   
                 
                   TGCTTATTTTTCTTTACGGTCTTTAAAAAGGC 
                 
                   
                   
               
               
                   
                 
                   CGTAATATCCAGCTGAACGGTCTGGTTATAG 
                 
                   
                   
               
               
                   
                 
                   GTACATTGAGCAACTGACTGAAATGCCTCAA 
                 
                   
                   
               
               
                   
                 
                   AATGTTCTTTACGATGCCATTGGGATATATC 
                 
                   
                   
               
               
                   
                 
                   AACGGTGGTATATCCAGTGATTTTTTTCTCC 
                 
                   
                   
               
               
                   
                 
                   ATTTTAGCTTCCTTAGCTCCTGAAAATCTCGA 
                 
                   
                   
               
               
                   
                 
                   CAACTCAAAAAATACGCCCGGTAGTGATCTT 
                 
                   
                   
               
               
                   
                 
                   ATTTCATTATGGTGAAAGTTGGAACCTCTTA 
                 
                   
                   
               
               
                   
                   CGTGCCGATCA ACGTCTCATTTTCGCCAAAA 
                   
                   
               
               
                   
                 GTTGGCCCAGGGCTTCCCGGTATCAACAGGG 
                   
                   
               
               
                   
                 ACACCAGGATTTATTTATTCTGCGAAGTGAT 
                   
                   
               
               
                   
                 CTTCCGTCACAGGTAGGCGCGCC   
                   
                   
               
               
                   
                 GGAATAG       
                   
                   
               
               
                   
                 GAACTAAGGAGGATATTCATATGGACCATGG 
                   
                   
               
               
                   
                 CTAATTCCCAGGTACCagttgttcttattggtggtgttgcttt 
                   
                   
               
               
                   
                 atggttgcatcgtagtaaatggttgtaacaaaagcaatttttccggctgtct 
                   
                   
               
               
                   
                 gtatacaaaaacgccgcaaagtttgagcgaagtcaataaactctctaccc 
                   
                   
               
               
                   
                 attcagggcaatatctctcttggatccaaagtgaa ctctagaaataattttg   
                   
                   
               
               
                   
                   tttaactttaagaaggagatatacat ATGTCTAACGTGCAG 
                   
                   
               
               
                   
                 GAGTGGCAACAGCTTGCCAACAAGGAATTGA 
                   
                   
               
               
                   
                 GCCGTCGGGAGAAAACTGTCGACTCGCTGGT 
                   
                   
               
               
                   
                 TCATCAAACCGCGGAAGGGATCGCCATCAAG 
                   
                   
               
               
                   
                 CCGCTGTATACCGAAGCCGATCTCGATAATC 
                   
                   
               
               
                   
                 TGGAGGTGACAGGTACCCTTCCTGGTTTGCC 
                   
                   
               
               
                   
                 GCCCTACGTTCGTGGCCCGCGTGCCACTATG 
                   
                   
               
               
                   
                 TATACCGCCCAACCGTGGACCATCCGTCAGT 
                   
                   
               
               
                   
                 ATGCTGGTTTTTCAACAGCAAAAGAGTCCAA 
                   
                   
               
               
                   
                 CGCTTTTTATCGCCGTAACCTGGCCGCCGGG 
                   
                   
               
               
                   
                 CAAAAAGGTCTTTCCGTTGCGTTTGACCTTG 
                   
                   
               
               
                   
                 CCACCCACCGTGGCTACGACTCCGATAACCC 
                   
                   
               
               
                   
                 GCGCGTGGCGGGCGACGTCGGCAAAGCGGG 
                   
                   
               
               
                   
                 CGTCGCTATCGACACCGTGGAAGATATGAAA 
                   
                   
               
               
                   
                 GTCCTGTTCGACCAGATCCCGCTGGATAAAA 
                   
                   
               
               
                   
                 TGTCGGTTTCGATGACCATGAATGGCGCAGT 
                   
                   
               
               
                   
                 GCTACCAGTACTGGCGTTTTATATCGTCGCC 
                   
                   
               
               
                   
                 GCAGAAGAGCAAGGTGTTACACCTGATAAAC 
                   
                   
               
               
                   
                 TGACCGGCACCATTCAAAACGATATTCTCAA 
                   
                   
               
               
                   
                 AGAGTACCTCTGCCGCAACACCTATATTTAC 
                   
                   
               
               
                   
                 CCACCAAAACCGTCAATGCGCATTATCGCCG 
                   
                   
               
               
                   
                 ACATCATCGCCTGGTGTTCCGGCAACATGCC 
                   
                   
               
               
                   
                 GCGATTTAATACCATCAGTATCAGCGGTTAC 
                   
                   
               
               
                   
                 CACATGGGTGAAGCGGGTGCCAACTGCGTG 
                   
                   
               
               
                   
                 CAGCAGGTAGCATTTACGCTCGCTGATGGGA 
                   
                   
               
               
                   
                 TTGAGTACATCAAAGCAGCAATCTCTGCCGG 
                   
                   
               
               
                   
                 ACTGAAAATTGATGACTTCGCTCCTCGCCTG 
                   
                   
               
               
                   
                 TCGTTCTTCTTCGGCATCGGCATGGATCTGT 
                   
                   
               
               
                   
                 TTATGAACGTCGCCATGTTGCGTGCGGCACG 
                   
                   
               
               
                   
                 TTATTTATGGAGCGAAGCGGTCAGTGGATTT 
                   
                   
               
               
                   
                 GGCGCACAGGACCCGAATCACTGGCGCTG 
                   
                   
               
               
                   
                 CGTACCCACTGCCAGACCTCAGGCTGGAGCC 
                   
                   
               
               
                   
                 TGACTGAACAGGATCCGTATAACAACGTTAT 
                   
                   
               
               
                   
                 CCGCACCACCATTGAAGCGCTGGCTGCGACG 
                   
                   
               
               
                   
                 CTGGGCGGTACTCAGTCACTGCATACCAACG 
                   
                   
               
               
                   
                 CCTTTGACGAAGCGCTTGGTTTGCCTACCGA 
                   
                   
               
               
                   
                 TTTCTCAGCACGCATTGCCCGCAACACCCAG 
                   
                   
               
               
                   
                 ATCATCATCCAGGAAGAATCAGAACTCTGCC 
                   
                   
               
               
                   
                 GCACCGTCGATCCACTGGCCGGATCCTATTA 
                   
                   
               
               
                   
                 CATTGAGTCGCTGACCGATCAAATCGTCAAA 
                   
                   
               
               
                   
                 CAAGCCAGAGCTATTATCCAACAGATCGACG 
                   
                   
               
               
                   
                 AAGCCGGTGGCATGGCGAAAGCGATCGAAG 
                   
                   
               
               
                   
                 CAGGTCTGCCAAAACGAATGATCGAAGAGGC 
                   
                   
               
               
                   
                 CTCAGCGCGCGAACAGTCGCTGATCGACCAG 
                   
                   
               
               
                   
                 GGCAAGCGTGTCATCGTTGGTGTCAACAAGT 
                   
                   
               
               
                   
                 ACAAACTGGATCACGAAGACGAAACCGATGT 
                   
                   
               
               
                   
                 ACTTGAGATCGACAACGTGATGGTGCGTAAC 
                   
                   
               
               
                   
                 GAGCAAATTGCTTCGCTGGAACGCATTCGCG 
                   
                   
               
               
                   
                 CCACCCGTGATGATGCCGCCGTAACCGCCGC 
                   
                   
               
               
                   
                 GTTGAACGCCCTGACTCACGCCGCACAGCAT 
                   
                   
               
               
                   
                 AACGAAAACCTGCTGGCTGCCGCTGTTAATG 
                   
                   
               
               
                   
                 CCGCTCGCGTTCGCGCCACCCTGGGTGAAAT 
                   
                   
               
               
                   
                 TTCCGATGCGCTGGAAGTCGCTTTCGACCGT 
                   
                   
               
               
                   
                 TATCTGGTGCCAAGCCAGTGTGTTACCGGCG 
                   
                   
               
               
                   
                 TGATTGCGCAAAGCTATCATCAGTCTGAGAA 
                   
                   
               
               
                   
                 ATCGGCCTCCGAGTTCGATGCCATTGTTGCG 
                   
                   
               
               
                   
                 CAAACGGAGCAGTTCCTTGCCGACAATGGTC 
                   
                   
               
               
                   
                 GTCGCCCGCGCATTCTGATCGCTAAGATGGG 
                   
                   
               
               
                   
                 CCAGGATGGACACGATCGCGGCGCGAAAGT 
                   
                   
               
               
                   
                 GATCGCCAGCGCCTATTCCGATCTCGGTTTC 
                   
                   
               
               
                   
                 GACGTAGATTTAAGCCCGATGTTCTCTACAC 
                   
                   
               
               
                   
                 CTGAAGAGATCGCCCGCCTGGCCGTAGAAAA 
                   
                   
               
               
                   
                 CGACGTTCACGTAGTGGGCGCATCCTCACTG 
                   
                   
               
               
                   
                 GCTGCCGGTCATAAAACGCTGATCCCGGAAC 
                   
                   
               
               
                   
                 TGGTCGAAGCGCTGAAAAAATGGGGACGCG 
                   
                   
               
               
                   
                 AAGATATCTGCGTGGTCGCGGGTGGCGTCAT 
                   
                   
               
               
                   
                 TCCGCCGCAGGATTACGCCTTCCTGCAAGAG 
                   
                   
               
               
                   
                 CGCGGCGTGGCGGCGATTTATGGTCCAGGT 
                   
                   
               
               
                   
                 ACACCTATGCTCGACAGTGTGCGCGACGTAC 
                   
                   
               
               
                   
                 TGAATCTGATAAGCCAGCATCATGATTAATG 
                   
                   
               
               
                   
                 AAGCCACGCTGGCAGAAAGTATTCGCCGCTT 
                   
                   
               
               
                   
                 ACGTCAGGGTGAGCGTGCCACACTCGCCCA 
                   
                   
               
               
                   
                 GGCCATGACGCTGGTGGAAAGCCGTCACCC 
                   
                   
               
               
                   
                 GCGTCATCAGGCACTAAGTACGCAGCTGCTT 
                   
                   
               
               
                   
                 GATGCCATTATGCCGTACTGCGGTAACACCC 
                   
                   
               
               
                   
                 TGCGACTGGGCGTTACCGGCACCCCCGGCG 
                   
                   
               
               
                   
                 CGGGGAAAAGTACCTTTCTTGAGGCCTTTGG 
                   
                   
               
               
                   
                 CATGTTGTTGATTCGAGAGGGATTAAAGGTC 
                   
                   
               
               
                   
                 GCGGTTATTGCGGTCGATCCCAGCAGCCCGG 
                   
                   
               
               
                   
                 TCACTGGCGGTAGCATTCTCGGGGATAAAAC 
                   
                   
               
               
                   
                 CCGCATGAATGACCTGGCGCGTGCCGAAGC 
                   
                   
               
               
                   
                 GGCGTTTATTCGCCCGGTACCATCCTCCGGT 
                   
                   
               
               
                   
                 CATCTGGGCGGTGCCAGTCAGCGAGCGCGG 
                   
                   
               
               
                   
                 GAATTAATGCTGTTATGCGAAGCAGCGGGTT 
                   
                   
               
               
                   
                 ATGACGTAGTGATTGTCGAAACGGTTGGCGT 
                   
                   
               
               
                   
                 CGGGCAGTCGGAAACAGAAGTCGCCCGCAT 
                   
                   
               
               
                   
                 GGTGGACTGTTTTATCTCGTTGCAAATTGCC 
                   
                   
               
               
                   
                 GGTGGCGGCGATGATCTGCAGGGCATTAAA 
                   
                   
               
               
                   
                 AAAGGGCTGATGGAAGTGGCTGATCTGATCG 
                   
                   
               
               
                   
                 TTATCAACAAAGACGATGGCGATAACCATAC 
                   
                   
               
               
                   
                 CAATGTCGCCATTGCCCGGCATATGTACGAG 
                   
                   
               
               
                   
                 AGTGCCCTGCATATTCTGCGACGTAAATACG 
                   
                   
               
               
                   
                 ACGAATGGCAGCCAGGGTTCTGACTTGTAG 
                   
                   
               
               
                   
                 CGCACTGGAAAAACGTGGAATCGATGAGATC 
                   
                   
               
               
                   
                 TGGCACGCCATCATCGACTTCAAAACCGCGC 
                   
                   
               
               
                   
                 TAACTGCCAGTGGTCGTTTACAACAAGTGCG 
                   
                   
               
               
                   
                 GCAACAACAATCGGTGGAATGGCTGCGTAAG 
                   
                   
               
               
                   
                 CAGACCGAAGAAGAAGTACTGAATCACCTGT 
                   
                   
               
               
                   
                 TCGCGAATGAAGATTTCGATCGCTATTACCG 
                   
                   
               
               
                   
                 CCAGACGCTTTTAGCGGTCAAAAACAATACG 
                   
                   
               
               
                   
                 CTCTCACCGCGCACCGGCCTGCGGCAGCTCA 
                   
                   
               
               
                   
                 GTGAATTTATCCAGACGCAATATTTTGATTA 
                   
                   
               
               
                   
                 AAGGAATTTTTATGTCTTATCAGTATGTTAAC 
                   
                   
               
               
                   
                 GTTGTCACTATCAACAAAGTGGCGGTCATTG 
                   
                   
               
               
                   
                 AGTTTAACTATGGCCGAAAACTTAATGCCTT 
                   
                   
               
               
                   
                 AAGTAAAGTCTTTATTGATGATCTTATGCAG 
                   
                   
               
               
                   
                 GCGTTAAGCGATCTCAACCGGCCGGAAATTC 
                   
                   
               
               
                   
                 GCTGTATCATTTTGCGCGCACCGAGTGGATC 
                   
                   
               
               
                   
                 CAAAGTCTTCTCCGCAGGTCACGATATTCAC 
                   
                   
               
               
                   
                 GAACTGCCGTCTGGCGGTCGCGATCCGCTCT 
                   
                   
               
               
                   
                 CCTATGATGATCCATTGCGTCAAATCACCCG 
                   
                   
               
               
                   
                 CATGATCCAAAAATTCCCGAAACCGATCATT 
                   
                   
               
               
                   
                 TCGATGGTGGAAGGTAGTGTTTGGGGTGGC 
                   
                   
               
               
                   
                 GCATTTGAAATGATCATGAGTTCCGATCTGA 
                   
                   
               
               
                   
                 TCATCGCCGCCAGTACCTCAACCTTCTCAAT 
                   
                   
               
               
                   
                 GASGCCTGTAAACCTCGGCGTCCCGTATAAC 
                   
                   
               
               
                   
                 CTGGTCGGCATTCACAACCTGACCCGCGACG 
                   
                   
               
               
                   
                 CGGGCTTCCACATTGTCAAAGAGCTGATTTT 
                   
                   
               
               
                   
                 TACCGCTTCGCCAATCACCGCCCAGCGCGCG 
                   
                   
               
               
                   
                 CTGGCTGTCGGCATCCTCAACCATGTTGTGG 
                   
                   
               
               
                   
                 AAGTGGAAGAACTGGAAGATTTCACCTTACA 
                   
                   
               
               
                   
                 AATGGCGCACCACATCTCTGAGAAAGCGCCG 
                   
                   
               
               
                   
                 TTAGCCATTGCCGTTATCAAAGAAGAGCTGC 
                   
                   
               
               
                   
                 GTGTACTGGGCGAAGCACACACCATGAACTC 
                   
                   
               
               
                   
                 CGATGAATTTGAACGTATTCAGGGGATGCGC 
                   
                   
               
               
                   
                 CGCGCGGTGTATGACAGCGAAGATTACCAG 
                   
                   
               
               
                   
                 GAAGGGATGAACGCTTTCCTCGAAAAACGTA 
                   
                   
               
               
                   
                 AACCTAATTTCGTTGGTCATTAATCCCTGCGA 
                   
                   
               
               
                   
                 ACGAAGGAGTAAAAATGGAAACTCAGTGGAC 
                   
                   
               
               
                   
                 AAGGATGACCGCCAATGAAGCGGCAGAAATT 
                   
                   
               
               
                   
                 ATCCAGCATAACGACATGGTGGCATTTAGCG 
                   
                   
               
               
                   
                 GCTTTACCCCGGCGGGTTCGCCGAAAGCCCT 
                   
                   
               
               
                   
                 ACCCACCGCGATTGCCCGCAGAGCTAACGAA 
                   
                   
               
               
                   
                 CAGCATGAGGCCAAAAAGCCGTATCAAATTC 
                   
                   
               
               
                   
                 GCCTTCTGACGGGTGCGTCAATCAGCGCCGC 
                   
                   
               
               
                   
                 CGCTGACGATGTACTTTCTGACGCCGATGCT 
                   
                   
               
               
                   
                 GTTTCCTGGCGTGCGCCATATCAAACATCGT 
                   
                   
               
               
                   
                 CCGGTTTACGTAAAAAGATCAATCAGGGCGC 
                   
                   
               
               
                   
                 GGTGAGTTTCGTTGACCTGCATTTGAGCGAA 
                   
                   
               
               
                   
                 GTGGCGCAAATGGTCAATTACGGTTTCTTCG 
                   
                   
               
               
                   
                 GCGACATTGATGTTGCCGTCATTGAAGCATC 
                   
                   
               
               
                   
                 GGCACTGGCACCGGATGGTCGAGTCTGGTTA 
                   
                   
               
               
                   
                 ACCAGCGGGATCGGTAATGCGCCGACCTGG 
                   
                   
               
               
                   
                 CTGCTGCGGGCGAAGAAAGTGATCATTGAAC 
                   
                   
               
               
                   
                 TCAATCACTATCACGATCCGCGCGTTGCAGA 
                   
                   
               
               
                   
                 ACTGGCGGATATTGTGATTCCTGGCGCGCCA 
                   
                   
               
               
                   
                 CCGCGGCGCAATAGCGTGTCGATCTTCCATG 
                   
                   
               
               
                   
                 CAATGGATCGCGTCGGTACCCGCTATGTGCA 
                   
                   
               
               
                   
                 AATCGATCCGAAAAAGATTGTCGCCGTCGTG 
                   
                   
               
               
                   
                 GAAACCAACTTGCCCGACGCCGGTAATATGC 
                   
                   
               
               
                   
                 TGGATAAGCAAAATCCCATGTGCCAGCAGAT 
                   
                   
               
               
                   
                 TGCCGATAACGTGGTCACGTTCTTATTGCAG 
                   
                   
               
               
                   
                 GAAATGGCGCATGGGCGTATTCCGCCGGAAT 
                   
                   
               
               
                   
                 TTCTGCCGCTGCAAAGTGGCGTGGGCAATAT 
                   
                   
               
               
                   
                 CAATAATGCGGTAATGGCGCGTCTGGGGGA 
                   
                   
               
               
                   
                 AAACCCGGTAATTCCTCCGTTTATGATGTAT 
                   
                   
               
               
                   
                 TCGGAAGTGCTACAGGAATCGGTGGTGCATT 
                   
                   
               
               
                   
                 TACTGGAAACCGGCAAAATCAGCGGGGCCA 
                   
                   
               
               
                   
                 GCGCCTCCAGCCTGACAATCTCGGCCGATTC 
                   
                   
               
               
                   
                 CCTGCGCAAGATTTACGACAATATGGATTAC 
                   
                   
               
               
                   
                 TTTGCCAGCCGCATTGTGTTGCGTCCGCAGG 
                   
                   
               
               
                   
                 AGATTTCCAATAACCCGGAAATCATCCGTCG 
                   
                   
               
               
                   
                 TCTGGGCGTCATCGCTCTGAACGTCGGCCTG 
                   
                   
               
               
                   
                 GAGTTTGATATTTACGGGCATGCCAACTCAA 
                   
                   
               
               
                   
                 CACACGTAGCCGGGGTCGATCTGATGAACG 
                   
                   
               
               
                   
                 GCATCGGCGGCAGCGGTGATTTTGAACGCAA 
                   
                   
               
               
                   
                 CGCGTATCTGTCGATCTTTATGGCCCCGTCG 
                   
                   
               
               
                   
                 ATTGCTAAAGAAGGCAAGATCTCAACCGTCG 
                   
                   
               
               
                   
                 TGCCAATGTGCAGCCATGTTGATCACAGCGA 
                   
                   
               
               
                   
                 ACACAGCGTCAAAGTGATCATCACCGAACAA 
                   
                   
               
               
                   
                 GGGATCGCCGATCTGCGCGGTCTTTCCCCGC 
                   
                   
               
               
                   
                 TTCAACGCGCCCGCACTATCATTGATAATTG 
                   
                   
               
               
                   
                 TGCACATCCTATGTATCGGGATTATCTGCAT 
                   
                   
               
               
                   
                 CGCTATCTGGAAAATGCGCCTGGCGGACATA 
                   
                   
               
               
                   
                 TTCACCACGATCTTAGCCACGTCTTCGACTT 
                   
                   
               
               
                   
                 ACACCGTAATTTAATTGCAACCGGCTCGATG 
                   
                   
               
               
                   
                 CTGGGTTAA 
                   
                   
               
               
                   
               
               
                 FNRS promoter bold lowercase, with 
                 agttgttcttattggtggtgttgctttatggttgcatcgtagtaaatggttgta 
                   
                   
               
               
                 RBS and leader region bold and 
                 acaaaagcaatttttccggctgtctgtatacaaaaacgccgcaaag   
                   
                   
               
               
                 underlined, and FNR binding site bold 
                    taaactctctacccattcagggcaatatctctcttggatcc 
                   
                   
               
               
                 and italics); sleeping beauty operon 
                 aaagtgaactctagaaataattttatttaaagaaggagatatcat 
                   
                   
               
               
                 (sbm ygflD, ygfG ygfH) bold and 
                 ATGTCTAACGTGCAGGAGTGGCAACAGCTTG 
                   
                   
               
               
                 uppercase 
                 CCAACAAGGAATTGAGCCGTCGGGAGAAAA 
                   
                   
               
               
                 (SEQ ID NO: 184) 
                 CTGTCGACTCGCTGGTTCATCAAACCGCGGA 
                   
                   
               
               
                   
                 AGGGATCGCCATCAAGCCGCTGTATACCGAA 
                   
                   
               
               
                   
                 GCCGATCTCGATAATCTGGAGGTGACAGGTA 
                   
                   
               
               
                   
                 CCCTTCCTGGTTTGCCGCCCTACGTTCGTGG 
                   
                   
               
               
                   
                 CCCGCGTGCCACTATGTATACCGCCCAACCG 
                   
                   
               
               
                   
                 TGGACCATCCGTCAGTATGCTGGTTTTTCAA 
                   
                   
               
               
                   
                 CAGCAAAAGAGTCCAACGCTTTTTATCGCCG 
                   
                   
               
               
                   
                 TAACCTGGCCGCCGGGCAAAAAGGTCTTTCC 
                   
                   
               
               
                   
                 GTTGCGTTTGACCTTGCCACCCACCGTGGCT 
                   
                   
               
               
                   
                 ACGACTCCGATAACCCGCGCGTGGCGGGCG 
                   
                   
               
               
                   
                 ACGTCGGCAAAGCGGGCGTCGCTATCGACA 
                   
                   
               
               
                   
                 CCGTGGAAGATATGAAAGTCCTGTTCGACCA 
                   
                   
               
               
                   
                 GATCCCGCTGGATAAAATGTCGGTTTCGATG 
                   
                   
               
               
                   
                 ACCATGAATGGCGCAGTGCTACCAGTACTGG 
                   
                   
               
               
                   
                 CGTTTTATATCGTCGCCGCAGAAGAGCAAGG 
                   
                   
               
               
                   
                 TGTTACACCTGATAAACTGACCGGCACCATT 
                   
                   
               
               
                   
                 CAAAACGATATTCTCAAAGAGTACCTCTGCC 
                   
                   
               
               
                   
                 GCAACACCTATATTTACCCACCAAAACCGTC 
                   
                   
               
               
                   
                 AATGCGCATTATCGCCGACATCATCGCCTGG 
                   
                   
               
               
                   
                 TGTTCCGGCAACATGCCGCGATTTAATACCA 
                   
                   
               
               
                   
                 TCAGTATCAGCGGTTACCACATGGGTGAAGC 
                   
                   
               
               
                   
                 GGGTGCCAACTGCGTGCAGCAGGTAGCATTT 
                   
                   
               
               
                   
                 ACGCTCGCTGATGGGATTGAGTACATCAAAG 
                   
                   
               
               
                   
                 CAGCAATCTCTGCCGGACTGAAAATTGATGA 
                   
                   
               
               
                   
                 CTTCGCTCCTCGCCTGTCGTTCTTCTTCGGC 
                   
                   
               
               
                   
                 ATCGGCATGGATCTGTTTATGAACGTCGCCA 
                   
                   
               
               
                   
                 TGTTGCGTGCGGCACGTTATTTATGGAGCGA 
                   
                   
               
               
                   
                 AGCGGTCAGTGGATTTGGCGCACAGGACCC 
                   
                   
               
               
                   
                 GAAATCACTGGCGCTGCGTACCCACTGCCAG 
                   
                   
               
               
                   
                 ACCTCAGGCTGGAGCCTGACTGAACAGGATC 
                   
                   
               
               
                   
                 CGTATAACAACGTTATCCGCACCACCATTGA 
                   
                   
               
               
                   
                 AGCGCTGGCTGCGACGCTGGGCGGTACTCA 
                   
                   
               
               
                   
                 GTCACTGCATACCAACGCCTTTGACGAAGCG 
                   
                   
               
               
                   
                 CTTGGTTTGCCTACCGATTTCTCAGCACGCA 
                   
                   
               
               
                   
                 TTGCCCGCAACACCCAGATCATCATCCAGGA 
                   
                   
               
               
                   
                 AGAATCAGAACTCTGCCGCACCGTCGATCCA 
                   
                   
               
               
                   
                 CTGGCCGGATCCTATTACATTGAGTCGCTGA 
                   
                   
               
               
                   
                 CCGATCAAATCGTCAAACAAGCCAGAGCTAT 
                   
                   
               
               
                   
                 TATCCAACAGATCGACGAAGCCGGTGGCATG 
                   
                   
               
               
                   
                 GCGAAAGCGATCGAAGCAGGTCTGCCAAAA 
                   
                   
               
               
                   
                 CGAATGATCGAAGAGGCCTCAGCGCGCGAA 
                   
                   
               
               
                   
                 CAGTCGCTGATCGACCAGGGCAAGCGTGTCA 
                   
                   
               
               
                   
                 TCGTTGGTGTCAACAAGTACAAACTGGATCA 
                   
                   
               
               
                   
                 CGAAGACGAAACCGATGTACTTGAGATCGAC 
                   
                   
               
               
                   
                 AACGTGATGGTGCGTAACGAGCAAATTGCTT 
                   
                   
               
               
                   
                 CGCTGGAACGCATTCGCGCCACCCGTGATGA 
                   
                   
               
               
                   
                 TGCCGCCGTAACCGCCGCGTTGAACGCCCTG 
                   
                   
               
               
                   
                 ACTCACGCCGCACAGCATAACGAAAACCTGC 
                   
                   
               
               
                   
                 TGGCTGCCGCTGTTAATGCCGCTCGCGTTCG 
                   
                   
               
               
                   
                 CGCCACCCTGGGTGAAATTTCCGATGCGCTG 
                   
                   
               
               
                   
                 GAAGTCGCTTTCGACCGTTATCTGGTGCCAA 
                   
                   
               
               
                   
                 GCCAGTGTGTTACCGGCGTGATTGCGCAAAG 
                   
                   
               
               
                   
                 CTATCATCAGTCTGAGAAATCGGCCTCCGAG 
                   
                   
               
               
                   
                 TTCGATGCCATTGTTGCGCAAACGGAGCAGT 
                   
                   
               
               
                   
                 TCCTTGCCGACAATGGTCGTCGCCCGCGCAT 
                   
                   
               
               
                   
                 TCTGATCGCTAAGATGGGCCAGGATGGACAC 
                   
                   
               
               
                   
                 GATCGCGGCGCGAAAGTGATCGCCAGCGCC 
                   
                   
               
               
                   
                 TATTCCGATCTCGGTTTCGACGTAGATTTAA 
                   
                   
               
               
                   
                 GCCCGATGTTCTCTACACCTGAAGAGATCGC 
                   
                   
               
               
                   
                 CCGCCTGGCCGTAGAAAACGACGTTCACGTA 
                   
                   
               
               
                   
                 GTGGGCGCATCCTCACTGGCTGCCGGTCATA 
                   
                   
               
               
                   
                 AAACGCTGATCCCGGAACTGGTCGAAGCGCT 
                   
                   
               
               
                   
                 GAAAAAATGGGGACGCGAAGATATCTGCGT 
                   
                   
               
               
                   
                 GGTCGCGGGTGGCGTCATTCCGCCGCAGGA 
                   
                   
               
               
                   
                 TTACGCCTTCCTGCAAGAGCGCGGCGTGGCG 
                   
                   
               
               
                   
                 GCGATTTATGGTCCAGGTACACCTATGCTCG 
                   
                   
               
               
                   
                 ACAGTGTGCGCGACGTACTGAATCTGATAAG 
                   
                   
               
               
                   
                 CCAGCATCATGATTAATGAAGCCACGCTGGC 
                   
                   
               
               
                   
                 AGAAAGTATTCGCCGCTTACGTCAGGGTGAG 
                   
                   
               
               
                   
                 CGTGCCACACTCGCCCAGGCCATGACGCTGG 
                   
                   
               
               
                   
                 TGGAAAGCCGTCACCCGCGTCATCAGGCACT 
                   
                   
               
               
                   
                 AAGTACGCAGCTGCTTGATGCCATTATGCCG 
                   
                   
               
               
                   
                 TACTGCGGTAACACCCTGCGACTGGGCGTTA 
                   
                   
               
               
                   
                 CCGGCACCCCCGGCGCGGGGAAAAGTACCT 
                   
                   
               
               
                   
                 TTCTTGAGGCCTTTGGCATGTTGTTGATTCG 
                   
                   
               
               
                   
                 AGAGGGATTAAAGGTCGCGGTTATTGCGGTC 
                   
                   
               
               
                   
                 GATCCCAGCAGCCCGGTCACTGGCGGTAGC 
                   
                   
               
               
                   
                 ATTCTCGGGGATAAAACCCGCATGAATGACC 
                   
                   
               
               
                   
                 TGGCGCGTGCCGAAGCGGCGTTTATTCGCCC 
                   
                   
               
               
                   
                 GGTACCATCCTCCGGTCATCTGGGCGGTGCC 
                   
                   
               
               
                   
                 AGTCAGCGAGCGCGGGAATTAATGCTGTTAT 
                   
                   
               
               
                   
                 GCGAAGCAGCGGGTTATGACGTAGTGATTGT 
                   
                   
               
               
                   
                 CGAAACGGTTGGCGTCGGGCAGTCGGAAAC 
                   
                   
               
               
                   
                 AGAAGTCGCCCGCATGGTGGACTGTTTTATC 
                   
                   
               
               
                   
                 TCGTTGCAAATTGCCGGTGGCGGCGATGATC 
                   
                   
               
               
                   
                 TGCAGGGCATTAAAAAAGGGCTGATGGAAGT 
                   
                   
               
               
                   
                 GGCTGATCTGATCGTTATCAACAAAGACGAT 
                   
                   
               
               
                   
                 GGCGATAACCATACCAATGTCGCCATTGCCC 
                   
                   
               
               
                   
                 GGCATATGTACGAGAGTGCCCTGCATATTCT 
                   
                   
               
               
                   
                 GCGACGTAAATACGACGAATGGCAGCCACG 
                   
                   
               
               
                   
                 GGTTCTGACTTGTAGCGCACTGGAAAAACGT 
                   
                   
               
               
                   
                 GGAATCGATGAGATCTGGCACGCCATCATCG 
                   
                   
               
               
                   
                 ACTTCAAAACCGCGCTAACTGCCAGTGGTCG 
                   
                   
               
               
                   
                 TTTACAACAAGTGCGGCAACAACAATCGGTG 
                   
                   
               
               
                   
                 GAATGGCTGCGTAAGCAGACCGAAGAAGAA 
                   
                   
               
               
                   
                 GTACTGAATCACCTGTTCGCGAATGAAGATT 
                   
                   
               
               
                   
                 TCGATCGCTATTACCGCCAGACGCTTTTAGC 
                   
                   
               
               
                   
                 GGTCAAAAACAATACGCTCTCACCGCGCACC 
                   
                   
               
               
                   
                 GGCCTGCGGCAGCTCAGTGAATTTATCCAGA 
                   
                   
               
               
                   
                 CGCAATATTTTGATTAAAGGAATTTTTATGTC 
                   
                   
               
               
                   
                 TTATCAGTATGTTAACGTTGTCACTATCAACA 
                   
                   
               
               
                   
                 AAGTGGCGGTCATTGAGTTTAACTATGGCCG 
                   
                   
               
               
                   
                 AAAACTTAATGCCTTAAGTAAAGTCTTTATTG 
                   
                   
               
               
                   
                 ATGATCTTATGCAGGCGTTAAGCGATCTCAA 
                   
                   
               
               
                   
                 CCGGCCGGAAATTCGCTGTATCATTTTGCGC 
                   
                   
               
               
                   
                 GCACCGAGTGGATCCAAAGTCTTCTCCGCAG 
                   
                   
               
               
                   
                 GTCACGATATTCACGAACTGCCGTCTGGCGG 
                   
                   
               
               
                   
                 TCGCGATCCGCTCTCCTATGATGATCCATTG 
                   
                   
               
               
                   
                 CGTCAAATCACCCGCATGATCCAAAAATTCC 
                   
                   
               
               
                   
                 CGAAACCGATCATTTCGATGGTGGAAGGTAG 
                   
                   
               
               
                   
                 TGTTTGGGGTGGCGCATTTGAAATGATCATG 
                   
                   
               
               
                   
                 AGTTCCGATCTGATCATCGCCGCCAGTACCT 
                   
                   
               
               
                   
                 CAACCTTCTCAATGACGCCTGTAAACCTCGG 
                   
                   
               
               
                   
                 CGTCCCGTATAACCTGGTCGGCATTCACAAC 
                   
                   
               
               
                   
                 CTGACCCGCGACGCGGGCTTCCACATTGTCA 
                   
                   
               
               
                   
                 AAGAGCTGATTTTTACCGCTTCGCCAATCAC 
                   
                   
               
               
                   
                 CGCCCAGCGCGCGCTGGCTGTCGGCATCCTC 
                   
                   
               
               
                   
                 AACCATGTTGTGGAAGTGGAAGAACTGGAAG 
                   
                   
               
               
                   
                 ATTTCACCTTACAAATGGCGCACCACATCTC 
                   
                   
               
               
                   
                 TGAGAAAGCGCCGTTAGCCATTGCCGTTATC 
                   
                   
               
               
                   
                 AAAGAAGAGCTGCGTGTACTGGGCGAAGCA 
                   
                   
               
               
                   
                 CACACCATGAACTCCGATGAATTTGAACGTA 
                   
                   
               
               
                   
                 TTCAGGGGATGCGCCGCGCGGTGTATGACA 
                   
                   
               
               
                   
                 GCGAAGATTACCAGGAAGGGATGAACGCTTT 
                   
                   
               
               
                   
                 CCTCGAAAAACGTAAACCTAATTTCGTTGGT 
                   
                   
               
               
                   
                 CATTAATCCCTGCGAACGAAGGAGTAAAAATG 
                   
                   
               
               
                   
                 GAAACTCAGTGGACAAGGATGACCGCCAATG 
                   
                   
               
               
                   
                 AAGCGGCAGAAATTATCCAGCATAACGACAT 
                   
                   
               
               
                   
                 GGTGGCATTTAGCGGCTTTACCCCGGCGGGT 
                   
                   
               
               
                   
                 TCGCCGAAAGCCCTACCCACCGCGATTGCCC 
                   
                   
               
               
                   
                 GCAGAGCTAACGAACAGCATGAGGCCAAAA 
                   
                   
               
               
                   
                 AGCCGTATCAAATTCGCCTTCTGACGGGTGC 
                   
                   
               
               
                   
                 GTCAATCAGCGCCGCCGCTGACGATGTACTT 
                   
                   
               
               
                   
                 TCTGACGCCGATGCTGTTTCCTGGCGTGCGC 
                   
                   
               
               
                   
                 CATATCAAACATCGTCCGGTTTACGTAAAAA 
                   
                   
               
               
                   
                 GATCAATCAGGGCGCGGTGAGTTTCGTTGAC 
                   
                   
               
               
                   
                 CTGCATTTGAGCGAAGTGGCGCAAATGGTCA 
                   
                   
               
               
                   
                 ATTACGGTTTCTTCGGCGACATTGATGTTGC 
                   
                   
               
               
                   
                 CGTCATTGAAGCATCGGCACTGGCACCGGAT 
                   
                   
               
               
                   
                 GGTCGAGTCTGGTTAACCAGCGGGATCGGTA 
                   
                   
               
               
                   
                 ATGCGCCGACCTGGCTGCTGCGGGCGAAGA 
                   
                   
               
               
                   
                 AAGTGATCATTGAACTCAATCACTATCACGA 
                   
                   
               
               
                   
                 TCCGCGCGTTGCAGAACTGGCGGATATTGTG 
                   
                   
               
               
                   
                 ATTCCTGGCGCGCCACCGCGGCGCAATAGC 
                   
                   
               
               
                   
                 GTGTCGATCTTCCATGCAATGGATCGCGTCG 
                   
                   
               
               
                   
                 GTACCCGCTATGTGCAAATCGATCCGAAAAA 
                   
                   
               
               
                   
                 GATTGTCGCCGTCGTGGAAACCAACTTGCCC 
                   
                   
               
               
                   
                 GACGCCGGTAATATGCTGGATAAGCAAAATC 
                   
                   
               
               
                   
                 CCATGTGCCAGCAGATTGCCGATAACGTGGT 
                   
                   
               
               
                   
                 CACGTTCTTATTGCAGGAAATGGCGCATGGG 
                   
                   
               
               
                   
                 CGTATTCCGCCGGAATTTCTGCCGCTGCAAA 
                   
                   
               
               
                   
                 GTGGCGTGGGCAATATCAATAATGCGGTAAT 
                   
                   
               
               
                   
                 GGCGCGTCTGGGGGAAAACCCGGTAATTCCT 
                   
                   
               
               
                   
                 CCGTTTATGATGTATTCGGAAGTGCTACAGG 
                   
                   
               
               
                   
                 AATCGGTGGTGCATTTACTGGAAACCGGCAA 
                   
                   
               
               
                   
                 AATCAGCGGGGCCAGCGCCTCCAGCCTGAC 
                   
                   
               
               
                   
                 AATCTCGGCCGATTCCCTGCGCAAGATTTAC 
                   
                   
               
               
                   
                 GACAATATGGATTACTTTGCCAGCCGCATTG 
                   
                   
               
               
                   
                 TGTTGCGTCCGCAGGAGATTTCCAATAACCC 
                   
                   
               
               
                   
                 GGAAATCATCCGTCGTCTGGGCGTCATCGCT 
                   
                   
               
               
                   
                 CTGAACGTCGGCCTGGAGTTTGATATTTACG 
                   
                   
               
               
                   
                 GGCATGCCAACTCAACACACGTAGCCGGGGT 
                   
                   
               
               
                   
                 CGATCTGATGAACGGCATCGGCGGCAGCGG 
                   
                   
               
               
                   
                 TGATTTTGAACGCAACGCGTATCTGTCGATC 
                   
                   
               
               
                   
                 TTTATGGCCCCGTCGATTGCTAAAGAAGGCA 
                   
                   
               
               
                   
                 AGATCTCAACCGTCGTGCCAATGTGCAGCCA 
                   
                   
               
               
                   
                 TGTTGATCACAGCGAACACAGCGTCAAAGTG 
                   
                   
               
               
                   
                 ATCATCACCGAACAAGGGATCGCCGATCTGC 
                   
                   
               
               
                   
                 GCGGTCTTTCCCCGCTTCAACGCGCCCGCAC 
                   
                   
               
               
                   
                 TATCATTGATAATTGTGCACATCCTATGTATC 
                   
                   
               
               
                   
                 GGGATTATCTGCATCGCTATCTGGAAAATGC 
                   
                   
               
               
                   
                 GCCTGGCGGACATATTCACCACGATCTTAGC 
                   
                   
               
               
                   
                 CACGTCTTCGACTTACACCGTAATTTAATTG 
                   
                   
               
               
                   
                 CAACCGGCTCGATGCTGGGTTAA 
               
               
                   
               
            
           
         
       
     
     Next, this strain was tested for propionate production. 
     Briefly, 3 ml LB (containing selective antibiotics (cam) where necessary was inoculated from frozen glycerol stocks with either wild type  E. coli  K12 or the genetically engineered bacteria comprising the chromosomal sleeping beauty mutase operon under the control of a FNR promoter. Bacteria were grown overnight at 37 C with shaking. Overnight cultures were diluted 1:100 into 10 ml LB in a 125 ml baffled flask. Cultures were grown aerobically at 37 C with shaking for about 1.5 h, and then transferred to the anaerobic chamber at 37 C for 4 h. Bacteria (2×10 8  CFU) were added to 1 ml M9 media containing 50 mM MOPS with 0.5% glucose in microcentrifuge tubes. Cells were plated to determine cell counts. The assay tubes were placed in the anaerobic chamber at 37 C. At 1, 2, and 24 hours, 120 ul of cells were removed and pelleted at 14,000 rpm for 1 min, and 100 ul of the supernatant was transferred to a 96-well assay plate and sealed with aluminum foil, and stored at −80 C until analysis by LC-MS for propionate concentrations, as described in 
     Results are depicted in  FIG. 29  and show that the genetically engineered strain produces ˜2.5 mM after 24 h, while very little or no propionate production was detected from the  E. coli  K12 wild type strain. Propionate was measured as described in Example 25. 
     Example 23. Evaluation of the Sleeping Beauty Mutase Pathway for the Production of Propionate in  E. coli  Nissle 
     Next, the SBM pathway is evaluated for propionate production in  E. coli  Nissle. Nissle does not have the full 4-gene sleeping beauty mutase operon; it only has the first gene and a partial gene of the second, and genes 3 and 4 are missing. Therefore, recombineering is used to introduce this pathway into Nissle. The frt-cam-frt-PfnrS-sbm, ygfD, ygfG, ygfH construct is inserted at the location of the endogenous, truncated Nissle SBM. Next, the construct is transformed into  E. coli  Nissle and tested for propionate production essentially as described above. 
     Example 24. Evaluation of the Acrylate Pathway from  Clostridium propionicum  for Propionate Production 
     The acrylate pathway from  Clostridium propionicum  is evaluated for adaptation to propionate production in  E. coli . A construct (Ptet-pct-lcdABC-acrABC), codon optimized for  E. coli , is synthesized by Genewiz and placed in a high copy plasmid (Logic51). Additionally, another construct is generated for side by side testing, in which the acrABC genes (which may be the rate limiting step of the pathway) are replaced with the acuI gene from  Rhodobacter sphaeroides  (Ptet-acuI-pct-lcdABC). Subsequently these constructs are transformed into BW25113 and are assessed for their ability to produce propionate, as compared to the type BW5113 strain as described above in Example 24. Propionate was measured as described in Example 27. 
     of Exemplary Propionate Cassette Sequences 
       
     
       
         
           
               
               
             
               
                 TABLE 59 
               
               
                   
               
               
                 Description and SEQ ID NO 
                 Sequence 
               
               
                   
               
             
            
               
                 Ptet-pct-lcdABC-acrABC; 
                 ttaagacccactttcacatttaagttgatttctaatccgcatatgatcaattcaaggccgaataagaa 
               
               
                 Ptet: lower case; tertR/tetA 
                 ggctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcatactatcag 
               
               
                 promoter within Ptet: 
                 tagtaggtgtuccattcactttagcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaa 
               
               
                 lower case bold, with tet 
                 tgccccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcataaaaaggctaa 
               
               
                 operator: lower case bold 
                 ttgattttcgagagtttcatactgtttttctgtaggccgtgtacctaaatgtacttttgctccatcgc 
               
               
                 underlined; ribosome 
                 gatgacttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgccagattcccctt 
               
               
                 binding site and leader:  
                 ctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagcccgc 
               
               
                 lower case italic; ribosome 
                 ttattttttacatgccaatacaatgtaggctgctctacacctagcttctgggcgagmacgggttgtta 
               
               
                 binding sites: lower case 
                 aaccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatcta 
               
               
                 underlined; coding regions: 
                 gacatcattaattcctaattttt gttgac     actctatcattgatagagagt     tattttaccac     tccctat     
               
               
                 upper case; (SEQ ID NO: 185) 
                     cagtgatagag     aa aagtgaa ctctagaaataattttgtttaactttaa     gaaggagatatacat   ATGCG 
               
               
                   
                 CAAAGTGCCGATTATCACGGCTGACGAGGCCGCAAAACTGATCAAGGACGGCGACACCGTGACAACTA 
               
               
                   
                 GCGGCTTTGTGGGTAACGCGATCCCTGAGGCCCTTGACCGTGCAGTCGAAAAGCGTTTCCTGGAAACG 
               
               
                   
                 GGCGAACCGAAGAACATTACTTATGTATATTGCGGCAGTCAGGGCAATCGCGACGGTCGTGGCGCAGA 
               
               
                   
                 ACATTTCGCGCATGAAGGCCTGCTGAAACGTTATATCGCTGGCCATTGGGCGACCGTCCCGGCGTTAG 
               
               
                   
                 GGAAAATGGCCATGGAGAATAAAATGGAGGCCTACAATGTCTCTCAGGGCGCCTTGTGTCATCTCTTT 
               
               
                   
                 CGCGATATTGCGAGCCATAAACCGGGTGTGTTCACGAAAGTAGGAATCGGCACCTTCATTGATCCACG 
               
               
                   
                 TAACGGTGGTGGGAAGGTCAACGATATTACCAAGGAAGATATCGTAGAACTGGTGGAAATTAAAGGGC 
               
               
                   
                 AGGAATACCTGTTTTATCCGGCGTTCCCGATCCATGTCGCGCTGATTCGTGGCACCTATGCGGACGAG 
               
               
                   
                 AGTGGTAACATCACCTTTGAAAAAGAGGTAGCGCCTTTGGAAGGGACTTCTGTCTGTCAAGCGGTGAA 
               
               
                   
                 GAACTCGGGTGGCATTGTCGTGGTTCAGGTTGAGCGTGTCGTCAAAGCAGGCACGCTGGATCCGCGCC 
               
               
                   
                 ATGTGAAAGTTCCGGGTATCTATGTAGATTACGTAGTCGTCGCGGATCCGGAGGACCATCAACAGTCC 
               
               
                   
                 CTTGACTGCGAATATGATCCTGCCCTTAGTGGAGAGCACCGTCGTCCGGAGGTGGTGGGTGAACCACT 
               
               
                   
                 GCCTTTATCCGCGAAGAAAGTCATCGGCCGCCGTGGCGCGATTGAGCTCGAGAAAGACGTTGCAGTGA 
               
               
                   
                 ACCTTGGGGTAGGTGCACCTGAGTATGTGGCCTCCGTGGCCGATGAAGAAGGCATTGTGGATTTTATG 
               
               
                   
                 ACTCTCACAGCGGAGTCCGGCGCTATCGGTGGCGTTCCAGCCGGCGGTGTTCGCTTTGGGGCGAGCTA 
               
               
                   
                 CAATGCTGACGCCTTGATCGACCAGGGCTACCAATTTGATTATTACGACGGTGGGGGTCTGGATCTTT 
               
               
                   
                 GTTACCTGGGTTTAGCTGAATGCGACGAAAAGGGTAATATCAATGTTAGCCGCTTCGGTCCTCGTATC 
               
               
                   
                 GCTGGGTGCGGCGGATTCATTAACATTACCCAAAACACGCCGAAAGTCTTCTTTTGTGGGACCTTTAC 
               
               
                   
                 AGCCGGGGGGCTGAAAGTGAAAATTGAAGATGGTAAGGTGATTATCGTTCAGGAAGGGAAACAGAAGA 
               
               
                   
                 AATTCCTTAAGGCAGTGGAGCAAATCACCTTTAATGGAGACGTGGCCTTAGCGAACAAGCAACAAGTT 
               
               
                   
                 ACCTACATCACGGAGCGTTGCGTCTTCCTCCTCAAAGAAGACGGTTTACACCTTTCGGAAATCGCGCC 
               
               
                   
                 AGGCATCGATCTGCAGACCCAGATTTTGGATGTTATGGACTTTGCCCCGATCATTGATCGTGACGCAA 
               
               
                   
                 ACGGGCAGATTAAACTGATGGACGCGGCGTTATTCGCAGAAGGGCTGATGGGCTTGAAAGAAATGAAG 
               
               
                   
                 TCTTGAtaa   gaaggagatatacat   ATGAGMAACCCAAGGCATGAAAGCTAAACAACTGTTAGCATACT 
               
               
                   
                 TTCAGGGTAAAGCCGATCAGGATGCACGTGAAGCGAAAGCCCGCGGTGAGCTGGTCTGCTGGTCGGCG 
               
               
                   
                 TCAGTCGCGCCGCCGGAATTTTGCGTAACAATGGGCATTGCCATGATCTACCCGGAGACTCATGCAGC 
               
               
                   
                 GGGCATCGGTGCCCGCAAAGGTGCGATGGACATGCTGGAAGTTGCGGACCGCAAAGGCTACAACGTGG 
               
               
                   
                 ATTGTTGTTCCTACGGCCGTGTAAATATGGGTTACATGGAATGTTTAAAAGAAGCCGCCATCACGGGC 
               
               
                   
                 GTCAAGCCGGAAGTTTTGGTTAATTCCCCTGCTGCTGACGTTCCGCTTCCCGATTTGGTGATTACGTG 
               
               
                   
                 TAATAATATCTGTAACACGCTGCTGAAATGGTACGAAAACTTAGCAGCAGAACTCGATATTCCTTGCA 
               
               
                   
                 TCGTGATCGACGTACCGTTTAATCATACCATGCCGATTCCGGAATATGCCAAGGCCTACATCGCGGAC 
               
               
                   
                 CAGTTCCGCAATGCAATTTCTCAGCTGGAAGTTATTTGTGGCCGTCCGTTCGATTGGAAGAAATTTAA 
               
               
                   
                 GGAGGTCAAAGATCAGACCCAGCGTAGCGTATACCACTGGAACCGCATTGCCGAGATGGCGAAATACA 
               
               
                   
                 AGCCTAGCCCGCTGAACGGCTTCGATCTGTTCAATTACATGGCGTTAATCGTGGCGTGCCGCAGCCTG 
               
               
                   
                 GATTATGCAGAAATTACCTTTAAAGCGTTCGCGGACGAATTAGAAGAGAATTTGAAGGCGGGTATCTA 
               
               
                   
                 CGCCTTTAAAGGTGCGGAAAAAACGCGCTTTCAATGGGAAGGTATCGCGGTGTGGCCACATTTAGGTC 
               
               
                   
                 ACACGTTTAAATCTATGAAGAATCTGAATTCGATTATGACCGGTACGGCATACCCCGCCCTTTGGGAC 
               
               
                   
                 CTGCACTATGACGCTAACGACGAATCTATGCACTCTATGGCTGAAGCGTACACCCGTATTTATATTAA 
               
               
                   
                 TACTTGTCTGCAGAACAAAGTAGAGGTCCTGCTTGGGATCATGGAAAAAGGCCAGGTGGATGGTACCG 
               
               
                   
                 TATATCATCTGAATCGCAGCTGCAAACTGATGAGTTTCCTGAACGTGGAAACGGCTGAAATTATTAAA 
               
               
                   
                 GAGAAGAACGGTCTTCCTTACGTCTCCATTGATGGCGATCAGACCGATCCTCGCGTTTTTTCTCCGGC 
               
               
                   
                 CCAGTTTGATACCCGTGTTCAGGCCCTGGTTGAGATGATGGAGGCCAATATGGCGGCAGCGGAATAAt 
               
               
                   
                 aa   gaaggagatatacat   ATGTCACGCGTGGAGGCAATCCTGTCGCAGCTGAAAGATGTCGCCGCGAAT 
               
               
                   
                 CCGAAAAAAGCCATGGATGACTATAAAGCTGAAACAGGTAAGGGCGCGGTTGGTATCATGCCGATCTA 
               
               
                   
                 CAGCCCCGAAGAAATGGTACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGCGCCCAGGGCAAAA 
               
               
                   
                 CGATTAGTAAAGCGCGCACCTATCTGCCTGCTTTTGCCTGCAGCGTAATGCAGCAGGTTATGGAATTA 
               
               
                   
                 CAGTGCGAGGGCGCGTATGATGACCTGTCCGCAGTTATTTTTAGCGTACCGTGCGACACTCTCAAATG 
               
               
                   
                 TCTTAGCCAGAAATGGAAAGGTACGTCCCCAGTGATTGTATTTACGCATCCGCAGAACCGCGGATTAG 
               
               
                   
                 AAGCGGCGAACCAATTCTTGGTTACCGAGTATGAACTGGTAAAAGCACAACTGGAATCAGTTCTGGGT 
               
               
                   
                 GTGAAAATTTCAAACGCCGCCCTGGAAAATTCGATTGCAATTTATAACGAGAATCGTGCCGTGATGCG 
               
               
                   
                 TGAGTTCGTGAAAGTGGCAGCGGACTATCCTCAAGTCATTGACGCAGTGAGCCGCCACGCGGTTTTTA 
               
               
                   
                 AAGCGCGCCAGTTTATGCTTAAGGAAAAACATACCGCACTTGTGAAAGAACTGATCGCTGAGATTAAA 
               
               
                   
                 GCAACGCCAGTCCAGCCGTGGGACGGAAAAAAGGTTGTAGTGACGGGCATTCTGTTGGAACCGAATGA 
               
               
                   
                 GTTATTAGATATCTTTAATGAGTTTAAGATCGCGATTGTTGATGATGATTTAGCGCAGGAAAGCCGTC 
               
               
                   
                 AGATCCGTGTTGACGTTCTGGACGGAGAAGGCGGACCGCTCTACCGTATGGCTAAAGCGTGGCAGCAA 
               
               
                   
                 ATGTATGGCTGCTCGCTGGCAACCGACACCAAGAAGGGTCGCGGCCCTATGTTAATTAACAAAACGAT 
               
               
                   
                 TCAGACCGGTGCGGACGCTATCGTAGTTGCAATGATGAAGTITTGCGACCCAGAAGAATGGGATTATC 
               
               
                   
                 CGGTAATGTACCGTGAATTTGAAGAAAAAGGGGTCAAATCACTTATGATTGAGGTGGATCAGGAAGTA 
               
               
                   
                 TCGTCTTTCGAACAGATTAAAACCCGTCTGCAGTCATTCGTCGAAATGCTTTAAtaag   aaggagatat     
               
               
                   
                     acat   ATGTATACCTTGGGGATTGATGTCGGTTCTGCCTCTAGTAAAGCGGTGATTCTGAAAGATGGAA 
               
               
                   
                 AAGATATTGTCGCTGCCGAGGTTGTCCAAGTCGGTACCGGCTCCTCGGGTCCCCAACGCGCACTGGAC 
               
               
                   
                 AAAGCCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACATCAGCTACACAGTAGCTACGGGCTATGGGCG 
               
               
                   
                 CTTCAATTTTAGCGACGCGGATAAACAGATTTCGGAAATTAGCTGTCATGCCAAAGGCATTTATTTCT 
               
               
                   
                 TAGTACCAACTGCGCGCACTATTATTGACATTGGCGGCCAAGATGCGAAAGCCATCCGCCTGGACGAC 
               
               
                   
                 AAGGGGGGTATTAAGCAATTCTTCATGAATGATAAATGCGCGGCGGGCACGGGGCGTTTCCTGGAAGT 
               
               
                   
                 CATGGCTCGCGTACTTGAAACCACCCTGGATGAAATGGCTGAACTGGATGAACAGGCGACTGACACCG 
               
               
                   
                 CTCCCATTTCAAGCACCTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAATTGAGCAATGGTGTC 
               
               
                   
                 TCACGCAACAACATCATTAAAGGTGTCCATCTGAGCGTTGCGTCACGTGCGTGTGGTCTGGCGTATCG 
               
               
                   
                 CGGCGGTTTGGAGAAAGATGTTGTTATGACAGGTGGCGTGGCAAAAAATGCAGGGGTGGTGCGCGCGG 
               
               
                   
                 TGGCGGGCGTTCTGAAGACCGATGTTATCGTTGCTCCGAATCCTCAGACGACCGGTGCACTGGGGGCA 
               
               
                   
                 GCGCTGTATGCTTATGAGGCCGCCCAGAAGAAGTAAtaa   gaaggagatatacat   ATGGCCTTCAATAG 
               
               
                   
                 CGCAGATATTAATTCTTTCCGCGATATTTGGTGTTTTGTGAACAGCGTGAGGGCAAACTGATTAACAC 
               
               
                   
                 CGATTTCGAATTAATTAGCGAAGGTCGTAAACTGGCTGACGAACGCGGAAGCAAACTGGTTGGAATTT 
               
               
                   
                 TGCTGGGGCACGAAGTTGAAGAAATCGCAAAAGAATTAGGCGGCTATGGTGCGGACAAGGTAATTGTG 
               
               
                   
                 TGCGATCATCCGGAACTTAAATTTTACACTACGGATGCTTATGCCAAAGTTTTATGTGACGTCGTGAT 
               
               
                   
                 GGAAGAGAAACCGGAGGTAATTTTGATCGGTGCCACCAACATTGGCCGTGATCTCGGACCGCGTTGTG 
               
               
                   
                 CTGCACGCTTGCACACGGGGCTGACGGCTGATTGCACGCACCTGGATATTGATATGAATAAATATGTG 
               
               
                   
                 GACTTTCTTAGCACCAGTAGCACCTTGGATATCTCGTCGATGACTTTCCCTATGGAAGATACAAACCT 
               
               
                   
                 TAAAATGACGCGCCCTGCATTTGGCGGACATCTGATGGCAACGATCATTTGTCCACGCTTCCGTCCCT 
               
               
                   
                 GTATGAGCACAGTGCGCCCCGGAGTGATGAAGAAAGCGGAGTTCTCGCAGGAGATGGCGCAAGCATGT 
               
               
                   
                 CAAGTAGTGACCCGTCACGTAAATTTGTCGGATGAAGACCTTAAAACTAAAGTAATTAATATCGTGAA 
               
               
                   
                 GGAAACGAAAAAGATTGTGGATCTGATCGGCGCAGAAATTATTGTGTCAGTTGGTCGTGGTATCTCGA 
               
               
                   
                 AAGATGTCCAAGGTGGAATTGCACTGGCTGAAAAACTTGCGGACGCATTTGGTAACGGTGTCGTGGGC 
               
               
                   
                 GGCTCGCGCGCAGTGATTGATTCCGGCTGGTTACCTGCGGATCATCAGGTTGGACAAACCGGTAAGAC 
               
               
                   
                 CGTGCACCCGAAAGTCTACGTGGCGCTGGGTATTAGTGGGGCTATCCAGCATAAGGCTGGGATGCAAG 
               
               
                   
                 ACTCTGAACTGATCATTGCCGTCAACAAAGACGAAACGGCGCCTATCTTCGACTGCGCCGATTATGGC 
               
               
                   
                 ATCACCGGTGATTTATTTAAAATCGTACCGATGATGATCGACGCGATCAAAGAGGGTAAAAACGCATG 
               
               
                   
                 Ataa   gaaggagatatacat   ATGCGCATCTATGTGTGTGTGAAACAAGTCCCAGATACGAGCGGCAAGG 
               
               
                   
                 TGGCCGTTAACCCTGATGGGACCCTTAACCGTGCCTCAATGGCAGCGATTATTAACCCGGACGATATG 
               
               
                   
                 TCCGCGATCGAACAGGCATTAAAACTGAAAGATGAAACCGGATGCCAGGTTACGGCGCTTACGATGGG 
               
               
                   
                 TCCTCCTCCTGCCGAGGGCATGTTGCGCGAAATTATTGCAATGGGGGCCGACGATGGTGTGCTGATTT 
               
               
                   
                 CGGCCCGTGAATTTGGGGGGTCCGATACCTTCGCAACCAGTCAAATTATTAGCGCGGCAATCCATAAA 
               
               
                   
                 TTAGGCTTAAGCAATGAAGACATGATCTTTTGCGGTCGTCAGGCCATTGACGGTGATACGGCCCAAGT 
               
               
                   
                 CGGCCCTCAAATTGCCGAAAAACTGAGCATCCCACAGGTAACCTATGGCGCAGGAATCAAAAAATCTG 
               
               
                   
                 GTGATTTAGTGCTGGTGAAGCGTATGTTGGAGGATGGTTATATGATGATCGAAGTCGAAACTCCATGT 
               
               
                   
                 CTGATTACCTGCATTCAGGATAAAGCGGTAAAACCACGTTACATGACTCTCAACGGTATTATGGAATG 
               
               
                   
                 CTACTCCAAGCCGCTCCTCGTTCTCGATTACGAAGCACTGAAAGATGAACCGCTGATCGAACTTGATA 
               
               
                   
                 CCATTGGGCTTAAAGGCTCCCCGACGAATATCTTTAAATCGTTTACGCCGCCTCAGAAAGGCGTTGGT 
               
               
                   
                 GTCATGCTCCAAGGCACCGATAAGGAAAAAGTCGAGGATCTGGTGGATAAGCTGATGCAGAAACATGT 
               
               
                   
                 CATCTAAtaa   gaaggagatatacat   ATGTTCTTACTGAAGATTAAAAAAGAACGTATGAAACGCATGG 
               
               
                   
                 ACTTTAGTTTAACGCGTGAACAGGAGATGTTAAAAAAACTGGCGCGTCAGTTTGCTGAGATCGAGCTG 
               
               
                   
                 GAACCGGTGGCCGAAGAGATTGATCGTGAGCACGTTTTTCCTGCAGAAAACTTTAAGAAGATGGCGGA 
               
               
                   
                 AATTGGCTTAACCGGCATTGGTATCCCGAAAGAATTTGGTGGCTCCGGTGGAGGCACCCTGGAGAAGG 
               
               
                   
                 TCATTGCCGTGTCAGAATTCGGCAAAAAGTGTATGGCCTCAGCTTCCATTTTAAGCATTCATCTTATC 
               
               
                   
                 GCGCCGCAGGCAATCTACAAATATGGGACCAAAGAACAGAAAGAGACGTACCTGCCGCGTCTTACCAA 
               
               
                   
                 AGGTGGTGAACTGGGCGCCTTTGCGCTGACAGAACCAAACGCCGGAAGCGATGCCGGCGCGGTAAAAA 
               
               
                   
                 CGACCGCGATTCTGGACAGCCAGACAAACGAGTACGTGCTGAATGGCACCAAATGCTTTATCAGCGGG 
               
               
                   
                 GGCGGGCGCGCGGGTGTTCTTGTAATTTTTGCGCTTACTGAACCGAAAAAAGGTCTGAAAGGGATGAG 
               
               
                   
                 CGCGATTATCGTGGAGAAAGGGACCCCGGGCTTCAGCATCGGCAAGGTGGAGAGCAAGATGGGGATCG 
               
               
                   
                 CAGGTTCGGAAACCGCGGAACTTATCTTCGAAGATTGTCGCGTTCCGGCTGCCAACCTTTTAGGTAAA 
               
               
                   
                 GAAGGCAAAGGCTTTAAAATTGCTATGGAAGCCCTGGATGGCGCCCGTATTGGCGTGGGCGCTCAAGC 
               
               
                   
                 AATCGGAATTGCCGAGGGGGCGATCGACCTGAGTGTGAAGTACGTTCACGAGCGCATTCAATTTGGTA 
               
               
                   
                 AACCGATCGCGAATCTGCAGGGAATTCAATGGTATATCGCGGATATGGCGACCAAAACCGCCGCGGCA 
               
               
                   
                 CGCGCACTTGTTGAGTTTGCAGCGTATCTTGAAGACGCGGGTAAACCGTTCACAAAGGAATCTGCTAT 
               
               
                   
                 GTGCAAGCTGAACGCCTCCGAAAACGCGCGTTTTGTGACAAATTTAGCTCTGCAGATTCACGGGGGTT 
               
               
                   
                 ACGGTTATATGAAAGATTATCCGTTAGAGCGTATGTATCGCGATGCTAAGATTACGGAAATTTACGAG 
               
               
                   
                 GGGACATCAGAAATCCATAAGGTGGTGATTGCGCGTGAAGTAATGAAACGCTAA 
               
               
                   
               
               
                 pct-lcdABC-acrABC 
                 ATGCGCAAAGTGCCGATTATCACGGCTGACGAGGCCGCAAAACTGATCAAGGACGGCGACACCGTGAC 
               
               
                 (ribosome binding sites: 
                 AACTAGCGGCTTTGTGGGTAACGCGATCCCTGAGGCCCTTGACCGTGCAGTCGAAAAGCGTTTCCTGG 
               
               
                 lower case underlined; 
                 AAACGGGCGAACCGAAGAACATTACTTATGTATATTGCGGCAGTCAGGGCAATCGCGACGGTCGTGGC 
               
               
                 coding regions: upper case) 
                 GCAGAACATTTCGCGCATGAAGGCCTGCTGAAACGTTATATCGCTGGCCATTGGGCGACCGTCCCGGC 
               
               
                 (SEQ ID NO: 186) 
                 GTTAGGGAAAATGGCCATGGAGAATAAAATGGAGGCCTACAATGTCTCTCAGGGCGCCTTGTGTCATC 
               
               
                   
                 TCTTTCGCGATATTGCGAGCCATAAACCGGGTGTGTTCACGAAAGTAGGAATCGGCACCTTCATTGAT 
               
               
                   
                 CCACGTAACGGTGGTGGGAAGGTCAACGATATTACCAAGGAAGATATCGTAGAACTGGTGGAAATTAA 
               
               
                   
                 AGGGCAGGAATACCTGTTTTATCCGGCGTTCCCGATCCATGTCGCGCTGATTCGTGGCACCTATGCGG 
               
               
                   
                 ACGAGAGTGGTAACATCACCTTTGAAAAAGAGGTAGCGCCTTTGGAAGGGACTTCTGTCTGTCAAGCG 
               
               
                   
                 GTGAAGAACTCGGGIGGCATTGTCGTGGTTCAGGTTGAGCGTGTCGTCAAAGCAGGCACGCTGGATCC 
               
               
                   
                 GCGCCATGTGAAAGTTCCGGGTATCTATGTAGATTACGTAGTCGTCGCGGATCCGGAGGACCATCAAC 
               
               
                   
                 AGTCCCTTGACTGCGAATATGATCCTGCCCTTAGTGGAGAGCACCGTCGTCCGGAGGTGGTGGGTGAA 
               
               
                   
                 CCACTGCCTTTATCCGCGAAGAAAGTCATCGGCCGCCGTGGCGCGATTGAGCTCGAGAAAGACGTTGC 
               
               
                   
                 AGTGAACCTTGGGGTAGGTGCACCTGAGTATGTGGCCTCCGTGGCCGATGAAGAAGGCATTGTGGATT 
               
               
                   
                 TTATGACTCTCACAGCGGAGTCCGGCGCTATCGGTGGCGTTCCAGCCGGCGGTGTTCGCTTTGGGGCG 
               
               
                   
                 AGCTACAATGCTGACGCCTTGATCGACCAGGGCTACCAATTTGATTATTACGACGGTGGGGGTCTGGA 
               
               
                   
                 TCTTTGTTACCTGGGTTTAGCTGAATGCGACGAAAAGGGTAATATCAATGTTAGCCGCTTCGGTCCTC 
               
               
                   
                 GTATCGCTGGGTGCGGCGGATTCATTAACATTACCCAAAACACGCCGAAAGTCTTCTTTTGTGGGACC 
               
               
                   
                 TTTACAGCCGGGGGGCTGAAAGTGAAAATTGAAGATGGTAAGGTGATTATCGTTCAGGAAGGGAAACA 
               
               
                   
                 GAAGAAATTCCTTAAGGCAGTGGAGCAAATCACCTTTAATGGAGACGTGGCCTTAGCGAACAAGCAAC 
               
               
                   
                 AAGTTACCTACATCACGGAGCGTTGCGTCTTCCTCCTCAAAGAAGACGGTTTACACCTTTCGGAAATC 
               
               
                   
                 GCGCCAGGCATCGATCTGCAGACCCAGATTTTGGATGTTATGGACTTTGCCCCGATCATTGATCGTGA 
               
               
                   
                 CGCAAACGGGCAGATTAAACTGATGGACGCGGCGTTATTCGCAGAAGGGCTGATGGGCTTGAAAGAAA 
               
               
                   
                 TGAAGTCTTGAtaa gaaggagatatacat ATGAGCTTAACCCAAGGCATGAAAGCTAAACAACTGTTA 
               
               
                   
                 GCATACTTTCAGGGTAAAGCCGATCAGGATGCACGTGAAGCGAAAGCCCGCGGTGAGCTGGTCTGCTG 
               
               
                   
                 GTCGGCGTCAGTCGCGCCGCCGGAATTTTGCGTAACAATGGGCATTGCCATGATCTACCCGGAGACTC 
               
               
                   
                 ATGCAGCGGGCATCGGTGCCCGCAAAGGTGCGATGGACATGCTGGAAGTTGCGGACCGCAAAGGCTAC 
               
               
                   
                 AACGTGGATTGTTGTTCCTACGGCCGTGTAAATATGGGTTACATGGAATGTTTAAAAGAAGCCGCCAT 
               
               
                   
                 CACGGGCGTCAAGCCGGAAGTTTTGGTTAATTCCCCTGCTGCTGACGTTCCGCTTCCCGATTTGGTGA 
               
               
                   
                 TTACGTGTAATAATATCTGTAACACGCTGCTGAAATGGTACGAAAACTTAGCAGCAGAACTCGATATT 
               
               
                   
                 CCTTGCATCGTGATCGACGTACCGTTTAATCATACCATGCCGATTCCGGAATATGCCAAGGCCTACAT 
               
               
                   
                 CGCGGACCAGTTCCGCAATGCAATTTCTCAGCTGGAAGTTATTTGTGGCCGTCCGTTCGATTGGAAGA 
               
               
                   
                 AATTTAAGGAGGTCAAAGATCAGACCCAGCGTAGCGTATACCACTGGAACCGCATTGCCGAGATGGCG 
               
               
                   
                 AAATACAAGCCTAGCCCGCTGAACGGCTTCGATCTGTTCAATTACATGGCGTTAATCGTGGCGTGCCG 
               
               
                   
                 CAGCCTGGATTATGCAGAAATTACCTTTAAAGCGTTCGCGGACGAATTAGAAGAGAATTTGAAGGCGG 
               
               
                   
                 GTATCTACGCCTTTAAAGGTGCGGAAAAAACGCGCTTTCAATGGGAAGGTATCGCGGTGTGGCCACAT 
               
               
                   
                 TTAGGTCACACGTTTAAATCTATGAAGAATCTGAATTCGATTATGACCGGTACGGCATACCCCGCCCT 
               
               
                   
                 TTGGGACCTGCACTATGACGCTAACGACGAATCTATGCACTCTATGGCTGAAGCGTACACCCGTATTT 
               
               
                   
                 ATATTAATACTTGTCTGCAGAACAAAGTAGAGGTCCTGCTTGGGATCATGGAAAAAGGCCAGGTGGAT 
               
               
                   
                 GGTACCGTATATCATCTGAATCGCAGCTGCAAACTGATGAGTTTCCTGAACGTGGAAACGGCTGAAAT 
               
               
                   
                 TATTAAAGAGAAGAACGGTCTTCCTTACGTCTCCATTGATGGCGATCAGACCGATCCTCGCGTTTTTT 
               
               
                   
                 CTCCGGCCCAGTTTGATACCCGTGTTCAGGCCCTGGTTGAGATGATGGAGGCCAATATGGCGGCAGCG 
               
               
                   
                 GAATAAtaa gaaggagatatacat ATGTCACGCGTGGAGGCAATCCTGTCGCAGCTGAAAGATGTCGC 
               
               
                   
                 CGCGAATCCGAAAAAAGCCATGGATGACTATAAAGCTGAAACAGGTAAGGGCGCGGTTGGTATCATGC 
               
               
                   
                 CGATCTACAGCCCCGAAGAAATGGTACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGCGCCCAG 
               
               
                   
                 GGCAAAACGATTAGTAAAGCGCGCACCTATCTGCCTGCTTTTGCCTGCAGCGTAATGCAGCAGGTTAT 
               
               
                   
                 GGAATTACAGTGCGAGGGCGCGTATGATGACCTGTCCGCAGTTATTTTTAGCGTACCGTGCGACACTC 
               
               
                   
                 TCAAATGTCTTAGCCAGAAATGGAAAGGTACGTCCCCAGTGATTGTATTTACGCATCCGCAGAACCGC 
               
               
                   
                 GGATTAGAAGCGGCGAACCAATTCTTGGTTACCGAGTATGAACTGGTAAAAGCACAACTGGAATCAGT 
               
               
                   
                 TCTGGGTGTGAAAATTTCAAACGCCGCCCTGGAAAATTCGATTGCAATTTATAACGAGAATCGTGCCG 
               
               
                   
                 TGATGCGTGAGTTCGTGAAAGTGGCAGCGGACTATCCTCAAGTCATTGACGCAGTGAGCCGCCACGCG 
               
               
                   
                 GTTTTTAAAGCGCGCCAGTTTATGCTTAAGGAAAAACATACCGCACTTGTGAAAGAACTGATCGCTGA 
               
               
                   
                 GATTAAAGCAACGCCAGTCCAGCCGTGGGACGGAAAAAAGGTTGTAGTGACGGGCATTCTGTTGGAAC 
               
               
                   
                 CGAATGAGTTATTAGATATCTTTAATGAGTTTAAGATCGCGATTGTTGATGATGATTTAGCGCAGGAC 
               
               
                   
                 AAGCCGTCAGATCCGTGTTGACGTTCTGGACGGAGAAGGCGGACCGCTCTACCGTATGGCTAAAGCGT 
               
               
                   
                 GGCAGCAAATGTATGGCTGCTCGCTGGCAACCGACACCAAGAAGGGTCGCGGCCGTATGTTAATTAAC 
               
               
                   
                 AAAACGATTCAGACCGGTGCGGACGCTATCGTAGTTGCAATGATGAAGTTTTGCGACCCAGAAGAATG 
               
               
                   
                 GGATTATCCGGTAATGTACCGTGAATTTGAAGAAAAAGGGGTCAAATCACTTATGATTGAGGTGGATC 
               
               
                   
                 AGGAAGTATCGTCTTTCGAACAGATTAAAACCCGTCTGCAGICATTCGTCGAAATGCTTTAAtaa gaa   
               
               
                   
                   ggagatatacat ATGTATACCTTGGGGATTGATGTCGGTTCTGCCTCTAGTAAAGCGGTGATTCTGAA 
               
               
                   
                 AGATGGAAAAGATATTGTCGCTGCCGAGGTTGTCCAAGTCGGTACCGGCTCCTCGGGTCCCCAACGCG 
               
               
                   
                 CACTGGACAAAGCCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACATCAGCTACACAGTAGCTACGGGC 
               
               
                   
                 TATGGGCGCTTCAATTTTAGCGACGCGGATAAACAGATTTCGGAAATTAGCTGTCATGCCAAAGGCAT 
               
               
                   
                 TTATTTCTTAGTACCAACTGCGCGCACTATTATTGACATTGGCGGCCAAGATGCGAAAGCCATCCGCC 
               
               
                   
                 TGGACGACAAGGGGGGTATTAAGCAATTCTTCATGAATGATAAATGCGCGGCGGGCACGGGGCGTTTC 
               
               
                   
                 CTGGAAGTCATGGCTCGCGTACTTGAAACCACCCTGGATGAAATGGCTGAACTGGATGAACAGGCGAC 
               
               
                   
                 TGACACCGCTCCCATTTCAAGCACCTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAATTGAGCA 
               
               
                   
                 ATGGTGTCTCACGCAACAACATCATTAAAGGTGTCCATCTGAGCGTTGCGTCACGTGCGTGTGGTCTG 
               
               
                   
                 GCGTATCGCGGCGGTTTGGAGAAAGATGTTGTTATGACAGGTGGCGTGGCAAAAAATGCAGGGGTGGT 
               
               
                   
                 GCGCGCGGTGGCGGGCGTTCTGAAGACCGATGTTATCGTTGCTCCGAATCCTCAGACGACCGGTGCAC 
               
               
                   
                 TGGGGGCAGCGCTGTATGCTTATGAGGCCGCCCAGAAGAAGTAAtaa gaaggagatatacat ATGGCC 
               
               
                   
                 TTCAATAGCGCAGATATTAATTCTTTCCGCGATATTTGGGTGTTTTGTGAACAGCGTGAGGGCAAACT 
               
               
                   
                 GATTAACACCGATTTCGAATTAATTAGCGAAGGTCGTAAACTGGCTGACGAACGCGGAAGCAAACTGG 
               
               
                   
                 TTGGAATTTTGCTGGGGCACGAAGTTGAAGAAATCGCAAAAGAATTAGGCGGCTATGGTGCGGACAAG 
               
               
                   
                 GTAATTGTGTGCGATCATCCGGAACTTAAATTTTACACTACGGATGCTTATGCCAAAGTTTTATGTGA 
               
               
                   
                 CGTCGTGATGGAAGAGAAACCGGAGGTAATTTTGATCGGTGCCACCAACATTGGCCGTGATCTCGGAC 
               
               
                   
                 CGCGTTGTGCTGCACGCTTGCACACGGGGCTGACGGCTGATTGCACGCACCTGGATATTGATATGAAT 
               
               
                   
                 AAATATGTGGACTTTCTTAGCACCAGTAGCACCTTGGATATCTCGTCGATGACTTTCCCTATGGAAGA 
               
               
                   
                 TACAAACCTTAAAATGACGCGCCCTGCATTTGGCGGACATCTGATGGCAACGATCATTTGTCCACGCT 
               
               
                   
                 TCCGTCCCTGTATGAGCACAGTGCGCCCCGGAGTGATGAAGAAAGCGGAGTTCTCGCAGGAGATGGCG 
               
               
                   
                 CAAGCATGTCAAGTAGTGACCCGTCACGTAAATTTGTCGGATGAAGACCTTAAAACTAAAGTAATTAA 
               
               
                   
                 TATCGTGAAGGAAACGAAAAAGATTGTGGATCTGATCGGCGCAGAAATTATTGTGTCAGTTGGTCGTG 
               
               
                   
                 GTATCTCGAAAGATGTCCAAGGTGGAATTGCACTGGCTGAAAAACTTGCGGACGCATTTGGTAACGGT 
               
               
                   
                 GTCGTGGGCGGCTCGCGCGCAGTGATTGATTCCGGCTGGTTACCTGCGGATCATCAGGTTGGACAAAC 
               
               
                   
                 CGGTAAGACCGTGCACCCGAAAGTCTACGTGGCGCTGGGTATTAGTGGGGCTATCCAGCATAAGGCTG 
               
               
                   
                 GGATGCAAGACTCTGAACTGATCATTGCCGTCAACAAAGACGAAACGGCGCCTATCTTCGACTGCGCC 
               
               
                   
                 GATTATGGCATCACCGGTGATTTATTTAAAATCGTACCGATGATGATCGACGCGATCAAAGAGGGTAA 
               
               
                   
                 AAACGCATGAtaa gaaaggagatatacat ATGCGCATCTATGTGTGTGTGAAACAAGTCCCAGATACG 
               
               
                   
                 AGCGGCAAGGTGGCCGTTAACCCTGATGGGACCCTTAACCGTGCCTCAATGGCAGCGATTATTAACCC 
               
               
                   
                 GGACGATATGTCCGCGATCGAACAGGCATTAAAACTGAAAGATGAAACCGGATGCCAGGTTACGGCGC 
               
               
                   
                 TTACGATGGGTCCTCCTCCTGCCGAGGGCATGTTGCGCGAAATTATTGCAATGGGGGCCGACGATGGT 
               
               
                   
                 GTGCTGATTTCGGCCCGTGAATTTGGGGGGTCCGATACCTTCGCAACCAGTCAAATTATTAGCGCGGC 
               
               
                   
                 AATCCATAAATTAGGCTTAAGCAATGAAGACATGATCTTTTGCGGTCGTCAGGCCATTGACGGTGATA 
               
               
                   
                 CGGCCCAAGTCGGCCCTCAAATTGCCGAAAAACTGAGCATCCCACAGGTAACCTATGGCGCAGGAATC 
               
               
                   
                 AAAAAATCTGGTGATTTAGTGCTGGTGAAGCGTATGTTGGAGGATGGTTATATGATGATCGAAGTCGA 
               
               
                   
                 AACTCCATGTCTGATTACCTGCATTCAGGATAAAGCGGTAAAACCACGTTACATGACTCTCAACGGTA 
               
               
                   
                 TTATGGAATGCTACTCCAAGCCGCTCCTCGTTCTCGATTACGAAGCACTGAAAGATGAACCGCTGATC 
               
               
                   
                 GAACTTGATACCATTGGGCTTAAAGGCTCCCCGACGAATATCTTTAAATCGTTTACGCCGCCTCAGAA 
               
               
                   
                 AGGCGTTGGTGTCATGCTCCAAGGCACCGATAAGGAAAAAGTCGAGGATCTGGTGGATAAGCTGATGC 
               
               
                   
                 AGAAACATGTCATCTAAtaa gaaggagatatacat ATGTTCTTACTGAAGATTAAAAAAGAACGTATG 
               
               
                   
                 AAACGCATGGACTTTAGTTTAACGCGTGAACAGGAGATGTTAAAAAAACTGGCGCGTCAGTTTGCTGA 
               
               
                   
                 GATCGAGCTGGAACCGGTGGCCGAAGAGATTGATCGTGAGCACGTTTTTCCTGCAGAAAACTTTAAGA 
               
               
                   
                 AGATGGCGGAAATTGGCTTAACCGGCATTGGTATCCCGAAAGAATTTGGTGGCTCCGGTGGAGGCACC 
               
               
                   
                 CTGGAGAAGGTCATTGCCGTGTCAGAATTCGGCAAAAAGTGTATGGCCTCAGCTTCCATTTTAAGCAT 
               
               
                   
                 TCATCTTATCGCGCCGCAGGCAATCTACAAATATGGGACCAAAGAACAGAAAGAGACGTACCTGCCGC 
               
               
                   
                 GTCTTACCAAAGGTGGTGAACTGGGCGCCTTTGCGCTGACAGAACCAAACGCCGGAAGCGATGCCGGC 
               
               
                   
                 GCGGTAAAAACGACCGCGATTCTGGACAGCCAGACAAACGAGTACGTGCTGAATGGCACCAAATGCTT 
               
               
                   
                 TATCAGCGGGGGCGGGCGCGCGGGTGTTCTTGTAATTTTTGCGCTTACTGAACCGAAAAAAGGTCTGA 
               
               
                   
                 AAGGGATGAGCGCGATTATCGTGGAGAAAGGGACCCCGGGCTTCAGCATCGGCAAGGTGGAGAGCAAG 
               
               
                   
                 ATGGGGATCGCAGGTTCGGAAACCGCGGAACTTATCTTCGAAGATTGTCGCGTTCCGGCTGCCAACCT 
               
               
                   
                 TTTAGGTAAAGAAGGCAAAGGCTTTAAAATTGCTATGGAAGCCCTGGATGGCGCCCGTATTGGCGTGG 
               
               
                   
                 GCGCTCAAGCAATCGGAATTGCCGAGGGGGCGATCGACCTGAGTGTGAAGTACGTTCACGAGCGCATT 
               
               
                   
                 CAATTTGGTAAACCGATCGCGAATCTGCAGGGAATTCAATGGTATATCGCGGATATGGCGACCAAAAC 
               
               
                   
                 CGCCGCGGCACGCGCACTTGTTGAGTTTGCAGCGTATCTTGAAGACGCGGGTAAACCGTTCACAAAGG 
               
               
                   
                 AATCTGCTATGTGCAAGCTGAACGCCTCCGAAAACGCGCGTTTTGTGACAAATTTAGCTCTGCAGATT 
               
               
                   
                 CACGGGGGTTACGGTTATATGAAAGATTATCCGTTAGAGCGTATGTATCGCGATGCTAAGATTACGGA 
               
               
                   
                 AATTTACGAGGGGACATCAGAAATCCATAAGGTGGTGATTGCGCGTGAAGTAATGAAACGCTAA 
               
               
                   
               
               
                 Ptet-acuI-pct-lcdABC 
                 caactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtg 
               
               
                 (Ptet: lower case; tetA/R 
                 ctgcaaggcgattaagttgggtaacgccaggcttcccagtcacgacgttgtaaaacgacggccagtga 
               
               
                 promoter within Ptet: 
                 attgacgcgtattgggatgtaaaacgacggccagtgaattcgttaagacccactttcacatttaagtt 
               
               
                 lower case bold, with tet 
                 gtttttctaatccgcatatgatcaattcaaggccgaataagaaggctggctctgcaccttggtgatca 
               
               
                 operator underlined; RBS 
                 aataattcgatagcttgtcgtaataatggcggcatactatcagtagtaggtgtttccctttcttcttt 
               
               
                 and leader region lower 
                 agcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgccccacagcgctgagtgcat 
               
               
                 case italic; ribosome 
                 ataatgcattctctagtgaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcatac 
               
               
                 binding site: lower case 
                 tgtttttctgtaggccgtgtacctaaatgtacattgctccatcgcgatgacttagtaaagcacatcta 
               
               
                 underlined italic; coding 
                 aaactutagcgttattacgtaaaaaatcttgccagctttccccttctaaagggcaaaagtgagtatgg 
               
               
                 region: upper case, rrnB T1  
                 tgcctatctaacatctcaatggctaaggcgtcgagcaaagcccgcttattttttacatgccaatacaa 
               
               
                 and T2 terminors: lower 
                 tgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaaccttcgattccgacctcat 
               
               
                 case bold underline italics) 
                 taagcagctctaatgcgctgttaatcactttacttuatctaatctagacatcattaattcctaatttt 
               
               
                 (SEQ ID NO: 187) 
                   gttgac     actctatcattgatagagt     tattttaccac     tccctatcagtgatagaga   aaagtgaa ctcta   
               
               
                   
                   gaaataattttgtttaactttaa     gaaggagatatacat   ATGCGTGCGGTACTGATCGAGAAGTCCGAT 
               
               
                   
                 GATACACAGTCCGTCTCTGTCACCGAACTGGCTGAAGATCAACTGCCGGAAGGCGACGTTTTGGTAGA 
               
               
                   
                 TGTTGCTTATTCAACACTGAACTACAAAGACGCCCTGGCAATTACCGGTAAAGCCCCCGTCGTTCGTC 
               
               
                   
                 GTTTTCCGATGGTACCTGGAATCGACTTTACGGGTACCGTGGCCCAGTCTTCCCACGCCGACTTCAAG 
               
               
                   
                 CCAGGTGATCGCGTAATCCTGAATGGTTGGGGTGTGGGGGAAAAACATTGGGGCGGTTTAGCGGAGCG 
               
               
                   
                 CGCTCGCGTGCGCGGAGACTGGCTTGTTCCCTTGCCAGCCCCCCTGGACTTACGCCAAGCGGCCATGA 
               
               
                   
                 TCGGTACAGCAGGATACACGGCGATGTTGTGCGTTCTGGCGCTTGAACGTCACGGAGTGGTGCCGGGT 
               
               
                   
                 AATGGGGAAATCGTGGTGTCCGGTGCAGCAGGCGGCGTCGGCTCCGTTGCGACGACCCTTCTTGCCGC 
               
               
                   
                 TAAGGGCTATGAGGTAGCGGCAGTGACTGGACGTGCGTCCGAAGCAGAATATCTGCGCGGTTTGGGGG 
               
               
                   
                 CGGCGAGCGTAATTGATCGTAACGAATTAACGGGGAAGGTACGCCCGCTGGGTCAGGAGCGTTGGGCT 
               
               
                   
                 GGCGGGATTGACGTGGCGGGATCAACCGTGCTTGCGAACATGCTTTCTATGATGAAGTATCGCGGGGT 
               
               
                   
                 AGTCGCTGCGTGTGGCCTGGCCGCGGGCATGGATCTGCCCGCGTCTGTCGCGCCCTTTATTCTTCGTG 
               
               
                   
                 GGATGACGCTGGCAGGGGTGGATAGCGTTATGTGCCCAAAGACAGATCGTTTAGCAGCGTGGGCCCGT 
               
               
                   
                 TTGGCGTCAGATCTTGACCCTGCCAAGCTGGAGGAGATGACTACAGAGTTGCCGTTTAGTGAAGTAAT 
               
               
                   
                 CGAGACAGCACCCAAATTCTTGGACGGGACGGTTCGTGGCCGCATTGTTATCCCCGTAACGCCCTAAg 
               
               
                   
                 aa ctctagaaataattttgtttaactttaa     gaaggagatatacat   ATGCGCAAAGTGCCGATTATCAC 
               
               
                   
                 GGCTGACGAGGCCGCAAAACTGATCAAGGACGGCGACACCGTGACAACTAGCGGCTTTGTGGGTAACG 
               
               
                   
                 CGATCCCTGAGGCCCTTGACCGTGCAGTCGAAAAGCGTTTCCTGGAAACGGGCGAACCGAAGAACATT 
               
               
                   
                 ACTTATGTATATTGCGGCAGTCAGGGCAATCGCGACGGTCGTGGCGCAGAACATTTCGCGCATGAAGG 
               
               
                   
                 CCTGCTGAAACGTTATATCGCTGGCCATTGGGCGACCGTCCCGGCGTTAGGGAAAATGGCCATGGAGA 
               
               
                   
                 ATAAAATGGAGGCCTACAATGTCTCTCAGGGCGCCTTGTGTCATCTCTTTCGCGATATTGCGAGCCAT 
               
               
                   
                 AAACCGGGTGTGTTCACGAAAGTAGGAATCGGCACCTTCATTGATCCACGTAACGGTGGTGGGAAGGT 
               
               
                   
                 CAACGATATTACCAAGGAAGATATCGTAGAACTGGTGGAAATTAAAGGGCAGGAATACCTGTTTTATC 
               
               
                   
                 CGGCGTTCCCGATCCATGTCGCGCTGATTCGTGGCACCTATGCGGACGAGAGTGGTAACATCACCTTT 
               
               
                   
                 GAAAAAGAGGTAGCGCCTTTGGAAGGGACTTCTGTCTGTCAAGCGGTGAAGAACTCGGGTGGCATTGT 
               
               
                   
                 CGTGGTTCAGGTTGAGCGTGTCGTCAAAGCAGGCACGCTGGATCCGCGCCATGTGAAAGTTCCGGGTA 
               
               
                   
                 TCTATGTAGATTACGTAGTCGTCGCGGATCCGGAGGACCATCAACAGTCCCTTGACTGCGAATATGAT 
               
               
                   
                 CCTGCCCTTAGTGGAGAGCACCGTCGTCCGGAGGTGGTGGGTGAACCACTGCCTTTATCCGCGAAGAA 
               
               
                   
                 AGTCATCGGCCGCCGTGGCGCGATTGAGCTCGAGAAAGACGTTGCAGTGAACCTTGGGGTAGGTGCAC 
               
               
                   
                 CTGAGTATGTGGCCTCCGTGGCCGATGAAGAAGGCATTGTGGATTTTATGACTCTCACAGCGGAGTCC 
               
               
                   
                 GGCGCTATCGGTGGCGTTCCAGCCGGCGGTGTTCGCTTTGGGGCGAGCTACAATGCTGACGCCTTGAT 
               
               
                   
                 CGACCAGGGCTACCAATTTGATTATTACGACGGTGGGGGTCTGGATCTTTGTTACCTGGGTTTAGCTG 
               
               
                   
                 AATGCGACGAAAAGGGTAATATCAATGTTAGCCGCTTCGGTCCTCGTATCGCTGGGTGCGGCGGATTC 
               
               
                   
                 ATTAACATTACCCAAAACACGCCGAAAGTCTTCTTTTGTGGGACCTTTACAGCCGGGGGGCTGAAAGT 
               
               
                   
                 GAAAATTGAAGATGGTAAGGTGATTATCGTTCAGGAAGGGAAACAGAAGAAATTCCTTAAGGCAGTGG 
               
               
                   
                 AGCAAATCACCTTTAATGGAGACGTGGCCTTAGCGAACAAGCAACAAGTTACCTACATCACGGAGCGT 
               
               
                   
                 TGCGTCTTCCTCCTCAAAGAAGACGGTTTACACCTTTCGGAAATCGCGCCAGGCATCGATCTGCAGAC 
               
               
                   
                 CCAGATTTTGGATGTTATGGACTTTGCCCCGATCATTGATCGTGACGCAAACGGGCAGATTAAACTGA 
               
               
                   
                 TGGACGCGGCGTTATTCGCAGAAGGGCTGATGGGCTTGAAAGAAATGAAGTCTTGAtaa   gaaggagat     
               
               
                   
                     atacat   ATGAGCTTAACCCAAGGCATGAAAGCTAAACAACTGTTAGCATACTTTCAGGGTAAAGCCGA 
               
               
                   
                 TCAGGATGCACGTGAAGCGAAAGCCCGCGGTGAGCTGGTCTGCTGGTCGGCGTCAGTCGCGCCGCCGG 
               
               
                   
                 AATTTTGCGTAACAATGGGCATTGCCATGATCTACCCGGAGACTCATGCAGCGGGCATCGGTGCCCGC 
               
               
                   
                 AAAGGTGCGATGGACATGCTGGAAGTTGCGGACCGCAAAGGCTACAACGTGGATTGTTGTTCCTACGG 
               
               
                   
                 CCGTGTAAATATGGGTTACATGGAATGTTTAAAAGAAGCCGCCATCACGGGCGTCAAGCCGGAAGTTT 
               
               
                   
                 TGGTTAATTCCCCTGCTGCTGACGTTCCGCTTCCCGATTTGGTGATTACGTGTAATAATATCTGTAAC 
               
               
                   
                 ACGCTGCTGAAATGGTACGAAAACTTAGCAGCAGAACTCGATATTCCTTGCATCGTGATCGACGTACC 
               
               
                   
                 GTTTAATCATACCATGCCGATTCCGGAATATGCCAAGGCCTACATCGCGGACCAGTTCCGCAATGCAA 
               
               
                   
                 TTTCTCAGCTGGAAGTTATTTGTGGCCGTCCGTTCGATTGGAAGAAATTTAAGGAGGTCAAAGATCAG 
               
               
                   
                 ACCCAGCGTAGCGTATACCACTGGAACCGCATTGCCGAGATGGCGAAATACAAGCCTAGCCCGCTGAA 
               
               
                   
                 CGGCTTCGATCTGTTCAATTACATGGCGTTAATCGTGGCGTGCCGCAGCCTGGATTATGCAGAAATTA 
               
               
                   
                 CCTTTAAAGCGTTCGCGGACGAATTAGAAGAGAATTTGAAGGCGGGTATCTACGCCTTTAAAGGTGCG 
               
               
                   
                 GAAAAAACGCGCTTTCAATGGGAAGGTATCGCGGTGTGGCCACATTTAGGTCACACGTTTAAATCTAT 
               
               
                   
                 GAAGAATCTGAATTCGATTATGACCGGTACGGCATACCCCGCCCTTTGGGACCTGCACTATGACGCTA 
               
               
                   
                 ACGACGAATCTATGCACTCTATGGCTGAAGCGTACACCCGTATTTATATTAATACTTGTCTGCAGAAC 
               
               
                   
                 AAAGTAGAGGTCCTGCTTGGGATCATGGAAAAAGGCCAGGTGGATGGTACCGTATATCATCTGAATCG 
               
               
                   
                 CAGCTGCAAACTGATGAGTTTCCTGAACGTGGAAACGGCTGAAATTATTAAAGAGAAGAACGGTCTTC 
               
               
                   
                 CTTACGTCTCCATTGATGGCGATCAGACCGATCCTCGCGTTTTTTCTCCGGCCCAGTTTGATACCCGT 
               
               
                   
                 GTTCAGGCCCTGGTTGAGATGATGGAGGCCAATATGGCGGCAGCGGAATAAtaa   gaaggagatataca     
               
               
                   
                     t   ATGTCACGCGTGGAGGCAATCCTGTCGCAGCTGAAAGATGTCGCCGCGAATCCGAAAAAAGCCATGG 
               
               
                   
                 ATGACTATAAAGCTGAAACAGGTAAGGGCGCGGTTGGTATCATGCCGATCTACAGCCCCGAAGAAATG 
               
               
                   
                 GTACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGCGCCCAGGGCAAAACGATTAGTAAAGCGCG 
               
               
                   
                 CACCTATCTGCCTGCTTTTGCCTGCAGCGTAATGCAGCAGGTTATGGAATTACAGTGCGAGGGCGCGT 
               
               
                   
                 ATGATGACCTGTCCGCAGTTATTTTTAGCGTACCGTGCGACACTCTCAAATGTCTTAGCCAGAAATGG 
               
               
                   
                 AAAGGTACGTCCCCAGTGATTGTATTTACGCATCCGCAGAACCGCGGATTAGAAGCGGCGAACCAATT 
               
               
                   
                 CTTGGTTACCGAGTATGAACTGGTAAAAGCACAACTGGAATCAGTTCTGGGTGTGAAAATTTCAAACG 
               
               
                   
                 CCGCCCTGGAAAATTCGATTGCAATTTATAACGAGAATCGTGCCGTGATGCGTGAGTTCGTGAAAGTG 
               
               
                   
                 GCAGCGGACTATCCTCAAGTCATTGACGCAGTGAGCCGCCACGCGGTTTTTAAAGCGCGCCAGTTTAT 
               
               
                   
                 GCTTAAGGAAAAACATACCGCACTTGTGAAAGAACTGATCGCTGAGATTAAAGCAACGCCAGTCCAGC 
               
               
                   
                 CGTGGGACGGAAAAAAGGTTGTAGTGACGGGCATTCTGTTGGAACCGAATGAGTTATTAGATATCTTT 
               
               
                   
                 AATGAGTTTAAGATCGCGATTGTTGATGATGATTTAGCGCAGGAAAGCCGTCGGATCCGTGTTGACGT 
               
               
                   
                 TCTGGACGGAGAAGGCGGACCGCTCTACCGTATGGCTAAAGCGTGGCAGCAAATGTATGGCTGCTCGC 
               
               
                   
                 TGGCAACCGACACCAAGAAGGGTCGCGGCCGTATGTTAATTAACAAAACGATTCAGACCGGTGCGGAC 
               
               
                   
                 GCTATCGTAGTTGCAATGATGAAGTTTTGCGACCCAGAAGAATGGGATTATCCGGTAATGTACCGTGA 
               
               
                   
                 ATTTGAAGAAAAAGGGGTCAAATCACTTATGATTGAGGTGGATCAGGAAGTATCGTCTTTCGAACAGA 
               
               
                   
                 TTAAAACCCGTCTGCAGTCATTCGTCGAAATGCTTTAAtaa   gaaggagatatacat   ATGTATACCTTG 
               
               
                   
                 GGGATTGATGTCGGTTCTGCCTCTAGTAAAGCGGTGATTCTGAAAGATGGAAAAGATATTGTCGCTGC 
               
               
                   
                 CGAGGTTGTCCAAGTCGGTACCGGCTCCTCGGGTCCCCAACGCGCACTGGACAAAGCCTTTGAAGTCT 
               
               
                   
                 CTGGCTTAAAAAAGGAAGACATCAGCTACACAGTAGCTACGGGCTATGGGCGCTTCAATTTTAGCGAC 
               
               
                   
                 GCGGATAAACAGATTTCGGAAATTAGCTGTCATGCCAAAGGCATTTATTTCTTAGTACCAACTGCGCG 
               
               
                   
                 CACTATTATTGACATTGGCGGCCAAGATGCGAAAGCCATCCGCCTGGACGACAAGGGGGGTATTAAGC 
               
               
                   
                 AATTCTTCATGAATGATAAATGCGCGGCGGGCACGGGGCGTTTCCTGGAAGTCATGGCTCGCGTACTT 
               
               
                   
                 GAAACCACCCTGGATGAAATGGCTGAACTGGATGAACAGGCGACTGACACCGCTCCCATTTCAAGCAC 
               
               
                   
                 CTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAATTGAGCAATGGTGTCTCACGCAACAACATCA 
               
               
                   
                 TTAAAGGTGTCCATCTGAGCGTTGCGTCACGTGCGTGTGGTCTGGCGTATCGCGGCGGTTTGGAGAAA 
               
               
                   
                 GATGTTGTTATGACAGGTGGCGTGGCAAAAAATGCAGGGGTGGTGCGCGCGGTGGCGGGCGTTCTGAA 
               
               
                   
                 GACCGATGTTATCGTTGCTCCGAATCCTCAGACGACCGGTGCACTGGGGGCAGCGCTGTATGCTTATG 
               
               
                   
                 AGGCCGCCCAGAAGAAGTAgatggtagtgtggggtctccccatgcgagagtagggaactgccaggcat 
               
               
                   
                 
                   
                   
                   
                   
                   
                 
               
               
                   
                    ccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgc 
               
               
                   
                 ccgccataaactgccaggcatcaaattaagc     
               
               
                   
               
               
                 acuI-pct-lcdABC  
                 ATGCGTGCGGTACTGATCGAGAAGTCCGATGATACACAGTCCGTCTCTGTCACCGAACTGGCTGAAGA 
               
               
                 (SEQ ID NO: 188) 
                 TCAACTGCCGGAAGGCGACGTTTTGGTAGATGTTGCTTATTCAACACTGAACTACAAAGACGCCCTGG 
               
               
                   
                 CAATTACCGGTAAAGCCCCCGTCGTTCGTCGTTTTCCGATGGTACCTGGAATCGACTTTACGGGTACC 
               
               
                   
                 GTGGCCCAGTCTTCCCACGCCGACTTCAAGCCAGGTGATCGCGTAATCCTGAATGGTTGGGGTGTGGG 
               
               
                   
                 GGAAAAACATTGGGGCGGTTTAGCGGAGCGCGCTCGCGTGCGCGGAGACTGGCTTGTTCCCTTGCCAG 
               
               
                   
                 CCCCCCTGGACTTACGCCAAGCGGCCATGATCGGTACAGCAGGATACACGGCGATGTTGTGCGTTCTG 
               
               
                   
                 GCGCTTGAACGTCACGGAGTGGTGCCGGGTAATGGGGAAATCGTGGTGTCCGGTGCAGCAGGCGGCGT 
               
               
                   
                 CGGCTTCCGTTGCGACGACCCTTCTTGCCGCTAAGGGCTATGAGGTAGCGGCAGTGACTGGACGTGCG 
               
               
                   
                 TCCGAAGCAGAATATCTGCGCGGTTTGGGGGCGGCGAGCGTAATTGATCGTAACGAATTAACGGGGAA 
               
               
                   
                 GGTACGCCCGCTGGGTCAGGAGCGTTGGGCTGGCGGGATTGACGTGGCGGGATCAACCGTGCTTGCGA 
               
               
                   
                 ACATGCTTTCTATGATGAAGTATCGCGGGGTAGTCGCTGCGTGTGGCCTGGCCGCGGGCATGGATCTG 
               
               
                   
                 CCCGCGTCTGTCGCGCCCTTTATTCTTCGTGGGATGACGCTGGCAGGGGTGGATAGCGTTATGTGCCC 
               
               
                   
                 AAAGACAGATCGTTTAGCAGCGTGGGCCCGTTTGGCGTCAGATCTTGACCCTGCCAAGCTGGAGGAGA 
               
               
                   
                 TGACTACAGAGTTGCCGTTTAGTGAAGTAATCGAGACAGCACCCAAATTCTTGGACGGGACGGTTCGT 
               
               
                   
                 GGCCGCATTGTTATCCCCGTAACGCCCTAAgaa ctctagaaataattttgtttaactttaa     gaaggag     
               
               
                   
                     atatacat   ATGCGCAAAGTGCCGATTATCACGGCTGACGAGGCCGCAAAACTGATCAAGGACGGCGAC 
               
               
                   
                 ACCGTGACAACTAGCGGCTTTGTGGGTAACGCGATCCCTGAGGCCCTTGACCGTGCAGTCGAAAAGCG 
               
               
                   
                 ACCTGGAAACGGGCGAACCGAAGAACATTACTTATGTATATTGCGGCAGTCAGGGCAATCGCGACGGT 
               
               
                   
                 CGTGGCGCAGAACATTTCGCGCATGAAGGCCTGCTGAAACGTTATATCGCTGGCCATTGGGCGACCGT 
               
               
                   
                 CCCGGCGTTAGGGAAAATGGCCATGGAGAATAAAATGGAGGCCTACAATGTCTCTCAGGGCGCCTTGT 
               
               
                   
                 GTCATCTCTTTCGCGATATTGCGAGCCATAAACCGGGTGTGTTCACGAAAGTAGGAATCGGCACCTTC 
               
               
                   
                 ATTGATCCACGTAACGGTGGTGGGAAGGTCAACGATATTACCAAGGAAGATATCGTAGAACTGGTGGA 
               
               
                   
                 AATTAAAGGGCAGGAATACCTGTTTTATCCGGCGTTCCCGATCCATGTCGCGCTGATTCGTGGCACCT 
               
               
                   
                 ATGCGGACGAGAGTGGTAACATCACCTTTGAAAAAGAGGTAGCGCCTTTGGAAGGGACTTCTGTCTGT 
               
               
                   
                 CAAGCGGTGAAGAACTCGGGTGGCATTGTCGTGGTTCAGGTTGAGCGTGTCGTCAAAGCAGGCACGCT 
               
               
                   
                 GGATCCGCGCCATGTGAAAGTTCCGGGTATCTATGTAGATTACGTAGTCGTCGCGGATCCGGAGGACC 
               
               
                   
                 ATCAACAGTCCCTTGACTGCGAATATGATCCTGCCCTTAGTGGAGAGCACCGTCGTCCGGAGGTGGTG 
               
               
                   
                 GGTGAACCACTGCCTTTATCCGCGAAGAAAGTCATCGGCCGCCGTGGCGCGATTGAGCTCGAGAAAGA 
               
               
                   
                 CGTTGCAGTGAACCTTGGGGTAGGTGCACCTGAGTATGTGGCCTCCGTGGCCGATGAAGAAGGCATTG 
               
               
                   
                 TGGATTTTATGACTCTCACAGCGGAGTCCGGCGCTATCGGTGGCGTTCCAGCCGGCGGTGTTCGCTTT 
               
               
                   
                 GGGGCGAGCTACAATGCTGACGCCTTGATCGACCAGGGCTACCAATTTGATTATTACGACGGTGGGGG 
               
               
                   
                 TCTGGATCTTTGTTACCTGGGTTTAGCTGAATGCGACGAAAAGGGTAATATCAATGTTAGCCGCTTCG 
               
               
                   
                 GTCCTCGTATCGCTGGGTGCGGCGGATTCATTAACATTACCCAAAACACGCCGAAAGTCTTCTTTTGT 
               
               
                   
                 GGGACCTTTACAGCCGGGGGGCTGAAAGTGAAAATTGAAGATGGTAAGGTGATTATCGTTCAGGAAGG 
               
               
                   
                 GAAACAGAAGAAATTCCTTAAGGCAGTGGAGCAAATCACCTTTAATGGAGACGTGGCCTTAGCGAACA 
               
               
                   
                 AGCAACAAGTTACCTACATCACGGAGCGTTGCGICTTCCTCCTCAAAGAAGACGGTTTACACCTTTCG 
               
               
                   
                 GAAATCGCGCCAGGCATCGATCTGCAGACCCAGATTTTGGATGTTATGGACTTTGCCCCGATCATTGA 
               
               
                   
                 TCGTGACGCAAACGGGCAGATTAAACTGATGGACGCGGCGTTATTCGCAGAAGGGCTGATGGGCTTGA 
               
               
                   
                 AAGAAATGAAGTCTTGAtaa   gaaggagatatacat   ATGAGCTTAACCCAAGGCATGAAAGCTAAACAA 
               
               
                   
                 CTGTTAGCATACTTTCAGGGTAAAGCCGATCAGGATGCACGTGAAGCGAAAGCCCGCGGTGAGCTGGT 
               
               
                   
                 CTGCTGGTCGGCGTCAGTCGCGCCGCCGGAATTTTGCGTAACAATGGGCATTGCCATGATCTACCCGG 
               
               
                   
                 AGACTCATGCAGCGGGCATCGGTGCCCGCAAAGGTGCGATGGACATGCTGGAAGTTGCGGACCGCAAA 
               
               
                   
                 GGCTACAACGTGGATTGTTGTTCCTACGGCCGTGTAAATATGGGTTACATGGAATGTTTAAAAGAAGC 
               
               
                   
                 CGCCATCACGGGCGTCAAGCCGGAAGTTTTGGTTAATTCCCCTGCTGCTGACGTTCCGCTTCCCGATT 
               
               
                   
                 TGGTGATTACGTGTAATAATATCTGTAACACGCTGCTGAAATGGTACGAAAACTTAGCAGCAGAACTC 
               
               
                   
                 GATATTCCTTGCATCGTGATCGACGTACCGTTTAATCATACCATGCCGATTCCGGAATATGCCAAGGC 
               
               
                   
                 CTACATCGCGGACCAGTTCCGCAATGCAATTTCTCAGCTGGAAGTTATTTGTGGCCGTCCGTTCGATT 
               
               
                   
                 GGAAGAAATTTAAGGAGGTCAAAGATCAGACCCAGCGTAGCGTATACCACTGGAACCGCATTGCCGAG 
               
               
                   
                 ATGGCGAAATACAAGCCTAGCCCGCTGAACGGCTTCGATCTGTTCAATTACATGGCGTTAATCGTGGC 
               
               
                   
                 GTGCCGCAGCCTGGATTATGCAGAAATTACCTTTAAAGCGTTCGCGGACGAATTAGAAGAGAATTTGA 
               
               
                   
                 AGGCGGGTATCTACGCCTTTAAAGGTGCGGAAAAAACGCGCTTTCAATGGGAAGGTATCGCGGTGTGG 
               
               
                   
                 CCACATTTAGGTCACACGTTTAAATCTATGAAGAATCTGAATTCGAITATGACCGGTACGGCATACCC 
               
               
                   
                 CGCCCTTTGGGACCTGCACTATGACGCTAACGACGAATCTATGCACTCTATGGCTGAAGCGTACACCC 
               
               
                   
                 GTATTTATATTAATACTTGTCTGCAGAACAAAGTAGAGGTCCTGCTTGGGATCATGGAAAAAGGCCAG 
               
               
                   
                 GTGGATGGTACCGTATATCATCTGAATCGCAGCTGCAAACTGATGAGTTTCCTGAACGTGGAAACGGC 
               
               
                   
                 TGAAATTATTAAAGAGAAGAACGGTCTTCCTTACGTCTCCATTGATGGCGATCAGACCGATCCTCGCG 
               
               
                   
                 TTMTCTCCGGCCCAGTTTGATACCCGTGTTCAGGCCCTGGTTGAGATGATGGAGGCCAATATGGCGGC 
               
               
                   
                 AGCGGAATAAtaa   gaaggagatatacat   ATGTCACGCGTGGAGGCAATCCTGTCGCAGCTGAAAGATG 
               
               
                   
                 TCGCCGCGAATCCGAAAAAAGCCATGGATGACTATAAAGCTGAAACAGGTAAGGGCGCGGTTGGTATC 
               
               
                   
                 ATGCCGATCTACAGCCCCGAAGAAATGGTACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGCGC 
               
               
                   
                 CCAGGGCAAAACGATTAGTAAAGCGCGCACCTATCTGCCTGCTTTTGCCTGCAGCGTAATGCAGCAGG 
               
               
                   
                 TTATGGAATTACAGTGCGAGGGCGCGTATGATGACCTGTCCGCAGTTATTTTTAGCGTACCGTGCGAC 
               
               
                   
                 ACTCTCAAATGTCTTAGCCAGAAATGGAAAGGTACGTCCCCAGTGATTGTATTTACGCATCCGCAGAA 
               
               
                   
                 CCGCGGATTAGAAGCGGCGAACCAATTCTTGGTTACCGAGTATGAACTGGTAAAAGCACAACTGGAAT 
               
               
                   
                 CAGTTCTGGGTGTGAAAATTTCAAACGCCGCCCTGGAAAATTCGATTGCAATTTATAACGAGAATCGT 
               
               
                   
                 GCCGTGATGCGTGAGTTCGTGAAAGTGGCAGCGGACTATCCTCAAGTCATTGACGCAGTGAGCCGCCA 
               
               
                   
                 CGCGGTTTTTAAAGCGCGCCAGTTTATGCTTAAGGAAAAACATACCGCACTTGTGAAAGAACTGATCG 
               
               
                   
                 CTGAGATTAAAGCAACGCCAGTCCAGCCGTGGGACGGAAAAAAGGTTGTAGTGACGGGCATTCTGTTG 
               
               
                   
                 GAACCGAATGAGTTATTAGATATCTTTAATGAGTTTAAGATCGCGATTGTTGATGATGATTTAGCGCA 
               
               
                   
                 GGAAAGCCGTCGGATCCGTGTTGACGTTCTGGACGGAGAAGGCGGACCGCTCTACCGTATGGCTAAAG 
               
               
                   
                 CGTGGCAGCAAATGTATGGCTGCTCGCTGGCAACCGACACCAAGAAGGGTCGCGGCCGTATGTTAATT 
               
               
                   
                 AACAAAACGATTCAGACCGGTGCGGACGCTATCGTAGTTGCAATGATGAAGTTTTGCGACCCAGAAGA 
               
               
                   
                 ATGGGATTATCCGGTAATGTACCGTGAATTTGAAGAAAAAGGGGTCAAATCACTTATGAITGAGGTGG 
               
               
                   
                 ATCAGGAAGTATCGTCTTTCGAACAGATTAAAACCCGTCTGCAGICATTCGTCGAAATGCTTTAAtaa 
               
               
                   
                     gaag   ga   g   atatacat   ATGTATACCTTGGGGATTGATGTCGGTTCTGCCTCTAGTAAAGCGGTGATTCT 
               
               
                   
                 GAAAGATGGAAAAGATATTGTCGCTGCCGAGGTTGTCCAAGTCGGTACCGGCTCCTCGGGTCCCCAAC 
               
               
                   
                 GCGCACTGGACAAAGCCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACATCAGCTACACAGTAGCTACG 
               
               
                   
                 GGCTATGGGCGCTTCAATTTTAGCGACGCGGATAAACAGATTTCGGAAATTAGCTGTCATGCCAAAGG 
               
               
                   
                 CATTTATTTCTTAGTACCAACTGCGCGCACTATTATTGACATTGGCGGCCAAGATGCGAAAGCCATCC 
               
               
                   
                 GCCTGGACGACAAGGGGGGTATTAAGCAATTCTTCATGAATGATAAATGCGCGGCGGGCACGGGGCGT 
               
               
                   
                 TTCCTGGAAGTCATGGCTCGCGTACTTGAAACCACCCTGGATGAAATGGCTGAACTGGATGAACAGGC 
               
               
                   
                 GACTGACACCGCTCCCATTTCAAGCACCTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAATTGA 
               
               
                   
                 GCAATGGTGTCTCACGCAACAACATCATTAAAGGTGTCCATCTGAGCGTTGCGTCACGTGCGTGTGGT 
               
               
                   
                 CTGGCGTATCGCGGCGGTTTGGAGAAAGATGTTGTTATGACAGGTGGCGTGGCAAAAAATGCAGGGGT 
               
               
                   
                 GGTGCGCGCGGTGGCGGGCGTTCTGAAGACCGATGTTATCGTTGCTCCGAATCCTCAGACGACCGGTG 
               
               
                   
                 CACTGGGGGCAGCGCTGTATGCTTATGAGGCCGCCCAGAAGAAGTA 
               
               
                   
               
            
           
         
       
     
     In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 185, 186, 187, or 188, or a functional fragment thereof. 
     Example 25. Quantification of Propionate by LC-MS/MS 
     Sample Preparation 
     First, fresh 1000, 500, 250, 100, 20, 4 and 0.8 μg/mL sodium propionate standards were prepared in water. Then, 25 μL of sample (bacterial supernatants and standards) were pipetted into a V-bottom polypropylene 96-well plate, and 75 μL of 60% ACN (45 uL ACN+30 uL water per reaction) with 10 ug/mL of butyrate-d5 (CDN isotope) internal standard in final solution were added to each sample. The plate was heat-sealed, mixed well, and centrifuged at 4000 rpm for 5 minutes. In a round-bottom 96-well polypropylene plate, 5 μL of diluted samples were added to 95 μL of a buffer containing 10 mM MES pH4.5, 20 mM EDC (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide), and 20 mM TFEA (2,2,2-trifluroethylamine). The plate was again heat-sealed and mixed well, and samples were incubated at room temperature for 1 hour 
     LC-MS/MS Method 
     Propionate was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. HPLC Details are listed in Table 60 and Table 61. Tandem Mass Spectrometry details are found in Table 62. 
     
       
         
           
               
             
               
                 TABLE 60 
               
               
                   
               
               
                 HPLC Details 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Column 
                 Thermo Aquasil C18 column, 5 μm (50 × 2.1 mm) 
               
               
                 Mobile Phase A 
                 100% H2O, 0.1% Formic Acid 
               
               
                 Mobile Phase B 
                 100% ACN, 0.1% Formic Acid 
               
               
                 Injection volume 
                 10 uL 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 61 
               
             
            
               
                   
               
               
                 HPLC Method 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Total Time (min) 
                 Flow Rate (μL/min) 
                 A % 
                 B % 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 0 
                 0.5 
                 100 
                 0 
               
               
                   
                 1 
                 0.5 
                 100 
                 0 
               
               
                   
                 2 
                 0.5 
                 10 
                 90 
               
               
                   
                 4 
                 0.5 
                 10 
                 90 
               
               
                   
                 4.01 
                 0.5 
                 100 
                 0 
               
               
                   
                 4.25 
                 0.5 
                 100 
                 0 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 62 
               
               
                   
               
               
                 Tandem Mass Spectrometry Details 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Ion Source 
                 HESI-II 
               
               
                   
                 Polarity 
                 Positive 
               
               
                   
                 SRM 
                 Propionate 156.2/57.1, 
               
               
                   
                 transitions 
                 Propionate-d5 161/62.1 
               
               
                   
                   
               
            
           
         
       
     
     Example 26. Generation of Constructs for Overproducing Therapeutic Molecules for Secretion 
     To produce strain capable of secreting anti-inflammatory or gut barrier enhancer polypeptides, e.g., GLP2, IL-22, IL-10 (viral or human), several constructs are designed employing different secretion strategies. The organization of exemplary constructs is shown in  FIG. 30A ,  FIG. 30B ,  FIG. 30C , and  FIG. 31A  and  FIG. 31B ,  FIG. 32A ,  FIG. 32B ,  FIG. 32C ,  FIG. 32D ,  FIG. 32E . Various GLP2, IL-22, IL-10 (viral or human) constructs are synthesized, and cloned into vector pBR322 for transformation of  E. coli . In some embodiments, the constructs encoding the effector molecules are integrated into the genome. In some embodiments, the constructs encoding the effector molecules are on a plasmid, e.g., a medium copy plasmid. Table 63. lists exemplary polypeptide coding sequences used in the constructs. 
     
       
         
           
               
             
               
                 TABLE 63  
               
             
            
               
                   
               
               
                 Polypeptide coding sequences 
               
            
           
           
               
               
               
            
               
                 Description 
                 Sequence 
                 SEQ ID NO 
               
               
                   
               
               
                 GLP2 
                 CATGCTGATGGTTCTTTCTCTGATGAGAT 
                 SEQ ID NO: 189 
               
               
                   
                 GAACACCATTCTTGATAATCTTGCCGCCA 
                   
               
               
                   
                 GGGACTTTATAAACTGGTTGATTCAGACC 
                   
               
               
                   
                 AAAATCACTGAC 
                   
               
               
                   
               
               
                 GLP2 codon 
                 CATGCTGACGGCTCTTTTTCTGACGAAAT 
                 SEQ ID NO: 190 
               
               
                 optimized 
                 GAATACCATCCTGGATAATCTGGCGGCG 
                   
               
               
                   
                 CGTGATTTTATTAATTGGCTGATCCAAAC 
                   
               
               
                   
                 TAAAATTACTGATTAA 
                   
               
               
                   
               
               
                 FliC20-GLP2 
                 
                   ATGGCACAAGTCATTAATACCAACAGCC 
                 
                 SEQ ID NO: 191 
               
               
                 (FliC20, start of 
                 
                   TCTCGCTGATCACTCAAAATAATATCAAC 
                 
                   
               
               
                 FliC gene preceding 
                   AAG CATGCTGACGGCTCTTTTTCTGACGA 
                   
               
               
                 GLP2 sequence 
                 AATGAATACCATCCTGGATAATCTGGCG 
                   
               
               
                 underlined) 
                 GCGCGTGATTTTATTAATTGGCTGATCCA 
                   
               
               
                   
                 AACTAAAATTACTGATTAA 
                   
               
               
                   
               
               
                 GLP2 codon 
                 ATGCATGCTGACGGCTCTTTTTCTGACGA 
                 SEQ ID NO: 192 
               
               
                 optimized 
                 AATGAATACCATCCTGGATAATCTGGCG 
                   
               
               
                 (e.g., used in fliC 
                 GCGCGTGATTTTATTAATTGGCTGATCCA 
                   
               
               
                 construct) 
                 AACTAAAATTACTGATTAA 
                   
               
               
                   
               
               
                 vIL10 codon 
                 ATGGGTACTGACCAATGTGATAATTTCCC 
                 SEQ ID NO: 193 
               
               
                 optimized 
                 ACAAATGCTGCGTGATTTGCGCGACGCTT 
                   
               
               
                 (e.g., used in fliC  
                 TCTCGCGTGTGAAAACTTTTTTTCAGACT 
                   
               
               
                 construct) 
                 AAAGATGAGGTGGATAATCTGCTGCTGA 
                   
               
               
                   
                 AAGAGAGCCTGTTGGAAGATTTTAAAGG 
                   
               
               
                   
                 CTACTTGGGCTGTCAAGCGCTGTCGGAG 
                   
               
               
                   
                 ATGATTCAATTTTATCTGGAAGAGGTGAT 
                   
               
               
                   
                 GCCGCAAGCTGAGAACCAAGATCCGGAA 
                   
               
               
                   
                 GCGAAAGATCACGTGAATTCGCTGGGCG 
                   
               
               
                   
                 AGAATCTGAAAACTCTGCGTCTGCGTCTG 
                   
               
               
                   
                 CGTCGTTGTCACCGTTTTTTGCCGTGCGA 
                   
               
               
                   
                 AAACAAAAGTAAAGCTGTTGAGCAAATT 
                   
               
               
                   
                 AAAAACGCTTTTAACAAACTGCAGGAAA 
                   
               
               
                   
                 AAGGTATCTATAAAGCGATGAGCGAATT 
                   
               
               
                   
                 TGATATTTTTATTAATTATATTGAAGCTT 
                   
               
               
                   
                 ATATGACTATTAAAGCTCGCTAA 
                   
               
               
                   
               
               
                 vIL10 
                 GGTACAGACCAATGTGACAATTTTCCCCA 
                 SEQ ID NO: 194 
               
               
                   
                 AATGTTGAGGGACCTAAGAGATGCCTTC 
                   
               
               
                   
                 AGTCGTGTTAAAACCTTTTTCCAGACAAA 
                   
               
               
                   
                 GGACGAGGTAGATAACCTTTTGCTCAAG 
                   
               
               
                   
                 GAGTCTCTGCTAGAGGACTTTAAGGGCT 
                   
               
               
                   
                 ACCTTGGATGCCAGGCCCTGTCAGAAAT 
                   
               
               
                   
                 GATCCAATTCTACCTGGAGGAAGTCATG 
                   
               
               
                   
                 CCACAGGCTGAAAACCAGGACCCTGAAG 
                   
               
               
                   
                 AATCTAAAGACCCTACGGCTCCGCCTGC 
                   
               
               
                   
                 GCCGTTGCCACAGGTTCCTGCCGTGTGAG 
                   
               
               
                   
                 AACAAGAGTAAAGCTGTGGAACAGATAA 
                   
               
               
                   
                 AAAATGCCTTTAACAAGCTGCAGGAAAA 
                   
               
               
                   
                 AGGAATTTACAAAGCCATGAGTGAATTT 
                   
               
               
                   
                 GACATTTTTATTAACTACATAGAAGCATA 
                   
               
               
                   
                 CATGACAATTAAAGCCAGG  
                   
               
               
                   
               
               
                 IL-22 codon 
                 GCACCGATCTCTTCCCACTGTCGCTTAGA 
                 SEQ ID NO: 195 
               
               
                 optimized  
                 TAAATCGAATTTTCAACAACCTTATATTA 
                   
               
               
                 (e.g., use with  
                 CGAATCGTACGTTTATGCTGGCTAAAGA 
                   
               
               
                 diffusible 
                 AGCGTCATTAGCTGATAACAACACTGAT 
                   
               
               
                 outer membrane 
                 GTTCGCCTGATTGGTGAGAAATTGTTTCA 
                   
               
               
                 construct) 
                 CGGTGTGTCTATGTCAGAACGTTGCTACC 
                   
               
               
                   
                 TGATGAAACAAGTTCTGAATTTCACCCTG 
                   
               
               
                   
                 GAAGAAGTGTTGTTTCCGCAATCTGACCG 
                   
               
               
                   
                 CTTTCAACCGTATATGCAAGAGGTTGTGC 
                   
               
               
                   
                 CGTTTCTGGCGCGCCTGAGTAATCGCCTG 
                   
               
               
                   
                 AGCACTTGTCATATTGAGGGCGACGACC 
                   
               
               
                   
                 TGCATATTCAACGAAATGTTCAAAAATTG 
                   
               
               
                   
                 AAAGATACGGTGAAGAAACTGGGTGAAA 
                   
               
               
                   
                 GTGGTGAAATCAAAGCGATTGGTGAGCT 
                   
               
               
                   
                 GGATCTGCTGTTTATGTCATTGCGCAATG 
                   
               
               
                   
                 CGTGCATTTAA 
                   
               
               
                   
               
               
                 IL-22 codon 
                 ATGGCACCGATCTCTTCCCACTGTCGCTT 
                 SEQ ID NO: 196 
               
               
                 optimized  
                 AGATAAATCGAATTTTCAACAACCTTATA 
                   
               
               
                 (e.g., used in fliC  
                 TTACGAATCGTACGTTTATGCTGGCTAAA 
                   
               
               
                 construct) 
                 GAAGCGTCATTAGCTGATAACAACACTG 
                   
               
               
                   
                 ATGTTCGCCTGATTGGTGAGAAATTGTTT 
                   
               
               
                   
                 CACGGTGTGTCTATGTCAGAACGTTGCTA 
                   
               
               
                   
                 CCTGATGAAACAAGTTCTGAATTTCACCC 
                   
               
               
                   
                 TGGAAGAAGTGTTGTTTCCGCAATCTGAC 
                   
               
               
                   
                 CGCTTTCAACCGTATATGCAAGAGGTTGT 
                   
               
               
                   
                 GCCGTTTCTGGCGCGCCTGAGTAATCGCC 
                   
               
               
                   
                 TGAGCACTTGTCATATTGAGGGCGACGA 
                   
               
               
                   
                 CCTGCATATTCAACGAAATGTTCAAAAAT 
                   
               
               
                   
                 TGAAAGATACGGTGAAGAAACTGGGTGA 
                   
               
               
                   
                 AAGTGGTGAAATCAAAGCGATTGGTGAG 
                   
               
               
                   
                 CTGGATCTGCTGTTTATGTCATTGCGCAA 
                   
               
               
                   
                 TGCGTGCATTTAA  
                   
               
               
                   
               
               
                 hIL-10 codon 
                 TCGCCAGGTCAAGGAACGCAGTCAGAGA 
                 SEQ ID NO: 197 
               
               
                 optimized 
                 ATTCATGCACTCACTTTCCGGGCAATCTG 
                   
               
               
                   
                 CCGAATATGCTGCGCGATCTGCGAGATG 
                   
               
               
                   
                 CATTCTCTCGCGTGAAAACGTTCTTTCAA 
                   
               
               
                   
                 ATGAAAGATCAACTGGATAATCTGCTGC 
                   
               
               
                   
                 TGAAGGAGTCGTTGTTGGAGGATTTTAA 
                   
               
               
                   
                 GGGGTATCTGGGTTGTCAAGCACTGTCTG 
                   
               
               
                   
                 AAATGATTCAATTTTACTTGGAGGAAGTT 
                   
               
               
                   
                 ATGCCGCAAGCGGAAAACCAAGATCCGG 
                   
               
               
                   
                 ATATTAAGGCGCACGTGAACTCACTGGG 
                   
               
               
                   
                 CGAAAACCTGAAAACTTTGCGCCTGCGT 
                   
               
               
                   
                 CTGAGACGATGTCACCGATTCCTGCCGTG 
                   
               
               
                   
                 TGAAAACAAGTCAAAGGCGGTTGAGCAA 
                   
               
               
                   
                 GTTAAGAATGCTTTCAATAAGCTGCAAG 
                   
               
               
                   
                 AAAAGGGCATCTATAAAGCGATGTCTGA 
                   
               
               
                   
                 ATTTGATATCTTTATAAACTACATAGAAG 
                   
               
               
                   
                 CTTATATGACTATGAAGATTCGAAATTAA 
                   
               
               
                   
               
               
                 Monomerized hIL-10  
                 TCGCCAGGTCAAGGAACGCAGTCAGAGA 
                 SEQ ID NO: 198 
               
               
                 (codon opt) 
                 ATTCATGCACTCACTTTCCGGGCAATCTG 
                   
               
               
                   
                 CCGAATATGCTGCGCGATCTGCGAGATG 
                   
               
               
                   
                 CATTCTCTCGCGTGAAAACGTTCTTTCAA 
                   
               
               
                   
                 ATGAAAGATCAACTGGATAATCTGCTGC 
                   
               
               
                   
                 TGAAGGAGTCGTTGTTGGAGGATTTTAA 
                   
               
               
                   
                 GGGGTATCTGGGTTGTCAAGCACTGTCTG 
                   
               
               
                   
                 AAATGATTCAATTTTACTTGGAGGAAGTT 
                   
               
               
                   
                 ATGCCGCAAGCGGAAAACCAAGATCCGG 
                   
               
               
                   
                 ATATTAAGGCGCACGTGAACTCACTGGG 
                   
               
               
                   
                 CGAAAACCTGAAAACTTTGCGCCTGCGT 
                   
               
               
                   
                 CTGAGACGATGTCACCGATTCCTGCCGTG 
                   
               
               
                   
                 TGAAAACGGAGGAGGAAGTGGTGGTAAG 
                   
               
               
                   
                 TCAAAGGCGGTTGAGCAAGTTAAGAATG 
                   
               
               
                   
                 CTTTCAATAAGCTGCAAGAAAAGGGCAT 
                   
               
               
                   
                 CTATAAAGCGATGTCTGAATTTGATATCT 
                   
               
               
                   
                 TTATAAACTACATAGAAGCTTATATGACT 
                   
               
               
                   
                 ATGAAGATTCGAAATTAA 
               
               
                   
               
            
           
         
       
     
     In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, or SEQ ID NO: 198 or a functional fragment thereof. 
     Table 64 lists exemplary secretion tags, which can be added at the N-terminus when the diffusible outer membrane (DOM) method or the fliC method is used. 
     
       
         
           
               
             
               
                 TABLE 64 
               
             
            
               
                   
               
               
                 Secretion Tags and FliC components 
               
            
           
           
               
               
               
            
               
                 Sequence Name 
                 Sequence 
                 SEQ ID NO 
               
               
                   
               
               
                 fliC-FliC20 (e.g., used in GLP2 
                 
                   tgacggcgattgagccgacgggtggaaaccc 
                 
                 SEQ ID NO: 199 
               
               
                 construct) 
                 
                   aaaacgtaatcaac 
                   
                     GTGGGTACTCCTTAAAT 
                   
                 
                   
               
               
                 FliC20: start of the fliC gene 
                 
                   
                     TGGGTTCGAATGGACC 
                   
                   atggcacaagtcatt 
                 
                   
               
               
                 which (in some constructs) 
                 
                   aataccaacagcctctcgcgatcactcaaaa 
                 
                   
               
               
                 precedes the effector polypeptide 
                 
                   taatatcaacaag 
                 
                   
               
               
                 sequence, see e.g., FIG 30B and 
                   
                   
               
               
                 FIG. 30C shown in italics 
                   
                   
               
               
                 fliC: native fliC UTR in bold, 
                   
                   
               
               
                 optimized RBS underlined 
                   
                   
               
               
                   
               
               
                 fliC-RBS (e.g., used in IL22 
                   tgacggcgattgagccgacgggtggaaaccc   
                 SEQ ID NO: 200 
               
               
                 construct) 
                 
                   aaaacgtaatcaact 
                   
                     acgaacacttacagga 
                   
                 
                   
               
               
                 fliC::native fliC UTR in bold, 
                 
                   
                     ggtaccca 
                   
                 
                   
               
               
                 optimized RBS underlined 
                   
                   
               
               
                   
               
               
                 fliC-RBS (e.g., used in GLP2 
                 
                   tgacggcgattgagccgacgggtggaaaccc 
                 
                   
               
               
                 construct) 
                 
                   aaaacgtaatcaac 
                   
                     aagtaaaactctggg 
                     a 
                   
                   g 
                 
                   
               
               
                 fliC: native fliC UTR in bold, 
                 
                   gttccta 
                 
                   
               
               
                 optimized RBS underlined 
                   
                   
               
               
                   
               
               
                 fliC-RBS (e.g., used in vIL10 
                 
                   tgacggcgattgagccgacgggtggaaaccc 
                 
                 SEQ ID NO: 201 
               
               
                 construct) 
                 
                   aaaacgtaatcaac 
                   
                     tcaaatcccttaataag 
                   
                 
                   
               
               
                 fliC: native fliC UTR in bold, 
                 
                   
                     gaggtaaa 
                   
                 
                   
               
               
                 optimized RBS underlined 
                   
                   
               
               
                   
               
               
                 RBS-phoA 
                 
                   Ctctagaaataattttgtttaactttaagaa 
                 
                 SEQ ID NO: 202 
               
               
                 RBS: underlined 
                   ggagatatacat atgaaacaaagcactattg 
                   
               
               
                   
                 cactggcactcttaccgttactgtttacccc 
                   
               
               
                   
                 tgtgacaaaagcg 
                   
               
               
                   
               
               
                 phoA 
                 atgaaacaaagcactattgcactggcactct 
                 SEQ ID NO: 203 
               
               
                   
                 taccgttactgtttacccctgtgacaaaagc 
                   
               
               
                   
                 g 
                   
               
               
                   
               
               
                 RBS-ompF 
                 
                   Ctctagaaataattttgtttaactttaagaa 
                 
                 SEQ ID NO: 204 
               
               
                 RBS: underlined 
                   ggagatatacat atgatgaagcgcaatattc 
                   
               
               
                   
                 tggcagtgatcgtccctgctctgttagtagc 
                   
               
               
                   
                 aggtactgcaaacgct 
                   
               
               
                   
               
               
                 ompF 
                 atgatgaagcgcaatattctggcagtgatcg 
                 SEQ ID NO: 205 
               
               
                   
                 tccctgctctgttagtagcaggtactgcaaa 
                   
               
               
                   
                 cgct 
                   
               
               
                   
               
               
                 RBS-cvaC 
                 
                   Ctctagaaataattttgtttaactttaagaa 
                 
                 SEQ ID NO: 206 
               
               
                 RBS: underlined 
                   ggagatatacat ATGAGAACTCTGACTCTAA 
                   
               
               
                   
                 ATGAATTAGATTCTGTTTCTGGTGGT 
                   
               
               
                   
               
               
                 cvaC 
                 ATGAGAACTCTGACTCTAAATGAATTAGATT 
                 SEQ ID NO: 207 
               
               
                   
                 CTGTTTCTGGTGGT 
                   
               
               
                   
               
               
                 RBS-phoA (Opimized, e.g., used 
                   GACGCCAGAGAGTTAAGGGGGTTAA ATGAAA 
                 SEQ ID NO: 208 
               
               
                 in IL10 construct) 
                 CAATCGACCATCGCATTGGCGCTGCTTCCTC 
                   
               
               
                 RBS: underlined  
                 TATTGTTCACACCGGTGACAAAGGCA 
                   
               
               
                   
               
               
                 Optimized phoA 
                 ATGAAACAATCGACCATCGCATTGGCGCTGC 
                 SEQ ID NO: 209 
               
               
                   
                 TTCCTCTATTGTTCACACCGGTGACAAAGGC 
                   
               
               
                   
                 A 
                   
               
               
                   
               
               
                 RBS-TorA 
                 
                   ctctagaaataattttgataactttaagaag 
                 
                 SEQ ID NO: 210 
               
               
                   
                   gagatatacat ATGAACAATAACGATCTCTT 
                   
               
               
                 RBS: underlined 
                 TCAGGCATCACGTCGGCGTTTTCTGGCACAA 
                   
               
               
                   
                 CTCGGCGGCTTAACCGTCGCCGGGATGCTGG 
                   
               
               
                   
                 GGCCGTCATTGTTAACGCCGCGACGTGCGAC 
                   
               
               
                   
                 TGCG 
                   
               
               
                   
               
               
                 TorA 
                 ATGAACAATAACGATCTCTTTCAGGCATCAC 
                 SEQ ID NO: 211 
               
               
                   
                 GTCGGCGTTTTCTGGCACAACTCGGCGGCTT 
                   
               
               
                   
                 AACCGTCGCCGGGATGCTGGGGCCGTCATTG 
                   
               
               
                   
                 TTAACGCCGCGACGTGCGACTGCG 
                   
               
               
                   
               
               
                 RBS-TorA alternate 
                   CCCACATTCGAGGTACTAA atgaacaataac 
                 SEQ ID NO: 212 
               
               
                   
                 gatctctttcaggcatcacgtcggcgttttc 
                   
               
               
                   
                 tggcacaactcggcggcttaaccgtcgccgg 
                   
               
               
                   
                 gatgctggggacgtcattgttaacgccgcgc 
                   
               
               
                   
                 cgtgcgactgcggcgcaagcggcg 
                   
               
               
                   
               
               
                 TorA (alternate) 
                 atgaacaataacgatctctttcaggcatcac 
                 SEQ ID NO: 213 
               
               
                   
                 gtcggcgttttctggcacaactcggcggctt 
                   
               
               
                   
                 aaccgtcgccgggatgctggggacgtcattg 
                   
               
               
                   
                 ttaacgccgcgccgtgcgactgcggcgcaag 
                   
               
               
                   
               
               
                 RBS-fdnG 
                 cggcgACCCTATTACACACCTAAGGAGGCCA 
                 SEQ ID NO: 214 
               
               
                   
                 AATACatggacgtcagtcgcagacaattttt 
                   
               
               
                   
                 taaaatctgcgcgggcggtatggcgggaaca 
                   
               
               
                   
                 acagtagcagcattgggctttgccccgaagc 
                   
               
               
                   
                 aagcactggct 
                   
               
               
                   
               
               
                 fdnG 
                 atggacgtcagtcgcagacaattttttaaaa 
                 SEQ ID NO: 215 
               
               
                   
                 cgcgggcggtatggcgggaacaacagtagca 
                   
               
               
                   
                 tctgcattgggctttgccccgaagcaagcac 
                   
               
               
                   
                 tggct 
                   
               
               
                   
               
               
                 RBS-dms 
                 ATACGCAAAAAACATAATTTAAGAGAGGATA 
                 SEQ ID NO: 216 
               
               
                   
                 AACatgaaaacgaaaatccctgatgcggtat 
                   
               
               
                   
                 tggctgctgaggtgagtcgccgtggtttggt 
                   
               
               
                   
                 aaaaacgacagcgatcggcggcctggcaatg 
                   
               
               
                   
                 gccagcagcgcattaacattaccttttagtc 
                   
               
               
                   
                 ggattgcgcacgct 
                   
               
               
                   
               
               
                 dmsA 
                 atgaaaacgaaaatccctgatgcggtattgg 
                 SEQ ID NO: 217 
               
               
                   
                 ctgctgaggtgagtcgccgtggtttggtaaa 
                   
               
               
                   
                 aacgacagcgatcggcggcctggcaatggcc 
                   
               
               
                   
                 agcagcgcattaacattaccttttagtcgga 
                   
               
               
                   
                 ttgcgcacgct 
               
               
                   
               
            
           
         
       
     
     In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, and SEQ ID NO: 217. Table 65 lists exemplary promoter sequences and miscellaneous construct sequences. 
     
       
         
           
               
             
               
                 TABLE 65 
               
             
            
               
                   
               
               
                 Promoter Sequences and Miscellaneous Construct Sequences 
               
            
           
           
               
               
               
            
               
                 Description 
                 Sequence 
                 SEQ ID NO 
               
               
                   
               
               
                 TetR/TetA 
                 gaattcgttaagacccactttcacatttaagttgtttactaatccgcat  
                 SEQ ID   
               
               
                 Promoter 
                 atgatcaattcaaggccgaataagaaggctggctctgcaccttggtgat 
                 NO: 218 
               
               
                   
                 caaataattcgatagcttgtcgtaataatggcggcatactatcagtagt 
                   
               
               
                   
                 aggtgtttccctttcactttagcgacttgatgctcttgatcttccaata 
                   
               
               
                   
                 cgcaacctaaagtaaaatgccccacagcgctgagtgcatataatgcatt 
                   
               
               
                   
                 ctctagtgaaaaaccttgttggcataaaaaggctaattgattttcgaga 
                   
               
               
                   
                 gtttcatactgtttttctgtaggccgtgtacctaaatgtacttttgctc 
                   
               
               
                   
                 catcgcgatgacttagtaaagcacatctaaaacttttagcgttattacg 
                   
               
               
                   
                 taaaaaatcttgccagctttccccttctaaagggcaaaagtgagtatgg 
                   
               
               
                   
                 tgcctatctaacatctcaatggctaaggcgtcgagcaaagcccgcttat 
                   
               
               
                   
                 tttttacatgccaatacaatgtaggctgctctacacctagcttctgggc 
                   
               
               
                   
                 gagtttacgggttgttaaaccttcgattccgacctcattaagcagctct 
                   
               
               
                   
                 aatgcgctgttaatcactttacttttatctaatctagacatcattaatt 
                   
               
               
                   
                 cctaatttttgttgacactctatcattgatagagttattttaccactcc 
                   
               
               
                   
                 ctatcagtgatagagaaaagtgaa 
                   
               
               
                   
               
               
                 fliC  
                 agcgggaataaggggcagagaaaagagtatttcgtcgactaacaaaaaa 
                 SEQ ID  
               
               
                 Promoter  
                 gtggctgtttgtaaaaaaattctaaaggttgttttacgacagacgataa 
                 NO: 219 
               
               
                   
                 cagggt 
                   
               
               
                   
               
               
                 FnrS 
                 ggtaccAGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGT 
                 SEQ ID  
               
               
                 Promoter 
                 AAATGGTIGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACG 
                 NO: 220 
               
               
                   
                 CCGCAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATTCAGGGNCCA 
                   
               
               
                   
                 ATATCTCTCTTggatcc 
                   
               
               
                   
               
               
                 DOM 
                 cacatttccccgaaaagtgccgatggccccccgatggtagtgtggccca 
                 SEQ ID  
               
               
                 Construct 
                 tgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtc 
                 NO: 221 
               
               
                 Terminator 
                 gaaagactgggccatcgttttatctgttgtttgtcggtgaacgctctcc 
                   
               
               
                   
                 tgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacg 
                   
               
               
                   
                 gcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaa 
                   
               
               
                   
                 attaagcagaaggccatcctgacggatggcctttttgcgtggccagtgc 
                   
               
               
                   
                 caagcttgcatgcagattgcagcattacacgtcttgagcgattgtgtag 
                   
               
               
                   
                 gctggagctgcttc 
                   
               
               
                   
               
               
                 FRT Site 
                 gaagttcctatactttctagagaataggaacttcggaataggaacttc 
                 SEQ ID  
               
               
                   
                   
                 NO: 222 
               
               
                   
               
               
                 Kanamycin 
                 aagatcccctcacgctgccgcaagcactcagggcgcaagggctgctaaa 
                 SEQ ID  
               
               
                 Resistance 
                 ggaagcggaacacgtagaaagccagtccgcagaaacggtgctgaccccg 
                 NO: 223 
               
               
                 Cassette   
                 gatgaatgtcagctactgggctatctggacaagggaaaacgcaagcgca 
                   
               
               
                 (for 
                 aagagaaagcaggtagcttgcagtgggcttacatggcgatagctagact 
                   
               
               
                 integration 
                 gggcggttttatggacagcaagcgaaccggaattgccagctggggcgcc 
                   
               
               
                 in between 
                 ctctggtaaggttgggaagccctgcaaagtaaactggatggctttcttg 
                   
               
               
                 FRT sites) 
                 ccgccaaggatctgatggcgcaggggatcaagatctgatcaagagacag 
                   
               
               
                   
                 gatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttc 
                   
               
               
                   
                 tccggccgcttgggtggagaggctattcggctatgactgggcacaacag 
                   
               
               
                   
                 acaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggc 
                   
               
               
                   
                 gcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaact 
                   
               
               
                   
                 gcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttcct 
                   
               
               
                   
                 tgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgc 
                   
               
               
                   
                 tattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcc 
                   
               
               
                   
                 tgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacg 
                   
               
               
                   
                 cttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcg 
                   
               
               
                   
                 agcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatct 
                   
               
               
                   
                 ggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctc 
                   
               
               
                   
                 aaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatg 
                   
               
               
                   
                 cctgcttgccgaatatcatggtggaaaatggccgcttttctggattcat 
                   
               
               
                   
                 cgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttg 
                   
               
               
                   
                 gctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgct 
                   
               
               
                   
                 tcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgcctt 
                   
               
               
                   
                 ctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaa 
                   
               
               
                   
                 tgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccac 
                   
               
               
                   
                 cgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgcc 
                   
               
               
                   
                 ggctggatgatcctccagcgcggggatctcatgctggagttcttcgccc 
                   
               
               
                   
                 accccagcttcaaaagcgctct 
               
               
                   
               
            
           
         
       
     
     In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, and SEQ ID NO: 223. Table 66 Lists exemplary secretion constructs. 
     
       
         
           
               
             
               
                 TABLE 66 
               
             
            
               
                   
               
               
                 Non-limiting Examples of Secretion Constructs 
               
            
           
           
               
               
               
            
               
                 Description 
                 Sequence 
                 SEQ ID NO: 
               
               
                   
               
               
                 FliC20-glp2; a human 
                 cgttccttgtagggcgtcatagcgttcgacggcattaagtaacccaatgcc 
                 SEQ ID NO: 
               
               
                 GLP2 construct 
                 gcccgcctgtagcagatcgtcaagttccacgctcgcgggcagtcgaacct 
                 224 
               
               
                 inserted into the FliC 
                 gcaggcgcaatgcttcgtgacgcaccagcgggacataacgctgccacag 
                   
               
               
                 locus, under the 
                 cgagtgtttatccattacaccttcagcggtatagagtgaattcacgataaaca 
                   
               
               
                 control of the native 
                 gccctgcgttatatgagttatcggcatgattatccgtttctgcagggtttttaat 
                   
               
               
                 FliC promoter (as 
                 cggacgattagtgggtgaaatgaggggttatttgggggttaccggtaaatt 
                   
               
               
                 shown in FIG. 32A) 
                 gcgggcagaaaaaaccccgccgttggcggggaagcacgttgctggcaa 
                   
               
               
                   
                 attaccattcatgttgccggatgcggcgtaaacgccttatccggcctacaaa 
                   
               
               
                   
                 aatgtgcaaattcaataaattgcaattccccttgtaggcctgataagcgcag 
                   
               
               
                   
                 cgcatcaggcaatttggcgttgccgtcagtctcagttaatcaggttacggcg 
                   
               
               
                   
                 attaatcagtaattttagtttggatcagccaattaataaaatcacgcgccgcc 
                   
               
               
                   
                 agattatccaggatggtattcatttcgtcagaaaaagagccgtcagcATG 
                   
               
               
                   
                 cattaggaacctcccagagtttatacttgttgattacgttttgggtttccaccc 
                   
               
               
                   
                 gtcggctcaatcgccgtcaaccctgttatcgtctgtcgtaaaacaacctttag 
                   
               
               
                   
                 aatttttttcacaaacagccattttttgttagtcgacgaaatactcttttctctgc 
                   
               
               
                   
                 cccttattcccgctattaaaaaaaacaattaaacgtaaactttgcgcaattca 
                   
               
               
                   
                 ggccgataaccccggtattcgttttacgtgtcgaaagataaaCGAAGT 
                   
               
               
                   
                 TCCTATACTTTCTAGAGAATAGGAACTTCGG 
                   
               
               
                   
                 AATAGGAACTTCATTTctcgttcgctgccacctaagaatact 
                   
               
               
                   
                 ctacggtcacatacAAATGGCGCGCCTTACGCCCCGC 
                   
               
               
                   
                 CCTGCCACTCATCGCAGTACTGTTGTATTCAT 
                   
               
               
                   
                 TAAGCATCTGCCGACATGGAAGCCATCACAA 
                   
               
               
                   
                 ACGGCATGATGAACCTGAATCGCCAGCGGCA 
                   
               
               
                   
                 TCAGCACCTTGTCGCCTTGCGTATAATATTTG 
                   
               
               
                   
                 CCCATGGTGAAAACGGGGGCGAAGAAGTTGT 
                   
               
               
                   
                 CCATATTGGCCACGTTTAAATCAAAACTGGT 
                   
               
               
                   
                 GAAACTCACCCAGGGATTGGCTGAGACGAAA 
                   
               
               
                   
                 AACATATTCTCAATAAACCCTTTAGGGAAAT 
                   
               
               
                   
                 AGGCCAGGTTTTCACCGTAACACGCCACATC 
                   
               
               
                   
                 TTGCGAATATATGTGTAGAAACTGCCGGAAA 
                   
               
               
                   
                 TCGTCGTGGTATTCACTCCAGAGCGATGAAA 
                   
               
               
                   
                 ACGTTTCAGTTTGCTCATGGAAAACGGTGTA 
                   
               
               
                   
                 ACAAGGGTGAACACTATCCCATATCACCAGC 
                   
               
               
                   
                 TCACCGTCTTTCATTGCCATACGTAATTCCGG 
                   
               
               
                   
                 ATGAGCATTCATCAGGCGGGCAAGAATGTGA 
                   
               
               
                   
                 ATAAAGGCCGGATAAAACTTGTGCTTATTTTT 
                   
               
               
                   
                 CTTTACGGTCTTTAAAAAGGCCGTAATATCC 
                   
               
               
                   
                 AGCTGAACGGTCTGGTTATAGGTACATTGAG 
                   
               
               
                   
                 CAACTGACTGAAATGCCTCAAAATGTTCTTT 
                   
               
               
                   
                 ACGATGCCATTGGGATATATCAACGGTGGTA 
                   
               
               
                   
                 TATCCAGTGATTTTTTTCTCCATTTTAGCTTCC 
                   
               
               
                   
                 TTAGCTCCTGAAAATCTCGACAACTCAAAAA 
                   
               
               
                   
                 ATACGCCCGGTAGTGATCTTATTTCATTATGG 
                   
               
               
                   
                 TGAAAGTTGGAACCTCTTACGTGCCGATCAA 
                   
               
               
                   
                 CGTCTCATTTTCGCCAAAAGTTGGCCCAGGG 
                   
               
               
                   
                 CTTCCCGGTATCAACAGGGACACCAGGATTT 
                   
               
               
                   
                 ATTTATTCTGCGAAGTGATCTTCCGTCACAGG 
                   
               
               
                   
                 TAGGCGCGCCGAAGTTCCTATACTTTCTAGA 
                   
               
               
                   
                 GAATAGGAACTTCGGAATAGGAACTctcaccgcc 
                   
               
               
                   
                 gcgcaaaaagcgacgctaacccctatttcaaatcagcaatcgtcgtttacc 
                   
               
               
                   
                 gctaaacttagcgcctacggtacgctgaaaagcgcgctgacgactttcca 
                   
               
               
                   
                 gaccgccaatactgcattgtctaaagccgatcttttttccgctaccagcacc 
                   
               
               
                   
                 accagcagcaccaccgcgttcagtgccaccaccgcgggtaatgccatcg 
                   
               
               
                   
                 ccgggaaatacaccatcagcgtcacccatctggcgcaggcgcaaaccct 
                   
               
               
                   
                 gacaacgcgcaccaccagagacgatacgaaaacggcgatcgccacca 
                   
               
               
                   
                 gcgacagcaaactcaccattcaacaaggcggcgacaaagatccgatttcc 
                   
               
               
                   
                 attgatatcagcgcggctaactcgtctttaagcgggatccgtgatgccatca 
                   
               
               
                   
                 acaacgcaaaagcaggcgtaagcgcaagcatcattaacgtgggtaacgg 
                   
               
               
                   
                 tgaatatcgtctgtcagtcacatcaaatgacaccggcct 
                   
               
               
                   
               
               
                 FliC20 with optimized 
                 attaatcagtaattttagtttggatcagccaattaataaaatcacgcgccgcc 
                 SEQ ID NO: 
               
               
                 RBS-GLP2 and UTR- 
                 agattatccaggatggtattcatttcgtcagaaaaagagccgtcagcATG 
                 225 
               
               
                 FliC (as shown in FIG. 
                 cattaggaacctcccagagtttatacttgttgattacgttttgggtttccaccc 
                   
               
               
                 32A, in reverse 
                 gtcggctcaatcgccgtca 
                   
               
               
                 orientation) 
                   
                   
               
               
                   
               
               
                 human GLP2 
                 cgttccttgtagggcgtcatagcgttcgacggcattaagtaacccaatgcc 
                 SEQ ID NO: 
               
               
                 construct, , including 
                 gcccgcctgtagcagatcgtcaagttccacgctcgcgggcagtcgaacct 
                 226 
               
               
                 the N terminal 20 
                 gcaggcgcaatgcttcgtgacgcaccagcgggacataacgctgccacag 
                   
               
               
                 amino acids of FliC 
                 cgagtgtttatccattacaccttcagcggtatagagtgaattcacgataaaca 
                   
               
               
                 (reverse orientation), 
                 gccctgcgttatatgagttatcggcatgattatccgtttctgcagggtttttaat 
                   
               
               
                 inserted into the FliC 
                 cggacgattagtgggtgaaatgaggggttatttgggggttaccggtaaatt 
                   
               
               
                 locus under the control 
                 gcgggcagaaaaaaccccgccgttggcggggaagcacgttgctggcaa 
                   
               
               
                 of a tet inducible 
                 attaccattcatgttgccggatgcggcgtaaacgccttatccggcctacaaa 
                   
               
               
                 promoter, with TetR 
                 aatgtgcaaattcaataaattgcaattccccttgtaggcctgataagcgcag 
                   
               
               
                 and chloramphenicol 
                 cgcatcaggcaatttggcgttgccgtcagtctcagttaatcaggttacggcg 
                   
               
               
                 resistance. 
                 attaatcagtaattttagtttggatcagccaattaataaaatcacgcgccgcc 
                   
               
               
                 (as shown in FIG. 
                 agattatccaggatggtattcatttcgtcagaaaaagagccgtcagcATG 
                   
               
               
                 32C) 
                 cttgttgatattattttgagtgatcagcgagaggctgttggtattaatgacttgt 
                   
               
               
                   
                 gccatGGTCCATTCGAACCCAATTTAAGGAGTA 
                   
               
               
                   
                 CCCACgttgattacgattttgggtttccacccgtcggctcaatcgccgtca 
                   
               
               
                   
                 ttctctatcactgatagggagtggtaaaataactctatcaatgatagagtgtc 
                   
               
               
                   
                 aacaaaaattaggaattaatgatgtctagattagataaaagtaaagtgattaa 
                   
               
               
                   
                 cagcgcattagagctgcttaatgaggtcggaatcgaaggtttaacaacccg 
                   
               
               
                   
                 taaactcgcccagaagctaggtgtagagcagcctacattgtattggcatgt 
                   
               
               
                   
                 aaaaaataagcgggctttgctcgacgccttagccattgagatgttagatag 
                   
               
               
                   
                 gcaccatactcacttttgccctttagaaggggaaagctggcaagattttttac 
                   
               
               
                   
                 gtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagca 
                   
               
               
                   
                 aaagtacatttaggtacacggcctacagaaaaacagtatgaaactctcgaa 
                   
               
               
                   
                 aatcaattagcctttttatgccaacaaggtttttcactagagaatgcattatatg 
                   
               
               
                   
                 cactcagcgctgtggggcattttactttaggttgcgtattggaagatcaaga 
                   
               
               
                   
                 gcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatgc 
                   
               
               
                   
                 cgccattattacgacaagctatcgaattatttgatcaccaaggtgcagagcc 
                   
               
               
                   
                 agccttcttattcggccttgaattgatcatatgcggattagaaaaacaactta 
                   
               
               
                   
                 aatgtgaaagtgggtcttaagaatttttttcacaaacagccattttttgttagtc 
                   
               
               
                   
                 gacgaaatactcttttctctgccccttattcccgctattaaaaaaaacaattaa 
                   
               
               
                   
                 acgtaaactttgcgcaattcaggccgataaccccggtattcgttttacgtgtc 
                   
               
               
                   
                 gaaagataaaCGAAGTTCCTATACTTTCTAGAGAA 
                   
               
               
                   
                 TAGGAACTTCGGAATAGGAACTTCATTTctcgtt 
                   
               
               
                   
                 cgctgccacctaagaatactctacggtcacatacAAATGGCGCG 
                   
               
               
                   
                 CCTTACGCCCCGCCCTGCCACTCATCGCAGTA 
                   
               
               
                   
                 CTGTTGTATTCATTAAGCATCTGCCGACATGG 
                   
               
               
                   
                 AAGCCATCACAAACGGCATGATGAACCTGAA 
                   
               
               
                   
                 TCGCCAGCGGCATCAGCACCTTGTCGCCTTG 
                   
               
               
                   
                 CGTATAATATTTGCCCATGGTGAAAACGGGG 
                   
               
               
                   
                 GCGAAGAAGTTGTCCATATTGGCCACGTTTA 
                   
               
               
                   
                 AATCAAAACTGGTGAAACTCACCCAGGGATT 
                   
               
               
                   
                 GGCTGAGACGAAAAACATATTCTCAATAAAC 
                   
               
               
                   
                 CCTTTAGGGAAATAGGCCAGGTTTTCACCGT 
                   
               
               
                   
                 AACACGCCACATCTTGCGAATATATGTGTAG 
                   
               
               
                   
                 AAACTGCCGGAAATCGTCGTGGTATTCACTC 
                   
               
               
                   
                 CAGAGCGATGAAAACGTTTCAGTTTGCTCAT 
                   
               
               
                   
                 GGAAAACGGTGTAACAAGGGTGAACACTATC 
                   
               
               
                   
                 CCATATCACCAGCTCACCGTCTTTCATTGCCA 
                   
               
               
                   
                 TACGTAATTCCGGATGAGCATTCATCAGGCG 
                   
               
               
                   
                 GGCAAGAATGTGAATAAAGGCCGGATAAAA 
                   
               
               
                   
                 CTTGTGCTTATTTTTCTTTACGGTCTTTAAAA 
                   
               
               
                   
                 AGGCCGTAATATCCAGCTGAACGGTCTGGTT 
                   
               
               
                   
                 ATAGGTACATTGAGCAACTGACTGAAATGCC 
                   
               
               
                   
                 TCAAAATGTTCTTTACGATGCCATTGGGATAT 
                   
               
               
                   
                 ATCAACGGTGGTATATCCAGTGATTTTTTTCT 
                   
               
               
                   
                 CCATTTTAGCTTCCTTAGCTCCTGAAAATCTC 
                   
               
               
                   
                 GACAACTCAAAAAATACGCCCGGTAGTGATC 
                   
               
               
                   
                 TTATTTCATTATGGTGAAAGTTGGAACCTCTT 
                   
               
               
                   
                 ACGTGCCGATCAACGTCTCATTTTCGCCAAA 
                   
               
               
                   
                 AGTTGGCCCAGGGCTTCCCGGTATCAACAGG 
                   
               
               
                   
                 GACACCAGGATTTATTTATTCTGCGAAGTGA 
                   
               
               
                   
                 TCTTCCGTCACAGGTAGGCGCGCCGAAGTTC 
                   
               
               
                   
                 CTATACTTTCTAGAGAATAGGAACTTCGGAA 
                   
               
               
                   
                 TAGGAACTctcaccgccgcgcaaaaagcgacgctaacccctattt 
                   
               
               
                   
                 caaatcagcaatcgtcgtttaccgctaaacttagcgcctacggtacgctga 
                   
               
               
                   
                 aaagcgcgctgacgactttccagaccgccaatactgcattgtctaaagccg 
                   
               
               
                   
                 atcttttttccgctaccagcaccaccagcagcaccaccgcgttcagtgcca 
                   
               
               
                   
                 ccaccgcgggtaatgccatcgccgggaaatacaccatcagcgtcaccca 
                   
               
               
                   
                 tctggcgcaggcgcaaaccctgacaacgcgcaccaccagagacgatac 
                   
               
               
                   
                 gaaaacggcgatcgccaccagcgacagcaaactcaccattcaacaagg 
                   
               
               
                   
                 cggcgacaaagatccgatttccattgatatcagcgcggctaactcgtcttta 
                   
               
               
                   
                 agcgggatccgtgatgccatcaacaacgcaaaagcaggcgtaagcgca 
                   
               
               
                   
                 agcatcattaacgtgggtaacggtgaatatcgtctgtcagtcacatcaaatg 
                   
               
               
                   
                 acaccggcct 
                   
               
               
                   
               
               
                 human GLP2 
                 ttaatcagtaattttagtttggatcagccaattaataaaatcacgcgccgcca 
                 SEQ ID NO: 
               
               
                 construct, , including 
                 gattatccaggatggtattcatttcgtcagaaaaagagccgtcagcATGc 
                 227 
               
               
                 the N terminal 20 
                 ttgttgatattattttgagtgatcagcgagaggctgttggtattaatgacttgtg 
                   
               
               
                 amino acids of FliC 
                 ccat 
                   
               
               
                 (reverse orientation) 
                   
                   
               
               
                   
               
               
                 human GLP2 
                 ttaagacccactttcacatttaagttgatttctaatccgcatatgatcaattcaa 
                 SEQ ID NO: 
               
               
                 construct with a N 
                 ggccgaataagaaggctggctctgcaccttggtgatcaaataattcgatag 
                 228 
               
               
                 terminal OmpF 
                 cttgtcgtaataatggcggcatactatcagtagtaggtgtttccctttcttcttt 
                   
               
               
                 secretion tag (sec- 
                 agcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgcccc 
                   
               
               
                 dependent secretion 
                 acagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcata 
                   
               
               
                 system) under the 
                 aaaaggctaattgattttcgagagtttcatactgtttttctgtaggccgtgtacc 
                   
               
               
                 control of a tet 
                 taaatgtacttttgctccatcgcgatgacttagtaaagcacatctaaaactttt 
                   
               
               
                 inducible promoter, 
                 agcgttattacgtaaaaaatcttgccagctttccccttctaaagggcaaaagt 
                   
               
               
                 includes TetR in 
                 gagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagccc 
                   
               
               
                 reverse direction 
                 gcttattttttacatgccaatacaatgtaggctgctctacacctagcttctggg 
                   
               
               
                 (as shown in FIG. 
                 cgagtttacgggttgttaaaccttcgattccgacctcattaagcagctctaat 
                   
               
               
                 32C) 
                 gcgctgttaatcactttacttttatctaatctagacatcattaattcctaatttttgt 
                   
               
               
                   
                 tgacactctatcattgatagagttattttaccactccctatcagtgatagagaa 
                   
               
               
                   
                 aagtgaactctagaaataattttgtttaactttaagaaggagatatacatatga 
                   
               
               
                   
                 tgaagcgcaatattctggcagtgatcgtccctgctctgttagtagcaggtac 
                   
               
               
                   
                 tgcaaacgctcatgctgatggttctttctctgatgagatgaacaccattcttga 
                   
               
               
                   
                 taatcttgccgccagggactttataaactggttgattcagaccaaaatcactg 
                   
               
               
                   
                 acaggtgacacatttccccgaaaagtgccgatggccccccgatggtagtg 
                   
               
               
                   
                 tggccccatgcgagagtagggaactgccaggcatcaaataaaacgaaag 
                   
               
               
                   
                 gctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgct 
                   
               
               
                   
                 ctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagca 
                   
               
               
                   
                 acggcccggagggtggcgggcaggacgcccgccataaactgccaggc 
                   
               
               
                   
                 atcaaattaagcagaaggccatcctgacggatggcctttttgcgtggccag 
                   
               
               
                   
                 tgccaagcttgcatgcagattgcagcattacacgtcttgagcgattgtgtag 
                   
               
               
                   
                 gctggagctgcttcgaagttcctatactttctagagaataggaacttcggaat 
                   
               
               
                   
                 aggaacttc 
                   
               
               
                   
               
               
                 human GLP2 
                 atgatgaagcgcaatattctggcagtgatcgtccctgctctgttagtagcag 
                 SEQ ID NO: 
               
               
                 construct with a N 
                 gtactgcaaacgctcatgctgatggttctttctctgatgagatgaacaccatt 
                 229 
               
               
                 terminal OmpF 
                 cttgataatcttgccgccagggactttataaactggttgattcagaccaaaat 
                   
               
               
                 secretion tag (sec- 
                 cactgacaggtga 
                   
               
               
                 dependent secretion 
                   
                   
               
               
                 system) (as shown in 
                   
                   
               
               
                 FIG. 32C) 
                   
                   
               
               
                   
               
               
                 human GLP2 
                 taagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaa 
                 SEQ ID NO: 
               
               
                 construct with a N 
                 ggccgaataagaaggctggctctgcaccttggtgatcaaataattcgatag 
                 230 
               
               
                 terminal TorA 
                 cttgtcgtaataatggcggcatactatcagtagtaggtgtttccctttcttcttt 
                   
               
               
                 secretion tag (tat 
                 agcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgcccc 
                   
               
               
                 secretion system) 
                 acagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcata 
                   
               
               
                 under the control of a 
                 aaaaggctaattgattttcgagagtttcatactgtttttctgtaggccgtgtacc 
                   
               
               
                 tet inducible promoter 
                 taaatgtacttttgctccatcgcgatgacttagtaaagcacatctaaaactttt 
                   
               
               
                 (as shown in FIG. 
                 agcgttattacgtaaaaaatcttgccagctttccccttctaaagggcaaaagt 
                   
               
               
                 32E) 
                 gagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagccc 
                   
               
               
                   
                 gcttattttttacatgccaatacaatgtaggctgctctacacctagcttctggg 
                   
               
               
                   
                 cgagtttacgggttgttaaaccttcgattccgacctcattaagcagctctaat 
                   
               
               
                   
                 gcgctgttaatcactttacttttatctaatctagacatcattaattcctaatttttgt 
                   
               
               
                   
                 tgacactctatcattgatagagttattttaccactccctatcagtgatagagaa 
                   
               
               
                   
                 aagtgaactctagaaataattttgtttaactttaagaaggagatatacatAT 
                   
               
               
                   
                 GAACAATAACGATCTCTTTCAGGCATCACGT 
                   
               
               
                   
                 CGGCGTTTTCTGGCACAACTCGGCGGCTTAA 
                   
               
               
                   
                 CCGTCGCCGGGATGCTGGGGCCGTCATTGTT 
                   
               
               
                   
                 AACGCCGCGACGTGCGACTGCGcatgctgatggttctt 
                   
               
               
                   
                 tctctgatgagatgaacaccattcttgataatcttgccgccagggactttata 
                   
               
               
                   
                 aactggttgattcagaccaaaatcactgactaataacacatttccccgaaaa 
                   
               
               
                   
                 gtgccgatggccccccgatggtagtgtggcccatgcgagagtagggaac 
                   
               
               
                   
                 tgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttc 
                   
               
               
                   
                 gttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccg 
                   
               
               
                   
                 ggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggca 
                   
               
               
                   
                 ggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcc 
                   
               
               
                   
                 tgacggatggcctttttgcgtggccagtgccaagcttgcatgcagattgca 
                   
               
               
                   
                 gcattacacgtcttgagcgattgtgtaggctggagctgcttcgaagttcctat 
                   
               
               
                   
                 actttctagagaataggaacttcggaataggaacttc 
                   
               
               
                   
               
               
                 GLP-2 with TORA tag 
                 ATGAACAATAACGATCTCTTTCAGGCATCAC 
                 SEQ ID NO: 
               
               
                   
                 GTCGGCGTTTTCTGGCACAACTCGGCGGCTT 
                 231 
               
               
                   
                 AACCGTCGCCGGGATGCTGGGGCCGTCATTG 
                   
               
               
                   
                 TTAACGCCGCGACGTGCGACTGCGcatgctgatggt 
                   
               
               
                   
                 tctttctctgatgagatgaacaccattcttgataatcttgccgccagggacttt 
                   
               
               
                   
                 ataaactggttgattcagaccaaaatcactgac 
               
               
                   
               
            
           
         
       
     
     In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, and SEQ ID NO: 231. Table 67 lists exemplary secretion constructs. 
     
       
         
           
               
             
               
                 TABLE 67 
               
             
            
               
                   
               
               
                 Non-limiting Examples of Secretion Constructs 
               
            
           
           
               
               
               
            
               
                 Description 
                 Sequences 
                 SEQ ID NO 
               
               
                   
               
               
                 Ptet-phoA-hIL10 
                 gaattcgttaagacccactttcacatttaagttgtttttctaatccgcatat 
                 SEQ ID NO: 
               
               
                   
                 gatcaattcaaggccgaataagaaggctggctctgcaccttggtgatca 
                 232 
               
               
                   
                 aataattcgatagcttgtcgtaataatggcggcatactatcagtagtagg 
                   
               
               
                   
                 tgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaac 
                   
               
               
                   
                 ctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctagt 
                   
               
               
                   
                 gaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcata 
                   
               
               
                   
                 ctgtttttctgtaggccgtgtacctaaatgtacttttgctccatcgcgatga 
                   
               
               
                   
                 cttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgc 
                   
               
               
                   
                 cagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatc 
                   
               
               
                   
                 tcaatggctaaggcgtcgagcaaagcccgcttattttttacatgccaatac 
                   
               
               
                   
                 aatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaa 
                   
               
               
                   
                 ccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttac 
                   
               
               
                   
                 ttttatctaatctagacatcattaattcctaatttttgttgacactctatcatt 
                   
               
               
                   
                 gatagagttattttaccactccctatcagtgatagagaaaagtgaa 
                   
               
               
                   
                   GACGCCAGAGAGTTAAGGGGGTTAA ATGAA 
                   
               
               
                   
                 ACAATCGACCATCGCATTGGCGCTGCTTCCTC 
                   
               
               
                   
                 TATTGTTCACACCGGTGACAAAGGCA 
                   
               
               
                   
                 TCGCCAGGTCAAGGAACGCAGTCAGAGAATT 
                   
               
               
                   
                 CATGCACTCACTTTCCGGGCAATCTGCCGAA 
                   
               
               
                   
                 TATGCTGCGCGATCTGCGAGATGCATTCTCTC 
                   
               
               
                   
                 GCGTGAAAACGTTCTTTCAAATGAAAGATCA 
                   
               
               
                   
                 ACTGGATAATCTGCTGCTGAAGGAGTCGTTG 
                   
               
               
                   
                 TTGGAGGATTTTAAGGGGTATCTGGGTTGTC 
                   
               
               
                   
                 AAGCACTGTCTGAAATGATTCAATTTTACTTG 
                   
               
               
                   
                 GAGGAAGTTATGCCGCAAGCGGAAAACCAA 
                   
               
               
                   
                 GATCCGGATATTAAGGCGCACGTGAACTCAC 
                   
               
               
                   
                 TGGGCGAAAACCTGAAAACTTTGCGCCTGCG 
                   
               
               
                   
                 TCTGAGACGATGTCACCGATTCCTGCCGTGT 
                   
               
               
                   
                 GAAAACAAGTCAAAGGCGGTTGAGCAAGTT 
                   
               
               
                   
                 AAGAATGCTTTCAATAAGCTGCAAGAAAAGG 
                   
               
               
                   
                 GCATCTATAAAGCGATGTCTGAATTTGATAT 
                   
               
               
                   
                 CTTTATAAACTACATAGAAGCTTATATGACT 
                   
               
               
                   
                 ATGAAGATTCGAAATTAA 
                   
               
               
                   
               
               
                 phoA-hIL10 
                   GACGCCAGAGAGTTAAGGGGGTTAA ATGAA 
                 SEQ ID NO: 
               
               
                   
                 ACAATCGACCATCGCATTGGCGCTGCTTCCTC 
                 233 
               
               
                   
                 TATTGTTCACACCGGTGACAAAGGCA 
                   
               
               
                   
                 TCGCCAGGTCAAGGAACGCAGTCAGAGAATT 
                   
               
               
                   
                 CATGCACTCACTTTCCGGGCAATCTGCCGAA 
                   
               
               
                   
                 TATGCTGCGCGATCTGCGAGATGCATTCTCTC 
                   
               
               
                   
                 GCGTGAAAACGTTCTTTCAAATGAAAGATCA 
                   
               
               
                   
                 ACTGGATAATCTGCTGCTGAAGGAGTCGTTG 
                   
               
               
                   
                 TTGGAGGATTTTAAGGGGTATCTGGGTTGTC 
                   
               
               
                   
                 AAGCACTGTCTGAAATGATTCAATTTTACTTG 
                   
               
               
                   
                 GAGGAAGTTATGCCGCAAGCGGAAAACCAA 
                   
               
               
                   
                 GATCCGGATATTAAGGCGCACGTGAACTCAC 
                   
               
               
                   
                 TGGGCGAAAACCTGAAAACTTTGCGCCTGCG 
                   
               
               
                   
                 TCTGAGACGATGTCACCGATTCCTGCCGTGT 
                   
               
               
                   
                 GAAAACAAGTCAAAGGCGGTTGAGCAAGTT 
                   
               
               
                   
                 AAGAATGCTTTCAATAAGCTGCAAGAAAAGG 
                   
               
               
                   
                 GCATCTATAAAGCGATGTCTGAATTTGATAT 
                   
               
               
                   
                 CTTTATAAACTACATAGAAGCTTATATGACT 
                   
               
               
                   
                 ATGAAGATTCGAAATTAA 
                   
               
               
                   
               
               
                 fliC UTR-RBS - 
                   tgacggcgattgagccgacgggtggaaacccaaaacgtaatcaac     t     
                 SEQ ID NO: 
               
               
                 pvIL10 
                     caaatcccttaataaggaggtaaa   ATGGGTACTGACCAA 
                 2334 
               
               
                   
                 TGTGATAATTTCCCACAAATGCTGCGTGATTT 
                   
               
               
                   
                 GCGCGACGCTTTCTCGCGTGTGAAAACTTTTT 
                   
               
               
                   
                 TTCAGACTAAAGATGAGGTGGATAATCTGCT 
                   
               
               
                   
                 GCTGAAAGAGAGCCTGTTGGAAGATTTTAAA 
                   
               
               
                   
                 GGCTACTTGGGCTGTCAAGCGCTGTCGGAGA 
                   
               
               
                   
                 TGATTCAATTTTATCTGGAAGAGGTGATGCC 
                   
               
               
                   
                 GCAAGCTGAGAACCAAGATCCGGAAGCGAA 
                   
               
               
                   
                 AGATCACGTGAATTCGCTGGGCGAGAATCTG 
                   
               
               
                   
                 AAAACTCTGCGTCTGCGTCTGCGTCGTTGTCA 
                   
               
               
                   
                 CCGTTTTTTGCCGTGCGAAAACAAAAGTAAA 
                   
               
               
                   
                 GCTGTTGAGCAAATTAAAAACGCTTTTAACA 
                   
               
               
                   
                 AACTGCAGGAAAAAGGTATCTATAAAGCGAT 
                   
               
               
                   
                 GAGCGAATTTGATATTTTTATTAATTATATTG 
                   
               
               
                   
                 AAGCTTATATGACTATTAAAGCTCGCTAA 
                   
               
               
                   
               
               
                 Ptet-phoA-vIL10 
                 
                   Gaattcgttaagacccactttcacatttaagttgtttttctaatccgcatat 
                 
                 SEQ ID NO: 
               
               
                   
                 
                   gatcaattcaaggccgaataagaaggctggctctgcaccttggtgatca 
                 
                 235 
               
               
                   
                 
                   aataattcgatagcttgtcgtaataatggcggcatactatcagtagtagg 
                 
                   
               
               
                   
                 
                   tgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaac 
                 
                   
               
               
                   
                 
                   ctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctagt 
                 
                   
               
               
                   
                 
                   gaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcata 
                 
                   
               
               
                   
                 
                   ctgtttttctgtaggccgtgtacctaaatgtacttttgctccatcgcgatga 
                 
                   
               
               
                   
                 
                   cttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgc 
                 
                   
               
               
                   
                 
                   cagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatc 
                 
                   
               
               
                   
                 
                   tcaatggctaaggcgtcgagcaaagcccgcttattttttacatgccaatac 
                 
                   
               
               
                   
                 
                   aatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaa 
                 
                   
               
               
                   
                 
                   ccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttac 
                 
                   
               
               
                   
                 
                   ttttatctaatctagacatcattaattcctaatttttgttgacactctatcatt 
                 
                   
               
               
                   
                 
                   gatagagttattttaccactccctatcagtgatagagaaaagtgaa 
                 
                   
               
               
                   
                   GACGCCAGAGAGTTAAGGGGGTTAA ATGAA 
                   
               
               
                   
                 ACAATCGACCATCGCATTGGCGCTGCTTCCTC 
                   
               
               
                   
                 TATTGTTCACACCGGTGACAAAGGCA 
                   
               
               
                   
                 GGTACAGACCAATGTGACAATTTTCCCCAAA 
                   
               
               
                   
                 TGTTGAGGGACCTAAGAGATGCCTTCAGTCG 
                   
               
               
                   
                 TGTTAAAACCTTTTTCCAGACAAAGGACGAG 
                   
               
               
                   
                 GTAGATAACCTTTTGCTCAAGGAGTCTCTGCT 
                   
               
               
                   
                 AGAGGACTTTAAGGGCTACCTTGGATGCCAG 
                   
               
               
                   
                 GCCCTGTCAGAAATGATCCAATTCTACCTGG 
                   
               
               
                   
                 AGGAAGTCATGCCACAGGCTGAAAACCAGG 
                   
               
               
                   
                 ACCCTGAAGCCAAAGACCATGTCAATTCTTT 
                   
               
               
                   
                 GGGTGAAAATCTAAAGACCCTACGGCTCCGC 
                   
               
               
                   
                 CTGCGCCGTTGCCACAGGTTCCTGCCGTGTG 
                   
               
               
                   
                 AGAACAAGAGTAAAGCTGTGGAACAGATAA 
                   
               
               
                   
                 AAAATGCCTTTAACAAGCTGCAGGAAAAAGG 
                   
               
               
                   
                 AATTTACAAAGCCATGAGTGAATTTGACATT 
                   
               
               
                   
                 TTTATTAACTACATAGAAGCATACATGACAA 
                   
               
               
                   
                 TTAAAGCCAGG 
                   
               
               
                   
               
               
                 phoA-vIL10 
                   GACGCCAGAGAGTTAAGGGGGTTAA ATGAA 
                 SEQ ID NO: 
               
               
                   
                 ACAATCGACCATCGCATTGGCGCTGCTTCCTC 
                 236 
               
               
                   
                 TATTGTTCACACCGGTGACAAAGGCA 
                   
               
               
                   
                 GGTACAGACCAATGTGACAATTTTCCCCAAA 
                   
               
               
                   
                 TGTTGAGGGACCTAAGAGATGCCTTCAGTCG 
                   
               
               
                   
                 TGTTAAAACCTTTTTCCAGACAAAGGACGAG 
                   
               
               
                   
                 GTAGATAACCTTTTGCTCAAGGAGTCTCTGCT 
                   
               
               
                   
                 AGAGGACTTTAAGGGCTACCTTGGATGCCAG 
                   
               
               
                   
                 GCCCTGTCAGAAATGATCCAATTCTACCTGG 
                   
               
               
                   
                 AGGAAGTCATGCCACAGGCTGAAAACCAGG 
                   
               
               
                   
                 ACCCTGAAGCCAAAGACCATGTCAATTCTTT 
                   
               
               
                   
                 GGGTGAAAATCTAAAGACCCTACGGCTCCGC 
                   
               
               
                   
                 CTGCGCCGTTGCCACAGGTTCCTGCCGTGTG 
                   
               
               
                   
                 AGAACAAGAGTAAAGCTGTGGAACAGATAA 
                   
               
               
                   
                 AAAATGCCTTTAACAAGCTGCAGGAAAAAGG 
                   
               
               
                   
                 AATTTACAAAGCCATGAGTGAATTTGACATT 
                   
               
               
                   
                 TTTATTAACTACATAGAAGCATACATGACAA 
                   
               
               
                   
                 TTAAAGCCAGG 
                   
               
               
                   
               
               
                 Ptet- PhoA-IL22 
                 
                   Gaattcgttaagacccactttcacatttaagttgtttttctaatccgcatat 
                 
                 SEQ ID NO: 
               
               
                   
                 
                   gatcaattcaaggccgaataagaaggctggctctgcaccttggtgatca 
                 
                 237 
               
               
                   
                 
                   aataattcgatagcttgtcgtaataatggcggcatactatcagtagtagg 
                 
                   
               
               
                   
                 
                   tgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaac 
                 
                   
               
               
                   
                 
                   ctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctagt 
                 
                   
               
               
                   
                 
                   gaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcata 
                 
                   
               
               
                   
                 
                   ctgtttttctgtaggccgtgtacctaaatgtacttttgctccatcgcgatga 
                 
                   
               
               
                   
                 
                   cttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgc 
                 
                   
               
               
                   
                 
                   cagctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatc 
                 
                   
               
               
                   
                 
                   tcaatggctaaggcgtcgagcaaagcccgcttattttttacatgccaatac 
                 
                   
               
               
                   
                 
                   aatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaa 
                 
                   
               
               
                   
                 
                   ccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttac 
                 
                   
               
               
                   
                 
                   ttttatctaatctagacatcattaattcctaatttttgttgacactctatcatt 
                 
                   
               
               
                   
                 
                   gatagagttattttaccactccctatcagtgatagagaaaagtgaa 
                 
                   
               
               
                   
                   GACGCCAGAGAGTTAAGGGGGTTAA ATGAA 
                   
               
               
                   
                 ACAATCGACCATCGCATTGGCGCTGCTTCCTC 
                   
               
               
                   
                 TATTGTTCACACCGGTGACAAAGGCA 
                   
               
               
                   
                 GCACCGATCTCTTCCCACTGTCGCTTAGATAA 
                   
               
               
                   
                 ATCGAATTTTCAACAACCTTATATTACGAATC 
                   
               
               
                   
                 GTACGTTTATGCTGGCTAAAGAAGCGTCATT 
                   
               
               
                   
                 AGCTGATAACAACACTGATGTTCGCCTGATT 
                   
               
               
                   
                 GGTGAGAAATTGTTTCACGGTGTGTCTATGTC 
                   
               
               
                   
                 AGAACGTTGCTACCTGATGAAACAAGTTCTG 
                   
               
               
                   
                 AATTTCACCCTGGAAGAAGTGTTGTTTCCGC 
                   
               
               
                   
                 AATCTGACCGCTTTCAACCGTATATGCAAGA 
                   
               
               
                   
                 GGTTGTGCCGTTTCTGGCGCGCCTGAGTAATC 
                   
               
               
                   
                 GCCTGAGCACTTGTCATATTGAGGGCGACGA 
                   
               
               
                   
                 CCTGCATATTCAACGAAATGTTCAAAAATTG 
                   
               
               
                   
                 AAAGATACGGTGAAGAAACTGGGTGAAAGT 
                   
               
               
                   
                 GGTGAAATCAAAGCGATTGGTGAGCTGGATC 
                   
               
               
                   
                 TGCTGTTTATGTCATTGCGCAATGCGTGCATT 
                   
               
               
                   
                 TAA 
                   
               
               
                   
               
               
                 PhoA-IL22 
                   GACGCCAGAGAGTTAAGGGGGTTAA ATGAA 
                 SEQ ID NO: 
               
               
                   
                 ACAATCGACCATCGCATTGGCGCTGCTTCCTC 
                 238 
               
               
                   
                 TATTGTTCACACCGGTGACAAAGGCA 
                   
               
               
                   
                 GCACCGATCTCTTCCCACTGTCGCTTAGATAA 
                   
               
               
                   
                 ATCGAATTTTCAACAACCTTATATTACGAATC 
                   
               
               
                   
                 GTACGTTTATGCTGGCTAAAGAAGCGTCATT 
                   
               
               
                   
                 AGCTGATAACAACACTGATGTTCGCCTGATT 
                   
               
               
                   
                 GGTGAGAAATTGTTTCACGGTGTGTCTATGTC 
                   
               
               
                   
                 AGAACGTTGCTACCTGATGAAACAAGTTCTG 
                   
               
               
                   
                 AATTTCACCCTGGAAGAAGTGTTGTTTCCGC 
                   
               
               
                   
                 AATCTGACCGCTTTCAACCGTATATGCAAGA 
                   
               
               
                   
                 GGTTGTGCCGTTTCTGGCGCGCCTGAGTAATC 
                   
               
               
                   
                 GCCTGAGCACTTGTCATATTGAGGGCGACGA 
                   
               
               
                   
                 CCTGCATATTCAACGAAATGTTCAAAAATTG 
                   
               
               
                   
                 AAAGATACGGTGAAGAAACTGGGTGAAAGT 
                   
               
               
                   
                 GGTGAAATCAAAGCGATTGGTGAGCTGGATC 
                   
               
               
                   
                 TGCTGTTTATGTCATTGCGCAATGCGTGCATT 
                   
               
               
                   
                 TAA 
                   
               
               
                   
               
               
                   
                   GACGCCAGAGAGTTAAGGGGGTTAA ATGAA 
                 SEQ ID NO: 
               
               
                   
                 ACAATCGACCATCGCATTGGCGCTGCTTCCTC 
                 239 
               
               
                   
                 TATTGTTCACACCGGTGACAAAGGCA 
               
               
                   
               
            
           
         
       
     
     In some embodiments, genetically engineered bacteria comprise a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% homologous to the DNA sequence of SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 2334, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, and SEQ ID NO: 239. 
     Example 27. Bacterial Secretion of hIL-10 and vIL-10 
     To determine whether the human IL-10 and vIL-10 expressed by engineered bacteria is secreted, the concentration of IL-10 in the bacterial supernatant from a selection of engineered strains comprising various hIL-10 and vIL-10 constructs/strains was measured (see Table 63, Table 64, Table 65, Table 66, Table 67 for components and sequences for hIL-10 and vIL-10 constructs/strains). 
       E. coli  Nissle comprising various tet-inducible constructs or constructs under the native fliC promoter were grown overnight in LB medium. Cultures were diluted 1:200 in LB and grown shaking (200 rpm) for 2 hours. Cultures were diluted to an optical density of 0.5 at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100 ng/mL to induce expression of hIL-10. No tetracycline was added to cultures harboring the fliC constructs. After 12 hours of induction, cells were spun down, and supernatant was collected. To generate cell free medium, the clarified supernatant was further filtered through a 0.22 micron filter to remove any remaining bacteria and placed on ice. Additionally, to detect intracellular recombinant protein production, pelleted were bacteria washed and resuspended in BugBuster™ (Millipore) with protease inhibitors and Ready-Lyse Lysozyme Solution (Epicentre), resulting in lysate concentrated 10-fold compared to original culture conditions. After incubation at room temperature for 10 minutes unsoluble debris is spun down at 20 min at 12,000 rcf at 4° C. then placed on ice until further processing. 
     The concentration of hIL-10 in the cell-free medium and in the bacterial cell extract was measured by hIL-10 ELISA (R&amp;D Systems DY217B), according to manufacturer&#39;s instructions. Similarly, to determine the concentrations of vIL-10 an Ultrasensitive ELISA kit (Alpco, 45-I10HUU-E01) was employed using commercially available recombinant vIL-10 (R&amp;D Systems, 915-VL-010). All samples were run in triplicate, and a standard curve was used to calculate secreted levels of IL-10. Standard curves were generated using both human and viral recombinant proteins. Wild type Nissle was included in the ELISA as a negative control, and no signal was observed. Table 68 and Table 69 summarize levels of hIL10 and vIL-10 measured in the supernatant and intracellularly Table 68 and extracellularly Table 69. The data show that both vIL-10 and hIL-10 are secreted at various levels from the different bacterial strains. 
     
       
         
           
               
             
               
                 TABLE 68 
               
             
            
               
                   
               
               
                 hIL-10 Secretion 
               
            
           
           
               
               
               
            
               
                   
                   
                 hu IL-10 (ng/ml) 
               
               
                   
                 Sample 
                 (extracellular) 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 WT 
                 0 
               
               
                   
                 IL-10 Plasmid (Nissle 
                 8.4 
               
               
                   
                 pUC57.Ptet-phoA-hIL10) 
               
               
                   
                 IL-10 plasmid/lpp (lpp::Cm 
                 19.3 
               
               
                   
                 pUC57.Ptet-phoA-hIL10) 
               
               
                   
                 2083 IL-10 plasmid/nlpI (nlpI::Cm 
                 20.5 
               
               
                   
                 pUC57.Ptet-phoA-hIL10) 
               
               
                   
                 2084 IL-10 plasmid/tolA (tolA::Cm 
                 21.4 
               
               
                   
                 pUC57.Ptet-phoA-hIL10) 
               
               
                   
                 2085 IL-10 plasmid/pal (PAL::Cm 
                 28.4 
               
               
                   
                 pUC57.Ptet-phoA-hIL10) 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 69 
               
             
            
               
                   
               
               
                 vIL-10 Secretion 
               
            
           
           
               
               
               
            
               
                   
                   
                 vIL-10 (ng/ml) 
               
               
                   
                 Sample 
                 (extracellular) 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 WT 
                 0 
               
               
                   
                 fliC-pvIL10 (Nissle pUN fli-vIL10 
                 29 
               
               
                   
                 Kan Cm) 
               
               
                   
                 fliC ::vIL10 (Nissle fliC::vIL10 
                 9 
               
               
                   
                 delta fliD CmR) 
               
               
                   
                 vIL-10 lpp (Nissle lpp mutant with 
                 527 
               
               
                   
                 vIL10 pBR3222 tet plasmid) 
               
               
                   
                 vIL-10 nlpI (Nissle delta nlpI::CmR 
                 982 
               
               
                   
                 pBR322.Ptet-phoA-vIL10) 
               
               
                   
                 vIL-10 tolA (Nissle delta tolA::CmR 
                 428 
               
               
                   
                 pBR322.Ptet-phoA-vIL10) 
               
               
                   
                 vIL-10 pal (Nissle delta PAL.:CmR 
                 1090 
               
               
                   
                 pBR322.Ptet-phoA-vIL10 
               
               
                   
                   
               
            
           
         
       
     
     Co-Culture Studies 
     To determine whether the hIL-10 and viral IL-10 expressed by the genetically engineered bacteria shown in Table 68 and Table 69 is biologically functional, in vitro experimentation is conducted, in which the bacterial supernatant containing secreted human or viral IL-10 is added to the growth medium of a Raji cells (a hematopoietic cell line) and J774a1 cells (a macrophage cell line). IL-10 is known to induce the phosphorylation of STAT3 in these cells Functional activity of bacterially secreted IL-10 is therefore assessed by its ability to phosphorylate STAT3 in Raji and J774a1 cells. 
     Raji cells are grown in RPMI 1640 supplemented with 10% FBS supplemented with 10% fetal bovine serum at 37° C. in a humidified incubator supplemented with 5% CO2. Prior to treatment with the bacterial supernatant, RPMI 1640 supplemented with 10% FBS (1e6/24 well) are serum starved overnight. Titrations of either recombinant human IL-10 diluted in LB or clarified supernatant from wild type Nissle or the engineered bacteria are added to cells for 30 minutes. Cells are harvested and resuspended in lysis buffer, and phospho-STAT3 ELISA (ELISA pSTAT3 (Tyr705) (Cell Signaling Technology)) is run in triplicate for all samples, according to manufacturer&#39;s instructions. PBS-treated cells and PBS are added as negative controls. Dilutions of samples are included to demonstrate linearity. 
     Competition Studies 
     As an additional control for specificity, a competition assay is performed. Titrations of anti-IL10 antibody are pre-incubated with constant concentrations of either rhIL10 (data not shown) or supernatants from the engineered bacteria expressing human or viral IL-10 for 15 min. Next, the supernatants/rhIL10 solutions are added to serum-starved Raji cells (1e6/well) and cells are incubated for 30 min followed by pSTAT3 ELISA as described above. 
     In other embodiments, similar studies are conducted with J774a1 cells. 
     Example 27. Bacterial Secretion of GLP-2 
     To determine whether the human GLP-2 expressed by engineered bacteria is secreted, the concentration of GLP-2 in the bacterial supernatant from two engineered strains comprising GLP-2 constructs/strains was measured. The first strain comprising a deletion in PAL and a plasmid expressing GLP-2 with an OmpF secretion tag from a tetracycline-inducible promoter and the second strain comprises the same PAL deletion and the same plasmid expressing GLP-2, further comprising a deletion in degP (see Table 74). 
       E. coli  Nissle comprising various tet-inducible constructs or constructs under the native fliC promoter were grown overnight in LB medium. Cultures were diluted 1:200 in LB and grown shaking (200 rpm) for 2 hours. Cultures were diluted to an optical density of 0.5 at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100 ng/mL to induce expression of hIL-10. No tetracycline was added to cultures harboring the fliC constructs. After 12 hours of induction, cells were spun down, and supernatant was collected. To generate cell free medium, the clarified supernatant was further filtered through a 0.22-micron filter to remove any remaining bacteria and placed on ice. Additionally, to detect intracellular recombinant protein production, pelleted were bacteria washed and resuspended in BugBuster™ (Millipore) with protease inhibitors and Ready-Lyse Lysozyme Solution (Epicentre), resulting in lysate concentrated 10-fold compared to original culture conditions. After incubation at room temperature for 10 minutes insoluble debris is spun down at 20 min at 12,000 rcf at 4° C. then placed on ice until further processing. 
     The concentration of GLP-2 in the cell-free medium and in the bacterial cell extract was measured by Human GLP2 ELISA Kit (Competitive EIA) (LSBio), according to manufacturer&#39;s instructions. All samples were run in triplicate, and a standard curve was used to calculate secreted levels of GLP-2. Standard curves were generated using recombinant GLP-2. Wild type Nissle was included in the ELISA as a negative control, and no signal was observed. As seen in Table 70, deletion of degP, a periplasmic protease, improved secretion levels over 3-fold. 
     
       
         
           
               
             
               
                 TABLE 70 
               
             
            
               
                   
               
               
                 GLP-2 Secretion 
               
            
           
           
               
               
               
            
               
                   
                 DOM mut 
                 ng/ml 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 WT 
                 1.14 
               
               
                   
                 PAL::CmR Ptet-ompF-GLP2 
                 1793.2 
               
               
                   
                 PAL::CmR ompT::Kan Ptet-ompF-GLP2 
                 1142.1 
               
               
                   
                 PAL::CmR ompT::Kan phoA-GLP2 fusion 
                 5360.4 
               
               
                   
                   
               
            
           
         
       
     
     Co-Culture Studies 
     To determine whether the hGLP-2 expressed by the genetically engineered bacteria is biologically functional, in vitro experimentation is conducted, in which the bacterial supernatant (from both strains shown above) containing secreted human GLP-2 is added to the growth medium of Caco-2 cells and CCD-18Co cells. The Caco-2 cell line is a continuous cell of heterogeneous human epithelial colorectal adenocarcinoma cells. As described e.g., in Jasleen et al. (Dig Dis Sci. 2002 May; 47(5): 1135-40) GLP-2 stimulates proliferation and [3H]thymidine incorporation in Caco-2 and T84 cells. Additionally, GLP-2 stimulates VEGFA secretion in these cells (see., e.g., Bulut et al, Eur J Pharmacol. 2008 Jan. 14; 578(2-3):279-85. 
     Functional activity of bacterially secreted GLP-2 is therefore assessed by its ability to induce proliferation and VEGF secretion. 
     Caco-2 are grown in Dulbecco&#39;s modified Eagle&#39;s medium supplemented with 10% fetal bovine serum at 37° C. in a humidified incubator supplemented with 5% CO2. Prior to treatment with the bacterial supernatant, Caco-2 cells (1e6/24 well) are serum starved overnight. Titrations of either recombinant human GLP-2 (50 and 250 nM) diluted in LB or clarified supernatant from wild type Nissle or the engineered bacteria are added to cells for a defined time. 
     For cell proliferation assays, cells are harvested and resuspended in lysis buffer. The cells are assayed after 12, 24, 48, and 72 hours of incubation. Cell proliferation is measured using a Cell proliferation assay kit according to manufacturers instruction (e.g., a Cell viability was assessed by a 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT)-assay). 
     For the measurements of VEFA secretion, cells are harvested and resuspended in lysis buffer, and concentrations of GLP-2 in the medium are determined ELISA 
     PBS-treated cells and PBS are added as negative controls. Dilutions of samples are included to demonstrate linearity. 
     Competition Studies 
     As an additional control for specificity, a competition assay is performed. Titrations of anti-GLP-2 antibody are pre-incubated with constant concentrations of either recombinant GLP-2 or supernatants from the engineered bacteria for 15 min. Next, the supernatants/rhIL2 solutions are added to serum-starved Cac-2 cells (1e6/well) and cells are incubated for 30 min followed by VEGFA ELISA as described above. 
     Example 28. Bacterial Secretion of IL-22 
     To determine whether the human IL-22 expressed by engineered bacteria is secreted, the concentration of IL-22 in the bacterial supernatant from a two engineered strains comprising IL-22 constructs/strains was measured. The first strain comprising a deletion in PAL and a plasmid expressing IL-22 with an OmpF secretion tag from a tetracycline-inducible promoter and the second strain comprises the same PAL deletion and the same plasmid expressing IL-22, further comprising a deletion in degP (Table 71). 
       E. coli  Nissle comprising various tet-inducible constructs or constructs under the native fliC promoter were grown overnight in LB medium. Cultures were diluted 1:200 in LB and grown shaking (200 rpm) for 2 hours. Cultures were diluted to an optical density of 0.5 at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100 ng/mL to induce expression of hIL-10. No tetracycline was added to cultures harboring the fliC constructs. After 12 hours of induction, cells were spun down, and supernatant was collected. To generate cell free medium, the clarified supernatant was further filtered through a 0.22 micron filter to remove any remaining bacteria and placed on ice. Additionally, to detect intracellular recombinant protein production, pelleted were bacteria washed and resuspended in BugBuster™ (Millipore) with protease inhibitors and Ready-Lyse Lysozyme Solution (Epicentre), resulting in lysate concentrated 10-fold compared to original culture conditions. After incubation at room temperature for 10 minutes unsoluble debris is spun down at 20 min at 12,000 rcf at 4° C. then placed on ice until further processing. 
     The concentration of IL-22 in the cell-free medium and in the bacterial cell extract was measured by hIL-22 ELISA (R&amp;D Systems (DY782) ELISA for hIL-22), according to manufacturer&#39;s instructions. All samples were run in triplicate, and a standard curve was used to calculate secreted levels of IL-22. Standard curves were generated using recombinant IL-22. Wild type Nissle was included in the ELISA as a negative control, and no signal was observed. Table 71 summarizes levels of IL-22 measured in the supernatant. The data show that both hIL-22 are secreted at various levels from the different bacterial strains. 
     
       
         
           
               
             
               
                 TABLE 71 
               
             
            
               
                   
               
               
                 IL-22 Secretion 
               
            
           
           
               
               
               
            
               
                   
                   
                 IL-22 Production/ 
               
               
                   
                   
                 Secretion Dilution 
               
               
                   
                 Genotype 
                 Corrected (ng/ml) 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 WT 
                 20.7 
               
               
                   
                 Lpp (delta lpp::CmR expressing 
                 87.6 
               
               
                   
                 PhoA-IL22 from Ptet) 
               
               
                   
                 nlpI (delta nlpI::CmR expressing 
                 105.4 
               
               
                   
                 PhoA-IL22 from Ptet) 
               
               
                   
                 tolA (delta tolA::CmR expressing 
                 623.2 
               
               
                   
                 PhoA-IL22 from Ptet) 
               
               
                   
                 PAL (delta pal::CmR expressing 
                 328.8 
               
               
                   
                 PhoA-IL22 from Ptet) 
               
               
                   
                   
               
            
           
         
       
     
     Example 29. Bacterial Secretion of IL-22 and Functional Assays 
     Generation of Bacterial Supernatant and Measurement of IL-22 Concentration 
     To determine whether the human IL-22 expressed by engineered bacteria is secreted, the concentration of IL-22 in the bacterial supernatant was measured. 
       E. coli  Nissle comprising a tet-inducible integrated construct (delta pal::CmR expressing PhoA-IL22 from Ptet) was grown overnight in LB medium. Cultures were diluted 1:200 in LB and grown shaking (200 rpm) for 2 hours. Cultures were diluted to an optical density of 0.5 at which time anhydrous tetracycline (ATC) was added to cultures at a concentration of 100 ng/mL to induce expression of hIL-22, After 12 hours of induction, cells were spun down, and supernatant was collected. To generate cell free medium, the supernatant was centrifuged, and filtered through a 0.22 micron filter to remove any remaining bacteria. 
     The concentration of hIL-22 in the cell-free medium was measured by hIL-22 ELISA (R&amp;D Systems (DY782) ELISA for hIL-22), according to manufacturer&#39;s instructions. All samples were run in triplicate, and a standard curve was used to calculate secreted levels of IL-22. Additionally, samples were diluted to ensure absence of matrix effects and to demonstrate linearity. Wild type Nissle was included in the ELISA as a negative control, and no signal was observed. The engineered bacteria comprising a PAL deletion and the integrated construct encoding hIL-22 with a phoA secretion tag were determined to be secreting at 199 ng/ml supernatant. 
     Co-Culture Studies 
     To determine whether the hIL-22 expressed by the genetically engineered bacteria is biologically functional, in vitro experimentation was conducted, in which the bacterial supernatant containing secreted human IL-22 was added to the growth medium of a mammalian colonic epithelial cell line. IL-22 is known to induce the phosphorylation of STAT1 and STAT3 in Colo205 cells (see, e.g., Nagalakshmi et al., Interleukin-22 activates STAT3 and induces IL-10 by colon epithelial cells. Int Immunopharmacol. 2004 May; 4(5):679-91). Functional activity of bacterially secreted IL-22 was therefore assessed by its ability to phosphorylate STAT3 in Colo205 cells. 
     Colo205 cells were grown in Dulbecco&#39;s modified Eagle&#39;s medium supplemented with 10% fetal bovine serum at 37° C. in a humidified incubator supplemented with 5% CO2. Prior to treatment with the bacterial supernatant, Colo205 (1e6/24 well) were serum starved overnight. Titrations of either recombinant human IL-22 diluted in LB or clarified supernatant from wild type Nissle or the engineered bacteria were added to cells for 30 minutes. Cells were harvested and resuspended in lysis buffer, and phospho-STAT3 ELISA (ELISA pSTAT3 (Tyr705) (Cell Signaling Technology)) was run in triplicate for all samples, according to manufacturer&#39;s instructions. PBS-treated cells and PBS were added as negative controls. Dilutions of samples were included to demonstrate linearity. No signal was observed for wild type Nissle. Results for the engineered strain comprising a PAL deletion and the integrated construct encoding hIL-22 with a phoA secretion tag are shown in  FIG. 33A , and demonstrate that hIL-22 secreted from the engineered bacteria is functionally active. 
     Competition Studies 
     As an additional control for specificity, a competition assay was performed. Titrations of anti-IL22 antibody (MAB7821, R&amp;D Systems) were pre-incubated with constant concentrations of either rhIL22 (data not shown) or supernatants from the engineered bacteria for 15 min. Next, the supernatants/rhIL2 solutions were added to serum-starved Colo205 cells (1e6/well) and cells were incubated for 30 min followed by pSTAT3 ELISA as described above. As shown in  FIG. 33B , the phospho-Stat3 signal induced by the secreted hIL-22 is competed by the hIL-22 antibody MAB7821. 
     Example 30. Generation of Indole Propionic Acid Strain and In Vitro Indole Production 
     To facilitate inducible production of indole propionic acid (IPA) in  Escherichia coli  Nissle, 6 genes allowing the production of indole propionic acid from tryptophan, as well as transcriptional and translational elements, are synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322 under a tet inducible promoter. In other embodiments, the IPA synthesis cassette is put under the control of an FNR, RNS or ROS promoter, e.g., described herein, or other promoter induced by conditions in the healthy or diseased gut, e.g., inflammatory conditions. For efficient translation of IPA synthesis genes, each synthetic gene in the cassette is separated by a 15 base pair ribosome binding site derived from the T7 promoter/translational start site. 
     The IPA synthesis cassette comprises TrpDH (tryptophan dehydrogenase from  Nostoc punctiforme  NIES-2108), FldH1/FldH2 (indole-3-lactate dehydrogenase from  Clostridium sporogenes ), FldA (indole-3-propionyl-CoA:indole-3-lactate CoA transferase from  Clostridium sporogenes ), FldBC (indole-3-lactate dehydratase from  Clostridium sporogenes ), FldD (indole-3-acrylyl-CoA reductase from  Clostridium sporogenes ), and AcuI (acrylyl-CoA reductase from  Rhodobacter sphaeroides ). 
     The tet inducible IPA construct described above is transformed into  E. coli  Nissle as described herein and production of IPA is assessed. In certain embodiments,  E. coli  Nissle strains containing the IPA synthesis cassette described further comprise a tryptophan synthesis cassette. In certain embodiments, the strains comprise a feedback resistant version of AroG and TrpE to achieve greater Trp production. In certain embodiments, additionally, the tnaA gene (tryptophanase converting Trp into indole) is deleted. 
     All incubations are performed at 37° C. LB-grown overnight cultures of  E. coli  Nissle transformed with the IPA biosynthesis construct alone or in combination with a tryptophan biosynthesis construct and feedback resistant AroG and TrpE are subcultured 1:100 into 10 mL of M9 minimal medium containing 0.5% glucose and grown shaking (200 rpm) for 2 h, at which time anhydrous tetracycline (ATC) is added to cultures at a concentration of 100 ng/mL to induce expression of the the IPA biosynthesis and tryptophan biosynthesis constructs. After 2 hours of induction, cells are spun down, supernatant is discarded, and the cells are resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant is then analyzed at predetermined time points (e.g., 0 up to 24 hours) by LC-MS to assess levels of IPA. 
     Production of IPA is also assessed in  E. coli  Nissle strains containing the IPA and tryptophan cassettes both driven by an RNS promoter e.g., a nsrR-norB-IPA biosynthesis construct) in order to assess nitrogen dependent induction of IPA production. Overnight bacterial cultures are diluted 1:100 into fresh LB and grown for 1.5 hrs to allow entry into early log phase. At this point, long half-life nitric oxide donor (DETA-NO; diethylenetriamine-nitric oxide adduct) wis added to cultures at a final concentration of 0.3 mM to induce expression from plasmid. After 2 hours of induction, cells are spun down, supernatant is discarded, and the cells are resuspended in M9 minimal media containing 0.5% glucose. Culture supernatant is then analyzed at predetermined time points (0 up to 24 hours, as shown in  FIG. 33 ) to assess IPA levels. 
     In alternate embodiments, production of IPA is also assessed in  E. coli  Nissle strains containing the IPA and tryptophan cassettes both driven by the low oxygen inducible FNR promoter, e.g., FNRS, or the the reactive oxygen regulated OxyS promoter. 
     Example 31. FNR Promoter Activity 
     In order to measure the promoter activity of different FNR promoters, the lacZ gene, as well as transcriptional and translational elements, were synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322. The lacZ gene was placed under the control of any of the exemplary FNR promoter sequences disclosed in Table 21. The nucleotide sequences of these constructs are shown in Table 72 through Table 76 ((SEQ ID NO: 240-244). However, as noted above, the lacZ gene may be driven by other inducible promoters in order to analyze activities of those promoters, and other genes may be used in place of the lacZ gene as a readout for promoter activity, exemplary results are shown in the figures. 
     Table 72 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, P fnr1  (SEQ ID NO: 240). The construct comprises a translational fusion of the Nissle nirB1 gene and the lacZ gene, in which the translational fusions are fused in frame to the 8 th  codon of the lacZ coding region. The P fnr1  sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case. 
     Table 73 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, P fnr2  ((SEQ ID NO: 241). The construct comprises a translational fusion of the Nissle ydfZ gene and the lacZ gene, in which the translational fusions are fused in frame to the 8 th  codon of the lacZ coding region. The P fnr2  sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case. 
     Table 74 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, P fnr3  ((SEQ ID NO: 242). The construct comprises a transcriptional fusion of the Nissle nirB gene and the lacZ gene, in which the transcriptional fusions use only the promoter region fused to a strong ribosomal binding site. The P fnr3  sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case. 
     Table 75 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pfnr4 ((SEQ ID NO: 243). The construct comprises a transcriptional fusion of the Nissle ydfZ gene and the lacZ gene. The P fnr4  sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case. 
     Table 76 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, PfnrS ((SEQ ID NO: 244). The construct comprises a transcriptional fusion of the anaerobically induced small RNA gene, fnrS1, fused to lacZ. The P fnrs  sequence is bolded lower case, and the predicted ribosome binding site within the promoter is underlined. The lacZ sequence is underlined upper case. ATG site is bolded upper case, and the cloning sites used to synthesize the construct are shown in regular upper case. 
     
       
         
           
               
             
               
                 TABLE 72 
               
               
                   
               
               
                 Pfnr1-lacZ construct Sequences 
               
               
                 Nucleotide sequences of Pfnr1-lacZ construct,  
               
               
                 low-copy (SEQ ID NO: 240) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 
                   GGTACCgtcagcataacaccctgacctctcattaattgttcatgccggg 
                 
               
               
                   
               
               
                 
                   cggcactatcgtcgtccggccttttcctctcttactctgctacgtacat 
                 
               
               
                   
               
               
                 
                   ctatttctataaatccgttcaatttgtctgttttttgcacaaacatgaa 
                 
               
               
                   
               
               
                 
                   atatcagacaattccgtgacttaagaaaatttatacaaatcagcaatat 
                 
               
               
                   
               
               
                 
                   accccttaaggagtatataaaggtgaatttgatttacatcaataagcgg 
                 
               
               
                   
               
               
                 
                   ggttgctgaatcgttaaggtaggcggtaatag 
                   
                     aaaagaaatcgaggcaa 
                   
                 
               
               
                   
               
               
                     aa     ATGagcaaagtcagactcgcaattat GGATCC TCTGGCCGTCGTATT   
               
               
                   
               
               
                 
                   ACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTT 
                 
               
               
                   
               
               
                 
                   GCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCA 
                 
               
               
                   
               
               
                 
                   CCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTT 
                 
               
               
                   
               
               
                 
                   TGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGC 
                 
               
               
                   
               
               
                 
                   GATCTTCCTGACGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGC 
                 
               
               
                   
               
               
                 
                   ACGGTTACGATGCGCCTATCTACACCAACGTGACCTATCCCATTACGGT 
                 
               
               
                   
               
               
                 
                   CAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTCGCTC 
                 
               
               
                   
               
               
                 
                   ACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTA 
                 
               
               
                   
               
               
                 
                   TTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTG 
                 
               
               
                   
               
               
                 
                   GGTCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGC 
                 
               
               
                   
               
               
                 
                   GCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCT 
                 
               
               
                   
               
               
                 
                   GGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGG 
                 
               
               
                   
               
               
                 
                   CATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGC 
                 
               
               
                   
               
               
                 
                   GATTTCCAAGTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTAC 
                 
               
               
                   
               
               
                 
                   TGGAGGCAGAAGTTCAGATGTACGGCGAGCTGCGCGATGAACTGCGGGT 
                 
               
               
                   
               
               
                 
                   GACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCG 
                 
               
               
                   
               
               
                 
                   CCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGCG 
                 
               
               
                   
               
               
                 
                   TCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAAT 
                 
               
               
                   
               
               
                 
                   CCCGAATCTCTATCGTGCAGTGGTTGAACTGCACACCGCCGACGGCACG 
                 
               
               
                   
               
               
                 
                   CTGATTGAAGCAGAAGCCTGCGACGTCGGTTTCCGCGAGGTGCGGATTG 
                 
               
               
                   
               
               
                 
                   AAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGCGGCGT 
                 
               
               
                   
               
               
                 
                   TAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAG 
                 
               
               
                   
               
               
                 
                   ACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCG 
                 
               
               
                   
               
               
                 
                   TGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGA 
                 
               
               
                   
               
               
                 
                   CCGCTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGC 
                 
               
               
                   
               
               
                 
                   ATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCTACCCGCGA 
                 
               
               
                   
               
               
                 
                   TGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCGAG 
                 
               
               
                   
               
               
                 
                   TGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCAC 
                 
               
               
                   
               
               
                 
                   GACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTAC 
                 
               
               
                   
               
               
                 
                   AGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCC 
                 
               
               
                   
               
               
                 
                   GATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAAA 
                 
               
               
                   
               
               
                 
                   TGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGA 
                 
               
               
                   
               
               
                 
                   TCCTTTGCGAATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGC 
                 
               
               
                   
               
               
                 
                   TAAATACTGGCAGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTC 
                 
               
               
                   
               
               
                 
                   GTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACGGCA 
                 
               
               
                   
               
               
                 
                   ACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCG 
                 
               
               
                   
               
               
                 
                   CCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCG 
                 
               
               
                   
               
               
                 
                   GCGCTGACGGAAGCAAAACACCAACAGCAGTATTTCCAGTTCCGTTTAT 
                 
               
               
                   
               
               
                 
                   CCGGGCGAACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGA 
                 
               
               
                   
               
               
                 
                   TAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAGCCGCTGGCA 
                 
               
               
                   
               
               
                 
                   AGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTG 
                 
               
               
                   
               
               
                 
                   AACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAAC 
                 
               
               
                   
               
               
                 
                   GGTACGCGTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCCGGACAC 
                 
               
               
                   
               
               
                 
                   ATCAGCGCCTGGCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGACAC 
                 
               
               
                   
               
               
                 
                   TCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAGCGGAACGGA 
                 
               
               
                   
               
               
                 
                   TTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCA 
                 
               
               
                   
               
               
                 
                   GGCTTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCC 
                 
               
               
                   
               
               
                 
                   CGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCGT 
                 
               
               
                   
               
               
                 
                   AAGTGAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAG 
                 
               
               
                   
               
               
                 
                   GCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAG 
                 
               
               
                   
               
               
                 
                   ATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCA 
                 
               
               
                   
               
               
                 
                   TCAGGGGAAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGG 
                 
               
               
                   
               
               
                 
                   CACGGTGAGATGGTCATCAATGTGGATGTTGCGGTGGCAAGCGATACAC 
                 
               
               
                   
               
               
                 
                   CGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGGCGCAGGTCTCAGA 
                 
               
               
                   
               
               
                 
                   GCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGACCGC 
                 
               
               
                   
               
               
                 
                   CTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGT 
                 
               
               
                   
               
               
                 
                   ATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCG 
                 
               
               
                   
               
               
                 
                   CGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAAC 
                 
               
               
                   
               
               
                 
                   ATCAGCCGCTACAGCCAACAACAACTGATGGAAACCAGCCATCGCCATC 
                 
               
               
                   
               
               
                 
                   TGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATAT 
                 
               
               
                   
               
               
                 
                   GGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTC 
                 
               
               
                   
               
               
                 
                   CAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAAT 
                 
               
               
                   
               
               
                 
                   AA 
                 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 73 
               
               
                   
               
               
                 Pfnr2-lacZ construct sequences 
               
               
                 Nucleotide sequences of Pfnr2-lacZ construct,  
               
               
                 low-copy (SEQ ID NO: 241) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 GGTACC catttcctctcatcccatccggggtgagagtcttttcccccg a 
               
               
                   
               
               
                 
                   cttatggctcatgcatgcatcaaaaaagatgtgagcttgatcaaaaaca 
                 
               
               
                   
               
               
                 
                   aaaaatatttcactcgacaggagtatttatattgcgcccgttacgtggg 
                 
               
               
                   
               
               
                 
                   cttcgactgtaaatc 
                   
                     agaaaggagaaaacacctATGacgacctacgatc 
                   
                 
               
               
                   
               
               
                 
                     g GGATCCTCTGGCCGTCGTATTACAACGTCGTGACTGGGAAAACCCTGG 
                 
               
               
                   
               
               
                 
                   CGTTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGCTGG 
                 
               
               
                   
               
               
                 
                   CGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCA 
                 
               
               
                   
               
               
                 
                   GCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGT 
                 
               
               
                   
               
               
                 
                   GCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATACTGTCGTC 
                 
               
               
                   
               
               
                 
                   GTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACACCA 
                 
               
               
                   
               
               
                 
                   ACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAA 
                 
               
               
                   
               
               
                 
                   TCCGACAGGTTGTTACTCGCTCACATTTAATATTGATGAAAGCTGGCTA 
                 
               
               
                   
               
               
                 
                   CAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTC 
                 
               
               
                   
               
               
                 
                   ATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGCCGTTT 
                 
               
               
                   
               
               
                 
                   GCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGC 
                 
               
               
                   
               
               
                 
                   CTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATC 
                 
               
               
                   
               
               
                 
                   AGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCA 
                 
               
               
                   
               
               
                 
                   TAAACCGACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTAAT 
                 
               
               
                   
               
               
                 
                   GATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACGGCG 
                 
               
               
                   
               
               
                 
                   AGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGAAAC 
                 
               
               
                   
               
               
                 
                   GCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAG 
                 
               
               
                   
               
               
                 
                   CGTGGCGGTTATGCCGATCGCGTCACACTACGCCTGAACGTTGAAAATC 
                 
               
               
                   
               
               
                 
                   CGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCAGTGGTTGA 
                 
               
               
                   
               
               
                 
                   ACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACGTC 
                 
               
               
                   
               
               
                 
                   GGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCA 
                 
               
               
                   
               
               
                 
                   AGCCGTTGCTGATTCGCGGCGTTAACCGTCACGAGCATCATCCTCTGCA 
                 
               
               
                   
               
               
                 
                   TGGTCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATG 
                 
               
               
                   
               
               
                 
                   AAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATTATCCGAACCATC 
                 
               
               
                   
               
               
                 
                   CGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGA 
                 
               
               
                   
               
               
                 
                   AGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGAT 
                 
               
               
                   
               
               
                 GATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACGCGGATGGTGC 
               
               
                   
               
               
                 
                   AGCGCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGA 
                 
               
               
                   
               
               
                 
                   ATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGATCAAATCT 
                 
               
               
                   
               
               
                 
                   GTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCA 
                 
               
               
                   
               
               
                 
                   CGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCA 
                 
               
               
                   
               
               
                 
                   GCCCTTCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTG 
                 
               
               
                   
               
               
                 
                   CCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATATGCCCACGCGATGG 
                 
               
               
                   
               
               
                 
                   GTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGTCAGTA 
                 
               
               
                   
               
               
                 
                   CCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTG 
                 
               
               
                   
               
               
                 
                   ATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATT 
                 
               
               
                   
               
               
                 
                   TTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTT 
                 
               
               
                   
               
               
                 
                   TGCCGACCGCACGCCGCATCCGGCGCTGACGGAAGCAAAACACCAACAG 
                 
               
               
                   
               
               
                 
                   CAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCG 
                 
               
               
                   
               
               
                 
                   AATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGC 
                 
               
               
                   
               
               
                 
                   ACTGGATGGCAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTTGGC 
                 
               
               
                   
               
               
                 
                   CCGCAAGGTAAGCAGTTGATTGAACTGCCTGAACTGCCGCAGCCGGAGA 
                 
               
               
                   
               
               
                 
                   GCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCAACCAAACGCGAC 
                 
               
               
                   
               
               
                 
                   CGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTG 
                 
               
               
                   
               
               
                 
                   GCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCCTC 
                 
               
               
                   
               
               
                 
                   AACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAATAAGCG 
                 
               
               
                   
               
               
                 
                   TTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGC 
                 
               
               
                   
               
               
                 
                   GATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTCACCCGTGCGC 
                 
               
               
                   
               
               
                 
                   CGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAA 
                 
               
               
                   
               
               
                 
                   CGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCG 
                 
               
               
                   
               
               
                 
                   GCGTTGTTGCAGTGCACGGCAGATACACTTGCCGACGCGGTGCTGATTA 
                 
               
               
                   
               
               
                 
                   CAACCGCCCACGCGTGGCAGCATCAGGGGAAAACCTTATTTATCAGCCG 
                 
               
               
                   
               
               
                 
                   GAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAATGTGGAT 
                 
               
               
                   
               
               
                 
                   GTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGACCT 
                 
               
               
                   
               
               
                 
                   GCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCC 
                 
               
               
                   
               
               
                 
                   GCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGG 
                 
               
               
                   
               
               
                 
                   GATCTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAA 
                 
               
               
                   
               
               
                 
                   ACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTG 
                 
               
               
                   
               
               
                 
                   GCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAACTG 
                 
               
               
                   
               
               
                 
                   ATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGC 
                 
               
               
                   
               
               
                 
                   TGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAG 
                 
               
               
                   
               
               
                 
                   CCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATTAC 
                 
               
               
                   
               
               
                 
                   AGTTGGTCTGGTGTCAAAAATAA 
                 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 74 
               
               
                   
               
               
                 Pfnr3-lacZ construct Sequences 
               
               
                 Nucleotide sequences of Pfnr3-lacZ construct,  
               
               
                 low-copy (SEQ ID NO: 242) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 GGTACC gtcagcataacaccctgacctctcattaattgttcatgccggg   
               
               
                   
               
               
                 
                   cggcactatcgtcgtccggccttttcctctcttactctgctacgtacat 
                 
               
               
                   
               
               
                 
                   ctatttctataaatccgttcaatttgtctgttttttgcacaaacatgaa 
                 
               
               
                   
               
               
                 
                   atatcagacaattccgtgacttaagaaaatttatacaaatcagcaatat 
                 
               
               
                   
               
               
                 accccttaaggagtatataaaggtgaatttgatttacatcaataagcgg 
               
               
                   
               
               
                   ggttgctgaatcgttaa GGATCC   ctctagaaataattttgtttaacttt     
               
               
                   
               
               
                 
                   
                     aagaaggagatatacat 
                   
                   ATG 
                   ACTATGATTACGGATTCTCTGGCCGTCGT 
                 
               
               
                   
               
               
                 
                   ATTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGC 
                 
               
               
                   
               
               
                 
                   CTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCC 
                 
               
               
                   
               
               
                 
                   GCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCG 
                 
               
               
                   
               
               
                 
                   CTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAG 
                 
               
               
                   
               
               
                 
                   TGCGATCTTCCTGACGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGA 
                 
               
               
                   
               
               
                 
                   TGCACGGTTACGATGCGCCTATCTACACCAACGTGACCTATCCCATTAC 
                 
               
               
                   
               
               
                 
                   GGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTCG 
                 
               
               
                   
               
               
                 
                   CTCACATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAA 
                 
               
               
                   
               
               
                 
                   TTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCG 
                 
               
               
                   
               
               
                 
                   CTGGGTCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTG 
                 
               
               
                   
               
               
                 
                   AGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGC 
                 
               
               
                   
               
               
                 
                   GCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAG 
                 
               
               
                   
               
               
                 
                   CGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATC 
                 
               
               
                   
               
               
                 
                   AGCGATTTCCAAGTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGG 
                 
               
               
                   
               
               
                 
                   TACTGGAGGCAGAAGTTCAGATGTACGGCGAGCTGCGCGATGAACTGCG 
                 
               
               
                   
               
               
                 
                   GGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCAGCGGCACC 
                 
               
               
                   
               
               
                 
                   GCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATC 
                 
               
               
                   
               
               
                 
                   GCGTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGA 
                 
               
               
                   
               
               
                 
                   AATCCCGAATCTCTATCGTGCAGTGGTTGAACTGCACACCGCCGACGGC 
                 
               
               
                   
               
               
                 
                   ACGCTGATTGAAGCAGAAGCCTGCGACGTCGGTTTCCGCGAGGTGCGGA 
                 
               
               
                   
               
               
                 
                   TTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGCGG 
                 
               
               
                   
               
               
                 
                   CGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAG 
                 
               
               
                   
               
               
                 
                   CAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACG 
                 
               
               
                   
               
               
                 
                   CCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTG 
                 
               
               
                   
               
               
                 
                   CGACCGCTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCAC 
                 
               
               
                   
               
               
                 
                   GGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCTACCCG 
                 
               
               
                   
               
               
                 
                   CGATGAGCGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCC 
                 
               
               
                   
               
               
                 
                   GAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAAT 
                 
               
               
                   
               
               
                 
                   CACGACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGG 
                 
               
               
                   
               
               
                 
                   TACAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTG 
                 
               
               
                   
               
               
                 
                   CCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCG 
                 
               
               
                   
               
               
                 
                   AAATGGTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGC 
                 
               
               
                   
               
               
                 
                   TGATCCTTTGCGAATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTT 
                 
               
               
                   
               
               
                 
                   CGCTAAATACTGGCAGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGC 
                 
               
               
                   
               
               
                 
                   TTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACG 
                 
               
               
                   
               
               
                 
                   GCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGA 
                 
               
               
                   
               
               
                 
                   TCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCAT 
                 
               
               
                   
               
               
                 
                   CCGGCGCTGACGGAAGCAAAACACCAACAGCAGTATTTCCAGTTCCGTT 
                 
               
               
                   
               
               
                 
                   TATCCGGGCGAACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATAG 
                 
               
               
                   
               
               
                 
                   CGATAACGAGTTCCTGCACTGGATGGTGGCACTGGATGGCAAGCCGCTG 
                 
               
               
                   
               
               
                 
                   GCAAGCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGA 
                 
               
               
                   
               
               
                 
                   TTGAACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCT 
                 
               
               
                   
               
               
                 
                   AACGGTACGCGTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCCGGA 
                 
               
               
                   
               
               
                 
                   CACATCAGCGCCTGGCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGA 
                 
               
               
                   
               
               
                 
                   CACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAGCGGAAC 
                 
               
               
                   
               
               
                 
                   GGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAG 
                 
               
               
                   
               
               
                 
                   TCAGGCTTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGA 
                 
               
               
                   
               
               
                 
                   CCCCGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACGACATTGG 
                 
               
               
                   
               
               
                 
                   CGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGG 
                 
               
               
                   
               
               
                 
                   AAGGCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGG 
                 
               
               
                   
               
               
                 
                   CAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCA 
                 
               
               
                   
               
               
                 
                   GCATCAGGGGAAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGAT 
                 
               
               
                   
               
               
                 
                   GGGCACGGTGAGATGGTCATCAATGTGGATGTTGCGGTGGCAAGCGATA 
                 
               
               
                   
               
               
                 
                   CACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGGCGCAGGTCTC 
                 
               
               
                   
               
               
                 
                   AGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGAC 
                 
               
               
                   
               
               
                 
                   CGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACA 
                 
               
               
                   
               
               
                 
                   TGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGAC 
                 
               
               
                   
               
               
                 
                   GCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTC 
                 
               
               
                   
               
               
                 
                   AACATCAGCCGCTACAGCCAACAACAACTGATGGAAACCAGCCATCGCC 
                 
               
               
                   
               
               
                 
                   ATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCA 
                 
               
               
                   
               
               
                 
                   TATGGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAA 
                 
               
               
                   
               
               
                 
                   TTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAAA 
                 
               
               
                   
               
               
                 
                   AATAA 
                 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 75 
               
               
                   
               
               
                 Pfnr4-lacZ construct Sequences 
               
               
                 Nucleotide sequences of Pfnr4-lacZ construct,  
               
               
                 low-copy (SEQ ID NO: 243) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 GGTACC catttcctctcatcccatccggggtgagagtcttttcccccga   
               
               
                   
               
               
                 
                   cttatggctcatgcatgcatcaaaaaagatgtgagcttgatcaaaaaca 
                 
               
               
                   
               
               
                   aaaaatatttcactcgacaggagtatttatattgcgccc GGATCC   ctct     
               
               
                   
               
               
                 
                   
                     agaaataattttgtttaactttaagaaggagatatacat 
                   
                   ATG 
                   ACTATGA 
                 
               
               
                   
               
               
                 
                   TTACGGATTCTCTGGCCGTCGTATTACAACGTCGTGACTGGGAAAACCC 
                 
               
               
                   
               
               
                 
                   TGGCGTTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGC 
                 
               
               
                   
               
               
                 
                   TGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGC 
                 
               
               
                   
               
               
                 
                   GCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGC 
                 
               
               
                   
               
               
                 
                   GGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATACTGTC 
                 
               
               
                   
               
               
                 
                   GTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCTACA 
                 
               
               
                   
               
               
                 
                   CCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGA 
                 
               
               
                   
               
               
                 
                   GAATCCGACAGGTTGTTACTCGCTCACATTTAATATTGATGAAAGCTGG 
                 
               
               
                   
               
               
                 
                   CTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACTCGGCGT 
                 
               
               
                   
               
               
                 
                   TTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGCCG 
                 
               
               
                   
               
               
                 
                   TTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAAC 
                 
               
               
                   
               
               
                 
                   CGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAG 
                 
               
               
                   
               
               
                 
                   ATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCT 
                 
               
               
                   
               
               
                 
                   GCATAAACCGACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTT 
                 
               
               
                   
               
               
                 AATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATGTACG 
               
               
                   
               
               
                 
                   GCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTGGCAGGGTGA 
                 
               
               
                   
               
               
                 
                   AACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGAT 
                 
               
               
                   
               
               
                 
                   GAGCGTGGCGGTTATGCCGATCGCGTCACACTACGCCTGAACGTTGAAA 
                 
               
               
                   
               
               
                 
                   ATCCGGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCAGTGGT 
                 
               
               
                   
               
               
                 
                   TGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGAC 
                 
               
               
                   
               
               
                 
                   GTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACG 
                 
               
               
                   
               
               
                 
                   GCAAGCCGTTGCTGATTCGCGGCGTTAACCGTCACGAGCATCATCCTCT 
                 
               
               
                   
               
               
                 
                   GCATGGTCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTG 
                 
               
               
                   
               
               
                 
                   ATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATTATCCGAACC 
                 
               
               
                   
               
               
                 
                   ATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGA 
                 
               
               
                   
               
               
                 
                   TGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACC 
                 
               
               
                   
               
               
                 
                   GATGATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACGCGGATGG 
                 
               
               
                   
               
               
                 
                   TGCAGCGCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAA 
                 
               
               
                   
               
               
                 
                   TGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGATCAAA 
                 
               
               
                   
               
               
                 
                   TCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACA 
                 
               
               
                   
               
               
                 
                   CCACGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGA 
                 
               
               
                   
               
               
                 
                   CCAGCCCTTCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGCTTTCG 
                 
               
               
                   
               
               
                 
                   CTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATATGCCCACGCGA 
                 
               
               
                   
               
               
                 
                   TGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTTTCGTCA 
                 
               
               
                   
               
               
                 
                   GTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCG 
                 
               
               
                   
               
               
                 
                   CTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTG 
                 
               
               
                   
               
               
                 
                   ATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGT 
                 
               
               
                   
               
               
                 
                   CTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAAGCAAAACACCAA 
                 
               
               
                   
               
               
                 
                   CAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCA 
                 
               
               
                   
               
               
                 
                   GCGAATACCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGT 
                 
               
               
                   
               
               
                 
                   GGCACTGGATGGCAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTT 
                 
               
               
                   
               
               
                 
                   GGCCCGCAAGGTAAGCAGTTGATTGAACTGCCTGAACTGCCGCAGCCGG 
                 
               
               
                   
               
               
                 
                   AGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGTGCAACCAAACGC 
                 
               
               
                   
               
               
                 
                   GACCGCATGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGT 
                 
               
               
                   
               
               
                 
                   CTGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCC 
                 
               
               
                   
               
               
                 
                   CTCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTAATAA 
                 
               
               
                   
               
               
                 
                   GCGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATT 
                 
               
               
                   
               
               
                 
                   GGCGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTCACCCGTG 
                 
               
               
                   
               
               
                 
                   CGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCC 
                 
               
               
                   
               
               
                 
                   TAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAA 
                 
               
               
                   
               
               
                 
                   GCGGCGTTGTTGCAGTGCACGGCAGATACACTTGCCGACGCGGTGCTGA 
                 
               
               
                   
               
               
                 
                   TTACAACCGCCCACGCGTGGCAGCATCAGGGGAAAACCTTATTTATCAG 
                 
               
               
                   
               
               
                 
                   CCGGAAAACCTACCGGATTGATGGGCACGGTGAGATGGTCATCAATGTG 
                 
               
               
                   
               
               
                 
                   GATGTTGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGA 
                 
               
               
                   
               
               
                 
                   CCTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGG 
                 
               
               
                   
               
               
                 
                   GCCGCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGC 
                 
               
               
                   
               
               
                 
                   TGGGATCTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCG 
                 
               
               
                   
               
               
                 
                   AAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCA 
                 
               
               
                   
               
               
                 
                   GTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCAACAACAA 
                 
               
               
                   
               
               
                 
                   CTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACAT 
                 
               
               
                   
               
               
                 
                   GGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCTG 
                 
               
               
                   
               
               
                 
                   GAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCAT 
                 
               
               
                   
               
               
                 
                   TACCAGTTGGTCTGGTGTCAAAAATAA 
                 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 76 
               
               
                   
               
               
                 Pfnrs-lacZ construct Sequences 
               
               
                 Nucleotide sequences of Pfnrs-lacZ construct, 
               
               
                 low-copy (SEQ ID NO: 244) 
               
               
                   
               
             
            
               
                 GGTACC agttgttcttattggtggtgttgctttatggttgcatcgtagt   
               
               
                   
               
               
                 
                   aaatggttgtaacaaaagcaatttttccggctgtctgtatacaaaaacg 
                 
               
               
                   
               
               
                 
                   ccgtaaagtttgagcgaagtcaataaactctctacccattcagggcaat 
                 
               
               
                   
               
               
                 
                   atctctcttGGATCC 
                   
                     ctctagaaataattttgtttaactttaagaagga 
                   
                 
               
               
                   
               
               
                 
                   
                     gatatacat 
                   
                   ATG 
                   CTATGATTACGGATTCTCTGGCCGTCGTATTACAACG 
                 
               
               
                   
               
               
                 
                   TCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCGGCA 
                 
               
               
                   
               
               
                 
                   CATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATC 
                 
               
               
                   
               
               
                 
                   GCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTG 
                 
               
               
                   
               
               
                 
                   GTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTT 
                 
               
               
                   
               
               
                 
                   CCTGACGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTT 
                 
               
               
                   
               
               
                 
                   ACGATGCGCCTATCTACACCAACGTGACCTATCCCATTACGGTCAATCC 
                 
               
               
                   
               
               
                 
                   GCGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTCGCTCACATTTA 
                 
               
               
                   
               
               
                 
                   ATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGA 
                 
               
               
                   
               
               
                 
                   TGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGT 
                 
               
               
                   
               
               
                 
                   TACGGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTT 
                 
               
               
                   
               
               
                 
                   TACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGA 
                 
               
               
                   
               
               
                 
                   CGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTTTC 
                 
               
               
                   
               
               
                 
                   CGTGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCC 
                 
               
               
                   
               
               
                 
                   AAGTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGGAGGC 
                 
               
               
                   
               
               
                 
                   AGAAGTTCAGATGTACGGCGAGCTGCGCGATGAACTGCGGGTGACGGTT 
                 
               
               
                   
               
               
                 
                   TCTTTGTGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCG 
                 
               
               
                   
               
               
                 
                   GCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGCGTCACACT 
                 
               
               
                   
               
               
                 
                   ACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAATCCCGAAT 
                 
               
               
                   
               
               
                 
                   CTCTATCGTGCAGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTG 
                 
               
               
                   
               
               
                 
                   AAGCAGAAGCCTGCGACGTCGGTTTCCGCGAGGTGCGGATTGAAAATGG 
                 
               
               
                   
               
               
                 
                   TCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGCGGCGTTAACCGT 
                 
               
               
                   
               
               
                 
                   CACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGATGG 
                 
               
               
                   
               
               
                 
                   TGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTG 
                 
               
               
                   
               
               
                 
                   TTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTAC 
                 
               
               
                   
               
               
                 
                   GGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGC 
                 
               
               
                   
               
               
                 
                   CAATGAATCGTCTGACCGATGATCCGCGCTGGCTACCCGCGATGAGCGA 
                 
               
               
                   
               
               
                 
                   ACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATC 
                 
               
               
                   
               
               
                 
                   ATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGC 
                 
               
               
                   
               
               
                 
                   TGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGA 
                 
               
               
                   
               
               
                 
                   AGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTAC 
                 
               
               
                   
               
               
                 GCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAAATGGTCCA 
               
               
                   
               
               
                 
                   TCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGCTGATCCTTTG 
                 
               
               
                   
               
               
                 
                   CGAATATGCCCACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATAC 
                 
               
               
                   
               
               
                 
                   TGGCAGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGG 
                 
               
               
                   
               
               
                 
                   ACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTG 
                 
               
               
                   
               
               
                 
                   GTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGCCAGTTC 
                 
               
               
                   
               
               
                 
                   TGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGA 
                 
               
               
                   
               
               
                 
                   CGGAAGCAAAACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCG 
                 
               
               
                   
               
               
                 
                   AACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAG 
                 
               
               
                   
               
               
                 
                   TTCCTGCACTGGATGGTGGCACTGGATGGCAAGCCGCTGGCAAGCGGTG 
                 
               
               
                   
               
               
                 
                   AAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACTGCC 
                 
               
               
                   
               
               
                 
                   TGAACTGCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGC 
                 
               
               
                   
               
               
                 
                   GTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCG 
                 
               
               
                   
               
               
                 
                   CCTGGCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTCCCCTC 
                 
               
               
                   
               
               
                 
                   CGCGTCCCACGCCATCCCTCAACTGACCACCAGCGGAACGGATTTTTGC 
                 
               
               
                   
               
               
                 
                   ATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTC 
                 
               
               
                   
               
               
                 
                   TTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGCTGCG 
                 
               
               
                   
               
               
                 
                   CGATCAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAA 
                 
               
               
                   
               
               
                 
                   GCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGG 
                 
               
               
                   
               
               
                 
                   GCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAGATACACT 
                 
               
               
                   
               
               
                 
                   TGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGG 
                 
               
               
                   
               
               
                 
                   AAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGTG 
                 
               
               
                   
               
               
                 
                   AGATGGTCATCAATGTGGATGTTGCGGTGGCAAGCGATACACCGCATCC 
                 
               
               
                   
               
               
                 
                   GGCGCGGATTGGCCTGACCTGCCAGCTGGCGCAGGTCTCAGAGCGGGTA 
                 
               
               
                   
               
               
                 
                   AACTGGCTCGGCCTGGGGCCGCAAGAAAACTATCCCGACCGCCTTACTG 
                 
               
               
                   
               
               
                 
                   CAGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCC 
                 
               
               
                   
               
               
                 
                   GTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTG 
                 
               
               
                   
               
               
                 
                   AATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCC 
                 
               
               
                   
               
               
                 
                   GCTACAGCCAACAACAACTGATGGAAACCAGCCATCGCCATCTGCTGCA 
                 
               
               
                   
               
               
                 
                   CGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATT 
                 
               
               
                   
               
               
                 
                   GGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGA 
                 
               
               
                   
               
               
                 
                   GCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAATAA 
                 
               
               
                   
               
            
           
         
       
     
     Example 32. Nitric Oxide-Inducible Reporter Constructs 
     ATC and nitric oxide-inducible reporter constructs were synthesized (Genewiz, Cambridge, Mass.). When induced by their cognate inducers, these constructs express GFP, which is detected by monitoring fluorescence in a plate reader at an excitation/emission of 395/509 nm, respectively. Nissle cells harboring plasmids with either the control, ATC-inducible Ptet-GFP reporter construct, or the nitric oxide inducible PnsrR-GFP reporter construct were first grown to early log phase (OD600 of about 0.4-0.6), at which point they were transferred to 96-well microtiter plates containing LB and two-fold decreased inducer (ATC or the long half-life NO donor, DETA-NO (Sigma)). Both ATC and NO were able to induce the expression of GFP in their respective constructs across a range of concentrations, as shown in the figures; promoter activity is expressed as relative florescence units. An exemplary sequence of a nitric oxide-inducible reporter construct is shown. The bsrR sequence is bolded. The gfp sequence is underlined. The PnsrR (NO regulated promoter and RBS) is italicized. The constitutive promoter and RBS are  . 
     
       
         
           
               
             
               
                 TABLE 77 
               
               
                   
               
               
                 SEQ ID NO: 245 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 SEQ ID NO: 245 
               
               
                 ttatta tcgcaccgcaatcgggattttcgattcataaagcaggtcgtaggtcggcttgtt   
               
               
                   
               
               
                 
                   gagcaggtcttgcagcgtgaaaccgtccagatacgtgaaaaacgacttcattgcaccgcc 
                 
               
               
                   
               
               
                 
                   gagtatgcccgtcagccggcaggacggcgtaatcaggcattcgttgttcgggcccataca 
                 
               
               
                   
               
               
                 
                   ctcgaccagctgcatcggttcgaggtggcggacgaccgcgccgatattgatgcgttcggg 
                 
               
               
                   
               
               
                 
                   cggcgcggccagcctcagcccgccgcctttcccgcgtacgctgtgcaagaacccgccttt 
                 
               
               
                   
               
               
                 
                   gaccagcgcggtaaccactttcatcaaatggcttttggaaatgccgtaggtcgaggcgat 
                 
               
               
                   
               
               
                 
                   ggtggcgatattgaccagcgcgtcgtcgttgacggcggtgtagatgaggacgcgcagccc 
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   caattaatcatcggctcgtataatgtataacattcatattttgtgaattttaaactctag 
                 
               
               
                   
               
               
                 
                   aaataattttgtttaactttaagaaggagatatacata 
                   
                     tggctagcaaaggcgaagaatt 
                   
                 
               
               
                   
               
               
                 
                   
                     gttcacgggcgttgttcctattttggttgaattggatggcgatgttaatggccataaatt 
                   
                 
               
               
                   
               
               
                 
                   
                     cagcgttagcggcgaaggcgaaggcgatgctacgtatggcaaattgacgttgaaattcat 
                   
                 
               
               
                   
               
               
                 
                   
                     ttgtacgacgggcaaattgcctgttccttggcctacgttggttacgacgttcagctatgg 
                   
                 
               
               
                   
               
               
                 
                   
                     cgttcaatgtttcagccgttatcctgatcatatgaaacgtcatgatttcttcaaaagcgc 
                   
                 
               
               
                   
               
               
                 
                   
                     tatgcctgaaggctatgttcaagaacgtacgattagcttcaaagatgatggcaattataa 
                   
                 
               
               
                   
               
               
                 
                   
                     aacgcgtgctgaagttaaattcgaaggcgatacgttggttaatcgtattgaattgaaagg 
                   
                 
               
               
                   
               
               
                 
                   
                     cattgatttcaaagaagatggcaatattttgggccataaattggaatataattataatag 
                   
                 
               
               
                   
               
               
                 
                   
                     ccataatgtttatattacggctgataaacaaaaaaatggcattaaagctaatttcaaaat 
                   
                 
               
               
                   
               
               
                 
                   
                     tcgtcataatattgaagatggcagcgttcaattggctgatcattatcaacaaaatacgcc 
                   
                 
               
               
                   
               
               
                 
                   
                     tattggcgatggccctgttttgttgcctgataatcattatttgagcacgcaaagcgcttt 
                   
                 
               
               
                   
               
               
                 
                   
                     gagcaaagatcctaatgaaaaacgtgatcatatggttttgttggaattcgttacggctgc 
                   
                 
               
               
                   
               
               
                     tggcattacgcatggcatggatgaattgtataaa   taataa 
               
               
                   
               
            
           
         
       
     
     These constructs, when induced by their cognate inducer, lead to high level expression of GFP, which is detected by monitoring fluorescence in a plate reader at an excitation/emission of 395/509 nm, respectively. Nissle cells harboring plasmids with either the ATC-inducible Ptet-GFP reporter construct or the nitric oxide inducible PnsrR-GFP reporter construct were first grown to early log phase (OD600=˜0.4-0.6), at which point they were transferred to 96-well microtiter plates containing LB and 2-fold decreases in inducer (ATC or the long half-life NO donor, DETA-NO (Sigma)). It was observed that both the ATC and NO were able to induce the expression of GFP in their respective construct across a wide range of concentrations. Promoter activity is expressed as relative florescence units. 
       FIG. 63D  NO-GFP constructs (the dot blot)  E. coli  Nissle harboring the nitric oxide inducible NsrR-GFP reporter fusion were grown overnight in LB supplemented with kanamycin. Bacteria were then diluted 1:100 into LB containing kanamycin and grown to an optical density of 0.4-0.5 and then pelleted by centrifugation. Bacteria were resuspended in phosphate buffered saline and 100 microliters were administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate for 7 days prior to bacterial gavage. At 4 hours post-gavage, mice were sacrificed and bacteria were recovered from colonic samples. Colonic contents were boiled in SDS, and the soluble fractions were used to perform a dot blot for GFP detection (induction of NsrR-regulated promoters). Detection of GFP was performed by binding of anti-GFP antibody conjugated to HRP (horse radish peroxidase). Detection was visualized using Pierce chemiluminescent detection kit. It is shown in the figure that NsrR-regulated promoters are induced in DSS-treated mice, but are not shown to be induced in untreated mice. This is consistent with the role of NsrR in response to NO, and thus inflammation. 
     Bacteria harboring a plasmid expressing NsrR under control of a constitutive promoter and the reporter gene gfp (green fluorescent protein) under control of an NsrR-inducible promoter were grown overnight in LB supplemented with kanamycin. Bacteria are then diluted 1:100 into LB containing kanamycin and grown to an optical density of about 0.4-0.5 and then pelleted by centrifugation. Bacteria are resuspended in phosphate buffered saline and 100 microliters were administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 2-3% dextran sodium sulfate for 7 days prior to bacterial gavage. At 4 hours post-gavage, mice were sacrificed and bacteria were recovered from colonic samples. Colonic contents were boiled in SDS, and the soluble fractions were used to perform a dot blot for GFP detection (induction of NsrR-regulated promoters) Detection of GFP was performed by binding of anti-GFP antibody conjugated to to HRP (horse radish peroxidase). Detection was visualized using Pierce chemiluminescent detection kit. The figures shows NsrR-regulated promoters are induced in DSS-treated mice, but not in untreated mice. 
     Example 33. Generation of ΔThyA 
     An auxotrophic mutation causes bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient. In order to generate genetically engineered bacteria with an auxotrophic modification, the thyA, a gene essential for oligonucleotide synthesis was deleted. Deletion of the thyA gene in  E. coli  Nissle yields a strain that cannot form a colony on LB plates unless they are supplemented with thymidine. 
     A thyA::cam PCR fragment was amplified using 3 rounds of PCR as follows. Sequences of the primers used at a 100 um concentration are found in Table 78. 
     
       
         
           
               
             
               
                 TABLE 78 
               
             
            
               
                   
               
               
                 Primer Sequences 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 SEQ ID 
               
               
                 Name 
                 Sequence 
                 Description 
                 NO 
               
               
                   
               
               
                 SR36 
                 tagaactgatgcaaaaagtgctcgacgaaggcacacagaTGTGTAGG 
                 Round 1: binds 
                 SEQ ID 
               
               
                   
                 CTGGAGCTGCTTC 
                 on pKD3 
                 NO: 246 
               
               
                   
               
               
                 SR38 
                 gtttcgtaattagatagccaccggcgctttaatgcccggaCATATGAAT 
                 Round 1: binds 
                 SEQ ID 
               
               
                   
                 ATCCTCCTTAG 
                 on pKD3 
                 NO: 247 
               
               
                   
               
               
                 SR33 
                 caacacgtttcctgaggaaccatgaaacagtatttagaact 
                 Round 2: binds to 
                 SEQ ID 
               
               
                   
                 gatgcaaaaag 
                 round 1 PCR 
                 NO: 248 
               
               
                   
                   
                 product 
                   
               
               
                   
               
               
                 SR34 
                 cgcacactggcgtcggctctggcaggatgtttcgtaattagatagc 
                 Round 2: binds to 
                 SEQ ID 
               
               
                   
                   
                 round 1 PCR 
                 NO: 249 
               
               
                   
                   
                 product 
                   
               
               
                   
               
               
                 SR43 
                 atatcgtcgcagcccacagcaacacgtttcctgagg 
                 Round 3: binds to 
                 SEQ ID 
               
               
                   
                   
                 round 2 PCR 
                 NO: 250 
               
               
                   
                   
                 product 
                   
               
               
                   
               
               
                 SR44 
                 aagaatttaacggagggcaaaaaaaaccgacgcacactggcgtcggc 
                 Round 3: binds to 
                 SEQ ID 
               
               
                   
                   
                 round 2 PCR 
                 NO: 251 
               
               
                   
                   
                 product 
               
               
                   
               
            
           
         
       
     
     For the first PCR round, 4×50 ul PCR reactions containing 1 ng pKD3 as template, 25 ul 2×phusion, 0.2 ul primer SR36 and SR38, and either 0, 0.2, 0.4 or 0.6 ul DMSO were brought up to 50 ul volume with nuclease free water and amplified under the following cycle conditions: 
     step1: 98c for 30 s 
     step2: 98c for 10 s 
     step3: 55c for 15 s 
     step4: 72c for 20 s 
     repeat step 2-4 for 30 cycles 
     step5: 72c for 5 min 
     Subsequently, 5 ul of each PCR reaction was run on an agarose gel to confirm PCR product of the appropriate size. The PCR product was purified from the remaining PCR reaction using a Zymoclean gel DNA recovery kit according to the manufacturer&#39;s instructions and eluted in 30 ul nuclease free water. 
     For the second round of PCR, 1 ul purified PCR product from round 1 was used as template, in 4×50 ul PCR reactions as described above except with 0.2 ul of primers SR33 and SR34. Cycle conditions were the same as noted above for the first PCR reaction. The PCR product run on an agarose gel to verify amplification, purified, and eluted in 30 ul as described above. 
     For the third round of PCR, 1 ul of purified PCR product from round 2 was used as template in 4×50 ul PCR reactions as described except with primer SR43 and SR44. Cycle conditions were the same as described for rounds 1 and 2. Amplification was verified, the PCR product purified, and eluted as described above. The concentration and purity was measured using a spectrophotometer. The resulting linear DNA fragment, which contains 92 bp homologous to upstream of thyA, the chloramphenicol cassette flanked by frt sites, and 98 bp homologous to downstream of the thyA gene, was transformed into a  E. coli  Nissle 1917 strain containing pKD46 grown for recombineering. Following electroporation, 1 ml SOC medium containing 3 mM thymidine was added, and cells were allowed to recover at 37 C for 2 h with shaking. Cells were then pelleted at 10,000×g for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in 100 ul LB containing 3 mM thymidine and spread on LB agar plates containing 3 mM thy and 20 ug/ml chloramphenicol. Cells were incubated at 37 C overnight. Colonies that appeared on LB plates were restreaked. +cam 20 ug/ml+ or −thy 3 mM. (thyA auxotrophs will only grow in media supplemented with thy 3 mM). 
     Next, the antibiotic resistance was removed with pCP20 transformation. pCP20 has the yeast Flp recombinase gene, FLP, chloramphenicol and ampicillin resistant genes, and temperature sensitive replication. Bacteria were grown in LB media containing the selecting antibiotic at 37° C. until OD600=0.4-0.6. 1 mL of cells were washed as follows: cells were pelleted at 16,000×g for 1 minute. The supernatant was discarded and the pellet was resuspended in 1 mL ice-cold 10% glycerol. This wash step was repeated 3× times. The final pellet was resuspended in 70 ul ice-cold 10% glycerol. Next, cells were electroporated with 1 ng pCP20 plasmid DNA, and 1 mL SOC supplemented with 3 mM thymidine was immediately added to the cuvette. Cells were resuspended and transferred to a culture tube and grown at 30° C. for 1 hours. Cells were then pelleted at 10,000×g for 1 minute, the supernatant was discarded, and the cell pellet was resuspended in 100 ul LB containing 3 mM thymidine and spread on LB agar plates containing 3 mM thy and 100 ug/ml carbenicillin and grown at 30° C. for 16-24 hours. Next, transformants were colony purified non-selectively (no antibiotics) at 42° C. 
     To test the colony-purified transformants, a colony was picked from the 42° C. plate with a pipette tip and resuspended in 10 μL LB. 3 μL of the cell suspension was pipetted onto a set of 3 plates: Cam, (37° C.; tests for the presence/absence of CamR gene in the genome of the host strain), Amp, (30° C., tests for the presence/absence of AmpR from the pCP20 plasmid) and LB only (desired cells that have lost the chloramphenicol cassette and the pCP20 plasmid), 37° C. Colonies were considered cured if there is no growth in neither the Cam or Amp plate, picked, and re-streaked on an LB plate to get single colonies, and grown overnight at 37° C. 
     Example 34. Nissle Residence 
     Unmodified  E. coli  Nissle and the genetically engineered bacteria of the invention may be destroyed, e.g., by defense factors in the gut or blood serum. The residence time of bacteria in vivo may be calculated. A non-limiting example using a streptomycin-resistant strain of  E. coli  Nissle is described below. In alternate embodiments, residence time is calculated for the genetically engineered bacteria of the invention. 
     C57BL/6 mice were acclimated in the animal facility for 1 week. After one week of acclimation (i.e., day 0), streptomycin-resistant Nissle (SYN-UCD103) was administered to the mice via oral gavage on days 1-3. Mice were not pre-treated with antibiotic. The amount of bacteria administered, i.e., the inoculant, is shown in Table 79. In order to determine the CFU of the inoculant, the inoculant was serially diluted, and plated onto LB plates containing streptomycin (300 μg/mL). The plates were incubated at 37° C. overnight, and colonies were counted. 
     
       
         
           
               
             
               
                 TABLE 79 
               
             
            
               
                   
               
               
                 CFU administered via oral gavage 
               
               
                 CFU administered via oral gavage 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Strain 
                 Day 1 
                 Day 2 
                 Day 3 
               
               
                   
                   
               
               
                   
                 SYN-UCD103 
                 1.30E+08 
                 8.50E+08 
                 1.90E+09 
               
               
                   
                   
               
            
           
         
       
     
     On days 2-10, fecal pellets were collected from up to 6 mice (ID NOs. 1-6; Table 80). The pellets were weighed in tubes containing PBS and homogenized. In order to determine the CFU of Nissle in the fecal pellet, the homogenized fecal pellet was serially diluted, and plated onto LB plates containing streptomycin (300 μg/mL). The plates were incubated at 37° C. overnight, and colonies were counted. 
     Fecal pellets from day 1 were also collected and plated on LB plates containing streptomycin (300 μg/mL) to determine if there were any strains native to the mouse gastrointestinal tract that were streptomycin resistant. The time course and amount of administered Nissle still residing within the mouse gastrointestinal tract is shown in Table 80. 
       FIG. 69  depicts a graph of Nissle residence in vivo. Streptomycin-resistant Nissle was administered to mice via oral gavage without antibiotic pre-treatment. Fecal pellets from six total mice were monitored post-administration to determine the amount of administered Nissle still residing within the mouse gastrointestinal tract. The bars represent the number of bacteria administered to the mice. The line represents the number of Nissle recovered from the fecal samples each day for 10 consecutive days. 
     
       
         
           
               
             
               
                 TABLE 80 
               
               
                   
               
               
                 Nissle residence in vivo 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 ID 
                 Day 2 
                 Day 3 
                 Day 4 
                 Day 5 
               
               
                   
               
               
                 1 
                 2.40E+05 
                 6.50E+03 
                 6.00E+04 
                 2.00E+03 
               
               
                 2 
                 1.00E+05 
                 1.00E+04 
                 3.30E+04 
                 3.00E+03 
               
               
                 3 
                 6.00E+04 
                 1.70E+04 
                 6.30E+04 
                 2.00E+02 
               
               
                 4 
                 3.00E+04 
                 1.50E+04 
                 1.10E+05 
                 3.00E+02 
               
               
                 5 
                   
                 1.00E+04 
                 3.00E+05 
                 1.50E+04 
               
               
                 6 
                   
                 1.00E+06 
                 4.00E+05 
                 2.30E+04 
               
               
                 Avg 
                 1.08E+05 
                 1.76E+05 
                 1.61E+05 
                 7.25E+03 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 ID 
                 Day 6 
                 Day 7 
                 Day 8 
                 Day 9 
                 Day 10 
               
               
                   
               
               
                 1 
                 9.10E+03 
                 1.70E+03 
                 4.30E+03 
                 6.40E+03 
                 2.77E+03 
               
               
                 2 
                 6.00E+03 
                 7.00E+02 
                 6.00E+02 
                 0.00E+00 
                 0.00E+00 
               
               
                 3 
                 1.00E+02 
                 2.00E+02 
                 0.00E+00 
                 0.00E+00 
                 0.00E+00 
               
               
                 4 
                 1.50E+03 
                 1.00E+02 
                   
                 0.00E+00 
                 0.00E+00 
               
               
                 5 
                 3.10E+04 
                 3.60E+03 
                   
                 0.00E+00 
                 0.00E+00 
               
               
                 6 
                 1.50E+03 
                 1.40E+03 
                 4.20E+03 
                 1.00E+02 
                 0.00E+00 
               
               
                 Avg 
                 8.20E+03 
                 1.28E+03 
                 2.28E+03 
                 1.08E+03 
                 4.62E+02 
               
               
                   
               
            
           
         
       
     
     Example 35. Intestinal Residence and Survival of Bacterial Strains In Vivo 
     Localization and intestinal residence time of streptomycin resistant Nissle,  FIG. 70 , was determined. Mice were gavaged, sacrificed at various time points, and effluents were collected from various areas of the small intestine cecum and colon. 
     Bacterial cultures were grown overnight and pelleted. The pellets were resuspended in PBS at a final concentration of approximately 10 10  CFU/mL. Mice (C57BL6/J, 10-12 weeks old) were gavaged with 100 μL of bacteria (approximately 10 9  CFU). Drinking water for the mice was changed to contain 0.1 mg/mL anhydrotetracycline (ATC) and 5% sucrose for palatability. At each timepoint (1, 4, 8, 12, 24, and 30 hours post-gavage), animals (n=4) were euthanized, and intestine, cecum, and colon were removed. The small intestine was cut into three sections, and the large intestine and colon each into two sections. Each section was flushed with 0.5 ml cold PBS and collected in separate 1.5 ml tubes. The cecum was harvested, contents were squeezed out, and flushed with 0.5 ml cold PBS and collected in a 1.5 ml tube. Intestinal effluents were placed on ice for serial dilution plating. 
     In order to determine the CFU of bacteria in each effluent, the effluent was serially diluted, and plated onto LB plates containing kanamycin. The plates were incubated at 37° C. overnight, and colonies were counted. The amount of bacteria and residence time in each compartment is shown in  FIG. 70 . 
     Example 36. Efficacy of Butyrate-Expressing Bacteria in a Mouse Model of IBD 
     Bacteria harboring the butyrate cassettes described above are grown overnight in LB. Bacteria are then diluted 1:100 into LB containing a suitable selection marker, e.g., ampicillin, and grown to an optical density of 0.4-0.5 and then pelleted by centrifugation. Bacteria are resuspended in phosphate buffered saline and 100 microliters is administered by oral gavage to mice. IBD is induced in mice by supplementing drinking water with 3% dextran sodium sulfate for 7 days prior to bacterial gavage. Mice are treated daily for 1 week and bacteria in stool samples are detected by plating stool homogenate on agar plates supplemented with a suitable selection marker, e.g., ampicillin. After 5 days of bacterial treatment, colitis is scored in live mice using endoscopy. Endoscopic damage score is determined by assessing colon translucency, fibrin attachment, mucosal and vascular pathology, and/or stool characteristics. Mice are sacrificed and colonic tissues are isolated. Distal colonic sections are fixed and scored for inflammation and ulceration. Colonic tissue is homogenized and measurements are made for myeloperoxidase activity using an enzymatic assay kit and for cytokine levels (IL-1β, TNF-α, IL-6, IFN-γ and IL-10). 
     Example 37. Generating a DSS-Induced Mouse Model of IBD 
     The genetically engineered bacteria described in Example 1 can be tested in the dextran sodium sulfate (DSS)-induced mouse model of colitis. The administration of DSS to animals results in chemical injury to the intestinal epithelium, allowing proinflammatory intestinal contents (e.g., luminal antigens, enteric bacteria, bacterial products) to disseminate and trigger inflammation (Low et al., 2013). To prepare mice for DSS treatment, mice are labeled using ear punch, or any other suitable labeling method. Labeling individual mice allows the investigator to track disease progression in each mouse, since mice show differential susceptibilities and responsiveness to DSS induction. Mice are then weighed, and if required, the average group weight is equilibrated to eliminate any significant weight differences between groups. Stool is also collected prior to DSS administration, as a control for subsequent assays. Exemplary assays for fecal markers of inflammation (e.g., cytokine levels or myeloperoxidase activity) are described below. 
     For DSS administration, a 3% solution of DSS (MP Biomedicals, Santa Ana, Calif.; Cat. No. 160110) in autoclaved water is prepared. Cage water bottles are then filled with 100 mL of DSS water, and control mice are given the same amount of water without DSS supplementation. This amount is generally sufficient for 5 mice for 2-3 days. Although DSS is stable at room temperature, both types of water are changed every 2 days, or when turbidity in the bottles is observed. 
     Acute, chronic, and resolving models of intestinal inflammation are achieved by modifying the dosage of DSS (usually 1-5%) and the duration of DSS administration (Chassaing et al., 2014). For example, acute and resolving colitis may be achieved after a single continuous exposure to DSS over one week or less, whereas chronic colitis is typically induced by cyclical administration of DSS punctuated with recovery periods (e.g., four cycles of DSS treatment for 7 days, followed by 7-10 days of water). 
       FIG. 14D  shows that butyrate produced in vivo in DSS mouse models under the control of an FNR promoter can be gut protective. LCN2 and calprotectin are both a measure of gut barrier disruption (measure by ELISA in this assay).  FIG. 14D  shows that SYN-501 (ter substitution) reduces inflammation and/or protects gut barrier as compared to wildtype Nissle. 
     Example 38. Monitoring Disease Progression In Vivo 
     Following initial administration of DSS, stool is collected from each animal daily, by placing a single mouse in an empty cage (without bedding material) for 15-30 min. However, as DSS administration progresses and inflammation becomes more robust, the time period required for collection increases. Stool samples are collected using sterile forceps, and placed in a microfuge tube. A single pellet is used to monitor occult blood according to the following scoring system: 0, normal stool consistency with negative hemoccult; 1, soft stools with positive hemoccult; 2, very soft stools with traces of blood; and 3, watery stools with visible rectal bleeding. This scale is used for comparative analysis of intestinal bleeding. All remaining stool is reserved for the measurement of inflammatory markers, and frozen at −20° C. 
     The body weight of each animal is also measured daily. Body weights may increase slightly during the first three days following initial DSS administration, and then begin to decrease gradually upon initiation of bleeding. For mouse models of acute colitis, DSS is typically administered for 7 days. However, this length of time may be modified at the discretion of the investigator. 
     Example 39. In Vivo Efficacy of Genetically Engineered Bacteria Following DSS Induction 
     The genetically engineered bacteria described in Example 1 can be tested in DSS-induced animal models of IBD. Bacteria are grown overnight in LB supplemented with the appropriate antibiotic. Bacteria are then diluted 1:100 in fresh LB containing selective antibiotic, grown to an optical density of 0.4-0.5, and pelleted by centrifugation. Bacteria are then resuspended in phosphate buffered saline (PBS). IBD is induced in mice by supplementing drinking water with 3% DSS for 7 days prior to bacterial gavage. On day 7 of DSS treatment, 100 μL of bacteria (or vehicle) is administered to mice by oral gavage. Bacterial treatment is repeated once daily for 1 week, and bacteria in stool samples are detected by plating stool homogenate on selective agar plates. 
     After 5 days of bacterial treatment, colitis is scored in live mice using the Coloview system (Karl Storz Veterinary Endoscopy, Goleta, Calif.). In mice under 1.5-2.0% isoflurane anesthesia, colons are inflated with air and approximately 3 cm of the proximal colon can be visualized (Chassaing et al., 2014). Endoscopic damage is scored by assessing colon translucency (score 0-3), fibrin attachment to the bowel wall (score 0-3), mucosal granularity (score 0-3), vascular pathology (score 0-3), stool characteristics (normal to diarrhea; score 0-3), and the presence of blood in the lumen (score 0-3), to generate a maximum score of 18. Mice are sacrificed and colonic tissues are isolated using protocols described in Examples 8 and 9. Distal colonic sections are fixed and scored for inflammation and ulceration. Remaining colonic tissue is homogenized and cytokine levels (e.g., IL-1β, TNF-α, IL-6, IFN-γ, and IL-10), as well as myeloperoxidase activity, are measured using methods described below. 
     Example 40. Euthanasia Procedures for Rodent Models of IBD 
     Four and 24 hours prior to sacrifice, 5-bromo-2′-deooxyuridine (BrdU) (Invitrogen, Waltham, Mass.; Cat. No. B23151) may be intraperitoneally administered to mice, as recommended by the supplier. BrdU is used to monitor intestinal epithelial cell proliferation and/or migration via immunohistochemistry with standard anti-BrdU antibodies (Abcam, Cambridge, Mass.). 
     On the day of sacrifice, mice are deprived of food for 4 hours, and then gavaged with FITC-dextran tracer (4 kDa, 0.6 mg/g body weight). Fecal pellets are collected, and mice are euthanized 3 hours following FITC-dextran administration. Animals are then cardiac bled to collect hemolysis-free serum. Intestinal permeability correlates with fluorescence intensity of appropriately diluted serum (excitation, 488 nm; emission, 520 nm), and is measured using spectrophotometry. Serial dilutions of a known amount of FITC-dextran in mouse serum are used to prepare a standard curve. 
     Alternatively, intestinal inflammation is quantified according to levels of serum keratinocyte-derived chemokine (KC), lipocalin 2, calprotectin, and/or CRP-1. These proteins are reliable biomarkers of inflammatory disease activity, and are measured using DuoSet ELISA kits (R&amp;D Systems, Minneapolis, Minn.) according to manufacturer&#39;s instructions. For these assays, control serum samples are diluted 1:2 or 1:4 for KC, and 1:200 for lipocalin 2. Samples from DSS-treated mice require a significantly higher dilution. 
     Example 41. Isolation and Preservation of Colonic Tissues 
     To isolate intestinal tissues from mice, each mouse is opened by ventral midline incision. The spleen is then removed and weighed. Increased spleen weights generally correlate with the degree of inflammation and/or anemia in the animal. Spleen lysates (100 mg/mL in PBS) plated on non-selective agar plates are also indicative of disseminated intestinal bacteria. The extent of bacterial dissemination should be consistent with any FITC-dextran permeability data. 
     Mesenteric lymph nodes are then isolated. These may be used to characterize immune cell populations and/or assay the translocation of gut bacteria. Lymph node enlargement is also a reliable indicator of DSS-induced pathology. Finally, the colon is removed by lifting the organ with forceps and carefully pulling until the cecum is visible. Colon dissection from severely inflamed DSS-treated mice is particularly difficult, since the inflammatory process causes colonic tissue to thin, shorten, and attach to extraintestinal tissues. 
     The colon and cecum are separated from the small intestine at the ileocecal junction, and from the anus at the distal end of the rectum. At this point, the mouse intestine (from cecum to rectum) may be imaged for gross analysis, and colonic length may be measured by straightening (but not stretching) the colon. The colon is then separated from the cecum at the ileocecal junction, and briefly flushed with cold PBS using a 5- or 10-mL syringe (with a feeding needle). Flushing removes any feces and/or blood. However, if histological staining for mucin layers or bacterial adhesion/translocation is ultimately anticipated, flushing the colon with PBS should be avoided. Instead, the colon is immersed in Carnoy&#39;s solution (60% ethanol, 30% chloroform, 10% glacial acetic acid; Johansson et al., 2008) to preserve mucosal architecture. The cecum can be discarded, as DSS-induced inflammation is generally not observed in this region. 
     After flushing, colon weights are measured. Inflamed colons exhibit reduced weights relative to normal colons due to tissue wasting, and reductions in colon weight correlate with the severity of acute inflammation. In contrast, in chronic models of colitis, inflammation is often associated with increased colon weight. Increased weight may be attributed to focal collections of macrophages, epithelioid cells, and multinucleated giant cells, and/or the accumulation of other cells, such as lymphocytes, fibroblasts, and plasma cells (Williams and Williams, 1983). 
     To obtain colon samples for later assays, colons are cut into the appropriate number of pieces. It is important to compare the same region of the colon from different groups of mice when performing any assay. For example, the proximal colon is frozen at −80° C. and saved for MPO analysis, the middle colon is stored in RNA later and saved for RNA isolation, and the rectal region is fixed in 10% formalin for histology. Alternatively, washed colons may be cultured ex vivo. Exemplary protocols for each of these assays are described below. 
     Example 42. Myeloperoxidase Activity Assay 
     Granulocyte infiltration in the rodent intestine correlates with inflammation, and is measured by the activity levels of myeloperoxidase, an enzyme abundantly expressed in neutrophil granulocytes. Myeloperoxidase (MPO) activity may be quantified using either o-dianisidine dihydrochloride (Sigma, St. Louis, Mo.; Cat. No. D3252) or 3,3′,5,5′-tetramethylbenzidine (Sigma; Cat. No. T2885) as a substrate. 
     Briefly, clean, flushed samples of colonic tissue (50-100 mg) are removed from storage at −80° C. and immediately placed on ice. Samples are then homogenized in 0.5% hexadecyltrimethylammonium bromide (Sigma; Cat. No. H6269) in 50 mM phosphate buffer, pH 6.0. Homogenates are then disrupted for 30 sec by sonication, snap-frozen in dry ice, and thawed for a total of three freeze-thaw cycles before a final sonication for 30 sec. 
     For assays with o-dianisidine dihydrochloride, samples are centrifuged for 6 min at high speed (13,400 g) at 4° C. MPO in the supernatant is then assayed in a 96-well plate by adding 1 mg/mL of o-dianisidine dihydrochloride and 0.5×10-4% H2O2, and measuring optical density at 450 nm. A brownish yellow color develops slowly over a period of 10-20 min; however, if color development is too rapid, the assay is repeated after further diluting the samples. Human neutrophil MPO (Sigma; Cat. No. M6908) is used as a standard, with a range of 0.5-0.015 units/mL. One enzyme unit is defined as the amount of enzyme needed to degrade 1.0 mol of peroxide per minute at 25° C. This assay is used to analyze MPO activity in rodent colonic samples, particularly in DSS-induced tissues. 
     For assays with 3,3′,5,5′-tetramethylbenzidine (TMB), samples are incubated at 60° C. for 2 hours and then spun down at 4,000 g for 12 min. Enzymatic activity in the supernatant is quantified photometrically at 630 nm. The assay mixture consists of 20 mL supernatant, 10 mL TMB (final concentration, 1.6 mM) dissolved in dimethylsulfoxide, and 70 mL H 2 O 2  (final concentration, 3.0 mM) diluted in 80 mM phosphate buffer, pH 5.4. One enzyme unit is defined as the amount of enzyme that produces an increase of one absorbance unit per minute. This assay is used to analyze MPO activity in rodent colonic samples, particularly in tissues induced by trinitrobenzene (TNBS) as described herein. 
     Example 43. RNA Isolation and Gene Expression Analysis 
     To gain further mechanistic insights into how the genetically engineered bacteria may reduce gut inflammation in vivo, gene expression is evaluated by semi-quantitative and/or real-time reverse transcription PCR. 
     For semi-quantitative analysis, total RNA is extracted from intestinal mucosal samples using the RNeasy isolation kit (Qiagen, Germantown, Md.; Cat. No. 74106). RNA concentration and purity are determined based on absorbency measurements at 260 and 280 nm. Subsequently, 1 μg of total RNA is reverse-transcribed, and cDNA is amplified for the following genes: tumor necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-2 (IL-2), or any other gene associated with inflammation. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as the internal standard. Polymerase chain reaction (PCR) reactions are performed with a 2-min melting step at 95° C., then 25 cycles of 30 sec at 94° C., 30 sec at 63° C., and 1 min at 75° C., followed by a final extension step of 5 min at 65° C. Reverse transcription (RT)-PCR products are separated by size on a 4% agarose gel and stained with ethidium bromide. Relative band intensities are analyzed using standard image analysis software. 
     For real-time, quantitative analysis, intestinal samples (50 mg) are stored in RNAlater solution (Sigma; Cat. No. R0901) until RNA extraction. Samples should be kept frozen at −20° C. for long-term storage. On the day of RNA extraction, samples are thawed, or removed from RNAlater, and total RNA is extracted using Trizol (Fisher Scientific, Waltham, Mass.; Cat. No. 15596026). Any suitable RNA extraction method may be used. When working with DSS-induced samples, it is necessary to remove all polysaccharides (including DSS) using the lithium chloride method (Chassaing et al., 2012). Traces of DSS in colonic tissues are known to interfere with PCR amplification in subsequent steps. 
     Primers are designed for various genes and cytokines associated with the immune response using Primer Express® software (Applied Biosystems, Foster City, Calif.). Following isolation of total RNA, reverse transcription is performed using random primers, dNTPs, and Superscript® II enzyme (Invitrogen; Ser. No. 18/064,014). cDNA is then used for real-time PCR with SYBR Green PCR Master Mix (Applied Biosystems; 4309155) and the ABI PRISM 7000 Sequence Detection System (Applied Biosystems), although any suitable detection method may be used. PCR products are validated by melt analysis. 
     Example 44. Histology 
     Standard histological stains are used to evaluate intestinal inflammation at the microscopic level. Hematoxylin-eosin (H&amp;E) stain allows visualization of the quality and dimension of cell infiltrates, epithelial changes, and mucosal architecture (Erben et al., 2014). Periodic Acid-Schiff (PAS) stain is used to stain for carbohydrate macromolecules (e.g., glycogen, glycoproteins, mucins). Goblet cells, for example, are PAS-positive due to the presence of mucin. 
     Swiss rolls are recommended for most histological stains, so that the entire length of the rodent intestine may be examined. This is a simple technique in which the intestine is divided into portions, opened longitudinally, and then rolled with the mucosa outwards (Moolenbeek and Ruitenberg, 1981). Briefly, individual pieces of colon are cut longitudinally, wrapped around a toothpick wetted with PBS, and placed in a cassette. Following fixation in 10% formalin for 24 hours, cassettes are stored in 70% ethanol until the day of staining. Formalin-fixed colonic tissue may be stained for BrdU using anti-BrdU antibodies (Abcam). Alternatively, Ki67 may be used to visualize epithelial cell proliferation. For stains using antibodies to more specific targets (e.g., immunohistochemistry, immunofluorescence), frozen sections are fixed in a cryoprotective embedding medium, such as Tissue-Tek® OCT (VWR, Radnor, Pa.; Cat. No. 25608-930). 
     For H&amp;E staining, stained colonic tissues are analyzed by assigning each section four scores of 0-3 based on the extent of epithelial damage, as well as inflammatory infiltration into the mucosa, submucosa, and muscularis/serosa. Each of these scores is multiplied by: 1, if the change is focal; 2, if the change is patchy; and 3, if the change is diffuse. The four individual scores are then summed for each colon, resulting in a total scoring range of 0-36 per animal. Average scores for the control and affected groups are tabulated. Alternative scoring systems are detailed herein. 
     Example 45. Ex Vivo Culturing of Rodent Colons 
     Culturing colons ex vivo may provide information regarding the severity of intestinal inflammation. Longitudinally-cut colons (approximately 1.0 cm) are serially washed three times in Hanks&#39; Balanced Salt Solution with 1.0% penicillin/streptomycin (Fisher; Cat. No. BP295950). Washed colons are then placed in the wells of a 24-well plate, each containing 1.0 mL of serum-free RPMI 1640 medium (Fisher; Cat. No. 11875093) with 1.0% penicillin/streptomycin, and incubated at 37° C. with 5.0% CO2 for 24 hours. Following incubation, supernatants are collected and centrifuged for 10 min at 4° C. Supernatants are stored at −80° C. prior to analysis for proinflammatory cytokines. 
     Example 46. In Vivo Efficacy of Genetically Engineered Bacteria Following TNBS Induction 
     Apart from DSS, the genetically engineered bacteria described in 1 can also be tested in other chemically induced animal models of IBD. Non-limiting examples include those induced by oxazolone (Boirivant et al., 1998), acetic acid (MacPherson and Pfeiffer, 1978), indomethacin (Sabiu et al., 2016), sulfhydryl inhibitors (Satoh et al., 1997), and trinitrobenzene sulfonic acid (TNBS) (Gurtner et al., 2003; Segui et al., 2004). To determine the efficacy of the genetically engineered bacteria in a TNBS-induced mouse model of colitis, bacteria are grown overnight in LB supplemented with the appropriate antibiotic. Bacteria are then diluted 1:100 in fresh LB containing selective antibiotic, grown to an optical density of 0.4-0.5, and pelleted by centrifugation. Bacteria are resuspended in PBS. IBD is induced in mice by intracolonic administration of 30 mg TNBS in 0.25 mL 50% (vol/vol) ethanol (Segui et al., 2004). Control mice are administered 0.25 mL saline. Four hours post-induction, 100 μL of bacteria (or vehicle) is administered to mice by oral gavage. Bacterial treatment is repeated once daily for 1 week. Animals are weighed daily. 
     After 7 days of bacterial treatment, mice are sacrificed via intraperitoneal administration of thiobutabarbital (100 mg/kg). Colonic tissues are isolated by blunt dissection, rinsed with saline, and weighed. Blood samples are collected by open cardiac puncture under aseptic conditions using a 1-mL syringe, placed in Eppendorf vials, and spun at 1,500 g for 10 min at 4° C. The supernatant serum is then pipetted into autoclaved Eppendorf vials and frozen at −80° C. for later assay of IL-6 levels using a quantitative, colorimetric commercial kit (R&amp;D Systems). 
     Macroscopic damage is examined under a dissecting microscope by a blinded observer. An established scoring system is used to account for the presence/severity of intestinal adhesions (score 0-2), strictures (score 0-3), ulcers (score 0-3), and wall thickness (score 0-2) (Mourelle et al., 1996). Two colon samples (50 mg) are then excised, snap-frozen in liquid nitrogen, and stored at −80° C. for subsequent myeloperoxidase activity assay. If desired, additional samples are preserved in 10% formalin for histologic grading. Formalin-fixed colonic samples are then embedded in paraffin, and 5 μm sections are stained with H&amp;E. Microscopic inflammation of the colon is assessed on a scale of 0 to 11, according to previously defined criteria (Appleyard and Wallace, 1995). 
     Example 47. Generating a Cell Transfer Mouse Model of IBD 
     The genetically engineered bacteria described in Example 1 can be tested in cell transfer animal models of IBD. One exemplary cell transfer model is the CD45RBHi T cell transfer model of colitis (Bramhall et al., 2015; Ostanin et al., 2009; Sugimoto et al., 2008). This model is generated by sorting CD4+ T cells according to their levels of CD45RB expression, and adoptively transferring CD4+ T cells with high CD45RB expression (referred to as CD45RBHi T cells) from normal donor mice into immunodeficient mice (e.g., SCID or RAG−/− mice). Specific protocols are described below. 
     Enrichment for (714 T Cells 
     Following euthanization of C57BL/6 wild-type mice of either sex (Jackson Laboratories, Bar Harbor, Me.), mouse spleens are removed and placed on ice in a 100 mm Petri dish containing 10-15 mL of FACS buffer (IX PBS without Ca2+/Mg2+, supplemented with 4% fetal calf serum). Spleens are teased apart using two glass slides coated in FACS buffer, until no large pieces of tissue remain. The cell suspension is then withdrawn from the dish using a 10-mL syringe (no needle), and expelled out of the syringe (using a 26-gauge needle) into a 50-mL conical tube placed on ice. The Petri dish is washed with an additional 10 mL of FACS buffer, using the same needle technique, until the 50-mL conical tube is full. Cells are pelleted by centrifugation at 400 g for 10 min at 4° C. After the cell pellet is gently disrupted with a stream of FACS buffer, cells are counted. Cells used for counting are kept on ice and saved for single-color staining described in the next section. All other cells (i.e., those remaining in the 50-mL conical tube) are transferred to new 50-mL conical tubes. Each tube should contain a maximum of 25×10 7  cells. 
     To enrich for CD4+ T cells, the Dynal® Mouse CD4 Negative Isolation kit (Invitrogen; Cat. No. 114-15D) is used as per manufacturer&#39;s instructions. Any comparable CD4+ T cell enrichment method may be used. Following negative selection, CD4+ cells remain in the supernatant. Supernatant is carefully pipetted into a new 50-mL conical tube on ice, and cells are pelleted by centrifugation at 400 g for 10 min at 4° C. Cell pellets from all 50-mL tubes are then resuspended, pooled into a single 15-mL tube, and pelleted once more by centrifugation. Finally, cells are resuspended in 1 mL of fresh FACS buffer, and stained with anti-CD4-APC and anti-CD45RB-FITC antibodies. 
     Fluorescent Labeling of CD4+ T Cells 
     To label CD4+ T cells, an antibody cocktail containing appropriate dilutions of pre-titrated anti-CD4-APC and anti-CD45RB-FITC antibodies in FACS buffer (approximately 1 mL cocktail/5×107 cells) is added to a 1.5-mL Eppendorf tube, and the volume is adjusted to 1 mL with FACS buffer. Antibody cocktail is then combined with cells in a 15-mL tube. The tube is capped, gently inverted to ensure proper mixing, and incubated on a rocking platform for 15 min at 4° C. 
     During the incubation period, a 96-well round-bottom staining plate is prepared by transferring equal aliquots of counted cells (saved from the previous section) into each well of the plate that corresponds to single-color control staining. These wells are then filled to 200 μL with FACs buffer, and the cells are pelleted at 300 g for 3 min at 4° C. using a pre-cooled plate centrifuge. Following centrifugation, the supernatant is discarded using a 21-gauge needle attached to a vacuum line, and 100 μL of anti-CD 16/32 antibody (Fc receptor-blocking) solution is added to each well to prevent non-specific binding. The plate is incubated on a rocking platform at 4° C. for 15 min. Cells are then washed with 200 μL FACS buffer and pelleted by centrifugation. Supernatant is aspirated, discarded, and 100 μL of the appropriate antibody (i.e., pre-titrated anti-CD4-APC or anti-CD45RB-FITC) is added to wells corresponding to each single-color control. Cells in unstained control wells are resuspended in 100 μL FACS buffer. The plate is incubated on a rocking platform at 4° C. for 15 min. After two washes, cells are resuspended in 200 μL of FACS buffer, transferred into twelve 75-mm flow tubes containing 150-200 μL of FACS buffer, and the tubes are placed on ice. 
     Following incubation, cells in the 15-mL tube containing antibody cocktail are pelleted by centrifugation at 400 g for 10 min at 4° C., and resuspended in FACS buffer to obtain a concentration of 25-50×10 6  cells/mL. 
     Purification of CD4+ CD45RBHi T Cells 
     Cell sorting of CD45RBHi and CD45RBLow populations is performed using flow cytometry. Briefly, a sample of unstained cells is used to establish baseline autofluorescence, and for forward scatter vs. side scatter gating of lymphoid cells. Single-color controls are used to set the appropriate levels of compensation to apply to each fluorochrome. However, with FITC and APC fluorochromes, compensation is generally not required. A single-parameter histogram (gated on singlet lymphoid cells) is then used to gate CD4+ (APC+) singlet cells, and a second singlet-parameter (gated on CD4+ singlet cells) is collected to establish sort gates. The CD45RBHi population is defined as the 40% of cells which exhibit the brightest CD45RB staining, whereas the CD45RBLow population is defined as the 15% of cells with the dimmest CD45RB expression. Each of these populations is sorted individually, and the CD45RBHi cells are used for adoptive transfer. 
     Adoptive Transfer 
     Purified populations of CD4+ CD45RBHi cells are adoptively transferred into 6- to 8-week-old RAG−/− male mice. The collection tubes containing sorted cells are filled with FACS buffer, and the cells are pelleted by centrifugation. The supernatant is then discarded, and cells are resuspended in 500 μL PBS. Resuspended cells are transferred into an injection tube, with a maximum of 5×106 cells per tube, and diluted with cold PBS to a final concentration of 1×106 cells/mL. Injection tubes are kept on ice. 
     Prior to injection, recipient mice are weighed and injection tubes are gently inverted several times to mix the cells. Mixed cells (0.5 mL, ˜0.5×106 cells) are carefully drawn into a 1-mL syringe with a 26G3/8 needle attached. Cells are then intraperitoneally injected into recipient mice. 
     Example 48. Efficacy of Genetically Engineered Bacteria in a CD45RBHi T Cell Transfer Model 
     To determine whether the genetically engineered bacteria of the disclosure are efficacious in CD45RBHi T cell transfer mice, disease progression following adoptive transfer is monitored by weighing each mouse on a weekly basis. Typically, modest weight increases are observed over the first 3 weeks post-transfer, followed by slow but progressive weight loss over the next 4-5 weeks. Weight loss is generally accompanied by the appearance of loose stools and diarrhea. 
     At weeks 4 or 5 post-transfer, as recipient mice begin to develop signs of disease, the genetically engineered bacteria described in Example 1 are grown overnight in LB supplemented with the appropriate antibiotic. Bacteria are then diluted 1:100 in fresh LB containing selective antibiotic, grown to an optical density of 0.4-0.5, and pelleted by centrifugation. Bacteria are resuspended in PBS and 100 μL of bacteria (or vehicle) is administered by oral gavage to CD45RBHi T cell transfer mice. Bacterial treatment is repeated once daily for 1-2 weeks before mice are euthanized. Murine colonic tissues are isolated and analyzed using the procedures described above. 
     Example 49. Efficacy of Genetically Engineered Bacteria in a Genetic Mouse Model of IBD 
     The genetically engineered bacteria described in Example 1 can be tested in genetic (including congenic and genetically modified) animal models of IBD. For example, IL-10 is an anti-inflammatory cytokine and the gene encoding IL-10 is a susceptibility gene for both Crohn&#39;s disease and ulcerative colitis (Khor et al., 2011). Functional impairment of IL-10, or its receptor, has been used to create several mouse models for the study of inflammation (Bramhall et al., 2015). IL-10 knockout (IL-10−/−) mice housed under normal conditions develop chronic inflammation in the gut (Iyer and Cheng, 2012). 
     To determine whether the genetically engineered bacteria of the disclosure are efficacious in IL-10−/− mice, bacteria are grown overnight in LB supplemented with the appropriate antibiotic. Bacteria are then diluted 1:100 in fresh LB containing selective antibiotic, grown to an optical density of 0.4-0.5, and pelleted by centrifugation. Bacteria are resuspended in PBS and 100 μL of bacteria (or vehicle) is administered by oral gavage to IL-10−/− mice. Bacterial treatment is repeated once daily for 1-2 weeks before mice are euthanized. Murine colonic tissues are isolated and analyzed using the procedures described above. 
     Protocols for testing the genetically engineered bacteria are similar for other genetic animal models of IBD. Such models include, but are not limited to, transgenic mouse models, e.g., SAMP1/YitFc (Pizarro et al., 2011), dominant negative N-cadherin mutant (NCAD delta; Hermiston and Gordon, 1995), TNFΔΔRE (Wagner et al., 2013), IL-7 (Watanabe et al., 1998), C3H/HeJBir (Elson et al., 2000), and dominant negative TGF-β receptor II mutant (Zhang et al., 2010); and knockout mouse models, e.g., TCRα−/− (Mombaerts et al., 1993; Sugimoto et al., 2008), WASP−/− (Nguyen et al., 2007), Mdr1a−/− (Wilk et al., 2005), IL-2 Rα−/− (Hsu et al., 2009), Gαi2−/− (Ohman et al., 2002), and TRUC (Tbet−/−Rag2−/−; Garrett et al., 2007). 
     Example 50. Efficacy of Genetically Engineered Bacteria in a Transgenic Rat Model of IBD 
     The genetically engineered bacteria described in Example 1 can be tested in non-murine animal models of IBD. The introduction of human leukocyte antigen B27 (HLA-B27) and the human β2-microglobulin gene into Fisher (F344) rats induces spontaneous, chronic inflammation in the GI tract (Alavi et al., 2000; Hammer et al., 1990). To investigate whether the genetically engineered bacteria of the invention are capable of ameliorating gut inflammation in this model, bacteria are grown overnight in LB supplemented with the appropriate antibiotic. Bacteria are then diluted 1:100 in fresh LB containing selective antibiotic, grown to an optical density of 0.4-0.5, and pelleted by centrifugation. Bacteria are resuspended in PBS and 100 μL of bacteria (or vehicle) is administered by oral gavage to transgenic F344-HLA-B27 rats. Bacterial treatment is repeated once daily for 2 weeks. 
     To determine whether bacterial treatment reduces the gross and histological intestinal lesions normally present in F344-HLA-B27 rats at 25 weeks of age, all animals are sacrificed at day 14 following the initial treatment. The GI tract is then resected from the ligament of Treitz to the rectum, opened along the antimesenteric border, and imaged using a flatbed scanner. Total mucosal damage, reported as a percent of the total surface area damaged, is quantified using standard image analysis software. 
     For microscopic analysis, samples (0.5-1.0 cm) are excised from both normal and diseased areas of the small and large intestine. Samples are fixed in formalin and embedded in paraffin before sections (5 lam) are processed for H&amp;E staining. The stained sections are analyzed and scored as follows: 0, no inflammation; 1, mild inflammation extending into the submucosa; 2, moderate inflammation extending into the muscularis propria; and 3, severe inflammation. The scores are combined and reported as mean±standard error. 
     Example 51. Synthesis of Constructs for Synthesis of Tryptophan, Tryptamine, and Other Indole Metabolites 
     Various constructs were synthesized, and cloned into vector pBR322 for transformation of  E. coli . In some embodiments, the constructs encoding the effector molecules are integrated into the genome according to methods described herein, e.g., Example 2. 
     
       
         
           
               
             
               
                 TABLE 81 
               
             
            
               
                   
               
               
                 Sequences 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 fbrAroG (RBS and 
                 
                   Ctctagaaataattttgtttaactttaagaaggagatatacat 
                 
               
               
                 leader region 
                 atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcg 
               
               
                 underlined) 
                 cattgctggaaaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccga 
               
               
                 SEQ ID NO: 255 
                 aaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtggtgattggccca 
               
               
                   
                 tgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgtg 
               
               
                   
                 aagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtacta 
               
               
                   
                 cggtgggctggaaagggctgattaacgatccgcatatggataacagcttccagatcaac 
               
               
                   
                 gacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagcg 
               
               
                   
                 gcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggc 
               
               
                   
                 gcaattggcgcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttc 
               
               
                   
                 ttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggctatcgatgccatta 
               
               
                   
                 atgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcgatt 
               
               
                   
                 gtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaa 
               
               
                   
                 ctacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgcc 
               
               
                   
                 agcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcagat 
               
               
                   
                 ggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgt 
               
               
                   
                 gatggtggaaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgct 
               
               
                   
                 ggcctacggtaagagcatcaccgatgcctgcattggctgggatgataccgatgctctgtt 
               
               
                   
                 acgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaa 
               
               
                   
               
               
                 fbrAroG 
                 atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcg 
               
               
                 SEQ ID NO: 256 
                 cattgctggaaaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccga 
               
               
                   
                 aaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtggtgattggccca 
               
               
                   
                 tgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgtg 
               
               
                   
                 aagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtacta 
               
               
                   
                 cggtgggctggaaagggctgattaacgatccgcatatggataacagatccagatcaac 
               
               
                   
                 gacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagcg 
               
               
                   
                 gcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggc 
               
               
                   
                 gcaattggcgcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtcttc 
               
               
                   
                 ttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggctatcgatgccatta 
               
               
                   
                 atgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcgatt 
               
               
                   
                 gtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaa 
               
               
                   
                 ctacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgcc 
               
               
                   
                 agcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcagat 
               
               
                   
                 ggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgt 
               
               
                   
                 gatggtggaaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgct 
               
               
                   
                 ggcctacggtaagagcatcaccgatgcctgcattggctgggatgataccgatgctctgtt 
               
               
                   
                 acgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaa 
               
               
                   
               
               
                 fbrAroG-serA 
                   Ctctagaaataattttgtttaactttaagaaggagatatacat atgaattatcagaacgacg 
               
               
                 (RBS and leader 
                 atttacgcatcaaagaaatcaaagagttacttcctcctgtcgcattgctggaaaaattcccc 
               
               
                 region underlined; 
                 gctactgaaaatgccgcgaatacggtcgcccatgcccgaaaagcgatccataagatcct 
               
               
                 SerA starts after 
                 gaaaggtaatgatgatcgcctgttggtggtgattggcccatgctcaattcatgatcctgtc 
               
               
                 second RBS) 
                 gcggctaaagagtatgccactcgcttgctgacgctgcgtgaagagctgcaagatgagct 
               
               
                 SEQ ID NO: 257 
                 ggaaatcgtgatgcgcgtctattttgaaaagccgcgtactacggtgggctggaaagggc 
               
               
                   
                 tgattaacgatccgcatatggataacagcttccagatcaacgacggtctgcgtattgccc 
               
               
                   
                 gcaaattgctgctcgatattaacgacagcggtctgccagcggcgggtgaattcctggata 
               
               
                   
                 tgatcaccctacaatatctcgctgacctgatgagctggggcgcaattggcgcacgtacca 
               
               
                   
                 ccgaatcgcaggtgcaccgcgaactggcgtctggtctttcttgtccggtaggtttcaaaa 
               
               
                   
                 atggcactgatggtacgattaaagtggctatcgatgccattaatgccgccggtgcgccgc 
               
               
                   
                 actgcttcctgtccgtaacgaaatgggggcattcggcgattgtgaataccagcggtaacg 
               
               
                   
                 gcgattgccatatcattctgcgcggcggtaaagagcctaactacagcgcgaagcacgtt 
               
               
                   
                 gctgaagtgaaagaagggctgaacaaagcaggcctgccagcgcaggtgatgatcgat 
               
               
                   
                 ttcagccatgctaactcgtcaaaacaattcaaaaagcagatggatgtttgtactgacgtttg 
               
               
                   
                 ccagcagattgccggtggcgaaaaggccattattggcgtgatggtggaaagccatctg 
               
               
                   
                 gtggaaggcaatcagagcctcgagagcggggaaccgctggcctacggtaagagcatc 
               
               
                   
                 accgatgcctgcattggctgggatgataccgatgctctgttacgtcaactggcgagtgca 
               
               
                   
                 gtaaaagcgcgtcgcgggtaaTACT 
               
               
                   
                   taagaaggagatatacat atggcaaaggtatcgctggagaaagacaagattaagtttctg 
               
               
                   
                 ctggtagaaggcgtgcaccaaaaggcgctggaaagccttcgtgcagctggttacacca 
               
               
                   
                 acatcgaatttcacaaaggcgcgctggatgatgaacaattaaaagaatccatccgcgat 
               
               
                   
                 gcccacttcatcggcctgcgatcccgtacccatctgactgaagacgtgatcaacgccgc 
               
               
                   
                 agaaaaactggtcgctattggctgtttctgtatcggaacaaatcaggttgatctggatgcg 
               
               
                   
                 gcggcaaagcgcgggatcccggtatttaacgcaccgttctcaaatacgcgctctgttgc 
               
               
                   
                 ggagctggtgattggcgaactgctgctgctattgcgcggcgtgccagaagccaatgcta 
               
               
                   
                 aagcgcatcgtggcgtgtggaacaaactggcggcgggttcttttgaagcgcgcggcaa 
               
               
                   
                 aaagctgggtatcatcggctacggtcatattggtacgcaattgggcattctggctgaatcg 
               
               
                   
                 ctgggaatgtatgtttacttttatgatattgaaaacaaactgccgctgggcaacgccactca 
               
               
                   
                 ggtacagcatctttctgacctgctgaatatgagcgatgtggtgagtctgcatgtaccagag 
               
               
                   
                 aatccgtccaccaaaaatatgatgggcgcgaaagagatttcgctaatgaagcccggctc 
               
               
                   
                 gctgctgattaatgcttcgcgcggtactgtggtggatattccagcgctgtgtgacgcgctg 
               
               
                   
                 gcgagcaaacatctggcgggggcggcaatcgacgtattcccgacggaaccggcgac 
               
               
                   
                 caatagcgatccatttacctctccgctgtgtgaattcgacaatgtccttctgacgccacaca 
               
               
                   
                 ttggcggttcgactcaggaagcgcaggagaatatcggcttggaagttgcgggtaaattg 
               
               
                   
                 atcaagtattctgacaatggctcaacgctctctgcggtgaacttcccggaagtctcgctgc 
               
               
                   
                 cactgcacggtgggcgtcgtctgatgcacatccacgaaaaccgtccgggcgtgctaact 
               
               
                   
                 gcgctcaacaaaatttttgccgagcagggcgtcaacatcgccgcgcaatatctacaaact 
               
               
                   
                 tccgcccagatgggttatgtagttattgatattgaagccgacgaagacgttgccgaaaaa 
               
               
                   
                 gcgctgcaggcaatgaaagctattccgggtaccattcgcgcccgtctgctgtactaa 
               
               
                   
               
               
                 SerA 
                 atggcaaaggtatcgctggagaaagacaagattaagtttctgctggtagaaggcgtgca 
               
               
                 SEQ ID NO: 258 
                 ccaaaaggcgctggaaagccttcgtgcagctggttacaccaacatcgaatttcacaaag 
               
               
                   
                 gcgcgctggatgatgaacaattaaaagaatccatccgcgatgcccacttcatcggcctg 
               
               
                   
                 cgatcccgtacccatctgactgaagacgtgatcaacgccgcagaaaaactggtcgctat 
               
               
                   
                 tggctgtttctgtatcggaacaaatcaggttgatctggatgcggcggcaaagcgcgggat 
               
               
                   
                 cccggtatttaacgcaccgttctcaaatacgcgctctgttgcggagctggtgattggcga 
               
               
                   
                 actgctgctgctattgcgcggcgtgccagaagccaatgctaaagcgcatcgtggcgtgt 
               
               
                   
                 ggaacaaactggcggcgggttcttttgaagcgcgcggcaaaaagctgggtatcatcgg 
               
               
                   
                 ctacggtcatattggtacgcaattgggcattctggctgaatcgctgggaatgtatgtttactt 
               
               
                   
                 ttatgatattgaaaacaaactgccgctgggcaacgccactcaggtacagcatctttctgac 
               
               
                   
                 ctgctgaatatgagcgatgtggtgagtctgcatgtaccagagaatccgtccaccaaaaat 
               
               
                   
                 atgatgggcgcgaaagagatttcgctaatgaagcccggctcgctgctgattaatgcttcg 
               
               
                   
                 cgcggtactgtggtggatattccagcgctgtgtgacgcgctggcgagcaaacatctggc 
               
               
                   
                 gggggcggcaatcgacgtattcccgacggaaccggcgaccaatagcgatccatttacc 
               
               
                   
                 tctccgctgtgtgaattcgacaatgtccttctgacgccacacattggcggttcgactcagg 
               
               
                   
                 aagcgcaggagaatatcggcttggaagttgcgggtaaattgatcaagtattctgacaatg 
               
               
                   
                 gctcaacgctctctgcggtgaacttcccggaagtctcgctgccactgcacggtgggcgt 
               
               
                   
                 cgtctgatgcacatccacgaaaaccgtccgggcgtgctaactgcgctcaacaaaattttt 
               
               
                   
                 gccgagcagggcgtcaacatcgccgcgcaatatctacaaacttccgcccagatgggtt 
               
               
                   
                 atgtagttattgatattgaagccgacgaagacgttgccgaaaaagcgctgcaggcaatg 
               
               
                   
                 aaagctattccgggtaccattcgcgcccgtctgctgtactaa 
               
               
                   
               
               
                 fbrAroG-Tdc (tdc 
                   ctctagaaataattttgtttaactttaagaaggagatatacat atgaattatcagaacgacga 
               
               
                 from C. roseus); 
                 tttacgcatcaaagaaatcaaagagttacttcctcctgtcgcattgctggaaaaattccccg 
               
               
                 RBS and leader 
                 ctactgaaaatgccgcgaatacggtcgcccatgcccgaaaagcgatccataagatcctg 
               
               
                 region underlined 
                 aaaggtaatgatgatcgcctgttggtggtgattggcccatgctcaattcatgatcctgtcgc 
               
               
                 SEQ ID NO: 259 
                 ggctaaagagtatgccactcgcttgctgacgctgcgtgaagagctgcaagatgagctgg 
               
               
                   
                 aaatcgtgatgcgcgtctattttgaaaagccgcgtactacggtgggctggaaagggctg 
               
               
                   
                 attaacgatccgcatatggataacagcttccagatcaacgacggtctgcgtattgcccgc 
               
               
                   
                 aaattgctgctcgatattaacgacagcggtctgccagcggcgggtgaattcctggatatg 
               
               
                   
                 atcaccctacaatatctcgctgacctgatgagctggggcgcaattggcgcacgtaccac 
               
               
                   
                 cgaatcgcaggtgcaccgcgaactggcgtctggtctttcttgtccggtaggtttcaaaaat 
               
               
                   
                 ggcactgatggtacgattaaagtggctatcgatgccattaatgccgccggtgcgccgca 
               
               
                   
                 ctgcttcctgtccgtaacgaaatgggggcattcggcgattgtgaataccagcggtaacg 
               
               
                   
                 gcgattgccatatcattctgcgcggcggtaaagagcctaactacagcgcgaagcacgtt 
               
               
                   
                 gctgaagtgaaagaagggctgaacaaagcaggcctgccagcgcaggtgatgatcgat 
               
               
                   
                 ttcagccatgctaactcgtcaaaacaattcaaaaagcagatggatgtttgtactgacgtttg 
               
               
                   
                 ccagcagattgccggtggcgaaaaggccattattggcgtgatggtggaaagccatctg 
               
               
                   
                 gtggaaggcaatcagagcctcgagagcggggaaccgctggcctacggtaagagcatc 
               
               
                   
                 accgatgcctgcattggctgggatgataccgatgctctgttacgtcaactggcgagtgca 
               
               
                   
                 gtaaaagcgcgtcgcgggtaaTACT taagaaggagatatacat ATGGGTTC 
               
               
                   
                 TATTGACTCGACGAATGTGGCCATGTCTAATTCTCCT 
               
               
                   
                 GTTGGCGAGTTTAAGCCCCTTGAAGCAGAAGAGTTCC 
               
               
                   
                 GTAAACAGGCACACCGCATGGTGGATTTTATTGCGGA 
               
               
                   
                 TTATTACAAGAACGTAGAAACATACCCGGTCCTTTCC 
               
               
                   
                 GAGGTTGAACCCGGCTATCTGCGCAAACGTATTCCCG 
               
               
                   
                 AAACCGCACCATACCTGCCGGAGCCACTTGATGATAT 
               
               
                   
                 TATGAAGGATATTCAAAAGGACATTATCCCCGGAAT 
               
               
                   
                 GACGAACTGGATGTCCCCGAACTTTTACGCCTTCTTC 
               
               
                   
                 CCGGCCACAGTTAGCTCAGCAGCTTTCTTGGGGGAAA 
               
               
                   
                 TGCTTTCAACGGCCCTTAACAGCGTAGGATTTACCTG 
               
               
                   
                 GGTCAGTTCCCCGGCAGCGACTGAATTAGAGATGATC 
               
               
                   
                 GTTATGGATTGGCTTGCGCAAATTTTGAAACTTCCAA 
               
               
                   
                 AAAGCTTTATGTTCTCCGGAACCGGGGGTGGTGTCAT 
               
               
                   
                 CCAAAACACTACGTCAGAGTCGATCTTGTGCACTATT 
               
               
                   
                 ATCGCGGCCCGTGAACGCGCCTTGGAAAAATTGGGC 
               
               
                   
                 CCTGATTCAATTGGTAAGCTTGTCTGCTATGGGTCCG 
               
               
                   
                 ATCAAACGCACACAATGTTTCCGAAAACCTGTAAGTT 
               
               
                   
                 AGCAGGAATTTATCCGAATAATATCCGCCTTATCCCT 
               
               
                   
                 ACCACGGTAGAAACCGACTTTGGCATCTCACCGCAG 
               
               
                   
                 GTACTTCGCAAGATGGTCGAAGACGACGTCGCTGCG 
               
               
                   
                 GGGTACGTTCCCTTATTTTTGTGTGCCACCTTGGGAA 
               
               
                   
                 CGACATCAACTACGGCAACAGATCCTGTAGATTCGCT 
               
               
                   
                 GTCCGAAATCGCAAACGAGTTTGGTATCTGGATTCAT 
               
               
                   
                 GTCGACGCCGCATATGCTGGATCGGCTTGCATCTGCC 
               
               
                   
                 CAGAATTTCGTCACTACCTTGATGGCATCGAACGTGT 
               
               
                   
                 GGATTCCTTATCGCTGTCTCCCCACAAATGGCTTTTA 
               
               
                   
                 GCATATCTGGATTGCACGTGCTTGTGGGTAAAACAAC 
               
               
                   
                 CTCACCTGCTGCTTCGCGCTTTAACGACTAATCCCGA 
               
               
                   
                 ATACTTGAAGAATAAACAGAGTGATTTAGATAAGGT 
               
               
                   
                 CGTGGATTTTAAGAACTGGCAGATCGCAACAGGACG 
               
               
                   
                 TAAGTTCCGCTCTTTAAAACTTTGGTTAATTCTGCGTT 
               
               
                   
                 CCTACGGGGTAGTTAACCTGCAAAGTCATATCCGTAG 
               
               
                   
                 TGATGTAGCGATGGGGAAGATGTTTGAGGAATGGGT 
               
               
                   
                 CCGTTCCGATAGCCGCTTTGAAATCGTCGTGCCACGT 
               
               
                   
                 AATTTTTCGCTTGTATGCTTTCGCTTGAAACCGGATGT 
               
               
                   
                 ATCTAGTTTACATGTCGAGGAGGTCAACAAGAAGTTG 
               
               
                   
                 TTGGATATGCTTAACTCCACCGGTCGCGTATATATGA 
               
               
                   
                 CGCATACAATTGTTGGCGGAATCTATATGTTACGTTT 
               
               
                   
                 GGCTGTAGGTAGCAGCTTGACAGAGGAACATCACGT 
               
               
                   
                 GCGCCGCGTTTGGGACTTGATCCAGAAGCTTACGGAC 
               
               
                   
                 GACCTGCTTAAAGAGGCGTGA 
               
               
                   
               
               
                 Tdc (tdc from C. 
                 ATGGGTTCTATTGACTCGACGAATGTGGCCATGTCTA 
               
               
                 roseus) 
                 ATTCTCCTGTTGGCGAGTTTAAGCCCCTTGAAGCAGA 
               
               
                 SEQ ID NO: 260 
                 AGAGTTCCGTAAACAGGCACACCGCATGGTGGATTTT 
               
               
                   
                 ATTGCGGATTATTACAAGAACGTAGAAACATACCCG 
               
               
                   
                 GTCCTTTCCGAGGTTGAACCCGGCTATCTGCGCAAAC 
               
               
                   
                 GTATTCCCGAAACCGCACCATACCTGCCGGAGCCACT 
               
               
                   
                 TGATGATATTATGAAGGATATTCAAAAGGACATTATC 
               
               
                   
                 CCCGGAATGACGAACTGGATGTCCCCGAACTTTTACG 
               
               
                   
                 CCTTCTTCCCGGCCACAGTTAGCTCAGCAGCTTTCTTG 
               
               
                   
                 GGGGAAATGCTTTCAACGGCCCTTAACAGCGTAGGA 
               
               
                   
                 TTTACCTGGGTCAGTTCCCCGGCAGCGACTGAATTAG 
               
               
                   
                 AGATGATCGTTATGGATTGGCTTGCGCAAATTTTGAA 
               
               
                   
                 ACTTCCAAAAAGCTTTATGTTCTCCGGAACCGGGGGT 
               
               
                   
                 GGTGTCATCCAAAACACTACGTCAGAGTCGATCTTGT 
               
               
                   
                 GCACTATTATCGCGGCCCGTGAACGCGCCTTGGAAAA 
               
               
                   
                 ATTGGGCCCTGATTCAATTGGTAAGCTTGTCTGCTAT 
               
               
                   
                 GGGTCCGATCAAACGCACACAATGTTTCCGAAAACCT 
               
               
                   
                 GTAAGTTAGCAGGAATTTATCCGAATAATATCCGCCT 
               
               
                   
                 TATCCCTACCACGGTAGAAACCGACTTTGGCATCTCA 
               
               
                   
                 CCGCAGGTACTTCGCAAGATGGTCGAAGACGACGTC 
               
               
                   
                 GCTGCGGGGTACGTTCCCTTATTTTTGTGTGCCACCTT 
               
               
                   
                 GGGAACGACATCAACTACGGCAACAGATCCTGTAGA 
               
               
                   
                 TTCGCTGTCCGAAATCGCAAACGAGTTTGGTATCTGG 
               
               
                   
                 ATTCATGTCGACGCCGCATATGCTGGATCGGCTTGCA 
               
               
                   
                 TCTGCCCAGAATTTCGTCACTACCTTGATGGCATCGA 
               
               
                   
                 ACGTGTGGATTCCTTATCGCTGTCTCCCCACAAATGG 
               
               
                   
                 CTTTTAGCATATCTGGATTGCACGTGCTTGTGGGTAA 
               
               
                   
                 AACAACCTCACCTGCTGCTTCGCGCTTTAACGACTAA 
               
               
                   
                 TCCCGAATACTTGAAGAATAAACAGAGTGATTTAGAT 
               
               
                   
                 AAGGTCGTGGATTTTAAGAACTGGCAGATCGCAACA 
               
               
                   
                 GGACGTAAGTTCCGCTCTTTAAAACTTTGGTTAATTC 
               
               
                   
                 TGCGTTCCTACGGGGTAGTTAACCTGCAAAGTCATAT 
               
               
                   
                 CCGTAGTGATGTAGCGATGGGGAAGATGTTTGAGGA 
               
               
                   
                 ATGGGTCCGTTCCGATAGCCGCTTTGAAATCGTCGTG 
               
               
                   
                 CCACGTAATTTTTCGCTTGTATGCTTTCGCTTGAAACC 
               
               
                   
                 GGATGTATCTAGTTTACATGTCGAGGAGGTCAACAAG 
               
               
                   
                 AAGTTGTTGGATATGCTTAACTCCACCGGTCGCGTAT 
               
               
                   
                 ATATGACGCATACAATTGTTGGCGGAATCTATATGTT 
               
               
                   
                 ACGTTTGGCTGTAGGTAGCAGCTTGACAGAGGAACA 
               
               
                   
                 TCACGTGCGCCGCGTTTGGGACTTGATCCAGAAGCTT 
               
               
                   
                 ACGGACGACCTGCTTAAAGAGGCGTGA 
               
               
                   
               
               
                 fbrAroG-Tdc (tdc 
                   ctctagaaataattttgtttaactttaagaaggagatatacat atgaattatcagaacgacga 
               
               
                 from Clostridium 
                 tttacgcatcaaagaaatcaaagagttacttcctcctgtcgcattgctggaaaaattccccg 
               
               
                 sporogenes); RBS 
                 ctactgaaaatgccgcgaatacggtcgcccatgcccgaaaagcgatccataagatcctg 
               
               
                 and leader region 
                 aaaggtaatgatgatcgcctgttggtggtgattggcccatgctcaattcatgatcctgtcgc 
               
               
                 underlined 
                 ggctaaagagtatgccactcgcttgctgacgctgcgtgaagagctgcaagatgagctgg 
               
               
                 SEQ ID NO: 261 
                 aaatcgtgatgcgcgtctattttgaaaagccgcgtactacggtgggctggaaagggctg 
               
               
                   
                 attaacgatccgcatatggataacagatccagatcaacgacggtctgcgtattgcccgc 
               
               
                   
                 aaattgctgctcgatattaacgacagcggtctgccagcggcgggtgaattcctggatatg 
               
               
                   
                 atcaccctacaatatctcgctgacctgatgagctggggcgcaattggcgcacgtaccac 
               
               
                   
                 cgaatcgcaggtgcaccgcgaactggcgtctggtctttcttgtccggtaggtttcaaaaat 
               
               
                   
                 ggcactgatggtacgattaaagtggctatcgatgccattaatgccgccggtgcgccgca 
               
               
                   
                 ctgcttcctgtccgtaacgaaatgggggcattcggcgattgtgaataccagcggtaacg 
               
               
                   
                 gcgattgccatatcattctgcgcggcggtaaagagcctaactacagcgcgaagcacgtt 
               
               
                   
                 gctgaagtgaaagaagggctgaacaaagcaggcctgccagcgcaggtgatgatcgat 
               
               
                   
                 ttcagccatgctaactcgtcaaaacaattcaaaaagcagatggatgtttgtactgacgtttg 
               
               
                   
                 ccagcagattgccggtggcgaaaaggccattattggcgtgatggtggaaagccatctg 
               
               
                   
                 gtggaaggcaatcagagcctcgagagcggggaaccgctggcctacggtaagagcatc 
               
               
                   
                 accgatgcctgcattggctgggatgataccgatgctctgttacgtcaactggcgagtgca 
               
               
                   
                 gtaaaagcgcgtcgcgggtaaTACT taagaaggagatatacat ATGAAATT 
               
               
                   
                 TTGGCGCAAGTATACGCAACAGGAGATGGATGAGAA 
               
               
                   
                 AATCACAGAATCGCTTGAGAAGACATTAAATTACGA 
               
               
                   
                 TAACACGAAAACCATCGGCATCCCAGGTACTAAGCT 
               
               
                   
                 GGATGATACTGTATTTTATGACGATCACTCCTTCGTT 
               
               
                   
                 AAGCACTCTCCCTATTTACGTACGTTCATCCAAAACC 
               
               
                   
                 CTAATCACATTGGTTGTCACACGTACGATAAAGCAGA 
               
               
                   
                 CATCTTGTTTGGCGGCACGTTTGACATCGAACGCGAA 
               
               
                   
                 CTGATTCAGCTTTTGGCCATCGATGTCTTAAACGGAA 
               
               
                   
                 ATGATGAGGAATTCGATGGATATGTGACACAGGGGG 
               
               
                   
                 GAACCGAGGCGAATATTCAGGCAATGTGGGTTTATC 
               
               
                   
                 GTAACTATTTCAAAAAAGAACGTAAAGCAAAACATG 
               
               
                   
                 AGGAAATCGCAATCATCACGAGCGCGGATACCCATT 
               
               
                   
                 ACAGTGCATATAAGGGGAGCGACTTGCTGAACATTG 
               
               
                   
                 ATATTATCAAGGTCCCAGTAGACTTCTATTCGCGTAA 
               
               
                   
                 GATCCAGGAGAACACGTTAGACTCGATTGTCAAGGA 
               
               
                   
                 GGCGAAGGAAATTGGAAAGAAGTACTTCATTGTCAT 
               
               
                   
                 CTCAAACATGGGTACGACTATGTTTGGCAGTGTAGAC 
               
               
                   
                 GACCCTGATCTTTATGCTAACATTTTTGATAAGTATA 
               
               
                   
                 ACTTAGAATACAAAATCCACGTCGATGGAGCTTTTGG 
               
               
                   
                 GGGTTTCATTTATCCTATCGATAATAAGGAGTGCAAA 
               
               
                   
                 ACAGATTTCTCGAACAAGAACGTCTCATCCATCACGC 
               
               
                   
                 TTGACGGTCACAAAATGCTTCAAGCCCCCTATGGGAC 
               
               
                   
                 TGGTATCTTCGTGTCACGTAAGAACTTGATCCATAAC 
               
               
                   
                 ACCCTGACAAAGGAAGCAACGTATATTGAAAACCTG 
               
               
                   
                 GACGTTACCCTGAGTGGGTCCCGCTCCGGATCCAACG 
               
               
                   
                 CCGTTGCGATCTGGATGGTTTTAGCCTCTTATGGCCC 
               
               
                   
                 CTACGGGTGGATGGAGAAGATTAACAAGTTGCGCAA 
               
               
                   
                 TCGCACTAAGTGGCTTTGCAAGCAGCTTAACGACATG 
               
               
                   
                 CGCATCAAATACTATAAGGAGGATAGCATGAATATC 
               
               
                   
                 GTCACGATTGAAGAGCAATACGTAAATAAAGAGATT 
               
               
                   
                 GCAGAGAAATACTTCCTTGTGCCTGAAGTACACAATC 
               
               
                   
                 CTACCAACAATTGGTACAAGATTGTAGTCATGGAACA 
               
               
                   
                 TGTTGAACTTGACATCTTGAACTCCCTTGTTTATGATT 
               
               
                   
                 TACGTAAATTCAACAAGGAGCACCTGAAGGCAATGT 
               
               
                   
                 GA 
               
               
                   
               
               
                 Tdc (tdc from 
                 ATGAAATTTTGGCGCAAGTATACGCAACAGGAGATG 
               
               
                 Clostridium 
                 GATGAGAAAATCACAGAATCGCTTGAGAAGACATTA 
               
               
                 sporogenes) 
                 AATTACGATAACACGAAAACCATCGGCATCCCAGGT 
               
               
                 SEQ ID NO: 262 
                 ACTAAGCTGGATGATACTGTATTTTATGACGATCACT 
               
               
                   
                 CCTTCGTTAAGCACTCTCCCTATTTACGTACGTTCATC 
               
               
                   
                 CAAAACCCTAATCACATTGGTTGTCACACGTACGATA 
               
               
                   
                 AAGCAGACATCTTGTTTGGCGGCACGTTTGACATCGA 
               
               
                   
                 ACGCGAACTGATTCAGCTTTTGGCCATCGATGTCTTA 
               
               
                   
                 AACGGAAATGATGAGGAATTCGATGGATATGTGACA 
               
               
                   
                 CAGGGGGGAACCGAGGCGAATATTCAGGCAATGTGG 
               
               
                   
                 GTTTATCGTAACTATTTCAAAAAAGAACGTAAAGCAA 
               
               
                   
                 AACATGAGGAAATCGCAATCATCACGAGCGCGGATA 
               
               
                   
                 CCCATTACAGTGCATATAAGGGGAGCGACTTGCTGA 
               
               
                   
                 ACATTGATATTATCAAGGTCCCAGTAGACTTCTATTC 
               
               
                   
                 GCGTAAGATCCAGGAGAACACGTTAGACTCGATTGT 
               
               
                   
                 CAAGGAGGCGAAGGAAATTGGAAAGAAGTACTTCAT 
               
               
                   
                 TGTCATCTCAAACATGGGTACGACTATGTTTGGCAGT 
               
               
                   
                 GTAGACGACCCTGATCTTTATGCTAACATTTTTGATA 
               
               
                   
                 AGTATAACTTAGAATACAAAATCCACGTCGATGGAG 
               
               
                   
                 CTTTTGGGGGTTTCATTTATCCTATCGATAATAAGGA 
               
               
                   
                 GTGCAAAACAGATTTCTCGAACAAGAACGTCTCATCC 
               
               
                   
                 ATCACGCTTGACGGTCACAAAATGCTTCAAGCCCCCT 
               
               
                   
                 ATGGGACTGGTATCTTCGTGTCACGTAAGAACTTGAT 
               
               
                   
                 CCATAACACCCTGACAAAGGAAGCAACGTATATTGA 
               
               
                   
                 AAACCTGGACGTTACCCTGAGTGGGTCCCGCTCCGGA 
               
               
                   
                 TCCAACGCCGTTGCGATCTGGATGGTTTTAGCCTCTT 
               
               
                   
                 ATGGCCCCTACGGGTGGATGGAGAAGATTAACAAGT 
               
               
                   
                 TGCGCAATCGCACTAAGTGGCTTTGCAAGCAGCTTAA 
               
               
                   
                 CGACATGCGCATCAAATACTATAAGGAGGATAGCAT 
               
               
                   
                 GAATATCGTCACGATTGAAGAGCAATACGTAAATAA 
               
               
                   
                 AGAGATTGCAGAGAAATACTTCCTTGTGCCTGAAGTA 
               
               
                   
                 CACAATCCTACCAACAATTGGTACAAGATTGTAGTCA 
               
               
                   
                 TGGAACATGTTGAACTTGACATCTTGAACTCCCTTGT 
               
               
                   
                 TTATGATTTACGTAAATTCAACAAGGAGCACCTGAAG 
               
               
                   
                 GCAATGTGA 
               
               
                   
               
               
                 fbrArG-trpDH- 
                 
                   Ctctagaaataattttgtttaactttaagaaggagatatacat 
                 
               
               
                 ipdC-iadl (RBS 
                 atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcg 
               
               
                 and leader region 
                 cattgctggaaaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccga 
               
               
                 underlined) 
                 aaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtggtgattggccca 
               
               
                 SEQ ID NO: 263 
                 tgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgtg 
               
               
                   
                 aagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtacta 
               
               
                   
                 cggtgggctggaaagggctgattaacgatccgcatatggataacagatccagatcaac 
               
               
                   
                 gacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagcg 
               
               
                   
                 gcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggc 
               
               
                   
                 gcaattggcgcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttc 
               
               
                   
                 ttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggctatcgatgccatta 
               
               
                   
                 atgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcgatt 
               
               
                   
                 gtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaa 
               
               
                   
                 ctacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgcc 
               
               
                   
                 agcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcagat 
               
               
                   
                 ggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgt 
               
               
                   
                 gatggtggaaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgct 
               
               
                   
                 ggcctacggtaagagcatcaccgatgcctgcattggctgggatgataccgatgctctgtt 
               
               
                   
                 acgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaaTACT taagaaggaga   
               
               
                   
                   tatacat ATGCTGTTATTCGAGACTGTGCGTGAAATGGGT 
               
               
                   
                 CATGAGCAAGTCCTTTTCTGTCATAGCAAGAATCCCG 
               
               
                   
                 AGATCAAGGCAATTATCGCAATCCACGATACCACCTT 
               
               
                   
                 AGGACCGGCTATGGGCGCAACTCGTATCTTACCTTAT 
               
               
                   
                 ATTAATGAGGAGGCTGCCCTGAAAGATGCATTACGTC 
               
               
                   
                 TGTCCCGCGGAATGACTTACAAAGCAGCCTGCGCCA 
               
               
                   
                 ATATTCCCGCCGGGGGCGGCAAAGCCGTCATCATCGC 
               
               
                   
                 TAACCCCGAAAACAAGACCGATGACCTGTTACGCGC 
               
               
                   
                 ATACGGCCGTTTCGTGGACAGCTTGAACGGCCGTTTC 
               
               
                   
                 ATCACCGGGCAGGACGTTAACATTACGCCCGACGAC 
               
               
                   
                 GTTCGCACTATTTCGCAGGAGACTAAGTACGTGGTAG 
               
               
                   
                 GCGTCTCAGAAAAGTCGGGAGGGCCGGCACCTATCA 
               
               
                   
                 CCTCTCTGGGAGTATTTTTAGGCATCAAAGCCGCTGT 
               
               
                   
                 AGAGTCGCGTTGGCAGTCTAAACGCCTGGATGGCAT 
               
               
                   
                 GAAAGTGGCGGTGCAAGGACTTGGGAACGTAGGAAA 
               
               
                   
                 AAATCTTTGTCGCCATCTGCATGAACACGATGTACAA 
               
               
                   
                 CTTTTTGTGTCTGATGTCGATCCAATCAAGGCCGAGG 
               
               
                   
                 AAGTAAAACGCTTATTCGGGGCGACTGTTGTCGAACC 
               
               
                   
                 GACTGAAATCTATTCTTTAGATGTTGATATTTTTGCAC 
               
               
                   
                 CGTGTGCACTTGGGGGTATTTTGAATAGCCATACCAT 
               
               
                   
                 CCCGTTCTTACAAGCCTCAATCATCGCAGGAGCAGCG 
               
               
                   
                 AATAACCAGCTGGAGAACGAGCAACTTCATTCGCAG 
               
               
                   
                 ATGCTTGCGAAAAAGGGTATTCTTTACTCACCAGACT 
               
               
                   
                 ACGTTATCAATGCAGGAGGACTTATCAATGTTTATAA 
               
               
                   
                 CGAAATGATCGGATATGACGAGGAAAAAGCATTCAA 
               
               
                   
                 ACAAGTTCATAACATCTACGATACGTTATTAGCGATT 
               
               
                   
                 TTCGAAATTGCAAAAGAACAAGGTGTAACCACCAAC 
               
               
                   
                 GACGCGGCCCGTCGTTTAGCAGAGGATCGTATCAAC 
               
               
                   
                 AACTCCAAACGCTCAAAGAGTAAAGCGATTGCGGCG 
               
               
                   
                 TGAAATG taagaaggagatatacat ATGCGTACACCCTACTGTG 
               
               
                   
                 TCGCCGATTATCTTTTAGATCGTCTGACGGACTGCGG 
               
               
                   
                 GGCCGATCACCTGTTTGGCGTACCGGGCGATTACAAC 
               
               
                   
                 TTGCAGTTTCTGGACCACGTCATTGACTCACCAGATA 
               
               
                   
                 TCTGCTGGGTAGGGTGTGCGAACGAGCTTAACGCGA 
               
               
                   
                 GCTACGCTGCTGACGGATATGCGCGTTGTAAAGGCTT 
               
               
                   
                 TGCTGCACTTCTTACTACCTTCGGGGTCGGTGAGTTA 
               
               
                   
                 TCGGCGATGAACGGTATCGCAGGCTCGTACGCTGAG 
               
               
                   
                 CACGTCCCGGTATTACACATTGTGGGAGCTCCGGGTA 
               
               
                   
                 CCGCAGCTCAACAGCGCGGAGAACTGTTACACCACA 
               
               
                   
                 CGCTGGGCGACGGAGAATTCCGCCACTTTTACCATAT 
               
               
                   
                 GTCCGAGCCAATTACTGTAGCCCAGGCTGTACTTACA 
               
               
                   
                 GAGCAAAATGCCTGTTACGAGATCGACCGTGTTTTGA 
               
               
                   
                 CCACGATGCTTCGCGAGCGCCGTCCCGGGTATTTGAT 
               
               
                   
                 GCTGCCAGCCGATGTTGCCAAAAAAGCTGCGACGCC 
               
               
                   
                 CCCAGTGAATGCCCTGACGCATAAACAAGCTCATGCC 
               
               
                   
                 GATTCCGCCTGTTTAAAGGCTTTTCGCGATGCAGCTG 
               
               
                   
                 AAAATAAATTAGCCATGTCGAAACGCACCGCCTTGTT 
               
               
                   
                 GGCGGACTTTCTGGTCCTGCGCCATGGCCTTAAACAC 
               
               
                   
                 GCCCTTCAGAAATGGGTCAAAGAAGTCCCGATGGCC 
               
               
                   
                 CACGCTACGATGCTTATGGGTAAGGGGATTTTTGATG 
               
               
                   
                 AACGTCAAGCGGGATTTTATGGAACTTATTCCGGTTC 
               
               
                   
                 GGCGAGTACGGGGGCGGTAAAGGAAGCGATTGAGGG 
               
               
                   
                 AGCCGACACAGTTCTTTGCGTGGGGACACGTTTCACC 
               
               
                   
                 GATACACTGACCGCTGGATTCACACACCAACTTACTC 
               
               
                   
                 CGGCACAAACGATTGAGGTGCAACCCCATGCGGCTC 
               
               
                   
                 GCGTGGGGGATGTATGGTTTACGGGCATTCCAATGAA 
               
               
                   
                 TCAAGCCATTGAGACTCTTGTCGAGCTGTGCAAACAG 
               
               
                   
                 CACGTCCACGCAGGACTGATGAGTTCGAGCTCTGGG 
               
               
                   
                 GCGATTCCTTTTCCACAACCAGATGGTAGTTTAACTC 
               
               
                   
                 AAGAAAACTTCTGGCGCACATTGCAAACCTTTATCCG 
               
               
                   
                 CCCAGGTGATATCATCTTAGCAGACCAGGGTACTTCA 
               
               
                   
                 GCCTTTGGAGCAATTGACCTGCGCTTACCAGCAGACG 
               
               
                   
                 TGAACTTTATTGTGCAGCCGCTGTGGGGGTCTATTGG 
               
               
                   
                 TTATACTTTAGCTGCGGCCTTCGGAGCGCAGACAGCG 
               
               
                   
                 TGTCCAAACCGTCGTGTGATCGTATTGACAGGAGATG 
               
               
                   
                 GAGCAGCGCAGTTGACCATTCAGGAGTTAGGCTCGA 
               
               
                   
                 TGTTACGCGATAAGCAGCACCCCATTATCCTGGTCCT 
               
               
                   
                 GAACAATGAGGGGTATACAGTTGAACGCGCCATTCA 
               
               
                   
                 TGGTGCGGAACAACGCTACAATGACATCGCTTTATGG 
               
               
                   
                 AATTGGACGCACATCCCCCAAGCCTTATCGTTAGATC 
               
               
                   
                 CCCAATCGGAATGTTGGCGTGTGTCTGAAGCAGAGC 
               
               
                   
                 AACTGGCTGATGTTCTGGAAAAAGTTGCTCATCATGA 
               
               
                   
                 ACGCCTGTCGTTGATCGAGGTAATGTTGCCCAAGGCC 
               
               
                   
                 GATATCCCTCCGTTACTGGGAGCCTTGACCAAGGCTT 
               
               
                   
                 TAGAAGCCTGCAACAACGCTTAAAGGT taagaaggagatata   
               
               
                   
                   cat ATGCCCACCTTGAACTTGGACTTACCCAACGGTAT 
               
               
                   
                 TAAGAGCACGATTCAGGCAGACCTTTTCATCAATAAT 
               
               
                   
                 AAGTTTGTGCCGGCGCTTGATGGGAAAACGTTCGCAA 
               
               
                   
                 CTATTAATCCGTCTACGGGGAAAGAGATCGGACAGG 
               
               
                   
                 TGGCAGAGGCTTCGGCGAAGGATGTGGATCTTGCAG 
               
               
                   
                 TTAAGGCCGCGCGTGAGGCGTTTGAAACTACTTGGGG 
               
               
                   
                 GGAAAACACGCCAGGTGATGCTCGTGGCCGTTTACTG 
               
               
                   
                 ATTAAGCTTGCTGAGTTGGTGGAAGCGAATATTGATG 
               
               
                   
                 AGTTAGCGGCAATTGAATCACTGGACAATGGGAAAG 
               
               
                   
                 CGTTCTCTATTGCTAAGTCATTCGACGTAGCTGCTGT 
               
               
                   
                 GGCCGCAAACTTACGTTACTACGGCGGTTGGGCTGAT 
               
               
                   
                 AAAAACCACGGTAAAGTCATGGAGGTAGACACAAAG 
               
               
                   
                 CGCCTGAACTATACCCGCCACGAGCCGATCGGGGTTT 
               
               
                   
                 GCGGACAAATCATTCCGTGGAATTTCCCGCTTTTGAT 
               
               
                   
                 GTTTGCATGGAAGCTGGGTCCCGCTTTAGCCACAGGG 
               
               
                   
                 AACACAATTGTGTTAAAGACTGCCGAGCAGACTCCCT 
               
               
                   
                 TAAGTGCTATCAAGATGTGTGAATTAATCGTAGAAGC 
               
               
                   
                 CGGCTTTCCGCCCGGAGTAGTTAATGTGATCTCGGGA 
               
               
                   
                 TTCGGACCGGTGGCGGGGGCCGCGATCTCGCAACAC 
               
               
                   
                 ATGGACATCGATAAGATTGCCTTTACAGGATCGACAT 
               
               
                   
                 TGGTTGGCCGCAACATTATGAAGGCAGCTGCGTCGAC 
               
               
                   
                 TAACTTAAAAAAGGTTACACTTGAGTTAGGAGGAAA 
               
               
                   
                 ATCCCCGAATATCATTTTCAAAGATGCCGACCTTGAC 
               
               
                   
                 CAAGCTGTTCGCTGGAGCGCCTTCGGTATCATGTTTA 
               
               
                   
                 ACCACGGACAATGCTGCTGCGCTGGATCGCGCGTATA 
               
               
                   
                 TGTGGAAGAATCCATCTATGACGCCTTCATGGAAAAA 
               
               
                   
                 ATGACTGCGCATTGTAAGGCGCTTCAAGTTGGAGATC 
               
               
                   
                 CTTTCAGCGCGAACACCTTCCAAGGACCACAAGTCTC 
               
               
                   
                 GCAGTTACAATACGACCGTATCATGGAATACATCGA 
               
               
                   
                 ATCAGGGAAAAAAGATGCAAATCTTGCTTTAGGCGG 
               
               
                   
                 CGTTCGCAAAGGGAATGAGGGGTATTTCATTGAGCC 
               
               
                   
                 AACTATTTTTACAGACGTGCCGCACGACGCGAAGATT 
               
               
                   
                 GCCAAAGAGGAGATCTTCGGTCCAGTGGTTGTTGTGT 
               
               
                   
                 CGAAATTTAAGGACGAAAAAGATCTGATCCGTATCG 
               
               
                   
                 CAAATGATTCTATTTATGGTTTAGCTGCGGCAGTCTTT 
               
               
                   
                 TCCCGCGACATCAGCCGCGCGATCGAGACAGCACAC 
               
               
                   
                 AAACTGAAAGCAGGCACGGTCTGGGTCAACTGCTAT 
               
               
                   
                 AATCAGCTTATTCCGCAGGTGCCATTCGGAGGGTATA 
               
               
                   
                 AGGCTTCCGGTATCGGCCGTGAGTTGGGGGAATATGC 
               
               
                   
                 CTTGTCTAATTACACAAATATCAAGGCCGTCCACGTT 
               
               
                   
                 AACCTTTCTCAACCGGCGCCCATTTGA 
               
               
                   
               
               
                 trpDH 
                 ATGCTGTTATTCGAGACTGTGCGTGAAATGGGTCATG 
               
               
                 SEQ ID NO: 264 
                 AGCAAGTCCTTTTCTGTCATAGCAAGAATCCCGAGAT 
               
               
                   
                 CAAGGCAATTATCGCAATCCACGATACCACCTTAGGA 
               
               
                   
                 CCGGCTATGGGCGCAACTCGTATCTTACCTTATATTA 
               
               
                   
                 ATGAGGAGGCTGCCCTGAAAGATGCATTACGTCTGTC 
               
               
                   
                 CCGCGGAATGACTTACAAAGCAGCCTGCGCCAATATT 
               
               
                   
                 CCCGCCGGGGGCGGCAAAGCCGTCATCATCGCTAAC 
               
               
                   
                 CCCGAAAACAAGACCGATGACCTGTTACGCGCATAC 
               
               
                   
                 GGCCGTTTCGTGGACAGCTTGAACGGCCGTTTCATCA 
               
               
                   
                 CCGGGCAGGACGTTAACATTACGCCCGACGACGTTC 
               
               
                   
                 GCACTATTTCGCAGGAGACTAAGTACGTGGTAGGCGT 
               
               
                   
                 CTCAGAAAAGTCGGGAGGGCCGGCACCTATCACCTC 
               
               
                   
                 TCTGGGAGTATTTTTAGGCATCAAAGCCGCTGTAGAG 
               
               
                   
                 TCGCGTTGGCAGTCTAAACGCCTGGATGGCATGAAA 
               
               
                   
                 GTGGCGGTGCAAGGACTTGGGAACGTAGGAAAAAAT 
               
               
                   
                 CTTTGTCGCCATCTGCATGAACACGATGTACAACTTT 
               
               
                   
                 TTGTGTCTGATGTCGATCCAATCAAGGCCGAGGAAGT 
               
               
                   
                 AAAACGCTTATTCGGGGCGACTGTTGTCGAACCGACT 
               
               
                   
                 GAAATCTATTCTTTAGATGTTGATATTTTTGCACCGTG 
               
               
                   
                 TGCACTTGGGGGTATTTTGAATAGCCATACCATCCCG 
               
               
                   
                 TTCTTACAAGCCTCAATCATCGCAGGAGCAGCGAATA 
               
               
                   
                 ACCAGCTGGAGAACGAGCAACTTCATTCGCAGATGC 
               
               
                   
                 TTGCGAAAAAGGGTATTCTTTACTCACCAGACTACGT 
               
               
                   
                 TATCAATGCAGGAGGACTTATCAATGTTTATAACGAA 
               
               
                   
                 ATGATCGGATATGACGAGGAAAAAGCATTCAAACAA 
               
               
                   
                 GTTCATAACATCTACGATACGTTATTAGCGATTTTCG 
               
               
                   
                 AAATTGCAAAAGAACAAGGTGTAACCACCAACGACG 
               
               
                   
                 CGGCCCGTCGTTTAGCAGAGGATCGTATCAACAACTC 
               
               
                   
                 CAAACGCTCAAAGAGTAAAGCGATTGCGGCGTGA 
               
               
                   
               
               
                 ipdC 
                 ATGCGTACACCCTACTGTGTCGCCGATTATCTTTTAG 
               
               
                 SEQ ID NO: 265 
                 ATCGTCTGACGGACTGCGGGGCCGATCACCTGTTTGG 
               
               
                   
                 CGTACCGGGCGATTACAACTTGCAGTTTCTGGACCAC 
               
               
                   
                 GTCATTGACTCACCAGATATCTGCTGGGTAGGGTGTG 
               
               
                   
                 CGAACGAGCTTAACGCGAGCTACGCTGCTGACGGAT 
               
               
                   
                 ATGCGCGTTGTAAAGGCTTTGCTGCACTTCTTACTAC 
               
               
                   
                 CTTCGGGGTCGGTGAGTTATCGGCGATGAACGGTATC 
               
               
                   
                 GCAGGCTCGTACGCTGAGCACGTCCCGGTATTACACA 
               
               
                   
                 TTGTGGGAGCTCCGGGTACCGCAGCTCAACAGCGCG 
               
               
                   
                 GAGAACTGTTACACCACACGCTGGGCGACGGAGAAT 
               
               
                   
                 TCCGCCACTTTTACCATATGTCCGAGCCAATTACTGT 
               
               
                   
                 AGCCCAGGCTGTACTTACAGAGCAAAATGCCTGTTAC 
               
               
                   
                 GAGATCGACCGTGTTTTGACCACGATGCTTCGCGAGC 
               
               
                   
                 GCCGTCCCGGGTATTTGATGCTGCCAGCCGATGTTGC 
               
               
                   
                 CAAAAAAGCTGCGACGCCCCCAGTGAATGCCCTGAC 
               
               
                   
                 GCATAAACAAGCTCATGCCGATTCCGCCTGTTTAAAG 
               
               
                   
                 GCTTTTCGCGATGCAGCTGAAAATAAATTAGCCATGT 
               
               
                   
                 CGAAACGCACCGCCTTGTTGGCGGACTTTCTGGTCCT 
               
               
                   
                 GCGCCATGGCCTTAAACACGCCCTTCAGAAATGGGTC 
               
               
                   
                 AAAGAAGTCCCGATGGCCCACGCTACGATGCTTATG 
               
               
                   
                 GGTAAGGGGATTTTTGATGAACGTCAAGCGGGATTTT 
               
               
                   
                 ATGGAACTTATTCCGGTTCGGCGAGTACGGGGGCGGT 
               
               
                   
                 AAAGGAAGCGATTGAGGGAGCCGACACAGTTCTTTG 
               
               
                   
                 CGTGGGGACACGTTTCACCGATACACTGACCGCTGGA 
               
               
                   
                 TTCACACACCAACTTACTCCGGCACAAACGATTGAGG 
               
               
                   
                 TGCAACCCCATGCGGCTCGCGTGGGGGATGTATGGTT 
               
               
                   
                 TACGGGCATTCCAATGAATCAAGCCATTGAGACTCTT 
               
               
                   
                 GTCGAGCTGTGCAAACAGCACGTCCACGCAGGACTG 
               
               
                   
                 ATGAGTTCGAGCTCTGGGGCGATTCCTTTTCCACAAC 
               
               
                   
                 CAGATGGTAGTTTAACTCAAGAAAACTTCTGGCGCAC 
               
               
                   
                 ATTGCAAACCTTTATCCGCCCAGGTGATATCATCTTA 
               
               
                   
                 GCAGACCAGGGTACTTCAGCCTTTGGAGCAATTGACC 
               
               
                   
                 TGCGCTTACCAGCAGACGTGAACTTTATTGTGCAGCC 
               
               
                   
                 GCTGTGGGGGTCTATTGGTTATACTTTAGCTGCGGCC 
               
               
                   
                 TTCGGAGCGCAGACAGCGTGTCCAAACCGTCGTGTG 
               
               
                   
                 ATCGTATTGACAGGAGATGGAGCAGCGCAGTTGACC 
               
               
                   
                 ATTCAGGAGTTAGGCTCGATGTTACGCGATAAGCAGC 
               
               
                   
                 ACCCCATTATCCTGGTCCTGAACAATGAGGGGTATAC 
               
               
                   
                 AGTTGAACGCGCCATTCATGGTGCGGAACAACGCTA 
               
               
                   
                 CAATGACATCGCTTTATGGAATTGGACGCACATCCCC 
               
               
                   
                 CAAGCCTTATCGTTAGATCCCCAATCGGAATGTTGGC 
               
               
                   
                 GTGTGTCTGAAGCAGAGCAACTGGCTGATGTTCTGGA 
               
               
                   
                 AAAAGTTGCTCATCATGAACGCCTGTCGTTGATCGAG 
               
               
                   
                 GTAATGTTGCCCAAGGCCGATATCCCTCCGTTACTGG 
               
               
                   
                 GAGCCTTGACCAAGGCTTTAGAAGCCTGCAACAACG 
               
               
                   
                 CTTAA 
               
               
                   
               
               
                 Iad1 
                 ATGCCCACCTTGAACTTGGACTTACCCAACGGTATTA 
               
               
                 SEQ ID NO: 266 
                 AGAGCACGATTCAGGCAGACCTTTTCATCAATAATAA 
               
               
                   
                 GTTTGTGCCGGCGCTTGATGGGAAAACGTTCGCAACT 
               
               
                   
                 ATTAATCCGTCTACGGGGAAAGAGATCGGACAGGTG 
               
               
                   
                 GCAGAGGCTTCGGCGAAGGATGTGGATCTTGCAGTT 
               
               
                   
                 AAGGCCGCGCGTGAGGCGTTTGAAACTACTTGGGGG 
               
               
                   
                 GAAAACACGCCAGGTGATGCTCGTGGCCGTTTACTGA 
               
               
                   
                 TTAAGCTTGCTGAGTTGGTGGAAGCGAATATTGATGA 
               
               
                   
                 GTTAGCGGCAATTGAATCACTGGACAATGGGAAAGC 
               
               
                   
                 GTTCTCTATTGCTAAGTCATTCGACGTAGCTGCTGTG 
               
               
                   
                 GCCGCAAACTTACGTTACTACGGCGGTTGGGCTGATA 
               
               
                   
                 AAAACCACGGTAAAGTCATGGAGGTAGACACAAAGC 
               
               
                   
                 GCCTGAACTATACCCGCCACGAGCCGATCGGGGTTTG 
               
               
                   
                 CGGACAAATCATTCCGTGGAATTTCCCGCTTTTGATG 
               
               
                   
                 TTTGCATGGAAGCTGGGTCCCGCTTTAGCCACAGGGA 
               
               
                   
                 ACACAATTGTGTTAAAGACTGCCGAGCAGACTCCCTT 
               
               
                   
                 AAGTGCTATCAAGATGTGTGAATTAATCGTAGAAGCC 
               
               
                   
                 GGCTTTCCGCCCGGAGTAGTTAATGTGATCTCGGGAT 
               
               
                   
                 TCGGACCGGTGGCGGGGGCCGCGATCTCGCAACACA 
               
               
                   
                 TGGACATCGATAAGATTGCCTTTACAGGATCGACATT 
               
               
                   
                 GGTTGGCCGCAACATTATGAAGGCAGCTGCGTCGACT 
               
               
                   
                 AACTTAAAAAAGGTTACACTTGAGTTAGGAGGAAAA 
               
               
                   
                 TCCCCGAATATCATTTTCAAAGATGCCGACCTTGACC 
               
               
                   
                 AAGCTGTTCGCTGGAGCGCCTTCGGTATCATGTTTAA 
               
               
                   
                 CCACGGACAATGCTGCTGCGCTGGATCGCGCGTATAT 
               
               
                   
                 GTGGAAGAATCCATCTATGACGCCTTCATGGAAAAA 
               
               
                   
                 ATGACTGCGCATTGTAAGGCGCTTCAAGTTGGAGATC 
               
               
                   
                 CTTTCAGCGCGAACACCTTCCAAGGACCACAAGTCTC 
               
               
                   
                 GCAGTTACAATACGACCGTATCATGGAATACATCGA 
               
               
                   
                 ATCAGGGAAAAAAGATGCAAATCTTGCTTTAGGCGG 
               
               
                   
                 CGTTCGCAAAGGGAATGAGGGGTATTTCATTGAGCC 
               
               
                   
                 AACTATTTTTACAGACGTGCCGCACGACGCGAAGATT 
               
               
                   
                 GCCAAAGAGGAGATCTTCGGTCCAGTGGTTGTTGTGT 
               
               
                   
                 CGAAATTTAAGGACGAAAAAGATCTGATCCGTATCG 
               
               
                   
                 CAAATGATTCTATTTATGGTTTAGCTGCGGCAGTCTTT 
               
               
                   
                 TCCCGCGACATCAGCCGCGCGATCGAGACAGCACAC 
               
               
                   
                 AAACTGAAAGCAGGCACGGTCTGGGTCAACTGCTAT 
               
               
                   
                 AATCAGCTTATTCCGCAGGTGCCATTCGGAGGGTATA 
               
               
                   
                 AGGCTTCCGGTATCGGCCGTGAGTTGGGGGAATATGC 
               
               
                   
                 CTTGTCTAATTACACAAATATCAAGGCCGTCCACGTT 
               
               
                   
                 AACCTTTCTCAACCGGCGCCCATTTGA 
               
               
                   
               
               
                 TrpEDCBA (RBS 
                 
                   Ctctagaaataattttgtttaactttaagaaggagatatacat 
                 
               
               
                 and leader region 
                 atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaa 
               
               
                 underlined) 
                 cccgactgcgctttttcaccagttgtgtggggatcgtccggcaacgctgctgctggaatc 
               
               
                 SEQ ID NO: 267 
                 cgcagatatcgacagcaaagatgatttaaaaagcctgctgctggtagacagtgcgctgc 
               
               
                   
                 gcattacagcattaagtgacactgtcacaatccaggcgctttccggcaatggagaagcc 
               
               
                   
                 ctgttgacactactggataacgccttgcctgcgggtgtggaaaatgaacaatcaccaaac 
               
               
                   
                 tgccgcgtactgcgcttcccgcctgtcagtccactgctggatgaagacgcccgcttatgc 
               
               
                   
                 tccctttcggtttttgacgctttccgcttattacagaatctgttgaatgtaccgaaggaagaa 
               
               
                   
                 cgagaagcaatgttcttcggcggcctgttctcttatgaccttgtggcgggatttgaaaattt 
               
               
                   
                 accgcaactgtcagcggaaaatagctgccctgatttctgtttttatctcgctgaaacgctga 
               
               
                   
                 tggtgattgaccatcagaaaaaaagcactcgtattcaggccagcctgtttgctccgaatg 
               
               
                   
                 aagaagaaaaacaacgtctcactgctcgcctgaacgaactacgtcagcaactgaccga 
               
               
                   
                 agccgcgccgccgctgccggtggtttccgtgccgcatatgcgttgtgaatgtaaccaga 
               
               
                   
                 gcgatgaagagttcggtggtgtagtgcgtttgttgcaaaaagcgattcgcgccggagaa 
               
               
                   
                 attttccaggtggtgccatctcgccgtttctctctgccctgcccgtcaccgctggcagccta 
               
               
                   
                 ttacgtgctgaaaaagagtaatcccagcccgtacatgttttttatgcaggataatgatttcac 
               
               
                   
                 cctgtttggcgcgtcgccggaaagttcgctcaagtatgacgccaccagccgccagattg 
               
               
                   
                 agatttacccgattgccggaacacgtccacgcggtcgtcgtgccgatggttcgctggac 
               
               
                   
                 agagacctcgacagccgcatcgaactggagatgcgtaccgatcataaagagctttctga 
               
               
                   
                 acatctgatgctggtggatctcgcccgtaatgacctggcacgcatttgcacacccggca 
               
               
                   
                 gccgctacgtcgccgatctcaccaaagttgaccgttactcttacgtgatgcacctagtctc 
               
               
                   
                 ccgcgttgttggtgagctgcgccacgatctcgacgccctgcacgcttaccgcgcctgtat 
               
               
                   
                 gaatatggggacgttaagcggtgcaccgaaagtacgcgctatgcagttaattgccgaag 
               
               
                   
                 cagaaggtcgtcgacgcggcagctacggcggcgcggtaggttattttaccgcgcatgg 
               
               
                   
                 cgatctcgacacctgcattgtgatccgctcggcgctggtggaaaacggtatcgccaccg 
               
               
                   
                 tgcaagccggtgctggcgtagtccttgattctgttccgcagtcggaagccgacgaaactc 
               
               
                   
                 gtaataaagcccgcgctgtactgcgcgctattgccaccgcgcatcatgcacaggagac 
               
               
                   
                 gttctaatggctgacattctgctgctcgataatatcgactcttttacgtacaacctggcagat 
               
               
                   
                 cagttgcgcagcaatggtcataacgtggtgatttaccgcaaccatattccggcgcagacc 
               
               
                   
                 ttaattgaacgcctggcgacgatgagcaatccggtgctgatgctttctcctggccccggt 
               
               
                   
                 gtgccgagcgaagccggttgtatgccggaactcctcacccgcttgcgtggcaagctgc 
               
               
                   
                 caattattggcatttgcctcggacatcaggcgattgtcgaagcttacgggggctatgtcgg 
               
               
                   
                 tcaggcgggcgaaattcttcacggtaaagcgtcgagcattgaacatgacggtcaggcg 
               
               
                   
                 atgtttgccggattaacaaacccgctgccagtggcgcgttatcactcgctggttggcagt 
               
               
                   
                 aacattccggccggtttaaccatcaacgcccattttaatggcatggtgatggcggtgcgtc 
               
               
                   
                 acgatgcagatcgcgtttgtggattccagttccatccggaatccattcttactacccaggg 
               
               
                   
                 cgctcgcctgctggaacaaacgctggcctgggcgcagcagaaactagagccaaccaa 
               
               
                   
                 cacgctgcaaccgattctggaaaaactgtatcaggcacagacgcttagccaacaagaa 
               
               
                   
                 agccaccagctgttttcagcggtggtacgtggcgagctgaagccggaacaactggcgg 
               
               
                   
                 cggcgctggtgagcatgaaaattcgcggtgaacacccgaacgagatcgccggggcag 
               
               
                   
                 caaccgcgctactggaaaacgccgcgccattcccgcgcccggattatctgtttgccgat 
               
               
                   
                 atcgtcggtactggcggtgacggcagcaacagcatcaatatttctaccgccagtgcgttt 
               
               
                   
                 gtcgccgcggcctgcgggctgaaagtggcgaaacacggcaaccgtagcgtctccagt 
               
               
                   
                 aaatccggctcgtcggatctgctggcggcgttcggtattaatcttgatatgaacgccgata 
               
               
                   
                 aatcgcgccaggcgctggatgagttaggcgtctgtttcctctttgcgccgaagtatcaca 
               
               
                   
                 ccggattccgccatgcgatgccggttcgccagcaactgaaaacccgcactctgttcaac 
               
               
                   
                 gtgctgggaccattgattaacccggcgcatccgccgctggcgctaattggtgtttatagtc 
               
               
                   
                 cggaactggtgctgccgattgccgaaaccttgcgcgtgctggggtatcaacgcgcggc 
               
               
                   
                 agtggtgcacagcggcgggatggatgaagtttcattacacgcgccgacaatcgttgccg 
               
               
                   
                 aactacatgacggcgaaattaagagctatcaattgaccgctgaagattttggcctgacac 
               
               
                   
                 cctaccaccaggagcaattggcaggcggaacaccggaagaaaaccgtgacattttaac 
               
               
                   
                 acgcttgttacaaggtaaaggcgacgccgcccatgaagcagccgtcgcggcgaatgtc 
               
               
                   
                 gccatgttaatgcgcctgcatggccatgaagatctgcaagccaatgcgcaaaccgttctt 
               
               
                   
                 gaggtactgcgcagtggttccgcttacgacagagtcaccgcactggcggcacgagggt 
               
               
                   
                 aaatgatgcaaaccgttttagcgaaaatcgtcgcagacaaggcgatttgggtagaaacc 
               
               
                   
                 cgcaaagagcagcaaccgctggccagttttcagaatgaggttcagccgagcacgcga 
               
               
                   
                 catttttatgatgcacttcagggcgcacgcacggcgtttattctggagtgtaaaaaagcgt 
               
               
                   
                 cgccgtcaaaaggcgtgatccgtgatgatttcgatccggcacgcattgccgccatttata 
               
               
                   
                 aacattacgcttcggcaatttcagtgctgactgatgagaaatattttcaggggagctttgatt 
               
               
                   
                 tcctccccatcgtcagccaaatcgccccgcagccgattttatgtaaagacttcattatcgat 
               
               
                   
                 ccttaccagatctatctggcgcgctattaccaggccgatgcctgcttattaatgctttcagta 
               
               
                   
                 ctggatgacgaacaatatcgccagcttgcagccgtcgcccacagtctggagatgggtgt 
               
               
                   
                 gctgaccgaagtcagtaatgaagaggaactggagcgcgccattgcattgggggcaaa 
               
               
                   
                 ggtcgttggcatcaacaaccgcgatctgcgcgatttgtcgattgatctcaaccgtacccg 
               
               
                   
                 cgagcttgcgccgaaactggggcacaacgtgacggtaatcagcgaatccggcatcaat 
               
               
                   
                 acttacgctcaggtgcgcgagttaagccacttcgctaacggctttctgattggttcggcgtt 
               
               
                   
                 gatggcccatgacgatttgaacgccgccgtgcgtcgggtgttgctgggtgagaataaag 
               
               
                   
                 tatgtggcctgacacgtgggcaagatgctaaagcagcttatgacgcgggcgcgatttac 
               
               
                   
                 ggtgggttgatttttgttgcgacatcaccgcgttgcgtcaacgttgaacaggcgcaggaa 
               
               
                   
                 gtgatggctgcagcaccgttgcagtatgttggcgtgttccgcaatcacgatattgccgatg 
               
               
                   
                 tggcggacaaagctaaggtgttatcgctggcggcagtgcaactgcatggtaatgaagat 
               
               
                   
                 cagctgtatatcgacaatctgcgtgaggctctgccagcacacgtcgccatctggaaggc 
               
               
                   
                 tttaagtgtcggtgaaactcttcccgcgcgcgattttcagcacatcgataaatatgtattcg 
               
               
                   
                 acaacggtcagggcgggagcggacaacgtttcgactggtcactattaaatggtcaatcg 
               
               
                   
                 cttggcaacgttctgctggcggggggcttaggcgcagataactgcgtggaagcggcac 
               
               
                   
                 aaaccggctgcgccgggcttgattttaattctgctgtagagtcgcaaccgggtatcaaag 
               
               
                   
                 acgcacgtcttttggcctcggttttccagacgctgcgcgcatattaaggaaaggaacaat 
               
               
                   
                 gacaacattacttaacccctattttggtgagtttggcggcatgtacgtgccacaaatcctga 
               
               
                   
                 tgcctgctctgcgccagctggaagaagcttttgtcagcgcgcaaaaagatcctgaatttc 
               
               
                   
                 aggctcagttcaacgacctgctgaaaaactatgccgggcgtccaaccgcgctgaccaa 
               
               
                   
                 atgccagaacattacagccgggacgaacaccacgctgtatctgaagcgcgaagatttg 
               
               
                   
                 ctgcacggcggcgcgcataaaactaaccaggtgctcggtcaggctttactggcgaagc 
               
               
                   
                 ggatgggtaaaactgaaattattgccgaaaccggtgccggtcagcatggcgtggcgtc 
               
               
                   
                 ggcccttgccagcgccctgctcggcctgaaatgccgaatttatatgggtgccaaagacg 
               
               
                   
                 ttgaacgccagtcgcccaacgttttccggatgcgcttaatgggtgcggaagtgatcccg 
               
               
                   
                 gtacatagcggttccgcgaccctgaaagatgcctgtaatgaggcgctacgcgactggtc 
               
               
                   
                 cggcagttatgaaaccgcgcactatatgctgggtaccgcagctggcccgcatccttacc 
               
               
                   
                 cgaccattgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgcagattctg 
               
               
                   
                 gaaagagaaggtcgcctgccggatgccgttatcgcctgtgttggcggtggttcgaatgc 
               
               
                   
                 catcggtatgtttgcagatttcatcaacgaaaccgacgtcggcctgattggtgtggagcct 
               
               
                   
                 ggcggccacggtatcgaaactggcgagcacggcgcaccgttaaaacatggtcgcgtg 
               
               
                   
                 ggcatctatttcggtatgaaagcgccgatgatgcaaaccgaagacgggcaaattgaaga 
               
               
                   
                 gtcttactccatttctgccgggctggatttcccgtccgtcggcccgcaacatgcgtatctca 
               
               
                   
                 acagcactggacgcgctgattacgtgtctattaccgacgatgaagccctggaagccttta 
               
               
                   
                 aaacgctttgcctgcatgaagggatcatcccggcgctggaatcctcccacgccctggcc 
               
               
                   
                 catgcgctgaaaatgatgcgcgaaaatccggaaaaagagcagctactggtggttaacct 
               
               
                   
                 ttccggtcgcggcgataaagacatcttcaccgttcacgatattttgaaagcacgagggga 
               
               
                   
                 aatctgatggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgc 
               
               
                   
                 attcgttccttcgtcaccctcggtgatccgggcattgagcagtcgttgaaaattatcgata 
               
               
                   
                 cgctaattgaagccggtgctgacgcgctggagttaggcatccccttctccgacccactg 
               
               
                   
                 gcggatggcccgacgattcaaaacgccacactgcgtgcttttgcggcgggagtaaccc 
               
               
                   
                 cggcgcagtgctttgagatgctggcactcattcgccagaagcacccgaccattcccatc 
               
               
                   
                 ggccttttgatgtatgccaacctggtgtttaacaaaggcattgatgagttttatgccgagtg 
               
               
                   
                 cgagaaagtcggcgtcgattcggtgctggttgccgatgtgcccgtggaagagtccgcg 
               
               
                   
                 cccttccgccaggccgcgttgcgtcataatgtcgcacctatctttatttgcccgccgaatg 
               
               
                   
                 ccgacgatgatttgctgcgccagatagcctcttacggtcgtggttacacctatttgctgtcg 
               
               
                   
                 cgagcgggcgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcatctggttg 
               
               
                   
                 cgaagctgaaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccg 
               
               
                   
                 gatcaggtaaaagccgcgattgatgcaggagctgcgggcgcgatttctggttcggccat 
               
               
                   
                 cgttaaaatcatcgagcaacatattaatgagccagagaaaatgctggcggcactgaaag 
               
               
                   
                 cttttgtacaaccgatgaaagcggcgacgcgcagtta 
               
               
                   
               
               
                 fbrS40FTrpE- 
                   ctctagaaataattttgtttaactttaagaaggagatatacat atgcaaacacaaaaaccga 
               
               
                 DCBA (leader 
                 ctctcgaactgctaacctgcgaaggcgcttatcgcgacaacccgactgcgctttttcacc 
               
               
                 region and RBS 
                 agttgtgtggggatcgtccggcaacgctgctgctggaattcgcagatatcgacagcaaa 
               
               
                 underlined) 
                 gatgatttaaaaagcctgctgctggtagacagtgcgctgcgcattacagcattaagtgac 
               
               
                 SEQ ID NO: 273 
                 actgtcacaatccaggcgctttccggcaatggagaagccctgttgacactactggataac 
               
               
                   
                 gccttgcctgcgggtgtggaaaatgaacaatcaccaaactgccgcgtactgcgcttccc 
               
               
                   
                 gcctgtcagtccactgctggatgaagacgcccgcttatgctccctttcggtttttgacgcttt 
               
               
                   
                 ccgcttattacagaatctgttgaatgtaccgaaggaagaacgagaagcaatgttcttcgg 
               
               
                   
                 cggcctgttctcttatgaccttgtggcgggatttgaaaatttaccgcaactgtcagcggaa 
               
               
                   
                 aatagctgccctgatttctgtttttatctcgctgaaacgctgatggtgattgaccatcagaaa 
               
               
                   
                 aaaagcactcgtattcaggccagcctgtttgctccgaatgaagaagaaaaacaacgtctc 
               
               
                   
                 actgctcgcctgaacgaactacgtcagcaactgaccgaagccgcgccgccgctgccg 
               
               
                   
                 gtggtttccgtgccgcatatgcgttgtgaatgtaaccagagcgatgaagagttcggtggt 
               
               
                   
                 gtagtgcgtttgttgcaaaaagcgattcgcgccggagaaattttccaggtggtgccatctc 
               
               
                   
                 gccgtttctctctgccctgcccgtcaccgctggcagcctattacgtgctgaaaaagagta 
               
               
                   
                 atcccagcccgtacatgttttttatgcaggataatgatttcaccctgtttggcgcgtcgccg 
               
               
                   
                 gaaagttcgctcaagtatgacgccaccagccgccagattgagatttacccgattgccgg 
               
               
                   
                 aacacgtccacgcggtcgtcgtgccgatggttcgctggacagagacctcgacagccgc 
               
               
                   
                 atcgaactggagatgcgtaccgatcataaagagctttctgaacatctgatgctggtggatc 
               
               
                   
                 tcgcccgtaatgacctggcacgcatttgcacacccggcagccgctacgtcgccgatctc 
               
               
                   
                 accaaagttgaccgttactcttacgtgatgcacctagtctcccgcgttgttggtgagctgc 
               
               
                   
                 gccacgatctcgacgccctgcacgcttaccgcgcctgtatgaatatggggacgttaagc 
               
               
                   
                 ggtgcaccgaaagtacgcgctatgcagttaattgccgaagcagaaggtcgtcgacgcg 
               
               
                   
                 gcagctacggcggcgcggtaggttattttaccgcgcatggcgatctcgacacctgcatt 
               
               
                   
                 gtgatccgctcggcgctggtggaaaacggtatcgccaccgtgcaagccggtgctggcg 
               
               
                   
                 tagtccttgattctgttccgcagtcggaagccgacgaaactcgtaataaagcccgcgctg 
               
               
                   
                 tactgcgcgctattgccaccgcgcatcatgcacaggagacgttctaatggctgacattct 
               
               
                   
                 gctgctcgataatatcgactcttttacgtacaacctggcagatcagttgcgcagcaatggt 
               
               
                   
                 cataacgtggtgatttaccgcaaccatattccggcgcagaccttaattgaacgcctggcg 
               
               
                   
                 acgatgagcaatccggtgctgatgctttctcctggccccggtgtgccgagcgaagccgg 
               
               
                   
                 ttgtatgccggaactcctcacccgcttgcgtggcaagctgccaattattggcatttgcctc 
               
               
                   
                 ggacatcaggcgattgtcgaagcttacgggggctatgtcggtcaggcgggcgaaattct 
               
               
                   
                 tcacggtaaagcgtcgagcattgaacatgacggtcaggcgatgtttgccggattaacaa 
               
               
                   
                 acccgctgccagtggcgcgttatcactcgctggttggcagtaacattccggccggtttaa 
               
               
                   
                 ccatcaacgcccattttaatggcatggtgatggcggtgcgtcacgatgcagatcgcgttt 
               
               
                   
                 gtggattccagttccatccggaatccattcttactacccagggcgctcgcctgctggaac 
               
               
                   
                 aaacgctggcctgggcgcagcagaaactagagccaaccaacacgctgcaaccgattc 
               
               
                   
                 tggaaaaactgtatcaggcacagacgcttagccaacaagaaagccaccagctgttttca 
               
               
                   
                 gcggtggtacgtggcgagctgaagccggaacaactggcggcggcgctggtgagcat 
               
               
                   
                 gaaaattcgcggtgaacacccgaacgagatcgccggggcagcaaccgcgctactgga 
               
               
                   
                 aaacgccgcgccattcccgcgcccggattatctgtttgccgatatcgtcggtactggcgg 
               
               
                   
                 tgacggcagcaacagcatcaatatttctaccgccagtgcgtttgtcgccgcggcctgcg 
               
               
                   
                 ggctgaaagtggcgaaacacggcaaccgtagcgtctccagtaaatccggctcgtcgga 
               
               
                   
                 tctgctggcggcgttcggtattaatcttgatatgaacgccgataaatcgcgccaggcgct 
               
               
                   
                 ggatgagttaggcgtctgtttcctctttgcgccgaagtatcacaccggattccgccatgcg 
               
               
                   
                 atgccggttcgccagcaactgaaaacccgcactctgttcaacgtgctgggaccattgatt 
               
               
                   
                 aacccggcgcatccgccgctggcgctaattggtgtttatagtccggaactggtgctgcc 
               
               
                   
                 gattgccgaaaccttgcgcgtgctggggtatcaacgcgcggcagtggtgcacagcggc 
               
               
                   
                 gggatggatgaagtttcattacacgcgccgacaatcgttgccgaactacatgacggcga 
               
               
                   
                 aattaagagctatcaattgaccgctgaagattttggcctgacaccctaccaccaggagca 
               
               
                   
                 attggcaggcggaacaccggaagaaaaccgtgacattttaacacgcttgttacaaggta 
               
               
                   
                 aaggcgacgccgcccatgaagcagccgtcgcggcgaatgtcgccatgttaatgcgcct 
               
               
                   
                 gcatggccatgaagatctgcaagccaatgcgcaaaccgttcttgaggtactgcgcagtg 
               
               
                   
                 gttccgcttacgacagagtcaccgcactggcggcacgagggtaaatgatgcaaaccgtt 
               
               
                   
                 ttagcgaaaatcgtcgcagacaaggcgatttgggtagaaacccgcaaagagcagcaac 
               
               
                   
                 cgctggccagttttcagaatgaggttcagccgagcacgcgacatttttatgatgcacttca 
               
               
                   
                 gggcgcacgcacggcgtttattctggagtgtaaaaaagcgtcgccgtcaaaaggcgtg 
               
               
                   
                 atccgtgatgatttcgatccggcacgcattgccgccatttataaacattacgcttcggcaat 
               
               
                   
                 ttcagtgctgactgatgagaaatattttcaggggagctttgatttcctccccatcgtcagcc 
               
               
                   
                 aaatcgccccgcagccgattttatgtaaagacttcattatcgatccttaccagatctatctg 
               
               
                   
                 gcgcgctattaccaggccgatgcctgcttattaatgctttcagtactggatgacgaacaat 
               
               
                   
                 atcgccagcttgcagccgtcgcccacagtctggagatgggtgtgctgaccgaagtcagt 
               
               
                   
                 aatgaagaggaactggagcgcgccattgcattgggggcaaaggtcgttggcatcaaca 
               
               
                   
                 accgcgatctgcgcgatttgtcgattgatctcaaccgtacccgcgagcttgcgccgaaac 
               
               
                   
                 tggggcacaacgtgacggtaatcagcgaatccggcatcaatacttacgctcaggtgcgc 
               
               
                   
                 gagttaagccacttcgctaacggctttctgattggttcggcgttgatggcccatgacgattt 
               
               
                   
                 gaacgccgccgtgcgtcgggtgttgctgggtgagaataaagtatgtggcctgacacgtg 
               
               
                   
                 ggcaagatgctaaagcagcttatgacgcgggcgcgatttacggtgggttgatttttgttgc 
               
               
                   
                 gacatcaccgcgttgcgtcaacgttgaacaggcgcaggaagtgatggctgcagcaccg 
               
               
                   
                 ttgcagtatgttggcgtgttccgcaatcacgatattgccgatgtggcggacaaagctaag 
               
               
                   
                 gtgttatcgctggcggcagtgcaactgcatggtaatgaagatcagctgtatatcgacaat 
               
               
                   
                 ctgcgtgaggctctgccagcacacgtcgccatctggaaggctttaagtgtcggtgaaact 
               
               
                   
                 cttcccgcgcgcgattttcagcacatcgataaatatgtattcgacaacggtcagggcggg 
               
               
                   
                 agcggacaacgtttcgactggtcactattaaatggtcaatcgcttggcaacgttctgctgg 
               
               
                   
                 cggggggcttaggcgcagataactgcgtggaagcggcacaaaccggctgcgccggg 
               
               
                   
                 cttgattttaattctgctgtagagtcgcaaccgggtatcaaagacgcacgtcttttggcctc 
               
               
                   
                 ggttttccagacgctgcgcgcatattaaggaaaggaacaatgacaacattacttaacccc 
               
               
                   
                 tattttggtgagtttggcggcatgtacgtgccacaaatcctgatgcctgctctgcgccagct 
               
               
                   
                 ggaagaagcttttgtcagcgcgcaaaaagatcctgaatttcaggctcagttcaacgacct 
               
               
                   
                 gctgaaaaactatgccgggcgtccaaccgcgctgaccaaatgccagaacattacagcc 
               
               
                   
                 gggacgaacaccacgctgtatctgaagcgcgaagatttgctgcacggcggcgcgcata 
               
               
                   
                 aaactaaccaggtgctcggtcaggctttactggcgaagcggatgggtaaaactgaaatt 
               
               
                   
                 attgccgaaaccggtgccggtcagcatggcgtggcgtcggcccttgccagcgccctgc 
               
               
                   
                 tcggcctgaaatgccgaatttatatgggtgccaaagacgttgaacgccagtcgcccaac 
               
               
                   
                 gttttccggatgcgcttaatgggtgcggaagtgatcccggtacatagcggttccgcgacc 
               
               
                   
                 ctgaaagatgcctgtaatgaggcgctacgcgactggtccggcagttatgaaaccgcgc 
               
               
                   
                 actatatgctgggtaccgcagctggcccgcatccttacccgaccattgtgcgtgagtttca 
               
               
                   
                 gcggatgattggcgaagaaacgaaagcgcagattctggaaagagaaggtcgcctgcc 
               
               
                   
                 ggatgccgttatcgcctgtgttggcggtggttcgaatgccatcggtatgtttgcagatttca 
               
               
                   
                 tcaacgaaaccgacgtcggcctgattggtgtggagcctggcggccacggtatcgaaac 
               
               
                   
                 tggcgagcacggcgcaccgttaaaacatggtcgcgtgggcatctatttcggtatgaaag 
               
               
                   
                 cgccgatgatgcaaaccgaagacgggcaaattgaagagtcttactccatttctgccggg 
               
               
                   
                 ctggatttcccgtccgtcggcccgcaacatgcgtatctcaacagcactggacgcgctgat 
               
               
                   
                 tacgtgtctattaccgacgatgaagccctggaagcctttaaaacgctttgcctgcatgaag 
               
               
                   
                 ggatcatcccggcgctggaatcctcccacgccctggcccatgcgctgaaaatgatgcg 
               
               
                   
                 cgaaaatccggaaaaagagcagctactggtggttaacctttccggtcgcggcgataaag 
               
               
                   
                 acatcttcaccgttcacgatattttgaaagcacgaggggaaatctgatggaacgctacga 
               
               
                   
                 atctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgttcctttcgtcaccctc 
               
               
                   
                 ggtgatccgggcattgagcagtcgttgaaaattatcgatacgctaattgaagccggtgct 
               
               
                   
                 gacgcgctggagttaggcatccccttctccgacccactggcggatggcccgacgattca 
               
               
                   
                 aaacgccacactgcgtgcttttgcggcgggagtaaccccggcgcagtgctttgagatgc 
               
               
                   
                 tggcactcattcgccagaagcacccgaccattcccatcggccttttgatgtatgccaacct 
               
               
                   
                 ggtgtttaacaaaggcattgatgagttttatgccgagtgcgagaaagtcggcgtcgattc 
               
               
                   
                 ggtgctggttgccgatgtgcccgtggaagagtccgcgcccttccgccaggccgcgttg 
               
               
                   
                 cgtcataatgtcgcacctatctttatttgcccgccgaatgccgacgatgatttgctgcgcca 
               
               
                   
                 gatagcctcttacggtcgtggttacacctatttgctgtcgcgagcgggcgtgaccggcgc 
               
               
                   
                 agaaaaccgcgccgcgttacccctcaatcatctggttgcgaagctgaaagagtacaacg 
               
               
                   
                 ctgcgcctccattgcagggatttggtatttccgccccggatcaggtaaaagccgcgattg 
               
               
                   
                 atgcaggagctgcgggcgcgatttctggttcggccatcgttaaaatcatcgagcaacata 
               
               
                   
                 ttaatgagccagagaaaatgctggcggcactgaaagcttttgtacaaccgatgaaagcg 
               
               
                   
                 gcgacgcgcagttaa 
               
               
                   
               
               
                 fbrTrpE 
                 atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaa 
               
               
                 SEQ ID NO: 274 
                 cccgactgcgctttttcaccagttgtgtggggatcgtccggcaacgctgctgctggaattc 
               
               
                   
                 gcagatatcgacagcaaagatgatttaaaaagcctgctgctggtagacagtgcgctgcg 
               
               
                   
                 cattacagcattaagtgacactgtcacaatccaggcgctttccggcaatggagaagccct 
               
               
                   
                 gttgacactactggataacgccttgcctgcgggtgtggaaaatgaacaatcaccaaactg 
               
               
                   
                 ccgcgtactgcgcttcccgcctgtcagtccactgctggatgaagacgcccgcttatgctc 
               
               
                   
                 cctttcggtttttgacgctttccgcttattacagaatctgttgaatgtaccgaaggaagaacg 
               
               
                   
                 agaagcaatgttcttcggcggcctgttctcttatgaccttgtggcgggatttgaaaatttac 
               
               
                   
                 cgcaactgtcagcggaaaatagctgccctgatttctgtttttatctcgctgaaacgctgatg 
               
               
                   
                 gtgattgaccatcagaaaaaaagcactcgtattcaggccagcctgtttgctccgaatgaa 
               
               
                   
                 gaagaaaaacaacgtctcactgctcgcctgaacgaactacgtcagcaactgaccgaag 
               
               
                   
                 ccgcgccgccgctgccggtggtttccgtgccgcatatgcgttgtgaatgtaaccagagc 
               
               
                   
                 gatgaagagttcggtggtgtagtgcgtttgttgcaaaaagcgattcgcgccggagaaatt 
               
               
                   
                 ttccaggtggtgccatctcgccgtttctctctgccctgcccgtcaccgctggcagcctatta 
               
               
                   
                 cgtgctgaaaaagagtaatcccagcccgtacatgttttttatgcaggataatgatttcaccc 
               
               
                   
                 tgtttggcgcgtcgccggaaagttcgctcaagtatgacgccaccagccgccagattgag 
               
               
                   
                 atttacccgattgccggaacacgtccacgcggtcgtcgtgccgatggttcgctggacag 
               
               
                   
                 agacctcgacagccgcatcgaactggagatgcgtaccgatcataaagagctttctgaac 
               
               
                   
                 atctgatgctggtggatctcgcccgtaatgacctggcacgcatttgcacacccggcagc 
               
               
                   
                 cgctacgtcgccgatctcaccaaagttgaccgttactcttacgtgatgcacctagtctccc 
               
               
                   
                 gcgttgttggtgagctgcgccacgatctcgacgccctgcacgcttaccgcgcctgtatga 
               
               
                   
                 atatggggacgttaagcggtgcaccgaaagtacgcgctatgcagttaattgccgaagca 
               
               
                   
                 gaaggtcgtcgacgcggcagctacggcggcgcggtaggttattttaccgcgcatggcg 
               
               
                   
                 atctcgacacctgcattgtgatccgctcggcgctggtggaaaacggtatcgccaccgtg 
               
               
                   
                 caagccggtgcggcgtagtccttgattctgttccgcagtcggaagccgacgaaactcgt 
               
               
                   
                 aataaagcccgcgctgtactgcgcgctattgccaccgcgcatcatgcacaggagacgtt 
               
               
                   
                 cta 
               
               
                   
               
               
                 trpDH- 
                   ctctagaaataattttgtttaactttaagaaggagatatacat atgaattatcagaacgacga 
               
               
                 fldABCDaculfldH 
                 tttacgcatcaaagaaatcaaagagttacttcctcctgtcgcattgctggaaaaattccccg 
               
               
                 (leader region and 
                 ctactgaaaatgccgcgaatacggtcgcccatgcccgaaaagcgatccataagatcctg 
               
               
                 RBS underlined) 
                 aaaggtaatgatgatcgcctgttggtggtgattggcccatgctcaattcatgatcctgtcgc 
               
               
                 SEQ ID NO: 275 
                 ggctaaagagtatgccactcgcttgctgacgctgcgtgaagagctgcaagatgagctgg 
               
               
                   
                 aaatcgtgatgcgcgtctattttgaaaagccgcgtactacggtgggctggaaagggctg 
               
               
                   
                 attaacgatccgcatatggataacagcttccagatcaacgacggtctgcgtattgcccgc 
               
               
                   
                 aaattgctgctcgatattaacgacagcggtctgccagcggcgggtgaattcctggatatg 
               
               
                   
                 atcaccctacaatatctcgctgacctgatgagctggggcgcaattggcgcacgtaccac 
               
               
                   
                 cgaatcgcaggtgcaccgcgaactggcgtctggtctttcttgtccggtaggtttcaaaaat 
               
               
                   
                 ggcactgatggtacgattaaagtggctatcgatgccattaatgccgccggtgcgccgca 
               
               
                   
                 ctgcttcctgtccgtaacgaaatgggggcattcggcgattgtgaataccagcggtaacg 
               
               
                   
                 gcgattgccatatcattctgcgcggcggtaaagagcctaactacagcgcgaagcacgtt 
               
               
                   
                 gctgaagtgaaagaagggctgaacaaagcaggcctgccagcgcaggtgatgatcgat 
               
               
                   
                 ttcagccatgctaactcgtcaaaacaattcaaaaagcagatggatgtttgtactgacgtttg 
               
               
                   
                 ccagcagattgccggtggcgaaaaggccattattggcgtgatggtggaaagccatctg 
               
               
                   
                 gtggaaggcaatcagagcctcgagagcggggaaccgctggcctacggtaagagcatc 
               
               
                   
                 accgatgcctgcattggctgggatgataccgatgctctgttacgtcaactggcgagtgca 
               
               
                   
                 gtaaaagcgcgtcgcgggtaaTACT taagaaggagatatacat ATGCTGTT 
               
               
                   
                 ATTCGAGACTGTGCGTGAAATGGGTCATGAGCAAGT 
               
               
                   
                 CCTTTTCTGTCATAGCAAGAATCCCGAGATCAAGGCA 
               
               
                   
                 ATTATCGCAATCCACGATACCACCTTAGGACCGGCTA 
               
               
                   
                 TGGGCGCAACTCGTATCTTACCTTATATTAATGAGGA 
               
               
                   
                 GGCTGCCCTGAAAGATGCATTACGTCTGTCCCGCGGA 
               
               
                   
                 ATGACTTACAAAGCAGCCTGCGCCAATATTCCCGCCG 
               
               
                   
                 GGGGCGGCAAAGCCGTCATCATCGCTAACCCCGAAA 
               
               
                   
                 ACAAGACCGATGACCTGTTACGCGCATACGGCCGTTT 
               
               
                   
                 CGTGGACAGCTTGAACGGCCGTTTCATCACCGGGCAG 
               
               
                   
                 GACGTTAACATTACGCCCGACGACGTTCGCACTATTT 
               
               
                   
                 CGCAGGAGACTAAGTACGTGGTAGGCGTCTCAGAAA 
               
               
                   
                 AGTCGGGAGGGCCGGCACCTATCACCTCTCTGGGAGT 
               
               
                   
                 ATTTTTAGGCATCAAAGCCGCTGTAGAGTCGCGTTGG 
               
               
                   
                 CAGTCTAAACGCCTGGATGGCATGAAAGTGGCGGTG 
               
               
                   
                 CAAGGACTTGGGAACGTAGGAAAAAATCTTTGTCGC 
               
               
                   
                 CATCTGCATGAACACGATGTACAACTTTTTGTGTCTG 
               
               
                   
                 ATGTCGATCCAATCAAGGCCGAGGAAGTAAAACGCT 
               
               
                   
                 TATTCGGGGCGACTGTTGTCGAACCGACTGAAATCTA 
               
               
                   
                 TTCTTTAGATGTTGATATTTTTGCACCGTGTGCACTTG 
               
               
                   
                 GGGGTATTTTGAATAGCCATACCATCCCGTTCTTACA 
               
               
                   
                 AGCCTCAATCATCGCAGGAGCAGCGAATAACCAGCT 
               
               
                   
                 GGAGAACGAGCAACTTCATTCGCAGATGCTTGCGAA 
               
               
                   
                 AAAGGGTATTCTTTACTCACCAGACTACGTTATCAAT 
               
               
                   
                 GCAGGAGGACTTATCAATGTTTATAACGAAATGATCG 
               
               
                   
                 GATATGACGAGGAAAAAGCATTCAAACAAGTTCATA 
               
               
                   
                 ACATCTACGATACGTTATTAGCGATTTTCGAAATTGC 
               
               
                   
                 AAAAGAACAAGGTGTAACCACCAACGACGCGGCCCG 
               
               
                   
                 TCGTTTAGCAGAGGATCGTATCAACAACTCCAAACGC 
               
               
                   
                 TCAAAGAGTAAAGCGATTGCGGCGTGAAATGtaagaagg 
               
               
                   
                 agatatacatATGGAAAACAACACCAATATGTTCTCTGGAG 
               
               
                   
                 TGAAGGTGATCGAACTGGCCAACTTTATCGCTGCTCC 
               
               
                   
                 GGCGGCAGGTCGCTTCTTTGCTGATGGGGGAGCAGA 
               
               
                   
                 AGTAATTAAGATCGAATCTCCAGCAGGCGACCCGCT 
               
               
                   
                 GCGCTACACGGCCCCATCAGAAGGACGCCCGCTTTCT 
               
               
                   
                 CAAGAGGAAAACACAACGTATGATTTGGAAAACGCG 
               
               
                   
                 AATAAGAAAGCAATTGTTCTGAACTTAAAATCGGAA 
               
               
                   
                 AAAGGAAAGAAAATTCTTCACGAGATGCTTGCTGAG 
               
               
                   
                 GCAGACATCTTGTTAACAAATTGGCGCACGAAAGCG 
               
               
                   
                 TTAGTCAAACAGGGGTTAGATTACGAAACACTGAAA 
               
               
                   
                 GAGAAGTATCCAAAATTGGTATTTGCACAGATTACAG 
               
               
                   
                 GATACGGGGAGAAAGGACCCGACAAAGACCTGCCTG 
               
               
                   
                 GTTTCGACTACACGGCGTTTTTCGCCCGCGGAGGAGT 
               
               
                   
                 CTCCGGTACATTATATGAAAAAGGAACTGTCCCTCCT 
               
               
                   
                 AATGTGGTACCGGGTCTGGGTGACCACCAGGCAGGA 
               
               
                   
                 ATGTTCTTAGCTGCCGGTATGGCTGGTGCGTTGTATA 
               
               
                   
                 AGGCCAAAACCACCGGACAAGGCGACAAAGTCACCG 
               
               
                   
                 TTAGTCTGATGCATAGCGCAATGTACGGCCTGGGAAT 
               
               
                   
                 CATGATTCAGGCAGCCCAGTACAAGGACCATGGGCT 
               
               
                   
                 GGTGTACCCGATCAACCGTAATGAAACGCCTAATCCT 
               
               
                   
                 TTCATCGTTTCATACAAGTCCAAAGATGATTACTTTG 
               
               
                   
                 TCCAAGTTTGCATGCCTCCCTATGATGTGTTTTATGAT 
               
               
                   
                 CGCTTTATGACGGCCTTAGGACGTGAAGACTTGGTAG 
               
               
                   
                 GTGACGAACGCTACAATAAGATCGAGAACTTGAAGG 
               
               
                   
                 ATGGTCGCGCAAAAGAAGTCTATTCCATCATCGAACA 
               
               
                   
                 ACAAATGGTAACGAAGACGAAGGACGAATGGGACA 
               
               
                   
                 AGATTTTTCGTGATGCAGACATTCCATTCGCTATTGC 
               
               
                   
                 CCAAACGTGGGAAGATCTTTTAGAAGACGAGCAGGC 
               
               
                   
                 ATGGGCCAACGACTACCTGTATAAAATGAAGTATCCC 
               
               
                   
                 ACAGGCAACGAACGTGCCCTGGTACGTTTACCTGTGT 
               
               
                   
                 TCTTCAAAGAAGCTGGACTTCCTGAATACAACCAGTC 
               
               
                   
                 GCCACAGATTGCTGAGAATACCGTGGAAGTGTTAAA 
               
               
                   
                 GGAGATGGGATATACCGAGCAAGAAATTGAGGAGCT 
               
               
                   
                 TGAGAAAGACAAAGACATCATGGTACGTAAAGAGAA 
               
               
                   
                 ATGAAGGT taagaaggagatatacat ATGTCAGACCGCAACAA 
               
               
                   
                 AGAAGTGAAAGAAAAGAAGGCTAAACACTATCTGCG 
               
               
                   
                 CGAGATCACAGCTAAACACTACAAGGAAGCGTTAGA 
               
               
                   
                 GGCTAAAGAGCGTGGGGAGAAAGTGGGTTGGTGTGC 
               
               
                   
                 CTCTAACTTCCCCCAAGAGATTGCAACCACGTTGGGT 
               
               
                   
                 GTAAAGGTTGTTTATCCCGAAAACCACGCCGCCGCCG 
               
               
                   
                 TAGCGGCACGTGGCAATGGGCAAAATATGTGCGAAC 
               
               
                   
                 ACGCGGAGGCTATGGGATTCAGTAATGATGTGTGTG 
               
               
                   
                 GATATGCACGTGTAAATTTAGCCGTAATGGACATCGG 
               
               
                   
                 CCATAGTGAAGATCAACCTATTCCAATGCCTGATTTC 
               
               
                   
                 GTTCTGTGCTGTAATAATATCTGCAATCAGATGATTA 
               
               
                   
                 AATGGTATGAACACATTGCAAAAACGTTGGATATTCC 
               
               
                   
                 TATGATCCTTATCGATATTCCATATAATACTGAGAAC 
               
               
                   
                 ACGGTGTCTCAGGACCGCATTAAGTACATCCGCGCCC 
               
               
                   
                 AGTTCGATGACGCTATCAAGCAACTGGAAGAAATCA 
               
               
                   
                 CTGGCAAAAAGTGGGACGAGAATAAATTCGAAGAAG 
               
               
                   
                 TGATGAAGATTTCGCAAGAATCGGCCAAGCAATGGT 
               
               
                   
                 TACGCGCCGCGAGCTACGCGAAATACAAACCATCAC 
               
               
                   
                 CGTTTTCGGGCTTTGACCTTTTTAATCACATGGCTGTA 
               
               
                   
                 GCCGTTTGTGCTCGCGGCACCCAGGAAGCCGCCGATG 
               
               
                   
                 CATTCAAAATGTTAGCAGATGAATATGAAGAGAACG 
               
               
                   
                 TTAAGACAGGAAAGTCTACTTATCGCGGCGAGGAGA 
               
               
                   
                 AGCAGCGTATCTTGTTCGAGGGCATCGCTTGTTGGCC 
               
               
                   
                 TTATCTGCGCCACAAGTTGACGAAACTGAGTGAATAT 
               
               
                   
                 GGAATGAACGTCACAGCTACGGTGTACGCCGAAGCT 
               
               
                   
                 TTTGGGGTTATTTACGAAAACATGGATGAACTGATGG 
               
               
                   
                 CCGCTTACAATAAAGTGCCTAACTCAATCTCCTTCGA 
               
               
                   
                 GAACGCGCTGAAGATGCGTCTTAATGCCGTTACAAGC 
               
               
                   
                 ACCAATACAGAAGGGGCTGTTATCCACATTAATCGCA 
               
               
                   
                 GTTGTAAGCTGTGGTCAGGATTCTTATACGAACTGGC 
               
               
                   
                 CCGTCGTTTGGAAAAGGAGACGGGGATCCCTGTTGTT 
               
               
                   
                 TCGTTCGACGGAGATCAAGCGGATCCCCGTAACTTCT 
               
               
                   
                 CCGAGGCTCAATATGACACTCGCATCCAAGGTTTAAA 
               
               
                   
                 TGAGGTGATGGTCGCGAAAAAAGAAGCAGAGTGAGC 
               
               
                   
                 TT taagaaggagatatacat ATGTCGAATAGTGACAAGTTTTTT 
               
               
                   
                 AACGACTTCAAGGACATTGTGGAAAACCCAAAGAAG 
               
               
                   
                 TATATCATGAAGCATATGGAACAAACGGGACAAAAA 
               
               
                   
                 GCCATCGGTTGCATGCCTTTATACACCCCAGAAGAGC 
               
               
                   
                 TTGTCTTAGCGGCGGGTATGTTTCCTGTTGGAGTATG 
               
               
                   
                 GGGCTCGAATACTGAGTTGTCAAAAGCCAAGACCTA 
               
               
                   
                 CTTTCCGGCTTTTATCTGTTCTATCTTGCAAACTACTT 
               
               
                   
                 TAGAAAACGCATTGAATGGGGAGTATGACATGCTGT 
               
               
                   
                 CTGGTATGATGATCACAAACTATTGCGATTCGCTGAA 
               
               
                   
                 ATGTATGGGACAAAACTTCAAACTTACAGTGGAAAA 
               
               
                   
                 TATCGAATTCATCCCGGTTACGGTTCCACAAAACCGC 
               
               
                   
                 AAGATGGAGGCGGGTAAAGAATTTCTGAAATCCCAG 
               
               
                   
                 TATAAAATGAATATCGAACAACTGGAAAAAATCTCA 
               
               
                   
                 GGGAATAAGATCACTGACGAGAGCTTGGAGAAGGCT 
               
               
                   
                 ATTGAAATTTACGATGAGCACCGTAAAGTCATGAAC 
               
               
                   
                 GATTTCTCTATGCTTGCGTCCAAGTACCCTGGTATCAT 
               
               
                   
                 TACGCCAACGAAACGTAACTACGTGATGAAGTCAGC 
               
               
                   
                 GTATTATATGGACAAGAAAGAACATACAGAGAAGGT 
               
               
                   
                 ACGTCAGTTGATGGATGAAATCAAGGCCATTGAGCCT 
               
               
                   
                 AAACCATTCGAAGGAAAACGCGTGATTACCACTGGG 
               
               
                   
                 ATCATTGCAGATTCGGAGGACCTTTTGAAAATCTTGG 
               
               
                   
                 AGGAGAATAACATTGCTATCGTGGGAGATGATATTG 
               
               
                   
                 CACACGAGTCTCGCCAATACCGCACTTTGACCCCGGA 
               
               
                   
                 GGCCAACACACCTATGGACCGTCTTGCTGAACAATTT 
               
               
                   
                 GCGAACCGCGAGTGTTCGACGTTGTATGACCCTGAAA 
               
               
                   
                 AAAAACGTGGACAGTATATTGTCGAGATGGCAAAAG 
               
               
                   
                 AGCGTAAGGCCGACGGAATCATCTTCTTCATGACAAA 
               
               
                   
                 ATTCTGCGATCCCGAAGAATACGATTACCCTCAGATG 
               
               
                   
                 AAAAAAGACTTCGAAGAAGCCGGTATTCCCCACGTT 
               
               
                   
                 CTGATTGAGACAGACATGCAAATGAAGAACTACGAA 
               
               
                   
                 CAAGCTCGCACCGCTATTCAAGCATTTTCAGAAACCC 
               
               
                   
                 TTTGACGCT taagaaggagatatacat ATGCGTGCTGTCTTAAT 
               
               
                   
                 CGAGAAGTCAGATGACACCCAGAGTGTTTCAGTTAC 
               
               
                   
                 GGAGTTGGCTGAAGACCAATTACCCGAAGGTGACGT 
               
               
                   
                 CCTTGTGGATGTCGCGTACAGCACATTGAATTACAAG 
               
               
                   
                 GATGCTCTTGCGATTACTGGAAAAGCACCCGTTGTAC 
               
               
                   
                 GCCGTTTTCCTATGGTCCCCGGAATTGACTTTACTGG 
               
               
                   
                 GACTGTCGCACAGAGTTCCCATGCTGATTTCAAGCCA 
               
               
                   
                 GGCGACCGCGTAATTCTGAACGGATGGGGAGTTGGT 
               
               
                   
                 GAGAAACACTGGGGCGGTCTTGCAGAACGCGCACGC 
               
               
                   
                 GTACGTGGGGACTGGCTTGTCCCGTTGCCAGCCCCCT 
               
               
                   
                 TAGACTTGCGCCAGGCTGCAATGATTGGCACTGCGGG 
               
               
                   
                 GTACACAGCTATGCTGTGCGTGCTTGCCCTTGAGCGC 
               
               
                   
                 CATGGAGTCGTACCTGGGAACGGCGAGATTGTCGTCT 
               
               
                   
                 CAGGCGCAGCAGGAGGGGTAGGTTCTGTAGCAACCA 
               
               
                   
                 CACTGTTAGCAGCCAAAGGCTACGAAGTGGCCGCCG 
               
               
                   
                 TGACCGGGCGCGCAAGCGAGGCCGAATATTTACGCG 
               
               
                   
                 GATTAGGCGCCGCGTCGGTCATTGATCGCAATGAATT 
               
               
                   
                 AACGGGGAAGGTGCGTCCATTAGGGCAGGAACGCTG 
               
               
                   
                 GGCAGGAGGAATCGATGTAGCAGGATCAACCGTACT 
               
               
                   
                 TGCTAATATGTTGAGCATGATGAAATACCGTGGCGTG 
               
               
                   
                 GTGGCGGCCTGTGGCCTGGCGGCTGGAATGGACTTGC 
               
               
                   
                 CCGCGTCTGTCGCCCCTTTTATTCTGCGTGGTATGACT 
               
               
                   
                 TTGGCAGGGGTAGATTCAGTCATGTGCCCCAAAACTG 
               
               
                   
                 ATCGTCTGGCTGCTTGGGCACGCCTGGCATCCGACCT 
               
               
                   
                 GGACCCTGCAAAGCTGGAAGAGATGACAACTGAATT 
               
               
                   
                 ACCGTTCTCTGAGGTGATTGAAACGGCTCCGAAGTTC 
               
               
                   
                 TTGGATGGAACAGTGCGTGGGCGTATTGTCATTCCGG 
               
               
                   
                 TAACACCTTGATACT taagaaggagatatacat ATGAAAATCTT 
               
               
                   
                 GGCATACTGCGTCCGCCCAGACGAGGTAGACTCCTTT 
               
               
                   
                 AAGAAATTTAGTGAAAAGTACGGGCATACAGTTGAT 
               
               
                   
                 CTTATTCCAGACTCTTTTGGACCTAATGTCGCTCATTT 
               
               
                   
                 GGCGAAGGGTTACGATGGGATTTCTATTCTGGGCAAC 
               
               
                   
                 GACACGTGTAACCGTGAGGCACTGGAGAAGATCAAG 
               
               
                   
                 GATTGCGGGATCAAATATCTGGCAACCCGTACAGCC 
               
               
                   
                 GGAGTGAACAACATTGACTTCGATGCAGCAAAGGAG 
               
               
                   
                 TTCGGTATTAACGTGGCTAATGTTCCCGCATATTCCC 
               
               
                   
                 CCAACTCGGTCAGCGAATTTACCATTGGATTGGCATT 
               
               
                   
                 AAGTCTGACGCGTAAGATTCCATTTGCCCTGAAACGC 
               
               
                   
                 GTGGAACTGAACAATTTTGCGCTTGGCGGCCTTATTG 
               
               
                   
                 GTGTGGAATTGCGTAACTTAACTTTAGGAGTCATCGG 
               
               
                   
                 TACTGGTCGCATCGGATTGAAAGTGATTGAGGGCTTC 
               
               
                   
                 TCTGGGTTTGGAATGAAAAAAATGATCGGTTATGACA 
               
               
                   
                 TTTTTGAAAATGAAGAAGCAAAGAAGTACATCGAAT 
               
               
                   
                 ACAAATCATTAGACGAAGTTTTTAAAGAGGCTGATAT 
               
               
                   
                 TATCACTCTGCATGCGCCTCTGACAGACGACAACTAT 
               
               
                   
                 CATATGATTGGTAAAGAATCCATTGCTAAAATGAAG 
               
               
                   
                 GATGGGGTATTTATTATCAACGCAGCGCGTGGAGCCT 
               
               
                   
                 TAATCGATAGTGAGGCCCTGATTGAAGGGTTAAAATC 
               
               
                   
                 GGGGAAGATT 
               
               
                   
               
               
                 fldA 
                 ATGGAAAACAACACCAATATGTTCTCTGGAGTGAAG 
               
               
                 SEQ ID NO: 276 
                 GTGATCGAACTGGCCAACTTTATCGCTGCTCCGGCGG 
               
               
                   
                 CAGGTCGCTTCTTTGCTGATGGGGGAGCAGAAGTAAT 
               
               
                   
                 TAAGATCGAATCTCCAGCAGGCGACCCGCTGCGCTAC 
               
               
                   
                 ACGGCCCCATCAGAAGGACGCCCGCTTTCTCAAGAG 
               
               
                   
                 GAAAACACAACGTATGATTTGGAAAACGCGAATAAG 
               
               
                   
                 AAAGCAATTGTTCTGAACTTAAAATCGGAAAAAGGA 
               
               
                   
                 AAGAAAATTCTTCACGAGATGCTTGCTGAGGCAGAC 
               
               
                   
                 ATCTTGTTAACAAATTGGCGCACGAAAGCGTTAGTCA 
               
               
                   
                 AACAGGGGTTAGATTACGAAACACTGAAAGAGAAGT 
               
               
                   
                 ATCCAAAATTGGTATTTGCACAGATTACAGGATACGG 
               
               
                   
                 GGAGAAAGGACCCGACAAAGACCTGCCTGGTTTCGA 
               
               
                   
                 CTACACGGCGTTTTTCGCCCGCGGAGGAGTCTCCGGT 
               
               
                   
                 ACATTATATGAAAAAGGAACTGTCCCTCCTAATGTGG 
               
               
                   
                 TACCGGGTCTGGGTGACCACCAGGCAGGAATGTTCTT 
               
               
                   
                 AGCTGCCGGTATGGCTGGTGCGTTGTATAAGGCCAAA 
               
               
                   
                 ACCACCGGACAAGGCGACAAAGTCACCGTTAGTCTG 
               
               
                   
                 ATGCATAGCGCAATGTACGGCCTGGGAATCATGATTC 
               
               
                   
                 AGGCAGCCCAGTACAAGGACCATGGGCTGGTGTACC 
               
               
                   
                 CGATCAACCGTAATGAAACGCCTAATCCTTTCATCGT 
               
               
                   
                 TTCATACAAGTCCAAAGATGATTACTTTGTCCAAGTT 
               
               
                   
                 TGCATGCCTCCCTATGATGTGTTTTATGATCGCTTTAT 
               
               
                   
                 GACGGCCTTAGGACGTGAAGACTTGGTAGGTGACGA 
               
               
                   
                 ACGCTACAATAAGATCGAGAACTTGAAGGATGGTCG 
               
               
                   
                 CGCAAAAGAAGTCTATTCCATCATCGAACAACAAAT 
               
               
                   
                 GGTAACGAAGACGAAGGACGAATGGGACAAGATTTT 
               
               
                   
                 TCGTGATGCAGACATTCCATTCGCTATTGCCCAAACG 
               
               
                   
                 TGGGAAGATCTTTTAGAAGACGAGCAGGCATGGGCC 
               
               
                   
                 AACGACTACCTGTATAAAATGAAGTATCCCACAGGC 
               
               
                   
                 AACGAACGTGCCCTGGTACGTTTACCTGTGTTCTTCA 
               
               
                   
                 AAGAAGCTGGACTTCCTGAATACAACCAGTCGCCAC 
               
               
                   
                 AGATTGCTGAGAATACCGTGGAAGTGTTAAAGGAGA 
               
               
                   
                 TGGGATATACCGAGCAAGAAATTGAGGAGCTTGAGA 
               
               
                   
                 AAGACAAAGACATCATGGTACGTAAAGAGAAATGA 
               
               
                   
               
               
                 fldB 
                 ATGTCAGACCGCAACAAAGAAGTGAAAGAAAAGAA 
               
               
                 SEQ ID NO: 277 
                 GGCTAAACACTATCTGCGCGAGATCACAGCTAAACA 
               
               
                   
                 CTACAAGGAAGCGTTAGAGGCTAAAGAGCGTGGGGA 
               
               
                   
                 GAAAGTGGGTTGGTGTGCCTCTAACTTCCCCCAAGAG 
               
               
                   
                 ATTGCAACCACGTTGGGTGTAAAGGTTGTTTATCCCG 
               
               
                   
                 AAAACCACGCCGCCGCCGTAGCGGCACGTGGCAATG 
               
               
                   
                 GGCAAAATATGTGCGAACACGCGGAGGCTATGGGAT 
               
               
                   
                 TCAGTAATGATGTGTGTGGATATGCACGTGTAAATTT 
               
               
                   
                 AGCCGTAATGGACATCGGCCATAGTGAAGATCAACC 
               
               
                   
                 TATTCCAATGCCTGATTTCGTTCTGTGCTGTAATAATA 
               
               
                   
                 TCTGCAATCAGATGATTAAATGGTATGAACACATTGC 
               
               
                   
                 AAAAACGTTGGATATTCCTATGATCCTTATCGATATT 
               
               
                   
                 CCATATAATACTGAGAACACGGTGTCTCAGGACCGC 
               
               
                   
                 ATTAAGTACATCCGCGCCCAGTTCGATGACGCTATCA 
               
               
                   
                 AGCAACTGGAAGAAATCACTGGCAAAAAGTGGGACG 
               
               
                   
                 AGAATAAATTCGAAGAAGTGATGAAGATTTCGCAAG 
               
               
                   
                 AATCGGCCAAGCAATGGTTACGCGCCGCGAGCTACG 
               
               
                   
                 CGAAATACAAACCATCACCGTTTTCGGGCTTTGACCT 
               
               
                   
                 TTTTAATCACATGGCTGTAGCCGTTTGTGCTCGCGGC 
               
               
                   
                 ACCCAGGAAGCCGCCGATGCATTCAAAATGTTAGCA 
               
               
                   
                 GATGAATATGAAGAGAACGTTAAGACAGGAAAGTCT 
               
               
                   
                 ACTTATCGCGGCGAGGAGAAGCAGCGTATCTTGTTCG 
               
               
                   
                 AGGGCATCGCTTGTTGGCCTTATCTGCGCCACAAGTT 
               
               
                   
                 GACGAAACTGAGTGAATATGGAATGAACGTCACAGC 
               
               
                   
                 TACGGTGTACGCCGAAGCTTTTGGGGTTATTTACGAA 
               
               
                   
                 AACATGGATGAACTGATGGCCGCTTACAATAAAGTG 
               
               
                   
                 CCTAACTCAATCTCCTTCGAGAACGCGCTGAAGATGC 
               
               
                   
                 GTCTTAATGCCGTTACAAGCACCAATACAGAAGGGG 
               
               
                   
                 CTGTTATCCACATTAATCGCAGTTGTAAGCTGTGGTC 
               
               
                   
                 AGGATTCTTATACGAACTGGCCCGTCGTTTGGAAAAG 
               
               
                   
                 GAGACGGGGATCCCTGTTGTTTCGTTCGACGGAGATC 
               
               
                   
                 AAGCGGATCCCCGTAACTTCTCCGAGGCTCAATATGA 
               
               
                   
                 CACTCGCATCCAAGGTTTAAATGAGGTGATGGTCGCG 
               
               
                   
                 AAAAAAGAAGCAGAGTGA 
               
               
                   
               
               
                 fldC 
                 ATGTCGAATAGTGACAAGTTTTTTAACGACTTCAAGG 
               
               
                 SEQ ID NO: 278 
                 ACATTGTGGAAAACCCAAAGAAGTATATCATGAAGC 
               
               
                   
                 ATATGGAACAAACGGGACAAAAAGCCATCGGTTGCA 
               
               
                   
                 TGCCTTTATACACCCCAGAAGAGCTTGTCTTAGCGGC 
               
               
                   
                 GGGTATGTTTCCTGTTGGAGTATGGGGCTCGAATACT 
               
               
                   
                 GAGTTGTCAAAAGCCAAGACCTACTTTCCGGCTTTTA 
               
               
                   
                 TCTGTTCTATCTTGCAAACTACTTTAGAAAACGCATT 
               
               
                   
                 GAATGGGGAGTATGACATGCTGTCTGGTATGATGATC 
               
               
                   
                 ACAAACTATTGCGATTCGCTGAAATGTATGGGACAA 
               
               
                   
                 AACTTCAAACTTACAGTGGAAAATATCGAATTCATCC 
               
               
                   
                 CGGTTACGGTTCCACAAAACCGCAAGATGGAGGCGG 
               
               
                   
                 GTAAAGAATTTCTGAAATCCCAGTATAAAATGAATAT 
               
               
                   
                 CGAACAACTGGAAAAAATCTCAGGGAATAAGATCAC 
               
               
                   
                 TGACGAGAGCTTGGAGAAGGCTATTGAAATTTACGA 
               
               
                   
                 TGAGCACCGTAAAGTCATGAACGATTTCTCTATGCTT 
               
               
                   
                 GCGTCCAAGTACCCTGGTATCATTACGCCAACGAAAC 
               
               
                   
                 GTAACTACGTGATGAAGTCAGCGTATTATATGGACAA 
               
               
                   
                 GAAAGAACATACAGAGAAGGTACGTCAGTTGATGGA 
               
               
                   
                 TGAAATCAAGGCCATTGAGCCTAAACCATTCGAAGG 
               
               
                   
                 AAAACGCGTGATTACCACTGGGATCATTGCAGATTCG 
               
               
                   
                 GAGGACCTTTTGAAAATCTTGGAGGAGAATAACATT 
               
               
                   
                 GCTATCGTGGGAGATGATATTGCACACGAGTCTCGCC 
               
               
                   
                 AATACCGCACTTTGACCCCGGAGGCCAACACACCTAT 
               
               
                   
                 GGACCGTCTTGCTGAACAATTTGCGAACCGCGAGTGT 
               
               
                   
                 TCGACGTTGTATGACCCTGAAAAAAAACGTGGACAG 
               
               
                   
                 TATATTGTCGAGATGGCAAAAGAGCGTAAGGCCGAC 
               
               
                   
                 GGAATCATCTTCTTCATGACAAAATTCTGCGATCCCG 
               
               
                   
                 AAGAATACGATTACCCTCAGATGAAAAAAGACTTCG 
               
               
                   
                 AAGAAGCCGGTATTCCCCACGTTCTGATTGAGACAGA 
               
               
                   
                 CATGCAAATGAAGAACTACGAACAAGCTCGCACCGC 
               
               
                   
                 TATTCAAGCATTTTCAGAAACCCTTTG 
               
               
                   
               
               
                 Acul 
                 ATGCGTGCTGTCTTAATCGAGAAGTCAGATGACACCC 
               
               
                 SEQ ID NO: 279 
                 AGAGTGTTTCAGTTACGGAGTTGGCTGAAGACCAATT 
               
               
                   
                 ACCCGAAGGTGACGTCCTTGTGGATGTCGCGTACAGC 
               
               
                   
                 ACATTGAATTACAAGGATGCTCTTGCGATTACTGGAA 
               
               
                   
                 AAGCACCCGTTGTACGCCGTTTTCCTATGGTCCCCGG 
               
               
                   
                 AATTGACTTTACTGGGACTGTCGCACAGAGTTCCCAT 
               
               
                   
                 GCTGATTTCAAGCCAGGCGACCGCGTAATTCTGAACG 
               
               
                   
                 GATGGGGAGTTGGTGAGAAACACTGGGGCGGTCTTG 
               
               
                   
                 CAGAACGCGCACGCGTACGTGGGGACTGGCTTGTCC 
               
               
                   
                 CGTTGCCAGCCCCCTTAGACTTGCGCCAGGCTGCAAT 
               
               
                   
                 GATTGGCACTGCGGGGTACACAGCTATGCTGTGCGTG 
               
               
                   
                 CTTGCCCTTGAGCGCCATGGAGTCGTACCTGGGAACG 
               
               
                   
                 GCGAGATTGTCGTCTCAGGCGCAGCAGGAGGGGTAG 
               
               
                   
                 GTTCTGTAGCAACCACACTGTTAGCAGCCAAAGGCTA 
               
               
                   
                 CGAAGTGGCCGCCGTGACCGGGCGCGCAAGCGAGGC 
               
               
                   
                 CGAATATTTACGCGGATTAGGCGCCGCGTCGGTCATT 
               
               
                   
                 GATCGCAATGAATTAACGGGGAAGGTGCGTCCATTA 
               
               
                   
                 GGGCAGGAACGCTGGGCAGGAGGAATCGATGTAGCA 
               
               
                   
                 GGATCAACCGTACTTGCTAATATGTTGAGCATGATGA 
               
               
                   
                 AATACCGTGGCGTGGTGGCGGCCTGTGGCCTGGCGG 
               
               
                   
                 CTGGAATGGACTTGCCCGCGTCTGTCGCCCCTTTTATT 
               
               
                   
                 CTGCGTGGTATGACTTTGGCAGGGGTAGATTCAGTCA 
               
               
                   
                 TGTGCCCCAAAACTGATCGTCTGGCTGCTTGGGCACG 
               
               
                   
                 CCTGGCATCCGACCTGGACCCTGCAAAGCTGGAAGA 
               
               
                   
                 GATGACAACTGAATTACCGTTCTCTGAGGTGATTGAA 
               
               
                   
                 ACGGCTCCGAAGTTCTTGGATGGAACAGTGCGTGGG 
               
               
                   
                 CGTATTGTCATTCCGGTAACACCTTGA 
               
               
                   
               
               
                 fldH1 
                 ATGAAAATCTTGGCATACTGCGTCCGCCCAGACGAG 
               
               
                 SEQ ID NO: 280 
                 GTAGACTCCTTTAAGAAATTTAGTGAAAAGTACGGGC 
               
               
                   
                 ATACAGTTGATCTTATTCCAGACTCTTTTGGACCTAAT 
               
               
                   
                 GTCGCTCATTTGGCGAAGGGTTACGATGGGATTTCTA 
               
               
                   
                 TTCTGGGCAACGACACGTGTAACCGTGAGGCACTGG 
               
               
                   
                 AGAAGATCAAGGATTGCGGGATCAAATATCTGGCAA 
               
               
                   
                 CCCGTACAGCCGGAGTGAACAACATTGACTTCGATGC 
               
               
                   
                 AGCAAAGGAGTTCGGTATTAACGTGGCTAATGTTCCC 
               
               
                   
                 GCATATTCCCCCAACTCGGTCAGCGAATTTACCATTG 
               
               
                   
                 GATTGGCATTAAGTCTGACGCGTAAGATTCCATTTGC 
               
               
                   
                 CCTGAAACGCGTGGAACTGAACAATTTTGCGCTTGGC 
               
               
                   
                 GGCCTTATTGGTGTGGAATTGCGTAACTTAACTTTAG 
               
               
                   
                 GAGTCATCGGTACTGGTCGCATCGGATTGAAAGTGAT 
               
               
                   
                 TGAGGGCTTCTCTGGGTTTGGAATGAAAAAAATGATC 
               
               
                   
                 GGTTATGACATTTTTGAAAATGAAGAAGCAAAGAAG 
               
               
                   
                 TACATCGAATACAAATCATTAGACGAAGTTTTTAAAG 
               
               
                   
                 AGGCTGATATTATCACTCTGCATGCGCCTCTGACAGA 
               
               
                   
                 CGACAACTATCATATGATTGGTAAAGAATCCATTGCT 
               
               
                   
                 AAAATGAAGGATGGGGTATTTATTATCAACGCAGCG 
               
               
                   
                 CGTGGAGCCTTAATCGATAGTGAGGCCCTGATTGAAG 
               
               
                   
                 GGTTAAAATCGGGGAAGATTGCGGGCGCGGCTCTGG 
               
               
                   
                 ATAGCTATGAGTATGAGCAAGGTGTCTTTCACAACAA 
               
               
                   
                 TAAGATGAATGAAATTATGCAGGATGATACCTTGGA 
               
               
                   
                 ACGTCTGAAATCTTTTCCCAACGTCGTGATCACGCCG 
               
               
                   
                 CATTTGGGTTTTTATACTGATGAGGCGGTTTCCAATA 
               
               
                   
                 TGGTAGAGATCACACTGATGAACCTTCAGGAATTCGA 
               
               
                   
                 GTTGAAAGGAACCTGTAAGAACCAGCGTGTTTGTAA 
               
               
                   
                 ATGA 
               
               
                   
               
               
                 fbrAroG-TrpDH- 
                 
                   Ctctagaaataattttgtttaactttaagaaggagatatacat 
                 
               
               
                 fldABCDH (RBS 
                 atgaattatcagaacgacgatttacgcatcaaagaaatcaaagagttacttcctcctgtcg 
               
               
                   
                 cattgctggaaaaattccccgctactgaaaatgccgcgaatacggtcgcccatgcccga 
               
               
                 and leader region 
                 aaagcgatccataagatcctgaaaggtaatgatgatcgcctgttggtggtgattggccca 
               
               
                 underlined) 
                 tgctcaattcatgatcctgtcgcggctaaagagtatgccactcgcttgctgacgctgcgtg 
               
               
                 SEQ ID NO: 281 
                 aagagctgcaagatgagctggaaatcgtgatgcgcgtctattttgaaaagccgcgtacta 
               
               
                   
                 cggtgggctggaaagggctgattaacgatccgcatatggataacagcttccagatcaac 
               
               
                   
                 gacggtctgcgtattgcccgcaaattgctgctcgatattaacgacagcggtctgccagcg 
               
               
                   
                 gcgggtgaattcctggatatgatcaccctacaatatctcgctgacctgatgagctggggc 
               
               
                   
                 gcaattggcgcacgtaccaccgaatcgcaggtgcaccgcgaactggcgtctggtctttc 
               
               
                   
                 ttgtccggtaggtttcaaaaatggcactgatggtacgattaaagtggctatcgatgccatta 
               
               
                   
                 atgccgccggtgcgccgcactgcttcctgtccgtaacgaaatgggggcattcggcgatt 
               
               
                   
                 gtgaataccagcggtaacggcgattgccatatcattctgcgcggcggtaaagagcctaa 
               
               
                   
                 ctacagcgcgaagcacgttgctgaagtgaaagaagggctgaacaaagcaggcctgcc 
               
               
                   
                 agcgcaggtgatgatcgatttcagccatgctaactcgtcaaaacaattcaaaaagcagat 
               
               
                   
                 ggatgtttgtactgacgtttgccagcagattgccggtggcgaaaaggccattattggcgt 
               
               
                   
                 gatggtggaaagccatctggtggaaggcaatcagagcctcgagagcggggaaccgct 
               
               
                   
                 ggcctacggtaagagcatcaccgatgcctgcattggctgggatgataccgatgctctgtt 
               
               
                   
                 acgtcaactggcgagtgcagtaaaagcgcgtcgcgggtaaTACT taagaaggaga   
               
               
                   
                   tatacat ATGCTGTTATTCGAGACTGTGCGTGAAATGGGT 
               
               
                   
                 CATGAGCAAGTCCTTTTCTGTCATAGCAAGAATCCCG 
               
               
                   
                 AGATCAAGGCAATTATCGCAATCCACGATACCACCTT 
               
               
                   
                 AGGACCGGCTATGGGCGCAACTCGTATCTTACCTTAT 
               
               
                   
                 ATTAATGAGGAGGCTGCCCTGAAAGATGCATTACGTC 
               
               
                   
                 TGTCCCGCGGAATGACTTACAAAGCAGCCTGCGCCA 
               
               
                   
                 ATATTCCCGCCGGGGGCGGCAAAGCCGTCATCATCGC 
               
               
                   
                 TAACCCCGAAAACAAGACCGATGACCTGTTACGCGC 
               
               
                   
                 ATACGGCCGTTTCGTGGACAGCTTGAACGGCCGTTTC 
               
               
                   
                 ATCACCGGGCAGGACGTTAACATTACGCCCGACGAC 
               
               
                   
                 GTTCGCACTATTTCGCAGGAGACTAAGTACGTGGTAG 
               
               
                   
                 GCGTCTCAGAAAAGTCGGGAGGGCCGGCACCTATCA 
               
               
                   
                 CCTCTCTGGGAGTATTTTTAGGCATCAAAGCCGCTGT 
               
               
                   
                 AGAGTCGCGTTGGCAGTCTAAACGCCTGGATGGCAT 
               
               
                   
                 GAAAGTGGCGGTGCAAGGACTTGGGAACGTAGGAAA 
               
               
                   
                 AAATCTTTGTCGCCATCTGCATGAACACGATGTACAA 
               
               
                   
                 CTTTTTGTGTCTGATGTCGATCCAATCAAGGCCGAGG 
               
               
                   
                 AAGTAAAACGCTTATTCGGGGCGACTGTTGTCGAACC 
               
               
                   
                 GACTGAAATCTATTCTTTAGATGTTGATATTTTTGCAC 
               
               
                   
                 CGTGTGCACTTGGGGGTATTTTGAATAGCCATACCAT 
               
               
                   
                 CCCGTTCTTACAAGCCTCAATCATCGCAGGAGCAGCG 
               
               
                   
                 AATAACCAGCTGGAGAACGAGCAACTTCATTCGCAG 
               
               
                   
                 ATGCTTGCGAAAAAGGGTATTCTTTACTCACCAGACT 
               
               
                   
                 ACGTTATCAATGCAGGAGGACTTATCAATGTTTATAA 
               
               
                   
                 CGAAATGATCGGATATGACGAGGAAAAAGCATTCAA 
               
               
                   
                 ACAAGTTCATAACATCTACGATACGTTATTAGCGATT 
               
               
                   
                 TTCGAAATTGCAAAAGAACAAGGTGTAACCACCAAC 
               
               
                   
                 GACGCGGCCCGTCGTTTAGCAGAGGATCGTATCAAC 
               
               
                   
                 AACTCCAAACGCTCAAAGAGTAAAGCGATTGCGGCG 
               
               
                   
                 TGAAATG taagaaggagatatacat ATGGAAAACAACACCAAT 
               
               
                   
                 ATGTTCTCTGGAGTGAAGGTGATCGAACTGGCCAACT 
               
               
                   
                 TTATCGCTGCTCCGGCGGCAGGTCGCTTCTTTGCTGA 
               
               
                   
                 TGGGGGAGCAGAAGTAATTAAGATCGAATCTCCAGC 
               
               
                   
                 AGGCGACCCGCTGCGCTACACGGCCCCATCAGAAGG 
               
               
                   
                 ACGCCCGCTTTCTCAAGAGGAAAACACAACGTATGA 
               
               
                   
                 TTTGGAAAACGCGAATAAGAAAGCAATTGTTCTGAA 
               
               
                   
                 CTTAAAATCGGAAAAAGGAAAGAAAATTCTTCACGA 
               
               
                   
                 GATGCTTGCTGAGGCAGACATCTTGTTAACAAATTGG 
               
               
                   
                 CGCACGAAAGCGTTAGTCAAACAGGGGTTAGATTAC 
               
               
                   
                 GAAACACTGAAAGAGAAGTATCCAAAATTGGTATTT 
               
               
                   
                 GCACAGATTACAGGATACGGGGAGAAAGGACCCGAC 
               
               
                   
                 AAAGACCTGCCTGGTTTCGACTACACGGCGTTTTTCG 
               
               
                   
                 CCCGCGGAGGAGTCTCCGGTACATTATATGAAAAAG 
               
               
                   
                 GAACTGTCCCTCCTAATGTGGTACCGGGTCTGGGTGA 
               
               
                   
                 CCACCAGGCAGGAATGTTCTTAGCTGCCGGTATGGCT 
               
               
                   
                 GGTGCGTTGTATAAGGCCAAAACCACCGGACAAGGC 
               
               
                   
                 GACAAAGTCACCGTTAGTCTGATGCATAGCGCAATGT 
               
               
                   
                 ACGGCCTGGGAATCATGATTCAGGCAGCCCAGTACA 
               
               
                   
                 AGGACCATGGGCTGGTGTACCCGATCAACCGTAATG 
               
               
                   
                 AAACGCCTAATCCTTTCATCGTTTCATACAAGTCCAA 
               
               
                   
                 AGATGATTACTTTGTCCAAGTTTGCATGCCTCCCTAT 
               
               
                   
                 GATGTGTTTTATGATCGCTTTATGACGGCCTTAGGAC 
               
               
                   
                 GTGAAGACTTGGTAGGTGACGAACGCTACAATAAGA 
               
               
                   
                 TCGAGAACTTGAAGGATGGTCGCGCAAAAGAAGTCT 
               
               
                   
                 ATTCCATCATCGAACAACAAATGGTAACGAAGACGA 
               
               
                   
                 AGGACGAATGGGACAAGATTTTTCGTGATGCAGACA 
               
               
                   
                 TTCCATTCGCTATTGCCCAAACGTGGGAAGATCTTTT 
               
               
                   
                 AGAAGACGAGCAGGCATGGGCCAACGACTACCTGTA 
               
               
                   
                 TAAAATGAAGTATCCCACAGGCAACGAACGTGCCCT 
               
               
                   
                 GGTACGTTTACCTGTGTTCTTCAAAGAAGCTGGACTT 
               
               
                   
                 CCTGAATACAACCAGTCGCCACAGATTGCTGAGAAT 
               
               
                   
                 ACCGTGGAAGTGTTAAAGGAGATGGGATATACCGAG 
               
               
                   
                 CAAGAAATTGAGGAGCTTGAGAAAGACAAAGACATC 
               
               
                   
                 ATGGTACGTAAAGAGAAATGAAGGT taagaaggagatatacat   
               
               
                   
                 ATGTCAGACCGCAACAAAGAAGTGAAAGAAAAGAA 
               
               
                   
                 GGCTAAACACTATCTGCGCGAGATCACAGCTAAACA 
               
               
                   
                 CTACAAGGAAGCGTTAGAGGCTAAAGAGCGTGGGGA 
               
               
                   
                 GAAAGTGGGTTGGTGTGCCTCTAACTTCCCCCAAGAG 
               
               
                   
                 ATTGCAACCACGTTGGGTGTAAAGGTTGTTTATCCCG 
               
               
                   
                 AAAACCACGCCGCCGCCGTAGCGGCACGTGGCAATG 
               
               
                   
                 GGCAAAATATGTGCGAACACGCGGAGGCTATGGGAT 
               
               
                   
                 TCAGTAATGATGTGTGTGGATATGCACGTGTAAATTT 
               
               
                   
                 AGCCGTAATGGACATCGGCCATAGTGAAGATCAACC 
               
               
                   
                 TATTCCAATGCCTGATTTCGTTCTGTGCTGTAATAATA 
               
               
                   
                 TCTGCAATCAGATGATTAAATGGTATGAACACATTGC 
               
               
                   
                 AAAAACGTTGGATATTCCTATGATCCTTATCGATATT 
               
               
                   
                 CCATATAATACTGAGAACACGGTGTCTCAGGACCGC 
               
               
                   
                 ATTAAGTACATCCGCGCCCAGTTCGATGACGCTATCA 
               
               
                   
                 AGCAACTGGAAGAAATCACTGGCAAAAAGTGGGACG 
               
               
                   
                 AGAATAAATTCGAAGAAGTGATGAAGATTTCGCAAG 
               
               
                   
                 AATCGGCCAAGCAATGGTTACGCGCCGCGAGCTACG 
               
               
                   
                 CGAAATACAAACCATCACCGTTTTCGGGCTTTGACCT 
               
               
                   
                 TTTTAATCACATGGCTGTAGCCGTTTGTGCTCGCGGC 
               
               
                   
                 ACCCAGGAAGCCGCCGATGCATTCAAAATGTTAGCA 
               
               
                   
                 GATGAATATGAAGAGAACGTTAAGACAGGAAAGTCT 
               
               
                   
                 ACTTATCGCGGCGAGGAGAAGCAGCGTATCTTGTTCG 
               
               
                   
                 AGGGCATCGCTTGTTGGCCTTATCTGCGCCACAAGTT 
               
               
                   
                 GACGAAACTGAGTGAATATGGAATGAACGTCACAGC 
               
               
                   
                 TACGGTGTACGCCGAAGCTTTTGGGGTTATTTACGAA 
               
               
                   
                 AACATGGATGAACTGATGGCCGCTTACAATAAAGTG 
               
               
                   
                 CCTAACTCAATCTCCTTCGAGAACGCGCTGAAGATGC 
               
               
                   
                 GTCTTAATGCCGTTACAAGCACCAATACAGAAGGGG 
               
               
                   
                 CTGTTATCCACATTAATCGCAGTTGTAAGCTGTGGTC 
               
               
                   
                 AGGATTCTTATACGAACTGGCCCGTCGTTTGGAAAAG 
               
               
                   
                 GAGACGGGGATCCCTGTTGTTTCGTTCGACGGAGATC 
               
               
                   
                 AAGCGGATCCCCGTAACTTCTCCGAGGCTCAATATGA 
               
               
                   
                 CACTCGCATCCAAGGTTTAAATGAGGTGATGGTCGCG 
               
               
                   
                 AAAAAAGAAGCAGAGTGAGCTT taagaaggagatatacat ATG 
               
               
                   
                 TCGAATAGTGACAAGTTTTTTAACGACTTCAAGGACA 
               
               
                   
                 TTGTGGAAAACCCAAAGAAGTATATCATGAAGCATA 
               
               
                   
                 TGGAACAAACGGGACAAAAAGCCATCGGTTGCATGC 
               
               
                   
                 CTTTATACACCCCAGAAGAGCTTGTCTTAGCGGCGGG 
               
               
                   
                 TATGTTTCCTGTTGGAGTATGGGGCTCGAATACTGAG 
               
               
                   
                 TTGTCAAAAGCCAAGACCTACTTTCCGGCTTTTATCT 
               
               
                   
                 GTTCTATCTTGCAAACTACTTTAGAAAACGCATTGAA 
               
               
                   
                 TGGGGAGTATGACATGCTGTCTGGTATGATGATCACA 
               
               
                   
                 AACTATTGCGATTCGCTGAAATGTATGGGACAAAACT 
               
               
                   
                 TCAAACTTACAGTGGAAAATATCGAATTCATCCCGGT 
               
               
                   
                 TACGGTTCCACAAAACCGCAAGATGGAGGCGGGTAA 
               
               
                   
                 AGAATTTCTGAAATCCCAGTATAAAATGAATATCGAA 
               
               
                   
                 CAACTGGAAAAAATCTCAGGGAATAAGATCACTGAC 
               
               
                   
                 GAGAGCTTGGAGAAGGCTATTGAAATTTACGATGAG 
               
               
                   
                 CACCGTAAAGTCATGAACGATTTCTCTATGCTTGCGT 
               
               
                   
                 CCAAGTACCCTGGTATCATTACGCCAACGAAACGTAA 
               
               
                   
                 CTACGTGATGAAGTCAGCGTATTATATGGACAAGAA 
               
               
                   
                 AGAACATACAGAGAAGGTACGTCAGTTGATGGATGA 
               
               
                   
                 AATCAAGGCCATTGAGCCTAAACCATTCGAAGGAAA 
               
               
                   
                 ACGCGTGATTACCACTGGGATCATTGCAGATTCGGAG 
               
               
                   
                 GACCTTTTGAAAATCTTGGAGGAGAATAACATTGCTA 
               
               
                   
                 TCGTGGGAGATGATATTGCACACGAGTCTCGCCAATA 
               
               
                   
                 CCGCACTTTGACCCCGGAGGCCAACACACCTATGGAC 
               
               
                   
                 CGTCTTGCTGAACAATTTGCGAACCGCGAGTGTTCGA 
               
               
                   
                 CGTTGTATGACCCTGAAAAAAAACGTGGACAGTATA 
               
               
                   
                 TTGTCGAGATGGCAAAAGAGCGTAAGGCCGACGGAA 
               
               
                   
                 TCATCTTCTTCATGACAAAATTCTGCGATCCCGAAGA 
               
               
                   
                 ATACGATTACCCTCAGATGAAAAAAGACTTCGAAGA 
               
               
                   
                 AGCCGGTATTCCCCACGTTCTGATTGAGACAGACATG 
               
               
                   
                 CAAATGAAGAACTACGAACAAGCTCGCACCGCTATT 
               
               
                   
                 CAAGCATTTTCAGAAACCCTTTGACGCT taagaaggagatata   
               
               
                   
                 catATGTTCTTTACGGAGCAACACGAACTTATTCGCAA 
               
               
                   
                 ACTGGCGCGTGACTTTGCCGAACAGGAAATCGAGCC 
               
               
                   
                 TATCGCAGACGAAGTAGATAAAACCGCAGAGTTCCC 
               
               
                   
                 AAAAGAAATCGTGAAGAAGATGGCTCAAAATGGATT 
               
               
                   
                 TTTCGGCATTAAAATGCCTAAAGAATACGGAGGGGC 
               
               
                   
                 GGGTGCGGATAACCGCGCTTATGTCACTATTATGGAG 
               
               
                   
                 GAAATTTCACGTGCTTCCGGGGTAGCGGGTATCTACC 
               
               
                   
                 TGAGCTCGCCGAACAGTTTGTTAGGAACTCCCTTCTT 
               
               
                   
                 ATTGGTCGGAACCGATGAGCAAAAAGAAAAGTACCT 
               
               
                   
                 TAAGCCTATGATCCGCGGCGAGAAGACTCTGGCGTTC 
               
               
                   
                 GCCCTGACAGAGCCTGGTGCTGGCTCTGATGCGGGTG 
               
               
                   
                 CGTTGGCTACTACTGCCCGTGAAGAGGGCGACTATTA 
               
               
                   
                 TATCTTAAATGGCCGCAAGACGTTTATTACAGGGGCT 
               
               
                   
                 CCTATTAGCGACAATATTATTGTGTTCGCAAAAACCG 
               
               
                   
                 ATATGAGCAAAGGGACCAAAGGTATCACCACTTTCA 
               
               
                   
                 TTGTGGACTCAAAGCAGGAAGGGGTAAGTTTTGGTA 
               
               
                   
                 AGCCAGAGGACAAAATGGGAATGATTGGTTGTCCGA 
               
               
                   
                 CAAGCGACATCATCTTGGAAAACGTTAAAGTTCATAA 
               
               
                   
                 GTCCGACATCTTGGGAGAAGTCAATAAGGGGTTTATT 
               
               
                   
                 ACCGCGATGAAAACACTTTCCGTTGGTCGTATCGGAG 
               
               
                   
                 TGGCGTCACAGGCGCTTGGAATTGCACAGGCCGCCGT 
               
               
                   
                 AGATGAGGCGGTAAAGTACGCCAAGCAACGTAAACA 
               
               
                   
                 ATTCAATCGCCCAATCGCGAAATTTCAGGCCATTCAA 
               
               
                   
                 TTTAAACTTGCCAATATGGAGACTAAATTAAATGCCG 
               
               
                   
                 CTAAACTTCTTGTTTATAACGCAGCGTACAAAATGGA 
               
               
                   
                 TTGTGGAGAAAAAGCCGACAAGGAAGCCTCTATGGC 
               
               
                   
                 TAAATACTTTGCTGCTGAATCAGCGATCCAAATCGTT 
               
               
                   
                 AACGACGCGCTGCAAATCCATGGCGGGTATGGCTAT 
               
               
                   
                 ATCAAAGACTACAAGATTGAACGTTTGTACCGCGATG 
               
               
                   
                 TGCGTGTGATCGCTATTTATGAGGGCACTTCCGAGGT 
               
               
                   
                 CCAACAGATGGTTATCGCGTCCAATCTGCTGAAGTAA 
               
               
                   
                 TACT taagaaggagatatacat ATGAAAATCTTGGCATACTGCG 
               
               
                   
                 TCCGCCCAGACGAGGTAGACTCCTTTAAGAAATTTAG 
               
               
                   
                 TGAAAAGTACGGGCATACAGTTGATCTTATTCCAGAC 
               
               
                   
                 TCTTTTGGACCTAATGTCGCTCATTTGGCGAAGGGTT 
               
               
                   
                 ACGATGGGATTTCTATTCTGGGCAACGACACGTGTAA 
               
               
                   
                 CCGTGAGGCACTGGAGAAGATCAAGGATTGCGGGAT 
               
               
                   
                 CAAATATCTGGCAACCCGTACAGCCGGAGTGAACAA 
               
               
                   
                 CATTGACTTCGATGCAGCAAAGGAGTTCGGTATTAAC 
               
               
                   
                 GTGGCTAATGTTCCCGCATATTCCCCCAACTCGGTCA 
               
               
                   
                 GCGAATTTACCATTGGATTGGCATTAAGTCTGACGCG 
               
               
                   
                 TAAGATTCCATTTGCCCTGAAACGCGTGGAACTGAAC 
               
               
                   
                 AATTTTGCGCTTGGCGGCCTTATTGGTGTGGAATTGC 
               
               
                   
                 GTAACTTAACTTTAGGAGTCATCGGTACTGGTCGCAT 
               
               
                   
                 CGGATTGAAAGTGATTGAGGGCTTCTCTGGGTTTGGA 
               
               
                   
                 ATGAAAAAAATGATCGGTTATGACATTTTTGAAAATG 
               
               
                   
                 AAGAAGCAAAGAAGTACATCGAATACAAATCATTAG 
               
               
                   
                 ACGAAGTTTTTAAAGAGGCTGATATTATCACTCTGCA 
               
               
                   
                 TGCGCCTCTGACAGACGACAACTATCATATGATTGGT 
               
               
                   
                 AAAGAATCCATTGCTAAAATGAAGGATGGGGTATTT 
               
               
                   
                 ATTATCAACGCAGCGCGTGGAGCCTTAATCGATAGTG 
               
               
                   
                 AGGCCCTGATTGAAGGGTTAAAATCGGGGAAGATTG 
               
               
                   
                 CGGGCGCGGCTCTGGATAGCTATGAGTATGAGCAAG 
               
               
                   
                 GTGTCTTTCACAACAATAAGATGAATGAAATTATGCA 
               
               
                   
                 GGATGATACCTTGGAACGTCTGAAATCTTTTCCCAAC 
               
               
                   
                 GTCGTGATCACGCCGCATTTGGGTTTTTATACTGATG 
               
               
                   
                 AGGCGGTTTCCAATATGGTAGAGATCACACTGATGA 
               
               
                   
                 ACCTTCAGGAATTCGAGTTGAAAGGAACCTGTAAGA 
               
               
                   
                 ACCAGCGTGTTTGTAAATGA 
               
               
                   
               
               
                 FldD 
                 ATGTTCTTTACGGAGCAACACGAACTTATTCGCAAAC 
               
               
                 SEQ ID NO: 282 
                 TGGCGCGTGACTTTGCCGAACAGGAAATCGAGCCTAT 
               
               
                   
                 CGCAGACGAAGTAGATAAAACCGCAGAGTTCCCAAA 
               
               
                   
                 AGAAATCGTGAAGAAGATGGCTCAAAATGGATTTTT 
               
               
                   
                 CGGCATTAAAATGCCTAAAGAATACGGAGGGGCGGG 
               
               
                   
                 TGCGGATAACCGCGCTTATGTCACTATTATGGAGGAA 
               
               
                   
                 ATTTCACGTGCTTCCGGGGTAGCGGGTATCTACCTGA 
               
               
                   
                 GCTCGCCGAACAGTTTGTTAGGAACTCCCTTCTTATT 
               
               
                   
                 GGTCGGAACCGATGAGCAAAAAGAAAAGTACCTTAA 
               
               
                   
                 GCCTATGATCCGCGGCGAGAAGACTCTGGCGTTCGCC 
               
               
                   
                 CTGACAGAGCCTGGTGCTGGCTCTGATGCGGGTGCGT 
               
               
                   
                 TGGCTACTACTGCCCGTGAAGAGGGCGACTATTATAT 
               
               
                   
                 CTTAAATGGCCGCAAGACGTTTATTACAGGGGCTCCT 
               
               
                   
                 ATTAGCGACAATATTATTGTGTTCGCAAAAACCGATA 
               
               
                   
                 TGAGCAAAGGGACCAAAGGTATCACCACTTTCATTGT 
               
               
                   
                 GGACTCAAAGCAGGAAGGGGTAAGTTTTGGTAAGCC 
               
               
                   
                 AGAGGACAAAATGGGAATGATTGGTTGTCCGACAAG 
               
               
                   
                 CGACATCATCTTGGAAAACGTTAAAGTTCATAAGTCC 
               
               
                   
                 GACATCTTGGGAGAAGTCAATAAGGGGTTTATTACCG 
               
               
                   
                 CGATGAAAACACTTTCCGTTGGTCGTATCGGAGTGGC 
               
               
                   
                 GTCACAGGCGCTTGGAATTGCACAGGCCGCCGTAGA 
               
               
                   
                 TGAGGCGGTAAAGTACGCCAAGCAACGTAAACAATT 
               
               
                   
                 CAATCGCCCAATCGCGAAATTTCAGGCCATTCAATTT 
               
               
                   
                 AAACTTGCCAATATGGAGACTAAATTAAATGCCGCTA 
               
               
                   
                 AACTTCTTGTTTATAACGCAGCGTACAAAATGGATTG 
               
               
                   
                 TGGAGAAAAAGCCGACAAGGAAGCCTCTATGGCTAA 
               
               
                   
                 ATACTTTGCTGCTGAATCAGCGATCCAAATCGTTAAC 
               
               
                   
                 GACGCGCTGCAAATCCATGGCGGGTATGGCTATATCA 
               
               
                   
                 AAGACTACAAGATTGAACGTTTGTACCGCGATGTGCG 
               
               
                   
                 TGTGATCGCTATTTATGAGGGCACTTCCGAGGTCCAA 
               
               
                   
                 CAGATGGTTATCGCGTCCAATCTGCTGAAGTAA 
               
               
                   
               
               
                 RBS 
                 taagaaggagatatacat 
               
               
                 SEQ ID NO: 283 
                   
               
               
                   
               
               
                 RBS 
                 ctctagaaataattttgtttaactttaagaaggagatatacat 
               
               
                 SEQ ID NO: 284 
               
               
                   
               
            
           
         
       
     
     In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with one or more sequences of Table 81. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with one or more sequences of Table 81. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with one or more sequences of Table 81. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with one or more sequences of Table 81. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with one or more sequences of Table 81. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one or more sequences of Table 81. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 263. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of with one or more sequences of Table 81. 
     In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 263. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 263. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 263. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 263. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 263. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 263. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 263. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 263. 
     In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 261. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 261. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 261. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 261. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 261. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 261. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 261. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 261. 
     In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 273. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 273. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 273. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 273. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 273. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 273. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 273. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 273. 
     In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 256. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 256. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 256. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 256. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 256. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 256. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 256. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 256. 
     In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80% identity with SEQ ID NO: 257. In another embodiment, the genetically engineered bacteria comprise a sequence which has at least about 85% identity with SEQ ID NO: 257. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 90% identity with SEQ ID NO: 257. In one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 95% identity with SEQ ID NO: 257. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 257. Accordingly, in one embodiment, the genetically engineered bacteria comprise a sequence which has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 257. In another embodiment, the genetically engineered bacteria comprise the sequence of SEQ ID NO: 257. In yet another embodiment the genetically engineered bacteria comprise a sequence which consists of the sequence of SEQ ID NO: 257. 
     Example 52. Tryptophan Production in an Engineered Strain of  E. coli  Nissle 
     A number of tryptophan metabolites, either host-derived (such as tryptamine or kynurenine) or intestinal bacteria-derived (such as indole acetate or indole), have been shown to downregulate inflammation and promote gut barrier health, via the activation of the AhR receptor. Other tryptophan metabolites, such as the bacteria-derived indole propionate, have been shown to help restore intestinal barrier integrity, in experimental models of colitis. In this example, the  E. coli  strain Nissle was engineered to produce tryptophan, the precursor to all those beneficial metabolites. 
     First, in order to remove the negative regulation of tryptophan biosynthetic genes mediated by the transcription factor TrpR, the trpR gene was deleted form the  E. coli  Nissle genome. The tryptophan operon trpEDCBA was amplified by PCR from the  E. coli  Nissle genomic DNA and cloned in the low-copy plasmid pSC101 under the control of the tet promoter, downstream of the tetR repressor gene. This tet-trpEDCBA plasmid was then transformed into the ΔtrpR mutant to obtain the ΔtrpR, tet-trpEDCBA strain. Subsequently, a feedback resistant version of the aroG gene (aroG fbr ) from  E. coli  Nissle, coding for the enzyme catalyzing the first committing step towards aromatic amino acid production, was synthetized and cloned into the medium copy plasmid p15A, under the control of the tet promoter, downstream of the tetR repressor. This plasmid was transformed into the ΔtrpR, tet-trpEDCBA strain to obtain the ΔtrpR, tet-trpEDCBA, tet-aroG fbr  strain. Finally, a feedback resistant version of the tet-trpEBCDA construct (tet-trpE fbr  BCDA) was generated from the tet-trpEBCDA. Both the tet-aroG fbr  and the tet-trpE fbr  BCDA constructs were transformed into the ΔtrpR mutant to obtain the ΔtrpR, tet-trpE fbr  DCBA, tet-aroG fbr  strain. 
     All generated strains were grown in LB overnight with the appropriate antibiotics and subcultured 1/100 in 3 mL LB with antibiotics in culture tubes. After two hours of growth at 37 C at 250 rpm, 100 ng/mL anhydrotetracycline (ATC) was added to the culture to induce expression of the constructs. Two hours after induction, the bacterial cells were pelleted by centrifugation at 4,000 rpm for 5 min and resuspended in 3 mL M9 minimal media. Cells were spun down again at 4,000 rpm for 5 min, resuspended in 3 mL M9 minimal media with 0.5% glucose and placed at 37 C at 250 rpm. 200 uL were collected at 2 h, 4 h and 16 h and tryptophan was quantified by LC-MS/MS in the bacterial supernatant.  FIG. 45A  shows that tryptophan is being produced and secreted by the ΔtrpR, tet-trpEDCBA, tet-aroG fbr  strain. The production of tryptophan is significantly enhanced by expressing the feedback resistant version of trpE. 
     Example 53. Improved Tryptophan by Using a Non-PTS Carbon Source and by Deleting the tnaA Gene Encoding Tryptophanase 
     One of the precursor molecule to tryptophan in  E. coli  is phosphoenolpyruvate (PEP). Only 3% of available PEP is normally used to produce aromatic acids (that include tryptophan, phenylalanine and tyrosine). When  E. coli  is grown using glucose as a sole carbon source, 50% of PEP is used to import glucose into the cell using the phosphotransferase system (PTS). In order to increase tryptophan production, a non-PTS oxidized sugar, glucuronate, was used to test tryptophan secretion by the engineered  E. coli  Nissle strain ΔtrpR, tet-trpE fbr  DCBA, tet-aroG fbr . In addition, the tnaA gene, encoding the tryptophanase enzyme, was deleted in the ΔtrpR, tet-trpE fbr  DCBA, tet-aroG fbr  strain in order to block the conversion of tryptophan into indole to obtain the ΔtrpRΔtnaA, tet-trp fbr  DCBA, tet-aroG fbr  strain. 
     The ΔtrpR, tet-trp fbr  DCBA, tet-aroG fbr  and ΔtrpRΔtnaA, tet-trpE fbr  DCBA, tet-aroG fbr  strains were grown in LB overnight with the appropriate antibiotics and subcultured 1/100 in 3 mL LB with antibiotics in culture tubes. After two hours of growth at 37 C at 250 rpm, 100 ng/mL anhydrotetracycline (ATC) was added to the culture to induce expression of the constructs. Two hours after induction, the bacterial cells were pelleted by centrifugation at 4,000 rpm for 5 min and resuspended in 3 mL M9 minimal media. Cells were spun down again at 4,000 rpm for 5 min, resuspended in 3 mL M9 minimal media with 1% glucose or 1% glucuronate and placed at 37 C at 250 rpm or at 37 C in an anaerobic chamber. 200 uL were collected at 3 h and 16 h and tryptophan was quantified by LC-MS/MS in the bacterial supernatant.  FIG. 45B  shows that tryptophan production is doubled in aerobic condition when the non-PTS oxidized sugar glucoronate was used. In addition, the deletion of tnaA had a positive effect on tryptophan production at the 3 h time point in both aerobic and anaerobic conditions and at the 16 h time point, only in anaerobic condition. 
     Example 54. Improved Tryptophan Production by Increasing the Rate of Serine Biosynthesis in  E. coli  Nissle 
     The last step in the tryptophan biosynthesis in  E. coli  consumes one molecule of serine. In this example, we demonstrate that serine availability is a limiting factor for tryptophan production and describe the construction of the tryptophan producing  E. coli  Nissle strains ΔtrpRΔtnaA, tet-trpE fbr  DCBA, tet-aroG fbr  serA and ΔtrpRΔtnaA, tet-trpE fbr  DCBA, tet-aroG fbr  serA fbr  strains. 
     The ΔtrpRΔtnaA, tet-trpE fbr  DCBA, tet-aroG fbr  strain was grown in LB overnight with the appropriate antibiotics and subcultured 1/100 in 3 mL LB with antibiotics in culture tubes. After two hours of growth at 37 C at 250 rpm, 100 ng/mL anhydrotetracycline (ATC) was added to the culture to induce expression of the constructs. Two hours after induction, the bacterial cells were pelleted by centrifugation at 4,000 rpm for 5 min and resuspended in 3 mL M9 minimal media. Cells were spun down again at 4,000 rpm for 5 min, resuspended in 3 mL M9 minimal media with 1% glucuronate or 1% glucuronate and 10 mM serine and placed at 37 C an anaerobic chamber. 200 uL were collected at 3 h and 16 h and tryptophan was quantified by LC-MS/MS in the bacterial supernatant.  FIG. 45C  shows that tryptophan production is improved three-fold by serine addition. 
     In order to increase the rate of serine biosynthesis in the ΔtrpRΔtnaA, tet-trpE fbr  DCBA, tet-aroG fbr  strain, the serA gene from  E. coli  Nissle encoding the enzyme catalyzing the first step in the serine biosynthetic pathway was amplified by PCR and cloned into the tet-aroG fbr  plasmid by Gibson assembly. The newly generated tet-aroG fbr -serA construct was then transformed into a ΔtrpRΔtnaA, tet-trpE fbr  DCBA strain to generate the ΔtrpRΔtnaA, tet-trpE fbr  DCBA, tet-aroG fbr -serA strain. The tet-aroG fbr -serA construct was further modified to encode a feedback resistant version of serA (serA fbr ). The newly generated tet-aroG fbr -serA fbr  construct was used to produce the ΔtrpRΔtnaA, tet-trpE fbr  DCBA, tet-aroG fbr -serA fbr  strain, optimized to improve the rate of serine biosynthesis and maximize tryptophan production. 
     Example 55. Comparison of Various Tryptophan Producing Strains 
     Compare the rates of tryptophan production in the different strains generated, the following constructs and strains were generated according to methods and sequences described herein (e.g. Example 43), and assayed for tryptophan production in the presence of glucuronate as a carbon source under aerobic conditions. SYN2126 comprises ΔtrpRΔtnaA (ΔtrpRΔtnaA). SYN2323 comprises ΔtrpRΔtnaA and a tetracycline inducible construct for the expression of feedback resistant aroG on a plasmid (ΔtrpRΔtnaA, tet-aroGfbr). SYN2339 comprises ΔtrpRΔtnaA and a first tetracycline inducible construct for the expression of feedback resistant aroG on a first plasmid and a second tetracycline inducible construct with the genes of the trp operon with a feedback resistant form of trpE on a second plasmid (ΔtrpRΔtnaA, tet-aroGfbr, tet-trpEfbrDCBA). SYN2473 comprises ΔtrpRΔtnaA and a first tetracycline inducible construct for the expression of feedback resistant aroG and SerA on a first plasmid and a second tetracycline inducible construct with the genes of the trp operon with a feedback resistant form of trpE on a second plasmid (ΔtrpRΔtnaA, tet-aroGfbr-serA, tet-trpEfbrDCBA). SYN2476 comprises ΔtrpRΔtnaA and a tetracycline inducible construct with the genes of the trp operon with a feedback resistant form of trpE on a plasmid (ΔtrpRΔtnaA, tet-trpEfbrDCBA). 
     Overnight cultures were diluted 1/100 in 3 mL LB plus antibiotics and grown for 2 hours (37 C, 250 rpm). Next, cells were induced with 100 ng/mL ATC for 2 hours (37 C, 250 rpm), spun down, washed with cmL M9, spun down again and resuspended in 3 mL M9+1% glucuronate. Cells were plated for CFU counting. For the assay, the cells were placed af 37 C with shaking at 250 rpm. Supernatants were collected at 1 h, 2 h, 3 h, 4 h 16 h for HPLC analysis for tryptophan. As seen in  FIG. 46 , results indicate that expressing aroG is not sufficient nor necessary under these conditions to get Trp production and that expressing serA is beneficial for tryptophan production. 
     Example 56. Bacterial Production of Indole Acetic Acid (IAA) 
     The ability of a strain comprising tryptophan production circuits and additionally Indole-3-pyruvate decarboxylase from  Enterobacter cloacae  (IpdC) and Indole-3-acetaldehyde dehydrogenase from  Ustilago maydis  (lad1) to produce indole acetic acid (IAA) was tested. The following strains were generated according to methods described herein and tested. 
     SYN2126: comprises ΔtrpR and ΔtnaA (ΔtrpRΔtnaA). SYN2339 comprises circuitry for the production of tryptophan; ΔtrpR and ΔtnaA, a first tetracline inducible trpEfbrDCBA construct on a first plasmid (pSC101), and a second tetracycline inducible aroGfbr construct on a second plasmid (ΔtrpRΔtnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr (p15A)) ( FIG. 40B ). SYN2342 comprises the same tryptophan production circuitry as the parental strain SYN2339, and additionally comprises trpDH-ipdC-iad1 incorporated at the end of the second construct (ΔtrpRΔtnaA, tetR-Ptet-trpEfbrDCBA (pSC101), tetR-Ptet-aroGfbr-trpDH-ipdC-iad1 (p15A))( FIG. 43B ). 
     Overnight cultures of the strains were diluted 1/100 in 3 mL LB plus antibiotics and grown for 2 hours (37 C, 250 rpm). Strains were then induced with 100 ng/mL ATC for 2 hours (37 C, 250 rpm). Cells were spun down, and resuspended in 1 mL M9+1% glucuronic acid and CFUs were quantified CFUs using the cellometer. Supernatants were collected at 1 h, 2.5 h and 18 h for LCMS analysis of tryptophan and indole acetic acid as described herein. 
     As seen in  FIG. 49 , SYN2126 produced no tryptophan, SYN2339 produces increasing tryptophan over the time points measured, and SYN2342 containing the additional IAA producing circuitry produces amounts of IAA that are comparable to the amounts of tryptophan produced in its parent SYN2339. No tryptophan is measured, indicating that all tryptophan produced in SYN2342 is efficiently converted into IAA. 
     Example 57. Tryptamine Production Comparing Two Tryptophan Decarboxylases 
     The efficacy of two tryptophan decarboxylases (tdc), one from  Catharanthus roseus  (tdc Cr ) and a second from  Clostridium sporogenes  (tdc Cs ) in producing tryptamine from tryptophan was tested. The following strains were generated according to methods described herein and tested. 
     SYN2339 comprises ΔtrpR and ΔtnaA and a tetracycline inducible trpE fbr DCBA construct on a plasmid and another tetracycline inducible construct expressing aroG fbr  on a second plasmid (ΔtrpRΔtnaA, tetR-P tet -trpE fbr DCBA (pSC101), tetR-P tet -aroG fbr  (p15A)). SYN2339 is used as a control which can produce tryptophan but cannot convert it to tryptamine. SYN2340 comprises ΔtrpR and ΔtnaA and a tetracycline inducible trpE fbr DCBA construct on a plasmid and another tetracycline inducible construct expressing aroG fbr  tdc Cr  on a second plasmid (ΔtrpRΔtnaA, tetR-P tet -trpE fbr DCBA (pSC101), tetR-P tet -aroG fbr -tdc Cr  (p15A)). SYN2794 comprises ΔtrpR and ΔtnaA and a tetracycline inducible trpE fbr DCBA construct on a plasmid and another tetracycline inducible construct expressing aroG fbr  tdc Cs  on a second plasmid (ΔtrpRΔtnaA, tetR-P tet -trpE fbr DCBA (pSC101), tetR-P tet -aroG fbr -tdc Cs  (p15A)). 
     Overnight cultures of the strains were diluted 1/100 in 3 mL LB plus antibiotics and grown for 2 hours (37 C, 250 rpm). Strains were then induced with 100 ng/mL ATC for 2 hours (37 C, 250 rpm). Cells were spun down, and resuspended in 1 mL M9+1% glucuronic acid and CFUs were quantified CFUs using the cellometer. Supernatants were collected at 3 h and 18 h for LCMS analysis of tryptophan and tryptamine, as described herein. 
     As seen in  FIG. 51 , Tdc Cs  from  Clostridium sporogenes  is more efficient than Tdc Cr  from  Catharanthus roseus  in tryptamine production and converts all the tryptophan produced into tryptamine 
     Example 58. Tryptophan and Anthranilic Acid Quantification in Bacterial Supernatant by LC-MS/MS 
     Tryptophan and Anthranilic acid stock (10 mg/mL) were prepared in 0.5N HCl, aliquoted in 1.5 mL microcentrifuge tubes (100 μL), and stored at −20° C. Standards (250, 100, 20, 4, 0.8, 0.16, 0.032 μg/mL) were prepared in water. Samples (10 pL) and standards were mixed with 90 μL of ACN/H2O (60:30, v/v) containing 1 μg/mL of Tryptophan-d5 in the final solution in a V-bottom 96-well plate. The plate was heat-sealed with a AlumASeal foil, mixed well, and centrifuged at 4000 rpm for 5 min. The solution (10 μL) was transferred into a round-bottom 96-well plate 90 uL 0.1% formic acid in water was added to the sample. The plate was again heat-sealed with a ClearASeal sheet and mixed well. 
     LC-MS/MS Method 
     Tryptophan and Anthranilic acid were measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. Table 82, Table 83, and Table 84 provide the summary of the LC-MS/MS method. 
     
       
         
           
               
             
               
                 TABLE 82 
               
               
                   
               
               
                 HPLC Method 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Column 
                 Accucore aQ column, 2.6 μm (100 × 2.1 mm) 
               
               
                 Mobile Phase A 
                 99.9% H2O, 0.1% Formic Acid 
               
               
                 Mobile Phase B 
                 99.9% ACN, 0.1% Formic Acid 
               
               
                 Injection volume 
                 10 uL 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 83 
               
             
            
               
                   
               
               
                 HPLC Method: 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Time (min) 
                 Flow Rate (μL/min) 
                 A % 
                 B % 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 −0.5 
                 350 
                 100 
                 0 
               
               
                   
                 0.5 
                 350 
                 100 
                 0 
               
               
                   
                 1.0 
                 350 
                 10 
                 90 
               
               
                   
                 2.5 
                 350 
                 10 
                 90 
               
               
                   
                 2.51 
                 350 
                 100 
                 10 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 84 
               
               
                   
               
               
                 Tandem Mass Spectrometry 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Ion Source 
                 HESI-II 
               
               
                   
                 Polarity 
                 Positive 
               
            
           
           
               
            
               
                 SRM transitions 
               
            
           
           
               
               
               
            
               
                   
                 Tryptophan 
                 205.1/118.2 
               
               
                   
                 Anthranilic acid 
                 138.1/92.2 
               
               
                   
                 Tryptophan-d5 
                 210.1/151.1 
               
               
                   
                   
               
            
           
         
       
     
     Example 59. Quantification of Tryptamine in Bacterial Supernatant by Liquid Chromatography-Mass Spectrometry (LC-MS) 
     Tryptamine acid stock (10 mg/mL) were prepared in 0.5N HCl, aliquoted in 1.5 mL microcentrifuge tubes (100 μL), and stored at −20° C. Standards (250, 100, 20, 4, 0.8, 0.16, 0.032 μg/mL) were prepared. Samples (10 μL) and standards were mixed with 90 μL of ACN/H2O (60:30, v/v) containing 1 μg/mL of tryptamine-d5 in the final solution in a V-bottom 96-well plate. The plate was heat-sealed with a AlumASeal foil, mixed well, and centrifuged at 4000 rpm for 5 min. The solution (10 μL) was transferred into a round-bottom 96-well plate 90 uL 0.1% formic acid in water was added to the sample. The plate was again heat-sealed with a ClearASeal sheet and mixed well. 
     LC-MS/MS Method 
     Tryptamine was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using a Thermo TSQ Quantum Max triple quadrupole mass spectrometer. Table 85, Table 86, and Table 87 provide the summary of the LC-MS/MS method. 
     
       
         
           
               
             
               
                 TABLE 85 
               
               
                   
               
               
                 HPLC Method 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Column 
                 Accucore aQ column, 2.6 μm (100 × 2.1 mm) 
               
               
                 Mobile Phase A 
                 99.9% H2O, 0.1% Formic Acid 
               
               
                 Mobile Phase B 
                 99.9% ACN, 0.1% Formic Acid 
               
               
                 Injection volume 
                 10 uL 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 86 
               
             
            
               
                   
               
               
                 HPLC Method: 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Time (min) 
                 Flow Rate (μL/min) 
                 A % 
                 B % 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 −0.5 
                 350 
                 100 
                 0 
               
               
                   
                 0.5 
                 350 
                 100 
                 0 
               
               
                   
                 1.0 
                 350 
                 10 
                 90 
               
               
                   
                 2.5 
                 350 
                 10 
                 90 
               
               
                   
                 2.51 
                 350 
                 100 
                 10 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 87 
               
               
                   
               
               
                 Tandem Mass Spectrometry 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Ion Source 
                 HESI-II 
               
               
                   
                 Polarity 
                 Positive 
               
            
           
           
               
            
               
                 SRM transitions 
               
            
           
           
               
               
               
            
               
                   
                 Tryptamine 
                 161.1/144.1 
               
               
                   
                   
               
            
           
         
       
     
     Example 60. Quantification of Tryptophan, Indole-3-Acetate, Indole-3-Lactate, Indole-3-Propionate in Bacterial Supernatant by High-Pressure Liquid Chromatography (HPLC) 
     Samples were thawed on ice and centrifuged at 3,220×g for 5 min at 4° C. 80 μL of the supernatant was pipetted, mixed with 20 μL 0.5% formic acid in water, and analyzed by HPLC using a Shimadzu Prominence-I. HPLC conditions used for the quantification of tryptophan, indole-3-acetate, indole-3-lactate and indole-3-propionate are described in Table 88. 
     
       
         
           
               
             
               
                 TABLE 88 
               
               
                   
               
               
                 HPLC Analysis 
               
               
                   
               
             
            
               
                 Chromatography 
               
            
           
           
               
               
            
               
                 Calibration standards 
                 250, 100, 20, 4, 0.8 μg/mL 
               
               
                 Column 
                 Luna 3 μm C18(2) 100 Å, 100 × 2 mm 
               
               
                   
                 (catalog# 00D-4251-B0) 
               
               
                 Column Temperature 
                 40° C. 
               
               
                 Injection Volume 
                 10 μL 
               
               
                 Autosampler Temperature 
                 10° C. 
               
               
                 Flow Rate 
                 0.5 mL/min 
               
               
                 Mobile Phases 
                 A: water, 0.1% FA 
               
               
                   
                 B: acetonitrile, 0.1% FA 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Gradient 
                 Time (min) 
                 % A 
                 % B 
               
               
                   
               
               
                   
                 0 
                 90 
                 10 
               
               
                   
                 0.5 
                 90 
                 10 
               
               
                   
                 3 
                 10 
                 90 
               
               
                   
                 5 
                 10 
                 90 
               
               
                   
                 5.01 
                 90 
                 10 
               
            
           
           
               
               
               
            
               
                   
                 7 
                 (end) 
               
            
           
           
               
            
               
                 Detection: Photodiode Array Detector (PDA) 
               
            
           
           
               
               
            
               
                 Polarity 
                 Positive 
               
               
                 Start Wavelength 
                 190 nm 
               
               
                 End Wavelength 
                 800 nm 
               
               
                 Spectrum resolution 
                 512 
               
               
                 Slit Width 
                 8 nm 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Compound 
                 Wavelength (nm) 
                 Retention time (min) 
               
               
                   
               
               
                 Tryptophan 
                 274 
                 1.3 
               
               
                 Indole-3-acetate 
                 274 
                 3.5 
               
               
                 Indole-lactate 
                 274 
                 3.3 
               
               
                 Indole-3-propionate 
                 274 
                 3.7 
               
               
                   
               
            
           
         
       
     
     Example 61. Biochemical Analysis of Butyrate Production in SYN1001 
     SYN1001 was assessed for its ability to produce butyrate in vitro. An overnight culture of LB-grown SYN1001 was diluted 1:100 into fresh LB (10 mL in a 125 mL baffled flask). The culture was grown aerobically with shaking at 250 rpm, 37° C. for 1.5 h. The culture was then moved into an anaerobic chamber (Coy Lab Products, MI) supplying an atmosphere of 85% N 2 , 10% CO 2 , and 5% H 2 . Anaerobic incubation commenced at 37° C. for 4 hours in order to induce the expression of the butyrate operon from the P fnrS  promoter. 
     After the 4 hour anaerobic induction of the butyrate operon, the culture was removed from the anaerobic chamber and approximately 2×10 8  activated cells were used to inoculate 1 mL of M9 minimal medium containing 0.5% glucose. Assay cultures were incubated statically at 37° C. for 18 hours in the presence of 02. For sample collection, 200 uL aliquots were removed from assay cultures and spun down at maximum speed for 1 min in a microcentrifuge. The culture supernatant was retained, and LC-MS-MS was used to determine the concentration of butyrate in the supernatant fraction (Table 89-data are average of assay performed in triplicate for three different manufacturing runs). 
     
       
         
           
               
             
               
                 TABLE 89 
               
             
            
               
                   
               
               
                 Butyrate production in SYN1001 from three different experiments 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Butyrate 
                   
                 Butyrate 
                   
                 Butyrate 
                   
               
               
                   
                 (mM) 
                   
                 (mM) 
                   
                 (mM) 
               
               
                 Strain 
                 Run 1 
                 SD 
                 Run 2 
                 SD 
                 Run 3 
                 SD 
               
               
                   
               
               
                 SYN94 
                 NA 
                 NA 
                 NA 
                 NA 
                 0.474 
                 0.002 
               
               
                 SYN2001 
                 NA 
                 NA 
                 NA 
                 NA 
                 0.389 
                 0.003 
               
               
                 SYN1001 
                 6.371 
                 0.530 
                 6.131 
                 0.100 
                 6.982 
                 0.577 
               
               
                   
               
            
           
         
       
     
     Equivalent concentrations of butyrate were obtained from 3 independent production runs of SYN1001. In production run 3, SYN94 and SYN2001 control strains were included and supernatants from these strains contained negligible amounts of butyrate (0.47 and 0.38 mM respectively) compared to SYN1001, which contained significantly higher levels (6.98 mM; n=3). SYN94 is a streptomycin-resistant version of the parental  E. coli  Nissle strain. SYN2001 is an engineered  E. coli  strain that has been modified to over-produce acetate and does not contain a synthetic butyrate operon, described elsewhere herein. Run 3 culture supernatants were used to generate bioactivity in cell based assays described below. 
     Example 62. Cell-Based Assay Development and In-Vitro Butyrate Strain Assessment 
     Methods 
     Mammalian Cell Culture: 
     HT-29 colon adenocarcinoma cells were obtained from ATCC (Cat #: HTB38). Cells were cultured at 37° C., 5% CO 2  in RPMI media supplemented with 10% FBS, 1% pen-strep (complete media). Cells were allowed to grow to ˜80% confluency before passaging for activity assays. 
     Alkaline Phosphatase (AP) Activity Assay: 
     HT-29 colon adenocarcinoma cells were plated in complete media at either 1×10 5  cells/well (24 well plates) or 1×10 4  cells/well (96 well plates) and allowed to recover overnight at 37° C., 5% CO 2 . The following day media was replaced with fresh complete media containing either PBS, synthetic acetate (SIGMA-Cat # S8750) or butyrate (SIGMA-Cat # B5887), or bacterial supernatants of interest. Cells were incubated for 4 days under these conditions and then media was removed and cellular lysates were prepared (10 min on ice with vendor-supplied lysis buffer (BioVision-see below) followed by clarification for 10 min @14K rpm, 4° C.). Lysates from each condition were then assessed for AP activity using an alkaline phosphatase activity kit (BioVision, Cat # K412-500) according to manufacturer&#39;s recommendations. 
     Cell Viability Assay: 
     HT-29 colon adenocarcinoma cells were plated in 2 separate plates in complete media at either 1×10 5  cells/well (24-well plates) or 1×10 4  cells/well (96-well plates) and allowed to recover overnight at 37° C., 5% CO 2 . The following day, one plate of cells, which served as the day 1 time point read out (input), was treated with trypsin (5 min at 37° C., 5% CO 2 ) and cells were counted using a Cellometer K2 instrument (Nexcelom). Live and dead cells were distinguished by trypan blue exclusion. For the remaining plate, media was replaced with fresh complete media containing either PBS, synthetic acetate or butyrate, or bacterial supernatants of interest. Cells were incubated for 4 days under these conditions and then media was removed. Cells were detached from plates with trypsin and counted using the Cellometer K2 as described above. 
     In Vitro Assessment of Engineered Butyrate-Producing Strain SYN1001 
     To assess the activity of the butyrate-producing strain SYN001 in vitro, we employed the AP cell-based assay. HT-29 cells were plated in triplicate at 1×10 4  cells/well 96-well plates in complete media and allowed to recover overnight. The following day, media was removed and fresh media containing a dilution series of exogenous synthetic butyrate (5 mM-0.3 mM), or culture supernatants from the SYN94 control (0.26 mM-0.016 mM), SYN1001 butyrate-producing strain (3.5 mM-0.11 mM) or SYN2001 acetate-producing strain (0.22 mM-0.01 mM) were added, and the cells were incubated for 4 days. After the incubation period, media was removed and the plates were processed for assessment of AP activity.  FIG. 19B  shows that incubation of HT-29 cells with the supernatants from the butyrate-producing SYN1001 strain demonstrated a similar AP activity profile to cells incubated with synthetic butyrate. In contrast, the unengineered strain SYN94 or the acetate-producing strain SYN2001 had little to no effect on AP activity at any concentration tested. To better visualize the similarity in AP activity induction between synthetic butyrate and SYN1001-produced butyrate, the values from the AP activity assay were fit to a non-linear equation algorithm and graphed. As shown in  FIG. 19C , the activity profile for butyrate produced by SYN1001 is comparable to synthetic butyrate. Incubation with synthetic butyrate, SYN94, SYN1001 or SYN2001 did not have any appreciable effect on cell viability (Data not shown) 
     Summary 
     The results describe the design and evaluation of an engineered, butyrate-producing strain, SYN1001, which contains a modified butyrate module comprised of the trans-2-enoyl-CoA reductase (ter) gene from  Treponema denticola , the thiolase (thiA1), 3-hydroxybutyryl-CoA dehydrogenase (hbd), and crotonase (crt2) genes from  Clostridium difficile , and the thioesterase B gene (tesB), which is endogenous to  E. coli . SYN1001 is capable of producing ˜7 mM butyrate in vitro under the conditions described here. This in vitro butyrate production translates to activity in a cell-based assay that is comparable on an equimolar basis to that observed with pure, synthetic butyrate. Table 90. summarizes the final pharmacological characteristics of the SYN1001. 
     
       
         
           
               
             
               
                 TABLE 90 
               
             
            
               
                   
               
               
                 Final characterization of the pharmacological 
               
               
                 characteristics of SYN1001 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Run 3 
                   
                   
                   
               
               
                   
                 [Butyrate] 
                   
                 AP Activity 
               
               
                   
                 secreted 
                   
                 at max dose 
               
               
                   
                 (in mM) 
                 SD 
                 (in U/mg prot) 
                 SD 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SYN94 
                 0.474 
                 0.002 
                 0.96 
                 0.052 
               
               
                   
                 SYN2001 
                 0.389 
                 0.003 
                 0.95 
                 0.047 
               
               
                   
                 SYN1001 
                 6.982 
                 0.577 
                 3.97 
                 0.36 
               
               
                   
                   
               
            
           
         
       
     
     Example 63. In Vitro Assessment of the Engineered Acetate-Producing Strain SYN2001 
     To evaluate the activity of acetate-producing strains, we employed a cell-based assay based on work by Cox et al. (Cox et al., WGJ, 15(44), 2009) where the authors demonstrated that the addition of acetate inhibited LPS-induced secretion of IFNγ in human PBMC cells. 
     To assess the activity of the acetate-producing strain SYN2001 in vitro, we employed the LPS-induction of IFNγ cell-based assay. Frozen normal human PBMCs from two independent donors (Lot #&#39;s A4956 and A4924) were plated in triplicate at 1×10 6  cells/mL in 96-well plates in complete media. The cells were then incubated for 15 minutes with media containing either a dilution series of synthetic acetate (40 mM-0.08 Mm), SYN2001 supernatant (30 mM-0.03 mM acetate concentrations based on LC-MS determination) or untreated (negative control). After the 15-minute incubation period, complete media containing LPS was added to the cells to a final concentration of 100 ng/mL and the cells were further incubated overnight under these conditions. The following day supernatants were harvested from each of the different conditions and the IFNγ levels assessed by ELISA.  FIG. 26G  and  FIG. 26H  show the results from 3 independent experiments (each performed in triplicate) with the two different donors (donor 1=D1; donor 2=D2) in which incubation of primary human PBMC cells with exogenous acetate that was either synthetic or derived from SYN2001 supernatants led to a dose-dependent decrease in the LPS-induced secretion of IFNγ by the cells. We noted that the absolute levels of IFNγ production in the SYN2001 experiments was higher than in the purified acetate experiments, likely due to residual additional LPS in the supernatants from the bacterially-derived acetate. Nonetheless, the IC50s observed for the two acetate sources were very similar. Table 91 summarizes the data from the 3 experiments using the 2 separate donors. 
     
       
         
           
               
             
               
                 TABLE 91 
               
             
            
               
                   
               
               
                 Summary of EC50&#39;s for SYN2001 on LPS-induced IFNγ 
               
               
                 secretion from 3 experiments performed in triplicate 
               
               
                 with human PBMC cells from 2 separate donors. 
               
            
           
           
               
               
               
            
               
                   
                 Donor 1 
                 Donor 2 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Acetate (mM) 
                 3.12 
                 2.18 
               
               
                   
                 SEM 
                 0.29 
                 0.38 
               
               
                   
                 SYN1592 (mM) 
                 5.44 
                 3.96 
               
               
                   
                 SEM 
                 0.92 
                 0.19 
               
               
                   
                   
               
            
           
         
       
     
     In conclusion, results presented describe the design and evaluation of an engineered, acetate-producing strain, SYN2001, which contains an enhanced acetate biosynthetic program resulting from deletion of the L-lactate dehydrogenase A (ldhA) gene to block the carbon flux from pyruvate to lactate, greatly improving acetate biosynthesis in  E. coli  Nissle. This strain is capable of producing &gt;30 mM acetate in vitro under the conditions described here. This in vitro acetate production translates to activity in a cell-based assay that is comparable on an equimolar basis to that observed with pure, synthetic acetate. The final pharmacological characterization of SYN2001 is summarized in Table 92. 
     
       
         
           
               
             
               
                 TABLE 92 
               
             
            
               
                   
               
               
                 Final pharmacological characterization of SYN2001 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Run 3 
                   
                   
                   
                   
               
               
                   
                 [Acetate] 
                 EC50- 
                   
                 EC50- 
               
               
                   
                 secreted 
                 donor 1 
                   
                 donor 2 
               
               
                   
                 (mM) 
                 (mM) 
                 SEM 
                 (mM) 
                 SEM 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 SYN2001 
                 31.58 
                 5.44 
                 0.92 
                 3.96 
                 0.19 
               
               
                 Acetate 
                 NA 
                 3.12 
                 0.39 
                 2.18 
                 0.28 
               
               
                 (synthetic) 
               
               
                   
               
            
           
         
       
     
     Example 64. Generation and Analysis of an Engineered IL-22-Producing  E. coli  Nissle Strain 
     Engineering and Production of IL-22 
     A synthetic construct was generated in which expression of IL-22 is controlled by the tetracycline-inducible promoter (P tet ), which is derepressed via the addition of the tetracycline analog anhydrotetracycline (aTc), and translation is driven by a strong ribosome binding site (RBS) located immediately upstream from the IL-22 coding sequence. To promote translocation to the periplasm, a 21-amino acid PhoA-secretion tag was added to the N-terminus of IL-22. 
     The corresponding engineered element was constructed using a synthetic DNA cassette encoding the IL-22 protein coding sequence (IDT Technologies, Coralville, Iowa) which was cloned into an initial plasmid vector, creating the plasmid Logic435. The IL-22 sequence was later amplified and cloned using Gibson assembly technology and the NEBuilder Hifi Mastermix (NEB). The final pBR322-based plasmid was sequence-verified by Sanger sequencing (Genewiz) and designated Logic522. 
     To create a Gram-negative bacterium capable of secreting bioactive proteins, a diffusible outer membrane (DOM) phenotype was engineered in the  E. coli  Nissle background. A series of DOM mutants were created by deleting different periplasmic proteins leading to a ‘leaky’ phenotype. Deletions of several different genes were tested including lpp, pal, tolA and nlpl. For example, the pal mutant (SYN3000) showed a good secretion phenotype with little-to-no deleterious effect on growth rate while supporting strong production of effectors in the extracellular medium. Logic522 was inserted into SYN3000 to create the IL-22 secretion strain, SYN3001. 
     To assay for production of IL-22, cultures were grown and induced, then supernatants were harvested and quantified using ELISA. Overnight cultures were harvested by centrifugation at 12.5K×g for 5 minutes. The supernatants of the cultures were removed from the cell pellet and filtered through a 0.22 μm filter to separate any remaining bacteria from the supernatant. This supernatant was run immediately in the ELISA, stored short-term at 4° C., or aliquoted and stored at −20° C. 
     To evaluate the production of IL-22 in the filtered supernatants, samples of SYN3000 and SYN3001 were diluted in triplicate and run on an R&amp;D Systems IL-22 Quantikine® ELISA Kit (Minneapolis, Minn.). The results from 3 independent production runs are shown in Table 93. The results demonstrated that the SYN3001 supernatants contained an average of 312 ng/ml (+/−11.38) of material that reacted positively in the IL-22 ELISA assay. In contrast, the SYN3000 supernatants had undetectable levels (not shown). Culture supernatant from run 3 was then used to generate the bioactivity results from the cell based assays described below. 
     
       
         
           
               
             
               
                 TABLE 93 
               
             
            
               
                   
               
               
                 SYN3001 supernatant results from three different production runs. 
               
            
           
           
               
               
               
               
            
               
                   
                 Run1 
                 Run 2 
                 Run 3 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 [IL-22] 
                   
                 [IL-22] 
                   
                 [IL-22] 
                   
               
               
                 Strain 
                 in ng/ml 
                 SD 
                 in ng/ml 
                 SD 
                 in ng/ml 
                 SD 
               
               
                   
               
               
                 SYN3001 
                 325.01 
                 8.40 
                 303.56 
                 2.94 
                 307.67 
                 6.21 
               
               
                   
               
            
           
         
       
     
     In Vitro Assessment of IL-22 Produced by the Engineered Strain SYN3001 
     To assess the biological activity of IL-22 produced by SYN3001 (IL-22 secreting strain), titrations of SYN3001 and SYN3000 (DOM mutant, non IL-22 secreting negative control strain) supernatants (starting at 150 ng/mL and titrated in 1:3 dilutions) were added to Colo205 cells and the activation of STAT3 was assessed.  FIG. 33C  shows the results from 5 independent experiments (each performed in triplicate). Supernatants from SYN3001 induced activation of STAT3 with an average EC50 of 4.8 ng/mL (+/−1.74 ng/mL). In contrast, SYN3000 had no effect on STAT3 activity. 
     To verify that the STAT3 activation elicited by supernatants from SYN3001 was indeed due to IL-22 signaling, Colo205 cells were stimulated with IL-22 supernatants derived from SYN3001 at 3 ng/mL in the presence of increasing concentrations of an anti-IL-22 neutralizing antibody. rLI-22 in the absence of the neutralizing antibody served as a positive control.  FIG. 33D  shows the results from 3 independent experiments (performed in triplicate), demonstrating that the anti-IL-22 antibody inhibited SYN3001-induced activation of STAT3 in a dose-dependent manner. The average IC50 for the anti-IL-22 antibody mediated inhibition of SYN3001-derived IL-22 was 3.45 ng/mL for SYN3001, in line with the value observed using rIL-22, 3.70 ng/mL. 
     Summary 
     The results describe the design and evaluation of an engineered IL-22 producing strain, SYN3001, which contains a tetracycline-inducible promoter driving the expression of IL-22 fused to a cleavable PhoA-secretion tag to mediate Sec-dependent secretion into the periplasm and a pal mutation to create a diffusible outer membrane phenotype (DOM) that facilitates extracellular secretion. This strain is capable of producing &gt;300 ng/mL IL-22 in vitro under the conditions described here. This in vitro IL-22 production translates to biological activity in a cell-based assay that is comparable to that observed with recombinant IL-22. In addition, the specific activity of the bacterially-produced IL-22 was verified by demonstrating that this signal could be inhibited by a neutralizing antibody against IL-22. Table 94 summarizes the final pharmacological characteristics of SYN3001. 
     
       
         
           
               
             
               
                 TABLE 94 
               
             
            
               
                   
               
               
                 Final characterization of the pharmacological 
               
               
                 characteristics of SYN300 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Run 3 
                   
                   
                   
                   
                   
               
               
                   
                 [IL-22] 
               
               
                   
                 secreted 
                   
                 EC50 
                   
                 IC50 
               
               
                   
                 (ng/mL) 
                 SD 
                 (ng/mL) 
                 SD 
                 (ng/mL) 
                 SD 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 SYN3001 
                 307.7 
                 6.21 
                 4.80 
                 1.74 
                 3.45 
                 0.37 
               
               
                 rIL-22 
                 NA 
                 NA 
                 1.56 
                 0.82 
                 3.70 
                 0.52 
               
               
                   
               
            
           
         
       
     
     Example 65. Generation and Testing of an IL-10 Producing Strain 
     Strain Construction 
     In order to generate strains which secrete human IL-10, a base strain was used which has a “leaky membrane” phenotype (SYN557), comprising delta PAL DOM background). For plasmid-based secretion construct, a codon optimized human IL10 sequence was combined with a number of secretion signals to determine the optimal configuration. Recently the Nissle genome was mined bioinformatically for signal sequences larger than the 21 AA PhoA tag. This yielded several candidates including the signal sequences from: ECOLIN_05715 (52 AA), ECOLIN_16495 (40 AA), ECOLIN_19410 (33 AA) and ECOLIN_19880 (53 AA). These signal sequences, were codon optimized and synthesized along with optimized RBS sequences, then inserted upstream of an optimized hIL10 sequence in a high copy pUC57 backbone. All of the candidate hIL10 constructs were then transformed into the delta PAL DOM background to test for secretion. 
     Production of hIL10 for In Vitro Quantification 
     To assay for production of bioactive hIL10 from  E. coli  Nissle, candidate strains were grown, induced and supernatants harvested and filter sterilized. These supernatants were then quantified via ELISA for hIL10 concentration corresponding to secreted hIL10. 
     Briefly, overnight cultures were used to inoculate 50 mL starter cultures of 2YT broth at a 1:50 dilution, and bacteria were grown for 2 hours and harvested by centrifugation at 12.8K×g for 5 minutes. The pellet was resuspended in 50 mL of fresh 2YT media with aTc (100 ug/mL) and appropriate antibiotic, and cells were induced at 30 C for an additional 4 hours to allow expression and secretion of hIL10. Supernatants were harvested via centrifugation and filtration through a 0.22 micron PVDF filter then used for Western blot analysis and in BD OptEIA Human IL10 ELISA Kit II (Cat. No. 550613) both according to manufacturers instruction.  FIG. 33E  depicts a Western blot analysis of bacterial supernatants from strain SYN2980 and SYN2982, using IL-10 antibody (IL-10 (D13A11) XP® Rabbit mAb #12163, Cell Signaling Technology). The secreted polypeptide has the same molecular weight as the standards, indicating that the signal sequence is cleaved from the native peptide. Results from the ELISA are shown in Table 95A. Selected secretion sequences are shown in Table 95B. 
     
       
         
           
               
             
               
                 TABLE 95A 
               
             
            
               
                   
               
               
                 ELISA results 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 [hIL10] 
               
               
                 STRAIN 
                 Genotype 
                 Circuit 
                 (ng/mL) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 SYN1557 
                 pal::Cm 
                 None 
                 0 
               
               
                 SYN2898 
                 pal::Cm 
                 Ptet-OmpFss-hIL10 p15a 
                 37 
               
               
                 SYN2890 
                 pal::Cm 
                 Ptet-ECOLIN_05715ss-hIL10 pUC57 
                 84 
               
               
                 SYN2892 
                 pal::Cm 
                 Ptet-ECOLIN_19410ss-hIL10 pUC57 
                 306 
               
               
                 SYN2893 
                 pal::Cm 
                 Ptet-ECOLIN_19880ss-hIL10 pUC57 
                 224 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 95B 
               
             
            
               
                   
               
               
                 Selected Sequences 
               
            
           
           
               
               
            
               
                 Description 
                 Sequences 
               
               
                   
               
               
                 Construct 
                 CAATATCCTCGTAATCCTAAGGACGCCACTATGAAACGCCATCTGAAC 
               
               
                 comprising RBS- 
                 ACCTCCTATCGCTTAGTCTGGAACCATATTACCGGTGCTTTCGTTGTTG 
               
               
                 ECOLIN_05715 
                 CATCAGAGCTGGCCCGCGCTCGTGGTAAGCGTGCAGGCGTGGCGGTA 
               
               
                 Secretion signal- 
                 GCCTTAAGCCTGGCAGCAGCAACATCTTTGCCTGCGTTAGCATCGCCA 
               
               
                 codon optimized 
                 GGTCAAGGAACGCAGTCAGAGAATTCATGCACTCACTTTCCGGGCAAT 
               
               
                 IL-10 
                 CTGCCGAATATGCTGCGCGATCTGCGAGATGCATTCTCTCGCGTGAAA 
               
               
                 SEQ ID NO: 
                 ACGTTCTTTCAAATGAAAGATCAACTGGATAATCTGCTGCTGAAGGAG 
               
               
                   
                 TCGTTGTTGGAGGATTTTAAGGGGTATCTGGGTTGTCAAGCACTGTCT 
               
               
                   
                 GAAATGATTCAATTTTACTTGGAGGAAGTTATGCCGCAAGCGGAAAAC 
               
               
                   
                 CAAGATCCGGATATTAAGGCGCACGTGAACTCACTGGGCGAAAACCT 
               
               
                   
                 GAAAACTTTGCGCCTGCGTCTGAGACGATGTCACCGATTCCTGCCGTG 
               
               
                   
                 TGAAAACAAGTCAAAGGCGGTTGAGCAAGTTAAGAATGCTTTCAATA 
               
               
                   
                 AGCTGCAAGAAAAGGGCATCTATAAAGCGATGTCTGAATTTGATATCT 
               
               
                   
                 TTATAAACTACATAGAAGCTTATATGACTATGAAGATTCGAAATTAA 
               
               
                   
               
               
                 RBS 
                 CAATATCCTCGTAATCCTAAGGACGCCACT 
               
               
                   
               
               
                 SEQ ID NO: 
                   
               
               
                   
               
               
                 ECOLIN_05715 
                 ATGAAACGCCATCTGAACACCTCCTATCGCTTAGTCTGGAACCATATT 
               
               
                 Secretion signal 
                 ACCGGTGCTTTCGTTGTTGCATCAGAGCTGGCCCGCGCTCGTGGTAAG 
               
               
                 SEQ ID NO: 
                 CGTGCAGGCGTGGCGGTAGCCTTAAGCCTGGCAGCAGCAACATCTTTG 
               
               
                   
                 CCTGCGTTAGCA 
               
               
                   
               
               
                 codon optimized 
                 TCGCCAGGTCAAGGAACGCAGTCAGAGAATTCATGCACTCACTTTCCG 
               
               
                 IL-10 
                 GGCAATCTGCCGAATATGCTGCGCGATCTGCGAGATGCATTCTCTCGC 
               
               
                 SEQ ID NO: 
                 GTGAAAACGTTCTTTCAAATGAAAGATCAACTGGATAATCTGCTGCTG 
               
               
                   
                 AAGGAGTCGTTGTTGGAGGATTTTAAGGGGTATCTGGGTTGTCAAGCA 
               
               
                   
                 CTGTCTGAAATGATTCAATTTTACTTGGAGGAAGTTATGCCGCAAGCG 
               
               
                   
                 GAAAACCAAGATCCGGATATTAAGGCGCACGTGAACTCACTGGGCGA 
               
               
                   
                 AAACCTGAAAACTTTGCGCCTGCGTCTGAGACGATGTCACCGATTCCT 
               
               
                   
                 GCCGTGTGAAAACAAGTCAAAGGCGGTTGAGCAAGTTAAGAATGCTT 
               
               
                   
                 TCAATAAGCTGCAAGAAAAGGGCATCTATAAAGCGATGTCTGAATTTG 
               
               
                   
                 ATATCTTTATAAACTACATAGAAGCTTATATGACTATGAAGATTCGAA 
               
               
                   
                 ATTAA 
               
               
                   
               
               
                 Construct 
                 TTAACAAAGATAGTTATCGCAGTAGGAGGCCCCCATGTTTTGGCGCGA 
               
               
                 comprising RBS- 
                 CATGACACTTTCGGTGTGGCGCAAAAAAACGACTGGCCTTAAAACTAA 
               
               
                 ECOLIN_16495 
                 GAAGCGTTTACTGGCTTTGGTATTGGCTGCTGCATTGTGCTCAAGCCCT 
               
               
                 Secretion signal- 
                 GTCTGGGCGTCGCCAGGTCAAGGAACGCAGTCAGAGAATTCATGCAC 
               
               
                 codon optimized 
                 TCACTTTCCGGGCAATCTGCCGAATATGCTGCGCGATCTGCGAGATGC 
               
               
                 IL-10 
                 ATTCTCTCGCGTGAAAACGTTCTTTCAAATGAAAGATCAACTGGATAA 
               
               
                 SEQ ID NO: 
                 TCTGCTGCTGAAGGAGTCGTTGTTGGAGGATTTTAAGGGGTATCTGGG 
               
               
                   
                 TTGTCAAGCACTGTCTGAAATGATTCAATTTTACTTGGAGGAAGTTAT 
               
               
                   
                 GCCGCAAGCGGAAAACCAAGATCCGGATATTAAGGCGCACGTGAACT 
               
               
                   
                 CACTGGGCGAAAACCTGAAAACTTTGCGCCTGCGTCTGAGACGATGTC 
               
               
                   
                 ACCGATTCCTGCCGTGTGAAAACAAGTCAAAGGCGGTTGAGCAAGTT 
               
               
                   
                 AAGAATGCTTTCAATAAGCTGCAAGAAAAGGGCATCTATAAAGCGAT 
               
               
                   
                 GTCTGAATTTGATATCTTTATAAACTACATAGAAGCTTATATGACTATG 
               
               
                   
                 AAGATTCGAAATTAA 
               
               
                   
               
               
                 RBS 
                 TTAACAAAGATAGTTATCGCAGTAGGAGGCCCCC 
               
               
                 SEQ ID NO: 
                   
               
               
                   
               
               
                 ECOLIN_16495 
                 ATGTTTTGGCGCGACATGACACTTTCGGTGTGGCGCAAAAAAACGACT 
               
               
                 Secretion signal 
                 GGCCTTAAAACTAAGAAGCGTTTACTGGCTTTGGTATTGGCTGCTGCA 
               
               
                 SEQ ID NO: 
                 TTGTGCTCAAGCCCTGTCTGGGCG 
               
               
                   
               
               
                 codon optimized 
                 TCGCCAGGTCAAGGAACGCAGTCAGAGAATTCATGCACTCACTTTCCG 
               
               
                 IL-10 
                 GGCAATCTGCCGAATATGCTGCGCGATCTGCGAGATGCATTCTCTCGC 
               
               
                 SEQ ID NO: 
                 GTGAAAACGTTCTTTCAAATGAAAGATCAACTGGATAATCTGCTGCTG 
               
               
                   
                 AAGGAGTCGTTGTTGGAGGATTTTAAGGGGTATCTGGGTTGTCAAGCA 
               
               
                   
                 CTGTCTGAAATGATTCAATTTTACTTGGAGGAAGTTATGCCGCAAGCG 
               
               
                   
                 GAAAACCAAGATCCGGATATTAAGGCGCACGTGAACTCACTGGGCGA 
               
               
                   
                 AAACCTGAAAACTTTGCGCCTGCGTCTGAGACGATGTCACCGATTCCT 
               
               
                   
                 GCCGTGTGAAAACAAGTCAAAGGCGGTTGAGCAAGTTAAGAATGCTT 
               
               
                   
                 TCAATAAGCTGCAAGAAAAGGGCATCTATAAAGCGATGTCTGAATTTG 
               
               
                   
                 ATATCTTTATAAACTACATAGAAGCTTATATGACTATGAAGATTCGAA 
               
               
                   
                 ATTAA 
               
               
                   
               
               
                 Construct 
                 TCAACTCAGTCTACAACATCGGAGGTTAAGAATGGGGTACAAAATGA 
               
               
                 comprising RBS- 
                 ACATTAGCTCGCTTCGCAAAGCATTCATTTTTATGGGGGCTGTTGCAG 
               
               
                 ECOLIN_19410 
                 CTTTAAGCCTTGTCAATGCCCAGTCAGCGCTTGCCTCGCCAGGTCAAG 
               
               
                 Secretion signal- 
                 GAACGCAGTCAGAGAATTCATGCACTCACTTTCCGGGCAATCTGCCGA 
               
               
                   
                 ATATGCTGCGCGATCTGCGAGATGCATTCTCTCGCGTGAAAACGTTCT 
               
               
                   
               
               
                 codon optimized 
                 TTCAAATGAAAGATCAACTGGATAATCTGCTGCTGAAGGAGTCGTTGT 
               
               
                 IL-10 
                 TGGAGGATTTTAAGGGGTATCTGGGTTGTCAAGCACTGTCTGAAATGA 
               
               
                 SEQ ID NO: 
                 TTCAATTTTACTTGGAGGAAGTTATGCCGCAAGCGGAAAACCAAGATC 
               
               
                   
                 CGGATATTAAGGCGCACGTGAACTCACTGGGCGAAAACCTGAAAACT 
               
               
                   
                 TTGCGCCTGCGTCTGAGACGATGTCACCGATTCCTGCCGTGTGAAAAC 
               
               
                   
                 AAGTCAAAGGCGGTTGAGCAAGTTAAGAATGCTTTCAATAAGCTGCA 
               
               
                   
                 AGAAAAGGGCATCTATAAAGCGATGTCTGAATTTGATATCTTTATAAA 
               
               
                   
                 CTACATAGAAGCTTATATGACTATGAAGATTCGAAATTAA 
               
               
                   
               
               
                 RBS 
                 TCAACTCAGTCTACAACATCGGAGGTTAAGA 
               
               
                 SEQ ID NO: 
                   
               
               
                   
               
               
                 ECOLIN_19410 
                 ATGGGGTACAAAATGAACATTAGCTCGCTTCGCAAAGCATTCATTTTT 
               
               
                 Secretion signal 
                 ATGGGGGCTGTTGCAGCTTTAAGCCTTGTCAATGCCCAGTCAGCGCTT 
               
               
                 SEQ ID NO: 
                 GCC 
               
               
                   
               
               
                 codon optimized 
                 TCGCCAGGTCAAGGAACGCAGTCAGAGAATTCATGCACTCACTTTCCG 
               
               
                 IL-10 
                 GGCAATCTGCCGAATATGCTGCGCGATCTGCGAGATGCATTCTCTCGC 
               
               
                 SEQ ID NO: 
                 GTGAAAACGTTCTTTCAAATGAAAGATCAACTGGATAATCTGCTGCTG 
               
               
                   
                 AAGGAGTCGTTGTTGGAGGATTTTAAGGGGTATCTGGGTTGTCAAGCA 
               
               
                   
                 CTGTCTGAAATGATTCAATTTTACTTGGAGGAAGTTATGCCGCAAGCG 
               
               
                   
                 GAAAACCAAGATCCGGATATTAAGGCGCACGTGAACTCACTGGGCGA 
               
               
                   
                 AAACCTGAAAACTTTGCGCCTGCGTCTGAGACGATGTCACCGATTCCT 
               
               
                   
                 GCCGTGTGAAAACAAGTCAAAGGCGGTTGAGCAAGTTAAGAATGCTT 
               
               
                   
                 TCAATAAGCTGCAAGAAAAGGGCATCTATAAAGCGATGTCTGAATTTG 
               
               
                   
                 ATATCTTTATAAACTACATAGAAGCTTATATGACTATGAAGATTCGAA 
               
               
                   
                 ATTAA 
               
               
                   
               
               
                 Construct 
                 CCCGTATAACACTAAGAGAGGCGAATTAGAGTATGAATAAGATTTTCA 
               
               
                 comprising RBS- 
                 AGGTCATTTGGAATCCTGCGACCGGAAGCTACACGGTTGCAAGTGAA 
               
               
                 ECOLIN_19880 
                 ACCGCTAAATCCCGTGGAAAAAAGAGTGGCCGCAGTAAATTACTTATC 
               
               
                 Secretion signal- 
                 TCTGCTTTGGTCGCAGGAGGGTTATTATCCTCATTCGGCGCTTCAGCGT 
               
               
                 codon optimized 
                 CGCCAGGTCAAGGAACGCAGTCAGAGAATTCATGCACTCACTTTCCGG 
               
               
                 IL-10 
                 GCAATCTGCCGAATATGCTGCGCGATCTGCGAGATGCATTCTCTCGCG 
               
               
                 SEQ ID NO: 
                 TGAAAACGTTCTTTCAAATGAAAGATCAACTGGATAATCTGCTGCTGA 
               
               
                   
                 AGGAGTCGTTGTTGGAGGATTTTAAGGGGTATCTGGGTTGTCAAGCAC 
               
               
                   
                 TGTCTGAAATGATTCAATTTTACTTGGAGGAAGTTATGCCGCAAGCGG 
               
               
                   
                 AAAACCAAGATCCGGATATTAAGGCGCACGTGAACTCACTGGGCGAA 
               
               
                   
                 AACCTGAAAACTTTGCGCCTGCGTCTGAGACGATGTCACCGATTCCTG 
               
               
                   
                 CCGTGTGAAAACAAGTCAAAGGCGGTTGAGCAAGTTAAGAATGCTTT 
               
               
                   
                 CAATAAGCTGCAAGAAAAGGGCATCTATAAAGCGATGTCTGAATTTG 
               
               
                   
                 ATATCTTTATAAACTACATAGAAGCTTATATGACTATGAAGATTCGAA 
               
               
                   
                 ATTAA 
               
               
                   
               
               
                 RBS 
                 CCCGTATAACACTAAGAGAGGCGAATTAGAGT 
               
               
                 SEQ ID NO: 
                   
               
               
                   
               
               
                 ECOL1N_19880 
                 ATGAATAAGATTTTCAAGGTCATTTGGAATCCTGCGACCGGAAGCTAC 
               
               
                 Secretion signal 
                 ACGGTTGCAAGTGAAACCGCTAAATCCCGTGGAAAAAAGAGTGGCCG 
               
               
                 SEQ ID NO: 
                 CAGTAAATTACTTATCTCTGCTTTGGTCGCAGGAGGGTTATTATCCTCA 
               
               
                   
                 TTCGGCGCTTCAGCG 
               
               
                   
               
               
                 codon optimized 
                 TCGCCAGGTCAAGGAACGCAGTCAGAGAATTCATGCACTCACTTTCCG 
               
               
                 IL-10 
                 GGCAATCTGCCGAATATGCTGCGCGATCTGCGAGATGCATTCTCTCGC 
               
               
                 SEQ ID NO: 
                 GTGAAAACGTTCTTTCAAATGAAAGATCAACTGGATAATCTGCTGCTG 
               
               
                   
                 AAGGAGTCGTTGTTGGAGGATTTTAAGGGGTATCTGGGTTGTCAAGCA 
               
               
                   
                 CTGTCTGAAATGATTCAATTTTACTTGGAGGAAGTTATGCCGCAAGCG 
               
               
                   
                 GAAAACCAAGATCCGGATATTAAGGCGCACGTGAACTCACTGGGCGA 
               
               
                   
                 AAACCTGAAAACTTTGCGCCTGCGTCTGAGACGATGTCACCGATTCCT 
               
               
                   
                 GCCGTGTGAAAACAAGTCAAAGGCGGTTGAGCAAGTTAAGAATGCTT 
               
               
                   
                 TCAATAAGCTGCAAGAAAAGGGCATCTATAAAGCGATGTCTGAATTTG 
               
               
                   
                 ATATCTTTATAAACTACATAGAAGCTTATATGACTATGAAGATTCGAA 
               
               
                   
                 ATTAA 
               
               
                   
               
            
           
         
       
     
     Functional Assays 
     Co-Culture Studies 
     To determine whether the hIL-10 expressed by the genetically engineered bacteria is biologically functional, in vitro experimentation is conducted, in which the bacterial supernatant containing secreted human IL-10 is added to the growth medium of THP-1 cells. IL-10 is known to induce the phosphorylation of STAT1 and STAT3 in these cells. Functional activity of bacterially secreted IL-10 is therefore assessed by its ability to phosphorylate STAT3 in THP-1 cells. 
     THP-1 cells are grown in Dulbecco&#39;s modified Eagle&#39;s medium supplemented with 10% fetal bovine serum at 37° C. in a humidified incubator supplemented with 5% CO2. Prior to treatment with the bacterial supernatant, THP-1 (1e6/24 well) are serum starved overnight. Titrations of either recombinant human IL-10 diluted in LB or clarified supernatant from wild type Nissle or the engineered bacteria are added to cells for 30 minutes. Cells are harvested and resuspended in lysis buffer, and phospho-STAT3 ELISA (ELISA pSTAT3 (Tyr705) (Cell Signaling Technology)) is run in triplicate for all samples, according to manufacturer&#39;s instructions. PBS-treated cells and PBS are added as negative controls. Dilutions of samples are included to demonstrate linearity. No signal is observed for wild type Nissle. Activity for the engineered strain comprising a PAL deletion and the integrated construct encoding hIL-10 with a various secretion tags as listed in Table 95 above are measured. 
     Competition Studies 
     As an additional control for specificity, a competition assay is performed. Titrations of anti-IL10 antibody are pre-incubated with constant concentrations of either rhIL10 (data not shown) or supernatants from the engineered bacteria for 15 min. Next, the supernatants/rhIL-10solutions are added to serum-starved THP-1 cells (1e6/well) and cells are incubated for 30 min followed by pSTAT3 ELISA as described above. 
     Example 66. Assessment of In Vitro and In Vivo Activity of Biosafety System Containing Strain 
     The activity of the following strains is tested: 
     SYN-1001 comprises a construct shown in  FIG. 74C  knocked into the dapA locus on the bacterial chromosome (low copy RBS; dapA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in  FIG. 74A , except that the bla gene is replaced with the construct of  FIG. 24C  (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used. 
     SYN-1002 comprises a construct shown in  FIG. 74C  knocked into the dapA locus on the bacterial chromosome (low copy RBS; dapA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in  FIG. 74A , except that the bla gene is replaced with the construct of  FIG. 24D  (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used. 
     SYN-1003 comprises a construct shown in  FIG. 74D  knocked into the dapA locus on the bacterial chromosome (medium copy RBS; dapA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in  FIG. 74A , except that the bla gene is replaced with the construct of  FIG. 24C  (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used. 
     SYN-1004 comprises a construct shown in  FIG. 74D  knocked into the dapA locus on the bacterial chromosome (medium copy RBS; dapA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in  FIG. 74A , except that the bla gene is replaced with the construct of  FIG. 24D  (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used. 
     SYN-1005 comprises a construct shown in  FIG. 74C  knocked into the thyA locus on the bacterial chromosome (low copy RBS; thyA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in  FIG. 74B , except that the bla gene is replaced with the construct of  FIG. 24C  (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used. 
     SYN-1006 comprises a construct shown in  FIG. 74C  knocked into the thyA locus on the bacterial chromosome (low copy RBS; thyA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in  FIG. 74B , except that the bla gene is replaced with the construct of  FIG. 24D  (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used. 
     SYN-1007 comprises a construct shown in  FIG. 74D  knocked into the thyA locus on the bacterial chromosome (medium copy RBS; thyA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in  FIG. 74B , except that the bla gene is replaced with the construct of  FIG. 24D  (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used. 
     SYN-1008 a construct shown in  FIG. 74D  knocked into the thyA locus on the bacterial chromosome (medium copy RBS; thyA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in  FIG. 74B , except that the bla gene is replaced with the construct of  FIG. 24D  (OmpF-hGLP-1). On other embodiments, other inducible or constitutive promoters are used. 
     SYN-1009 a construct shown inf  FIG. 74C  knocked into the dapA locus on the bacterial chromosome (low copy RBS; dapA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in  FIG. 74A , except that the bla gene is replaced with the construct of  FIG. 8A  (FNR-ter/pbt-buk butyrate cassette). On other embodiments, other inducible or constitutive promoters are used. 
     SYN-1011 comprises a construct shown in  FIG. 74D  knocked into the dapA locus on the bacterial chromosome (medium copy RBS; dapA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in  FIG. 74A , except that the bla gene is replaced with the construct of  FIG. 8A  (FNR-ter/pbt-buk butyrate cassette). On other embodiments, other inducible or constitutive promoters are used. 
     SYN-1013 comprises a construct shown in  FIG. 74C  knocked into the thyA locus on the bacterial chromosome (low copy RBS; thyA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in  FIG. 74B , except that the bla gene is replaced with the construct of  FIG. 8A  (FNR-ter/pbt-buk butyrate cassette). On other embodiments, other inducible or constitutive promoters are used. 
     SYN-1014 comprises a construct shown in  FIG. 74D  knocked into the thyA locus on the bacterial chromosome (medium copy RBS; thyA::constitutive prom1 (BBA_J26100)-Pi(R6K)-constitutive promoter 2(P1)-Kis antitoxin). The strain further comprises a plasmid shown in  FIG. 74B , except that the bla gene is replaced with the construct of  FIG. 8A  (FNR-ter/pbt-buk butyrate cassette). On other embodiments, other inducible or constitutive promoters are used. 
     
       
         
           
               
             
               
                 TABLE 96 
               
             
            
               
                   
               
               
                 Biosafety System Constructs and Sequence Components 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 SEQ ID  
                   
               
               
                 Description 
                 Sequence 
                 NO 
               
               
                   
               
               
                 Biosafety Plasmid 
                 ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAG 
                 352 
                   
               
               
                 System 
                 GGTTATTGTCTCATGAGCGGATACATATTTGAATGT 
                   
                   
               
               
                 Component—dap 
                 ATTTAGAAAAATAAACAAATAGGGGAATTAAAAAA 
                   
                   
               
               
                 A 
                 AAGCCCGCTCATTAGGCGGGCTACTACCTAGGCCG 
                   
                   
               
               
                 Biosafety Plasmid 
                 CGGCCGCGCGAATTCGAGCTCGGTACCCGGGGATC 
                   
                   
               
               
                 System Vector 
                 CTCTAGAGTCGACCTGCAGGCATGCAAGCTTGCGG 
                   
                   
               
               
                 sequences, 
                 CCGCGTCGTGACTGGGAAAACCCTGGCGACTAGTC 
                   
                   
               
               
                 comprising dapA, 
                 TTGGACTCCTGTTGATAGATCCAGTAATGACCTCAG 
                   
                   
               
               
                 Kid Toxin and 
                 AACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCC 
                   
                   
               
               
                 R6K minimal ori 
                 GCCGGGCGTTTTTTATTGGTGAGAATCCAGGGGTCC 
                   
                   
               
               
                 and promoter 
                 CCAATAATTACGATTTAAATCACAGCAAACACCAC 
                   
                   
               
               
                 elements driving 
                 GTCGGCCCTATCAGCTGCGTGCTTTCTATGAGTCGT 
                   
                   
               
               
                 expression of these 
                 TGCTGCATAACTTGACAATTAACATCCGGCTCGTAG 
                   
                   
               
               
                 components, as 
                 GGTTTGTGGAGGGCCCAAGTTCACTTAAAAAGGAG 
                   
                   
               
               
                 shown in FIG. 
                 ATCAACAATGAAAGCAATTTTCGTACTGAAACATCT 
                   
                   
               
               
                 74A 
                 TAATCATGCTGGGGAGGGTTTCTAATGTTCACGGGA 
                   
                   
               
               
                   
                 AGTATTGTCGCGATTGTTACTCCGATGGATGAAAAA 
                   
                   
               
               
                   
                 GGTAATGTCTGTCGGGCTAGCTTGAAAAAACTGATT 
                   
                   
               
               
                   
                 GATTATCATGTCGCCAGCGGTACTTCGGCGATCGTT 
                   
                   
               
               
                   
                 TCTGTTGGCACCACTGGCGAGTCCGCTACCTTAAAT 
                   
                   
               
               
                   
                 CATGACGAACATGCTGATGTGGTGATGATGACGCT 
                   
                   
               
               
                   
                 GGATCTGGCTGATGGGCGCATTCCGGTAATTGCCGG 
                   
                   
               
               
                   
                 GACCGGCGCTAACGCTACTGCGGAAGCCATTAGCC 
                   
                   
               
               
                   
                 TGACGCAGCGCTTCAATGACAGTGGTATCGTCGGCT 
                   
                   
               
               
                   
                 GCCTGACGGTAACCCCTTACTACAATCGTCCGTCGC 
                   
                   
               
               
                   
                 AAGAAGGTTTGTATCAGCATTTCAAAGCCATCGCTG 
                   
                   
               
               
                   
                 AGCATACTGACCTGCCGCAAATTCTGTATAATGTGC 
                   
                   
               
               
                   
                 CGTCCCGTACTGGCTGCGATCTGCTCCCGGAAACGG 
                   
                   
               
               
                   
                 TGGGCCGTCTGGCGAAAGTAAAAAATATTATCGGA 
                   
                   
               
               
                   
                 ATCAAAGAGGCAACAGGGAACTTAACGCGTGTAAA 
                   
                   
               
               
                   
                 CCAGATCAAAGAGCTGGTTTCAGATGATTTTGTTCT 
                   
                   
               
               
                   
                 GCTGAGCGGCGATGATGCGAGCGCGCTGGACTTCA 
                   
                   
               
               
                   
                 TGCAATTGGGCGGTCATGGGGTTATTTCCGTTACGG 
                   
                   
               
               
                   
                 CTAACGTCGCAGCGCGTGATATGGCCCAGATGTGC 
                   
                   
               
               
                   
                 AAACTGGCAGCAGAAGGGCATTTTGCCGAGGCACG 
                   
                   
               
               
                   
                 CGTTATTAATCAGCGTCTGATGCCATTACACAACAA 
                   
                   
               
               
                   
                 ACTATTTGTCGAACCCAATCCAATCCCGGTGAAATG 
                   
                   
               
               
                   
                 GGCATGTAAGGAACTGGGTCTTGTGGCGACCGATA 
                   
                   
               
               
                   
                 CGCTGCGCCTGCCAATGACACCAATCACCGACAGT 
                   
                   
               
               
                   
                 GGCCGTGAGACGGTCAGAGCGGCGCTTAAACATGC 
                   
                   
               
               
                   
                 CGGTTTGCTGTAAGACTTTTGTCAGGTTCCTACTGT 
                   
                   
               
               
                   
                 GACGACTACCACCGATAGACTGGAGTGTTGCTGCG 
                   
                   
               
               
                   
                 AAAAAACCCCGCCGAAGCGGGGTTTTTTGCGAGAA 
                   
                   
               
               
                   
                 GTCACCACGATTGTGCTTTACACGGAGTAGTCGGCA 
                   
                   
               
               
                   
                 GTTCCTTAAGTCAGAATAGTGGACAGGCGGCCAAG 
                   
                   
               
               
                   
                 AACTTCGTTCATGATAGTCTCCGGAACCCGTTCGAG 
                   
                   
               
               
                   
                 TCGTTTTCCGCCCCGTGCTTTCATATCAATTGTCCGG 
                   
                   
               
               
                   
                 GGTTGATCGCAACGTACAACACCTGTGGTACGTATG 
                   
                   
               
               
                   
                 CCAACACCATCCAACGACACCGCAAAGCCGGCAGT 
                   
                   
               
               
                   
                 GCGGGCAAAATTGCCTCCGCTGGTTACGGGCACAA 
                   
                   
               
               
                   
                 CAACAGGCAGGCGGGTCACGCGATTAAAGGCCGCC 
                   
                   
               
               
                   
                 GGTGTGACAATCAGCACCGGCCGCGTTCCCTGCTGC 
                   
                   
               
               
                   
                 TCATGACCTGCGGTAGGATCAAGCGAGACAAGCCA 
                   
                   
               
               
                   
                 GATTTCCCCTCTTTCCATCTAGTATAACTATTGTTTC 
                   
                   
               
               
                   
                 TCTAGTAACATTTATTGTACAACACGAGCCCATTTT 
                   
                   
               
               
                   
                 TGTCAAATAAATTTTAAATTATATCAACGTTAATAA 
                   
                   
               
               
                   
                 GACGTTGTCAATAAAATTATTTTGACAAAATTGGCC 
                   
                   
               
               
                   
                 GGCCGGCGCGCCGATCTGAAGATCAGCAGTTCAAC 
                   
                   
               
               
                   
                 CTGTTGATAGTACGTACTAAGCTCTCATGTTTCACG 
                   
                   
               
               
                   
                 TACTAAGCTCTCATGTTTAACGTACTAAGCTCTCAT 
                   
                   
               
               
                   
                 GTTTAACGAACTAAACCCTCATGGCTAACGTACTAA 
                   
                   
               
               
                   
                 GCTCTCATGGCTAACGTACTAAGCTCTCATGTTTCA 
                   
                   
               
               
                   
                 CGTACTAAGCTCTCATGTTTGAACAATAAAATTAAT 
                   
                   
               
               
                   
                 ATAAATCAGCAACTTAAATAGCCTCTAAGGTTTTAA 
                   
                   
               
               
                   
                 GTTTTATAAGAAAAAAAAGAATATATAAGGCTTTT 
                   
                   
               
               
                   
                 AAAGCCTTTAAGGTTTAACGGTTGTGGACAACAAG 
                   
                   
               
               
                   
                 CCAGGGATGTAACGCACTGAGAAGCCCTTAGAGCC 
                   
                   
               
               
                   
                 TCTCAAAGCAATTTTGAGTGACACAGGAACACTTA 
                   
                   
               
               
                   
                 ACGGCTGACATGGGGCGCGCCCAGCTGTCTAGGGC 
                   
                   
               
               
                   
                 GGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGAC 
                   
                   
               
               
                   
                 AAACAACAGATAAAACGAAAGGCCCAGTCTTTCGA 
                   
                   
               
               
                   
                 CTGAGCCTTTCGTTTTATTTGATGCCT 
                   
                   
               
               
                   
               
               
                 Biosafety Plasmid 
                 ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAG 
                 353 
                   
               
               
                 System 
                 GGTTATTGTCTCATGAGCGGATACATATTTGAATGT 
                   
                   
               
               
                   
                 ATTTAGAAAAATAAACAAATAGGGGAATTAAAAAA 
                   
                   
               
               
                   
                   
                   
                   
               
               
                 Component— 
                 AAGCCCGCTCATTAGGCGGGCTACTACCTAGGCCG 
                   
                   
               
               
                 ThyA 
                 CGGCCGCGCGAATTCGAGCTCGGTACCCGGGGATC 
                   
                   
               
               
                 Biosafety Plasmid 
                 CTCTAGAGTCGACCTGCAGGCATGCAAGCTTGCGG 
                   
                   
               
               
                 System Vector 
                 CCGCGTCGTGACTGGGAAAACCCTGGCGACTAGTC 
                   
                   
               
               
                 sequences, 
                 TTGGACTCCTGTTGATAGATCCAGTAATGACCTCAG 
                   
                   
               
               
                 comprising ThyA, 
                 AACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCC 
                   
                   
               
               
                 Kid Toxin and 
                 GCCGGGCGTTTTTTATTGGTGAGAATCCAGGGGTCC 
                   
                   
               
               
                 R6K minimal ori, 
                 CCAATAATTACGATTTAAATCACAGCAAACACCAC 
                   
                   
               
               
                 and promoter 
                 GTCGGCCCTATCAGCTGCGTGCTTICTATGAGTCGT 
                   
                   
               
               
                 elements driving 
                 TGCTGCATAACTTGACAATTAATCATCCGGCTCGTA 
                   
                   
               
               
                 expression of these 
                 GGGTTTGTGGAGGGCCCAAGTTCACTTAAAAAGGA 
                   
                   
               
               
                 components, as 
                 GATCAACAATGAAAGCAATTTTCGTACTGAAACAT 
                   
                   
               
               
                 shown in FIG. 
                 CTTAATCATGCTGGGGAGGGTTTCTAATGAAACAGT 
                   
                   
               
               
                 74B 
                 ATTTAGAACTGATGCAAAAAGTGCTCGACGAAGGC 
                   
                   
               
               
                   
                 ACACAGAAAAACGACCGTACCGGAACCGGAACGCT 
                   
                   
               
               
                   
                 TTCCATTTTTGGTCATCAGATGCGTTTTAACCTGCA 
                   
                   
               
               
                   
                 AGATGGATTCCCGCTGGTGACAACTAAACGTTGCC 
                   
                   
               
               
                   
                 ACCTGCGTTCCATCATCCATGAACTGCTGTGGTTTC 
                   
                   
               
               
                   
                 TTCAGGGCGACACTAACATTGCTTATCTACACGAAA 
                   
                   
               
               
                   
                 ACAATGTCACCATCTGGGACGAATGGGCCGATGAA 
                   
                   
               
               
                   
                 AACGGCGACCTCGGGCCAGTGTATGGTAAACAGTG 
                   
                   
               
               
                   
                 GCGTGCCTGGCCAACGCCAGATGGTCGTCATATTGA 
                   
                   
               
               
                   
                 CCAGATCACTACGGTACTGAACCAGCTGAAAAACG 
                   
                   
               
               
                   
                 ACCCGGATTCGCGCCGCATTATTGTTTCAGCGTGGA 
                   
                   
               
               
                   
                 ACGTAGGCGAACTGGATAAAATGGCGCTGGCACCG 
                   
                   
               
               
                   
                 TGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGC 
                   
                   
               
               
                   
                 AAACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGAC 
                   
                   
               
               
                   
                 GTCTTCCTCGGCCTGCCGTTCAACATTGCCAGCTAC 
                   
                   
               
               
                   
                 GCGTTATTGGTGCATATGATGGCGCAGCAGTGCGAT 
                   
                   
               
               
                   
                 CTGGAAGTGGGTGATTTTGTCTGGACCGGTGGCGAC 
                   
                   
               
               
                   
                 ACGCATCTGTACAGCAACCATATGGATCAAACTCAT 
                   
                   
               
               
                   
                 CTGCAATTAAGCCGCGAACCGCGTCCGCTGCCGAA 
                   
                   
               
               
                   
                 GTTGATTATCAAACGTAAACCCGAATCCATCTTCGA 
                   
                   
               
               
                   
                 CTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGA 
                   
                   
               
               
                   
                 TCCGCATCCGGGCATTAAAGCGCCGGTGGCTATCTA 
                   
                   
               
               
                   
                 AGACTTTTGTCAGGTTCCTACTGTGACGACTACCAC 
                   
                   
               
               
                   
                 CGATAGACTGGAGTGTTGCTGCGAAAAAACCCCGC 
                   
                   
               
               
                   
                 CGAAGCGGGGTTTTTTGCGAGAAGTCACCACGATT 
                   
                   
               
               
                   
                 GTGCTTTACACGGAGTAGTCGGCAGTTCCTTAAGTC 
                   
                   
               
               
                   
                 AGAATAGTGGACAGGCGGCCAAGAACTTCGTTCAT 
                   
                   
               
               
                   
                 GATAGTCTCCGGAACCCGTTCGAGTCGTTTTCCGCC 
                   
                   
               
               
                   
                 CCGTGCTTTCATATCAATTGTCCGGGGTTGATCGCA 
                   
                   
               
               
                   
                 ACGTACAACACCTGTGGTACGTATGCCAACACCATC 
                   
                   
               
               
                   
                 CAACGACACCGCAAAGCCGGCAGTGCGGGCAAAAT 
                   
                   
               
               
                   
                 TGCCTCCGCTGGTTACGGGCACAACAACAGGCAGG 
                   
                   
               
               
                   
                 CGGGTCACGCGATTAAAGGCCGCCGGTGTGACAAT 
                   
                   
               
               
                   
                 CAGCACCGGCCGCGTTCCCTGCTGCTCATGACCTGC 
                   
                   
               
               
                   
                 GGTAGGATCAAGCGAGACAAGCCAGATTTCCCCTC 
                   
                   
               
               
                   
                 TTTCCATCTAGTATAACTATTGTTTCTCTAGTAACAT 
                   
                   
               
               
                   
                 TTATTGTACAACACGAGCCCATTTTTGTCAAATAAA 
                   
                   
               
               
                   
                 TTTTAAATTATATCAACGTTAATAAGACGTTGTCAA 
                   
                   
               
               
                   
                 TAAAATTATTTTGACAAAATTGGCCGGCCGGCGCGC 
                   
                   
               
               
                   
                 CGATCTGAAGATCAGCAGTTCAACCTGTTGATAGTA 
                   
                   
               
               
                   
                 CGTACTAAGCTCTCATGTTTCACGTACTAAGCTCTC 
                   
                   
               
               
                   
                 ATGTTTAACGTACTAAGCTCTCATGTTTAACGAACT 
                   
                   
               
               
                   
                 AAACCCTCATGGCTAACGTACTAAGCTCTCATGGCT 
                   
                   
               
               
                   
                 AACGTACTAAGCTCTCATGTTTCACGTACTAAGCTC 
                   
                   
               
               
                   
                 TCATGTTTGAACAATAAAATTAATATAAATCAGCAA 
                   
                   
               
               
                   
                 CTTAAATAGCCTCTAAGGTTTTAAGTTTTATAAGAA 
                   
                   
               
               
                   
                 AAAAAAGAATATATAAGGCTTTTAAAGCCTTTAAG 
                   
                   
               
               
                   
                 GTTTAACGGTTGTGGACAACAAGCCAGGGATGTAA 
                   
                   
               
               
                   
                 CGCACTGAGAAGCCCTTAGAGCCTCTCAAAGCAAT 
                   
                   
               
               
                   
                 TTTGAGTGACACAGGAACACTTAACGGCTGACATG 
                   
                   
               
               
                   
                 GGGCGCGCCCAGCTGTCTAGGGCGGCGGATTTGTC 
                   
                   
               
               
                   
                 CTACTCAGGAGAGCGTTCACCGACAAACAACAGAT 
                   
                   
               
               
                   
                 AAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCG 
                   
                   
               
               
                   
                 TTTTATTTGATGCCT 
                   
                   
               
               
                   
               
               
                 Kid toxin (reverse 
                 TTAAGTCAGAATAGTGGACAGGCGGCCAAGAACTT 
                 354 
                   
               
               
                 orientation) 
                 CGTTCATGATAGTCTCCGGAACCCGTTCGAGTCGTT 
                   
                   
               
               
                   
                 TTCCGCCCCGTGCTTTCATATCAATTGTCCGGGGTT 
                   
                   
               
               
                   
                 GATCGCAACGTACAACACCTGTGGTACGTATGCCA 
                   
                   
               
               
                   
                 ACACCATCCAACGACACCGCAAAGCCGGCAGTGCG 
                   
                   
               
               
                   
                 GGCAAAATTGCCTCCGCTGGTTACGGGCACAACAA 
                   
                   
               
               
                   
                 CAGGCAGGCGGGTCACGCGATTAAAGGCCGCCGGT 
                   
                   
               
               
                   
                 GTGACAATCAGCACCGGCCGCGTTCCCTGCTGCTCA 
                   
                   
               
               
                   
                 TGACCTGCGGTAGGATCAAGCGAGACAAGCCAGAT 
                   
                   
               
               
                   
                 TTCCCCTCTTTCCAT 
                   
                   
               
               
                   
               
               
                 dapA 
                 ATGTTCACGGGAAGTATTGTCGCGATTGTTACTCCG 
                 355 
                   
               
               
                   
                 ATGGATGAAAAAGGTAATGTCTGTCGGGCTAGCTT 
                   
                   
               
               
                   
                 GAAAAAACTGATTGATTATCATGTCGCCAGCGGTA 
                   
                   
               
               
                   
                 CTTCGGCGATCGTTTCTGTTGGCACCACTGGCGAGT 
                   
                   
               
               
                   
                 CCGCTACCTTAAATCATGACGAACATGCTGATGTGG 
                   
                   
               
               
                   
                 TGATGATGACGCTGGATCTGGCTGATGGGCGCATTC 
                   
                   
               
               
                   
                 CGGTAATTGCCGGGACCGGCGCTAACGCTACTGCG 
                   
                   
               
               
                   
                 GAAGCCATTAGCCTGACGCAGCGCTTCAATGACAG 
                   
                   
               
               
                   
                 TGGTATCGTCGGCTGCCTGACGGTAACCCCTTACTA 
                   
                   
               
               
                   
                 CAATCGTCCGTCGCAAGAAGGTTTGTATCAGCATTT 
                   
                   
               
               
                   
                 CAAAGCCATCGCTGAGCATACTGACCTGCCGCAAA 
                   
                   
               
               
                   
                 TTCTGTATAATGTGCCGTCCCGTACTGGCTGCGATC 
                   
                   
               
               
                   
                 TGCTCCCGGAAACGGTGGGCCGTCTGGCGAAAGTA 
                   
                   
               
               
                   
                 AAAAATATTATCGGAATCAAAGAGGCAACAGGGAA 
                   
                   
               
               
                   
                 CTTAACGCGTGTAAACCAGATCAAAGAGCTGGTTTC 
                   
                   
               
               
                   
                 AGATGATTTTGTTCTGCTGAGCGGCGATGATGCGAG 
                   
                   
               
               
                   
                 CGCGCTGGACTTCATGCAATTGGGCGGTCATGGGGT 
                   
                   
               
               
                   
                 TATTTCCGTTACGGCTAACGTCGCAGCGCGTGATAT 
                   
                   
               
               
                   
                 GGCCCAGATGTGCAAACTGGCAGCAGAAGGGCATT 
                   
                   
               
               
                   
                 TTGCCGAGGCACGCGTTATTAATCAGCGTCTGATGC 
                   
                   
               
               
                   
                 CATTACACAACAAACTATTTGTCGAACCCAATCCAA 
                   
                   
               
               
                   
                 TCCCGGTGAAATGGGCATGTAAGGAACTGGGTCTT 
                   
                   
               
               
                   
                 GTGGCGACCGATACGCTGCGCCTGCCAATGACACC 
                   
                   
               
               
                   
                 AATCACCGACAGTGGCCGTGAGACGGTCAGAGCGG 
                   
                   
               
               
                   
                 CGCTTAAACATGCCGGTTTGCTGTAA 
                   
                   
               
               
                   
               
               
                 thyA 
                 ATGAAACAGTATTTAGAACTGATGCAAAAAGTGCT 
                 356 
                   
               
               
                   
                 CGACGAAGGCACACAGAAAAACGACCGTACCGGA 
                   
                   
               
               
                   
                 ACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGT 
                   
                   
               
               
                   
                 TTTAACCTGCAAGATGGATTCCCGCTGGTGACAACT 
                   
                   
               
               
                   
                 AAACGTTGCCACCTGCGTTCCATCATCCATGAACTG 
                   
                   
               
               
                   
                 CTGTGGTTTCTTCAGGGCGACACTAACATTGCTTAT 
                   
                   
               
               
                   
                 CTACACGAAAACAATGTCACCATCTGGGACGAATG 
                   
                   
               
               
                   
                 GGCCGATGAAAACGGCGACCTCGGGCCAGTGTATG 
                   
                   
               
               
                   
                 GTAAACAGTGGCGTGCCTGGCCAACGCCAGATGGT 
                   
                   
               
               
                   
                 CGTCATATTGACCAGATCACTACGGTACTGAACCAG 
                   
                   
               
               
                   
                 CTGAAAAACGACCCGGATTCGCGCCGCATTATTGTT 
                   
                   
               
               
                   
                 TCAGCGTGGAACGTAGGCGAACTGGATAAAATGGC 
                   
                   
               
               
                   
                 GCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGT 
                   
                   
               
               
                   
                 GGCAGACGGCAAACTCTCTTGCCAGCTTTATCAGCG 
                   
                   
               
               
                   
                 CTCCTGTGACGTCTTCCTCGGCCTGCCGTTCAACAT 
                   
                   
               
               
                   
                 TGCCAGCTACGCGTTATTGGTGCATATGATGGCGCA 
                   
                   
               
               
                   
                 GCAGTGCGATCTGGAAGTGGGTGATTTTGTCTGGAC 
                   
                   
               
               
                   
                 CGGTGGCGACACGCATCTGTACAGCAACCATATGG 
                   
                   
               
               
                   
                 ATCAAACTCATCTGCAATTAAGCCGCGAACCGCGTC 
                   
                   
               
               
                   
                 CGCTGCCGAAGTTGATTATCAAACGTAAACCCGAA 
                   
                   
               
               
                   
                 TCCATCTTCGACTACCGTTTCGAAGACTTTGAGATT 
                   
                   
               
               
                   
                 GAAGGCTACGATCCGCATCCGGGCATTAAAGCGCC 
                   
                   
               
               
                   
                 GGTGGCTATCTAA 
                   
                   
               
               
                   
               
               
                 Kid toxin 
                 MERGEIWLVSLDPTAGHEQQGTRPVLIVTPAAFNRVT 
                 357 
                   
               
               
                 polypeptide 
                 RLPVVVPVTSGGNFARTAGFAVSLDGVGIRTTGVVRC 
                   
                   
               
               
                   
                 DQPRTIDMKARGGKRLERVPETIMNEVLGRLSTILT* 
                   
                   
               
               
                   
               
               
                 dapA polypeptide 
                 MFTGSIVAIVTPMDEKGNVCRASLKKLIDYHVASGTS 
                 358 
                   
               
               
                   
                 AIVSVGTTGESATLNHDEHADVVMMTLDLADGRIPVI 
                   
                   
               
               
                   
                 AGTGANATAEAISLTQRFNDSGIVGCLTVTPYYNRPS 
                   
                   
               
               
                   
                 QEGLYQHFKAIAEHTDLPQILYNVPSRTGCDLLPETVG 
                   
                   
               
               
                   
                 RLAKVKNIIGIKEATGNLTRVNQIKELVSDDFVLLSGD 
                   
                   
               
               
                   
                 DASALDFMQLGGHGVISVTANVAARDMAQMCKLAA 
                   
                   
               
               
                   
                 EGHFAEARVINQRLMPLHNKLFVEPNPIPVKWACKEL 
                   
                   
               
               
                   
                 GLVATDTLRLPMTPITDSGRETVRAALKHAGLL 
                   
                   
               
               
                   
               
               
                 ThyA polypeptide 
                 MKQYLELMQKVLDEGTQKNDRTGTGTLSIFGHQMRF 
                 359 
                   
               
               
                   
                 NLQDGFPLVTTKRCHLRSIIHELLWFLQGDTNIAYLHE 
                   
                   
               
               
                   
                 NNVTIWDEWADENGDLGPVYGKQWRAWPTPDGRHI 
                   
                   
               
               
                   
                 DQITTVLNQLKNDPDSRRIIVSAWNVGELDKMALAPC 
                   
                   
               
               
                   
                 HAFFQFYVADGKLSCQLYQRSCDVFLGLPFNIASYAL 
                   
                   
               
               
                   
                 LVHMMAQQCDLEVGDFVWTGGDTHLYSNHMDQTH 
                   
                   
               
               
                   
                 LQLSREPRPLPKLIIKRKPESIFDYRFEDFEIEGYDPHPG 
                   
                   
               
               
                   
                 IKAPVAI* 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 97 
               
             
            
               
                   
               
               
                 Chromosomally Inserted Biosafety System Constructs 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ 
               
               
                   
                   
                 ID 
               
               
                 Description 
                 Sequence 
                 NO 
               
               
                   
               
               
                 Biosafety 
                 TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCGGAT 
                 360 
               
               
                 Chromosomal 
                 CTGCTGGAACAGGTGGTGAGACTCAAGGTCATGATGGA 
                   
               
               
                 Construct—low 
                 CGTGAACAAAAAAACGAAAATTCGCCACCGAAACGAGC 
                   
               
               
                 copy Rep (Pi) 
                 TAAATCACACCCTGGCTCAACTTCCTTTGCCCGCAAAGC 
                   
               
               
                 and Kis antitoxin 
                 GAGTGATGTATATGGCGCTTGCTCCCATTGATAGCAAAG 
                   
               
               
                 (as shown in 
                 AACCTCTTGAACGAGGGCGAGTTTTCAAAATTAGGGCTG 
                   
               
               
                 FIG. 74C) 
                 AAGACCTTGCAGCGCTCGCCAAAATCACCCCATCGCTTG 
                   
               
               
                   
                 CTTATCGACAATTAAAAGAGGGTGGTAAATTACTTGGTG 
                   
               
               
                   
                 CCAGCAAAATTTCGCTAAGAGGGGATGATATCATTGCTT 
                   
               
               
                   
                 TAGCTAAAGAGCTTAACCTGCTCTTTACTGCTAAAAACT 
                   
               
               
                   
                 CCCCTGAAGAGTTAGACCTTAACATTATTGAGTGGATAG 
                   
               
               
                   
                 CTTATTCAAATGATGAAGGATACTTGTCTTTAAAATTCA 
                   
               
               
                   
                 CCAGAACCATAGAACCATATATCTCTAGCCTTATTGGGA 
                   
               
               
                   
                 AAAAAAATAAATTCACAACGCAATTGTTAACGGCAAGC 
                   
               
               
                   
                 TTACGCTTAAGTAGCCAGTATTCATCTTCTCTTTATCAAC 
                   
               
               
                   
                 TTATCAGGAAGCATTACTCTAATTTTAAGAAGAAAAATT 
                   
               
               
                   
                 ATTTTATTATTTCCGTTGATGAGTTAAAGGAAGAGTTAA 
                   
               
               
                   
                 TAGCTTATACTTTTGATAAAGATGGAAATATTGAGTACA 
                   
               
               
                   
                 AATACCCTGACTTTCCTATTTTTAAAAGGGATGTGTTAA 
                   
               
               
                   
                 ATAAAGCCATTGCTGAAATTAAAAAGAAAACAGAAATA 
                   
               
               
                   
                 TCGTTTGTTGGCTTCACTGTTCATGAAAAAGAAGGAAGA 
                   
               
               
                   
                 AAAATTAGTAAGCTGAAGTTCGAATTTGTCGTTGATGAA 
                   
               
               
                   
                 GATGAATTTTCTGGCGATAAAGATGATGAAGCTTTTTTT 
                   
               
               
                   
                 ATGAATTTATCTGAAGCTGATGCAGCTTTTCTCAAGGTA 
                   
               
               
                   
                 TTTGATGAAACCGTACCTCCCAAAAAAGCTAAGGGGTGA 
                   
               
               
                   
                 GGATCTCCAGGCATCAAATAAAACGAAAGGCTCAGTCG 
                   
               
               
                   
                 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGA 
                   
               
               
                   
                 ACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTG 
                   
               
               
                   
                 GGCCTTTCTGCGTTTATACCCGGGAAAAAGAGTATTGAC 
                   
               
               
                   
                 TtaaagtctaacctataggTATAATGTGTGGAGACCAGAGGTAAGG 
                   
               
               
                   
                 AGGTAACAACCATGCGAGTGTTGAAGAAACATCTTAATC 
                   
               
               
                   
                 ATGCTAAGGAGGTTTTCTAATGCATACCACCCGACTGAA 
                   
               
               
                   
                 GAGGGTTGGCGGCTCAGTTATGCTGACCGTCCCACCGGC 
                   
               
               
                   
                 ACTGCTGAATGCGCTGTCTCTGGGCACAGATAATGAAGT 
                   
               
               
                   
                 TGGCATGGTCATTGATAATGGCCGGCTGATTGTTGAGCC 
                   
               
               
                   
                 GTACAGACGCCCGCAATATTCACTGGCTGAGCTACTGGC 
                   
               
               
                   
                 ACAGTGTGATCCGAATGCTGAAATATCAGCTGAAGAAC 
                   
               
               
                   
                 GAGAATGGCTGGATGCACCGGCGACTGGTCAGGAGGAA 
                   
               
               
                   
                 ATCTGA 
                   
               
               
                   
               
               
                 Biosafety 
                 TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCGGAT 
                 361 
               
               
                 Chromosomal 
                 CTTCCGGAAGACTAGGTGAGACTCAAGGTCATGATGGAC 
                   
               
               
                 Construct— 
                 GTGAACAAAAAAACGAAAATTCGCCACCGAAACGAGCT 
                   
               
               
                 medium copy 
                 AAATCACACCCTGGCTCAACTTCCTTTGCCCGCAAAGCG 
                   
               
               
                 Rep (Pi) and Kis 
                 AGTGATGTATATGGCGCTTGCTCCCATTGATAGCAAAGA 
                   
               
               
                 antitoxin (as 
                 ACCTCTTGAACGAGGGCGAGTTTTCAAAATTAGGGCTGA 
                   
               
               
                 shown in FIG. 
                 AGACCTTGCAGCGCTCGCCAAAATCACCCCATCGCTTGC 
                   
               
               
                 74D) 
                 TTATCGACAATTAAAAGAGGGTGGTAAATTACTTGGTGC 
                   
               
               
                   
                 CAGCAAAATTTCGCTAAGAGGGGATGATATCATTGCTTT 
                   
               
               
                   
                 AGCTAAAGAGCTTAACCTGCTCTTTACTGCTAAAAACTC 
                   
               
               
                   
                 CCCTGAAGAGTTAGACCTTAACATTATTGAGTGGATAGC 
                   
               
               
                   
                 TTATTCAAATGATGAAGGATACTTGTCTTTAAAATTCAC 
                   
               
               
                   
                 CAGAACCATAGAACCATATATCTCTAGCCTTATTGGGAA 
                   
               
               
                   
                 AAAAAATAAATTCACAACGCAATTGTTAACGGCAAGCTT 
                   
               
               
                   
                 ACGCTTAAGTAGCCAGTATTCATCTTCTCTTTATCAACTT 
                   
               
               
                   
                 ATCAGGAAGCATTACTCTAATTTTAAGAAGAAAAATTAT 
                   
               
               
                   
                 TTTATTATTTCCGTTGATGAGTTAAAGGAAGAGTTAATA 
                   
               
               
                   
                 GCTTATACTTTTGATAAAGATGGAAATATTGAGTACAAA 
                   
               
               
                   
                 TACCCTGACTTTCCTATTTTTAAAAGGGATGTGITAAATA 
                   
               
               
                   
                 AAGCCATTGCTGAAATTAAAAAGAAAACAGAAATATCG 
                   
               
               
                   
                 TTTGTTGGCTTCACTGTTCATGAAAAAGAAGGAAGAAAA 
                   
               
               
                   
                 ATTAGTAAGCTGAAGTTCGAATTTGTCGTTGATGAAGAT 
                   
               
               
                   
                 GAATTTTCTGGCGATAAAGATGATGAAGCTTTTTTTATG 
                   
               
               
                   
                 AATTTATCTGAAGCTGATGCAGCTTTTCTCAAGGTATTTG 
                   
               
               
                   
                 ATGAAACCGTACCTCCCAAAAAAGCTAAGGGGTGAGGA 
                   
               
               
                   
                 TCTCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAA 
                   
               
               
                   
                 GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACG 
                   
               
               
                   
                 CTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGC 
                   
               
               
                   
                 CTTTCTGCGTTTATACCCGGGAAAAAGAGTATTGACTtaaa 
                   
               
               
                   
                 gtctaacctataggTATAATGTGTGGAGACCAGAGGTAAGGAGG 
                   
               
               
                   
                 TAACAACCATGCGAGTGTTGAAGAAACATCTTAATCATG 
                   
               
               
                   
                 CTAAGGAGGTTTTCTAATGCATACCACCCGACTGAAGAG 
                   
               
               
                   
                 GGTTGGCGGCTCAGTTATGCTGACCGTCCCACCGGCACT 
                   
               
               
                   
                 GCTGAATGCGCTGTCTCTGGGCACAGATAATGAAGTTGG 
                   
               
               
                   
                 CATGGTCATTGATAATGGCCGGCTGATTGTTGAGCCGTA 
                   
               
               
                   
                 CAGACGCCCGCAATATTCACTGGCTGAGCTACTGGCACA 
                   
               
               
                   
                 GTGTGATCCGAATGCTGAAATATCAGCTGAAGAACGAG 
                   
               
               
                   
                 AATGGCTGGATGCACCGGCGACTGGTCAGGAGGAAATC 
                   
               
               
                   
                 TGA 
                   
               
               
                   
               
               
                 Rep (Pi) 
                 TGAGACTCAAGGTCATGATGGACGTGAACAAAAAAACG 
                 362 
               
               
                   
                 AAAATTCGCCACCGAAACGAGCTAAATCACACCCTGGCT 
                   
               
               
                   
                 CAACTTCCTTTGCCCGCAAAGCGAGTGATGTATATGGCG 
                   
               
               
                   
                 CTTGCTCCCATTGATAGCAAAGAACCTCTTGAACGAGGG 
                   
               
               
                   
                 CGAGTTTTCAAAATTAGGGCTGAAGACCTTGCAGCGCTC 
                   
               
               
                   
                 GCCAAAATCACCCCATCGCTTGCTTATCGACAATTAAAA 
                   
               
               
                   
                 GAGGGTGGTAAATTACTTGGTGCCAGCAAAATTTCGCTA 
                   
               
               
                   
                 AGAGGGGATGATATCATTGCTTTAGCTAAAGAGCTTAAC 
                   
               
               
                   
                 CTGCTCTTTACTGCTAAAAACTCCCCTGAAGAGTTAGAC 
                   
               
               
                   
                 CTTAACATTATTGAGTGGATAGCTTATTCAAATGATGAA 
                   
               
               
                   
                 GGATACTTGTCTTTAAAATTCACCAGAACCATAGAACCA 
                   
               
               
                   
                 TATATCTCTAGCCTTATTGGGAAAAAAAATAAATTCACA 
                   
               
               
                   
                 ACGCAATTGTTAACGGCAAGCTTACGCTTAAGTAGCCAG 
                   
               
               
                   
                 TATTCATCTTCTCTTTATCAACTTATCAGGAAGCATTACT 
                   
               
               
                   
                 CTAATTTTAAGAAGAAAAATTATTTTATTATTTCCGTTGA 
                   
               
               
                   
                 TGAGTTAAAGGAAGAGTTAATAGCTTATACTTTTGATAA 
                   
               
               
                   
                 AGATGGAAATATTGAGTACAAATACCCTGACTTTCCTAT 
                   
               
               
                   
                 TTTTAAAAGGGATGTGTTAAATAAAGCCATTGCTGAAAT 
                   
               
               
                   
                 TAAAAAGAAAACAGAAATATCGTTTGTTGGCTTCACTGT 
                   
               
               
                   
                 TCATGAAAAAGAAGGAAGAAAAATTAGTAAGCTGAAGT 
                   
               
               
                   
                 TCGAATTTGTCGTTGATGAAGATGAATTTTCTGGCGATA 
                   
               
               
                   
                 AAGATGATGAAGCTTTTTTTATGAATTTATCTGAAGCTG 
                   
               
               
                   
                 ATGCAGCTTTTCTCAAGGTATTTGATGAAACCGTACCTC 
                   
               
               
                   
                 CCAAAAAAGCTAAGGGGTGA 
                   
               
               
                   
               
               
                 Kis antitoxin 
                 CATACCACCCGACTGAAGAGGGTTGGCGGCTCAGTTATG 
                 363 
               
               
                   
                 CTGACCGTCCCACCGGCACTGCTGAATGCGCTGTCTCTG 
                   
               
               
                   
                 GGCACAGATAATGAAGTTGGCATGGTCATTGATAATGGC 
                   
               
               
                   
                 CGGCTGATTGTTGAGCCGTACAGACGCCCGCAATATTCA 
                   
               
               
                   
                 CTGGCTGAGCTACTGGCACAGTGTGATCCGAATGCTGAA 
                   
               
               
                   
                 ATATCAGCTGAAGAACGAGAATGGCTGGATGCACCGGC 
                   
               
               
                   
                 GACTGGTCAGGAGGAAATCTGA 
                   
               
               
                   
               
               
                 RBS (low copy) 
                 GCTGGAACAGGTGG 
                 364 
               
               
                 RBS (medium 
                 TCCGGAAGACTAGG 
                 365 
               
               
                 copy) 
               
               
                   
               
            
           
         
       
     
     Example 67 
       
     
       
         
           
               
             
               
                 TABLE 98 
               
               
                   
               
               
                 Other Sequences of interest 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Wild-type clbA 
                 caaatatcacataatcttaacatatcaataaacacagtaaagtttcatgtgaaaaacat 
               
               
                 (SEQ ID NO: 350) 
                 caaacataaaatacaagctcggaatacgaatcacgctatacacattgctaacagga 
               
               
                   
                 atgagattatctaaatgaggattgatatattaattggacatactagtttttttcatcaaac 
               
               
                   
                 cagtagagataacttecttcactatctcaatgaggaagaaataaaacgctatgatca 
               
               
                   
                 gtttcattttgtgagtgataaagaactctatattttaagccgtatcctgctcaaaacagc 
               
               
                   
                 actaaaaagatatcaacctgatgtctcattacaatcatggcaatttagtacgtgcaaat 
               
               
                   
                 atggcaaaccatttatagtttttcctcagttggcaaaaaagattttttttaacctttcccat 
               
               
                   
                 actatagatacagtagccgttgctattagttctcactgcgagcttggtgtcgatattga 
               
               
                   
                 acaaataagagatttagacaactcttatctgaatatcagtcagcatttttttactccaca 
               
               
                   
                 ggaagctactaacatagtttcacttcctcgttatgaaggtcaattacttttttggaaaat 
               
               
                   
                 gtggacgctcaaagaagcttacatcaaatatcgaggtaaaggcctatctttaggact 
               
               
                   
                 ggattgtattgaatttcatttaacaaataaaaaactaacttcaaaatatagaggttcacc 
               
               
                   
                 tgtttatttctctcaatggaaaatatgtaactcatttctcgcattagcctctccactcatca 
               
               
                   
                 cccctaaaataactattgagctatttcctatgcagtcccaactttatcaccacgactatc 
               
               
                   
                 agctaattcattcgtcaaatgggcagaattgaatcgccacggataatctagacacttc 
               
               
                   
                 tgagccgtcgataatattgattttcatattccgtcggtggtgtaagtatcccgcataatc 
               
               
                   
                 gtgccattcacatttag 
               
               
                   
               
               
                 clbA knock-out 
                 ggatggggggaaacatggataagttcaaagaaaaaaacccgttatctctgcgtgaaa 
               
               
                 (SEQ ID NO: 351) 
                 gacaagtattgcgcatgctggcacaaggtgatgagtactctcaaatatcacataatctt 
               
               
                   
                 aacatatcaataaacacagtaaagtttcatgtgaaaaacatcaaacataaaatacaagc 
               
               
                   
                 tcggaatacgaatcacgctatacacattgctaacaggaatgagattatctaaatgagga 
               
               
                   
                 ttgaTGTGTAGGCTGGAGCTGCTTCGAAGTTCCTATAC 
               
               
                   
                 TTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCG 
               
               
                   
                 GAATAGGAACTAAGGAGGATATTCATATGtcgtcaaatggg 
               
               
                   
                 cagaattgaatcgccacggataatctagacacttctgagccgtcgataatattgattttc 
               
               
                   
                 atattccgtcggtgg