Patent Publication Number: US-2022226395-A1

Title: Bacteria engineered to treat metabolic diseases

Description:
The present application is a continuation application based upon U.S. patent application Ser. No. 15/738,174, filed Dec. 20, 2017, which is a 35 U.S.C. § 371 national stage filing of International Application No. PCT/US2016/039444, filed Jun. 24, 2016, which in turn claims the benefit of priority to U.S. Provisional Patent Application No. 62/184,777, filed Jun. 25, 2015, U.S. Provisional Patent Application No. 62/347,576, filed Jun. 8, 2016, U.S. Provisional Patent Application No. 62/348,620, filed Jun. 10, 2016, U.S. Provisional Patent Application No. 62/277,346, filed Jan. 11, 2016, U.S. Provisional Patent Application No. 62/336,012, filed May 13, 2016, U.S. Provisional Patent Application No. 62/293,695, filed Feb. 10, 2016, U.S. Provisional Patent Application No. 62/347,554, filed Jun. 8, 2016, U.S. Provisional Patent Application No. 62/348,416, filed Jun. 10, 2016, U.S. Provisional Patent Application No. 62/354,681, filed Jun. 24, 2016, U.S. Provisional Patent Application No. 62/347,508, filed Jun. 8, 2016, and U.S. Provisional Patent Application No. 62/354,682, filed Jun. 24, 2016, and is a continuation-in-part of PCT International Application No. PCT/US2016/032565, filed May 13, 2016. The contents of each of the foregoing applications are hereby incorporated by reference herein in their entirety. 
     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 Jan. 26, 2022, is named 126046-01806_SL.txt and is 692,691 bytes in size. 
     Compositions and therapeutic methods for treating metabolic diseases are provided. In certain aspects, the compositions of the invention comprise bacteria that are genetically engineered to treat, modulate, and/or ameliorate metabolic diseases, particularly in low-oxygen environments, such as in the mammalian gut. In certain aspects, the compositions and methods of the invention as disclosed herein may be used for treating metabolic diseases such as obesity and type 2 diabetes. Obesity is caused by an imbalance between energy intake and expenditure, leading to the accumulation of unused energy in the form of fat. The World Health Organization considers obesity to be a global epidemic, and the United States Centers for Disease Control and Prevention estimates that nearly one third of adult Americans are obese. Diet and exercise may help reduce obesity and its associated pathologies, but adherence to a strict diet and exercise regime is challenging. Obesity may also be caused by other factors, e.g., mutations in genes regulating metabolic pathways (e.g., satiety, fatty acid oxidation, and mitochondrial function), which can contribute to energy imbalance. For example, congenital deficits in the signaling pathways for leptin, a satiety hormone, are known to cause obesity in humans and animal models. 
     Patients suffering from obesity are at increased risk of developing adverse physiological conditions, e.g., non-alcoholic fatty liver, cardiovascular diseases, type 2 diabetes mellitus (T2DM). The incidence of T2DM has increased 300% in the last three decades in the United States. T2DM patients are resistant to the effects of insulin, a hormone that regulates blood glucose levels, and frequently experience hyperglycemia, a condition in which blood glucose is above physiologically tolerable levels. When left untreated, hyperglycemia can result in severe complications such as hypertension, cardiovascular disease, inflammatory disease, blood vessel damage, nerve damage, cancer, and diabetes-induced coma. 
     T2DM involves the dysregulation of multiple metabolic organs, such as the pancreas, liver, skeletal muscle, adipose tissue, and brain, and it has been challenging to design therapeutics that target multiple tissue while avoiding systemic side effects. Insulin has been the first-line treatment for T2DM for decades. However, patients with severe T2DM may not respond to the insulin as a result of chronic insulin resistance. In addition, insulin must be administered multiple times throughout the day, which can adversely affect quality of life. Multiple therapies have been developed to treat T2DM, but not without limitations and sometimes life-threatening side effects. For example, thiazolidinedione was once widely used in order to increase the glucose metabolism in patients. However, the compound has been pulled from certain markets due to an increased association with heart failure (Nissen et al., 2007). Likewise, inhibitors of dipeptidyl peptidase-4 (DPP-4) have shown therapeutic promise, but may be linked to increased risk of pancreatic diseases (Karagiannis, et al., 2014). 
     Recently, researchers have demonstrated the close relationship between gut bacteria and metabolic disease (Harley et al., 2012). In obese mice, the ratio of firmicutes to bacteroidetes bacteria is increased (Harley et al., 2012; Mathur et al., 2015). These bacteria extract different amounts of energy from food, which may contribute to changes in energy balance. Similar changes have been also been observed in human studies (Harley et al., 2012; Mathur et al., 2015). Several molecules that are produced by gut bacteria have been shown to be metabolic regulators. For example, gut bacteria digest and break down dietary fiber into molecules such as acetate, butyrate, and propionate. These molecules are absorbed through intestinal cells, transferred to organs such as the liver and brain, and produce physiological changes, such as de novo glucose production and lipid synthesis (Brussow et al., 2014; De Vadder et al., 2014; Lin et al., 2012). There has been an effort to engineer bacteria that produce N-acylphosphatidylethanolamines (NAPEs) (Chen et al., 2014). However, these bacteria express NAPEs constitutively and systemically, and NAPEs may be capable of “displac[ing] cholesterol from raft-like structures [and] may have dramatic implications for neural cell membrane function during stress and injury” (Terova et al., 2005). Thus, there is significant unmet need for effective, reliable, and/or long-term treatment for metabolic diseases, including obesity and T2DM. 
     In some embodiments, the invention provides genetically engineered bacteria that are capable of producing a metabolic and/or satiety effector molecule, and/or a modulator of inflammation, and/or a molecule which reduces excess bile salt levels, particularly in low-oxygen environments, e.g., the gut. In certain embodiments, the genetically engineered bacteria are non-pathogenic and may be introduced into the gut in order to treat metabolic diseases. In certain embodiments, the metabolic and/or satiety effector molecule and/or modulator of inflammation or/and or effector of excess bile salt reduction 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 modulating and treating metabolic diseases. 
    
    
     SUMMARY 
     The disclosure provides genetically engineered bacteria that are capable of treating metabolic diseases, including but not limited to, type 2 diabetes, obesity-related symptoms, Nonalcoholic Steatohepatitis (NASH), Prader Willi Syndrome, and cardiovascular disorders. The genetically engineered bacteria comprise one or more gene(s) or gene cassette(s), for the production of molecules which, inter alia, act as metabolic and/or satiety effectors and/or modulators of the inflammatory status and/or are able convert excess bile salts into non-toxic molecules, as described herein. 
     Another aspect of the invention provides methods for selecting or targeting genetically engineered bacteria based on increased levels of metabolite consumption, or production of certain metabolites. The invention also provides pharmaceutical compositions comprising the genetically engineered bacteria, and methods of modulating and treating disorders associated with metabolic disorders. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) or circuit(s), containing one or more native or non-native component(s), which mediate one or more mechanisms of action. The genetically engineered bacteria harbor these genes or gene cassettes or circuits on a plasmid or, alternatively, the genes/gene cassettes have been inserted into the chromosome at certain regions, where they do not interfere with essential gene expression. Additionally, one or more endogenous genes or regulatory regions within the bacterial chromosome may be mutated or deleted. 
     These gene(s)/gene cassette(s) may be under the control of constitutive or inducible promoters. Exemplary inducible promoters described herein include oxygen level-dependent promoters (e.g., FNR-inducible promoter), promoters induced by molecules or metabolites indicative of liver damage (e.g., bilirubin) and/or metabolic disease, 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. 
     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 one or more of GLP-1 and GLP-1 analogs, as described herein (4) one or more gene(s) or gene cassette(s) for the production of one or more bile salt hydrolases, as described herein (5) one or more gene(s) or gene cassette(s) for the production of one or more transporters, e.g. for the import of bile salts and/or metabolites, e.g. tryptophan and/or tryptophan metabolites, as described herein (6) one or more polypetides for secretion, including but not limited to.GLP-1 and its analogs, bile salt hydrolases, and tryptophan synthesis and/or catabolic enzymes of the tryptophan degradation pathways, in wild type or in mutated form (for increased stability or metabolic activity) (3) one or more components of secretion machinery, as described herein (4) one or more auxotrophies, e.g., deltaThyA (5) one more more antibiotic resistances, including but not limited to, kanamycin or chloramphenicol resistance (6) 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 (7) one or more mutations/deletions in one or more genes of the endogenous metabolic pathways, e.g., tryptophan synthesis pathway. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  depicts a schematic of an  E. coli  that is genetically engineered to express a kynurenine biosynthesis cassette and/or a tryptophan biosynthesis cassette and/or tryptophan catabolic cassette which produces bioactive tryptophan metabolites described herein and/or GLP-1 and/or a propionate gene cassette and/or a butyrate gene cassette under the control of a FNR-responsive promoter and further comprising a secretion system and a metabolite transporter system. 
         FIG. 2A  depicts a metabolic pathway for butyrate production  FIGS. 2B and 2C  depict two schematics of two different butyrate producing circuits (found in SYN-UCD503 and SYN-UCD504), both under the control of a tetracycline inducible promoter.  FIG. 2D  depicts a schematic of a third butyrate gene cassette (found in SYN-UCD505) under the control of a tetracycline inducible promoter. SYN-UCD503 comprises 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. SYN-UCD504 comprises 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 cassete that comprises at least the following genes: ter, thiA1, hbd, crt2, pbt, buk. SYN-UCD505 comprises 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 HE-specific molecules or metabolites indicative of liver damage (e.g., bilirubin), 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. 3  depicts the gene organization of exemplary engineered bacteria of the disclosure and their induction under anaerobic or inflammatory conditions for the production of butyrate.  FIGS. 3A and 3B  depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions.  FIG. 3A  depicts relatively low butyrate production under aerobic conditions in which oxygen (O2) prevents (indicated by “X”) FNR (grey boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiA1, hbd, crt2, pbt, and buk; black boxes) is expressed.  FIG. 3B  depicts increased butyrate production under low-oxygen conditions due to FNR dimerizing (two grey boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate.  FIGS. 3C and 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 (gray 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; black boxes) 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 gray arrows and black squiggles) and ultimately to the production of butyrate.  FIGS. 3E  and F 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 (gray 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; black boxes) is expressed. In  FIG. 3F , in the presence of H2O2, 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 gray arrows and black squiggles) and ultimately to the production of butyrate. 
         FIG. 4  depicts the gene organization of exemplary recombinant bacteria of the disclosure and their induction under anaerobic or inflammatory conditions for the production of butyrate.  FIGS. 4A and 4B  depict the gene organization of an exemplary recombinant bacterium of the invention and its induction under low-oxygen conditions.  FIG. 4A  depicts relatively low butyrate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by “X”) FNR (grey 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; black boxes) is expressed.  FIG. 4B  depicts increased butyrate production under low-oxygen conditions due to FNR dimerizing (two grey boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate.  FIGS. 4C and 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 (gray circle, “NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, pbt, buk; black boxes) 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 gray arrows and black squiggles) and ultimately to the production of butyrate.  FIGS. 4E and 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 (gray circle, “OxyR”) binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, pbt, buk; black boxes) 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 oxyS promoter. This leads to expression of the butyrate biosynthesis enzymes (indicated by gray arrows and black squiggles) and ultimately to the production of butyrate. 
         FIG. 5  depicts the gene organization of exemplary recombinant bacteria of the disclosure and their induction under anaerobic or inflammatory conditions for the production of butyrate.  FIGS. 5A and 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 (grey 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; black boxes) is expressed.  FIG. 5B  depicts increased butyrate production under low-oxygen conditions due to FNR dimerizing (two grey boxed “FNR”s), binding to the FNR-responsive promoter, and inducing expression of the butyrate biosynthesis enzymes, which leads to the production of butyrate.  FIGS. 5C and 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 (gray circle, “NsrR”) binds to and represses a corresponding regulatory region. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, tesB; black boxes) 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 gray arrows and black squiggles) and ultimately to the production of butyrate.  FIGS. 5E and 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 (gray circle, “OxyR”) binds to, but does not induce, the oxyS promoter. Therefore, none of the butyrate biosynthesis enzymes (ter, thiA1, hbd, crt2, tesB; black boxes) is expressed. In  FIG. 5F , 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 gray arrows and black squiggles) and ultimately to the production of butyrate. 
         FIG. 6  depicts a graph of butyrate production using the circuits shown in  FIG. 48 . Cells were grown in M9 minimal media containing 0.2% glucose and induced with ATC at early log phase. As seen in  FIG. 6A , similar amounts of butyrate were produced for each construct under aerobic vs anaerobic conditions. The ter strain produces more butyrate overall. pLogic031 comprises (bdc2 butyrate cassette under control of tet promoter on a plasmid) and pLogic046 comprises (ter butyrate cassette under control of tet promoter on a plasmid).  FIG. 6B  depicts butyrate production of pLogic046 (ter butyrate cassette under control of tet promoter on a plasmid)) and a Nissle strain comprising plasmid pLOGIC046-delta pbt.buk/tesB+, an ATC-inducible ter-comprising butyrate construct with a deletion in the pbt-buk genes and their replacement with the tesB gene. The tesB construct results in greater butyrate production. 
         FIG. 7  depicts a graph of butyrate production using different butyrate-producing circuits comprising a nuoB gene deletion. Strains depicted are SYN-UCD503, SYN-UCD504, SYN-UCD510 (SYN-UCD510 is the same as SYN-UCD503 except that it further comprises a nuoB deletion), and SYN-UCD511 (SYN-UCD511 is the same as SYN-UCD504 except that it further comprises a nuoB deletion). 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. 8A  depicts a schematic of a butyrate producing circuit under the control of an FNR promoter.  FIG. 8B  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/0.5% glucose and incubated microaerobically to monitor butyrate production over time. SYN-UCD501 led to significant butyrate production under anaerobic conditions.  FIG. 8C  depicts a bar graph of butyrate production in strains comprising an FNR-butyrate cassette SYN501 (having the ter substitution) in the presence/absence of glucose and oxygen. 
         FIG. 9  depicts butyrate production by genetically engineered Nissle comprising the pLogic031-nsrR-norB-butyrate construct (SYN-507) or the pLogic046-nsrR-norB-butyrate construct (SYN-508), which produce more butyrate as compared to wild-type Nissle. 
         FIG. 10  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-&gt;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. 11  depicts a bar graph showing butyrate concentrations produced in vitro by strains comprising chromsolmally integrated butyrate copies as compared to plasmid cpopies. Integrated butyrate strains, SYN1001 and SYN1002 gave comparable butyrate production to the plasmid strain SYN501. 
         FIG. 12  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. 
         FIGS. 13A-13B  depicts the gene organization of an exemplary engineered bacterium of the invention and its induction under low-oxygen conditions for the production of propionate.  FIG. 13A  depicts relatively low propionate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by “X”) FNR (grey boxed “FNR”) from dimerizing and activating the FNR-responsive promoter (“FNR promoter”). Therefore, none of the propionate biosynthesis enzymes (pct, lcdA, lcdB, cdC, etfA, acrB, acrC; black boxes) are expressed.  FIG. 13B  depicts increased propionate production under low-oxygen conditions due to FNR dimerizing (two grey 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. 14  depicts an exemplary propionate biosynthesis gene cassette. 
         FIGS. 15A, 15B and 15C  depict the gene organization of an exemplary engineered bacterium and its induction under low-oxygen conditions for the production of propionate.  FIG. 15A  depicts relatively low propionate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by “X”) FNR (grey 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; black boxes) are expressed.  FIG. 15B  depicts increased propionate production under low-oxygen conditions due to FNR dimerizing (two grey 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. 15C  depicts an exemplary propionate biosynthesis gene cassette. 
         FIGS. 16A and 16B  depict the gene organization of an exemplary engineered bacterium and its induction under low-oxygen conditions for the production of propionate.  FIG. 16A  depicts relatively low propionate production under aerobic conditions in which oxygen (O 2 ) prevents (indicated by “X”) FNR (grey 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; black boxes) are expressed.  FIG. 16B  depicts increased propionate production under low-oxygen conditions due to FNR dimerizing (two grey 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. 17  depicts a schematic of an exemplary propionate biosynthesis gene cassette. 
         FIG. 18  depicts a schematic of an exemplary propionate biosynthesis gene cassette. 
         FIG. 19  depicts a schematic of a genetically engineered sleeping beauty metabolic pathway from  E. coli  for propionate production. Glucose and glycerol dissimilation pathways are shown under microaerobic conditions. In vivo, e.g., in a mammal, glycerol is not a substrate, and therefore only the glucose pathway is utilized. 
         FIGS. 20A-20B  depicts a propionate production strategy.  FIG. 20A  a schematic of a construct comprising the sleeping beauty mutase operon from  E. coli  under the control of a heterologous FnrS promoter.  FIG. 20B  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. 20A . 
         FIG. 21  depicts a schematic of a construct comprising GLP-1 (1-37) under the control of the FliC promoter and 5′UTR containing the N-terminal flagellar secretion signal for secretion. 
         FIG. 22  depicts bile salt metabolism. Bile salts are synthesized from cholesterol in the liver and stored in the gallbladder. After release into the duodenum, microbial bile salt hydrolase activity in the small intestine deconjugates the glycine or taurine molecules to produce primary bile acids (also known as unconjugated bile acids). Most bile acids are reabsorbed into the enterohepatic portal system, but some enter the large intestine where they are further metabolized by microbial 7α-dehydroxylase to produce secondary bile acids. Excess bile acids are also lost in the stool (200 mg-600 mg per day). 
         FIG. 23  depicts the structure of bile salts and the location at which bile salt hydrolase enzymes deconjugate the bile salts. BSH activity has been detected in  Lactobacillus  spp,  Bifidobacterium  spp,  Enterococcus  spp,  Clostridium  spp, and  Bacteroides  spp. BSH positive bacteria are gram positive with the exception of two  Bacteroides  strains. BSH in has been detected in pathogenic bacteria, e.g.,  Listeria monocytogenes  and  Enterococcus feacalis. E. coli  does not demonstrate BSH actvity nor contain bsh homolog in genome 
         FIG. 24  depicts the state of one non-limiting embodiment of the bile salt hydrolase enzyme construct under inducing conditions. Expression of the bile salt hydrolase enzyme and a bile salt transporter are both induced by the FNR promoter in the absence of oxygen. The thyA gene has been mutated in the  E. coli  Nissle genome, so thymidine must be supplied in the culture medium to support growth. The recombinant bacterial cell may further comprise an auxotrophic mutation, a type III secretion system, and/or a kill switch, as further described herein. 
         FIG. 25  depicts schematic of the  E. coli  tryptophan synthesis pathway, including genes, enzymes, and reactions involved. The seven genes, or genetic segments, seven enzymes, or enzyme domains, and seven reactions, involved in tryptophan formation are shown. Only one of the reactions is reversible. The products of four other pathways contribute carbon and/or nitrogen during tryptophan formation. Two of the tryptophan pathway enzymes often function as polypeptide complexes: anthranilate synthase, consisting of the TrpG and TrpE polypeptides, and tryptophan synthase, consisting of the TrpB and TrpA polypeptides. 
         FIG. 26  depicts a schematic of one embodiment of the disclosure. In this embodiment, tryptophan is synthesized from kynurenine. Through this conversion, a kynurenine can be removed from the external environment, and tryptophan is generated. Kynureninase from  Pseudomonas fluorescens  converts KYN to AA (Anthranillic acid), which then can be converted to tryptophan through the enzymes of the  E. coli  trp operon. Optionally, the trpE gene may be deleted as it is not needed for the generation of tryptophan from kynurenine. In alternate embodiments, the trpE gene is not deleted, in order to maximize tryptophan production by using both kynurenine and chorismate as a substrate. In some embodiments, a new strain is generated through adaptive laboratory evolution. The ability of this strain to metabolize kynurenine is improved (through lowering of kynurenine substrate). Additionally, the ability or preference of the strain take up tryptophan is lowered (due to selection pressure imposed by toxic tryptophan analogs. As a result, this strain has improved therapeutic properties in a number of applications, including but not limited to immunoncology. 
         FIG. 27  shows a schematic depicting an exemplary Tryptophan circuit. Tryptophan is produced from the Chorismate precursor through expression of the trpE, trpG-D, trpC-F, trpB and trpA genes. Optional knockout of the tryptophan Repressor trpR is also depicted. Optional production of the Chorismate precursor through expression of aroG/F/H and aroB, aroD, aroE, aroK and aroC genes is also shown. All of these genes are optionally expressed from an inducible promoter, e.g., a FNR-inducible promoter. The bacteria may also include an auxotrophy, e.g., deletion of thyA (A thyA; thymidine dependence). The bacteria may also include gene sequence(s) for yddG to express YddG to assist in the exportation of tryptophan. Non limiting example of a bacterial strain is listed. 
         FIG. 28  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. 29  depicts a schematic of tryptophan metabolism 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. In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes comprising one or more of the tryptophan metabolism enzymes depicted in  FIG. 26 , or bacterial functional homologs thereof. In certain embodiments of the disclosure, the genetically engineered bacteria comprise gene cassettes which produce one or more of the tryptophan metabolites depicted in  FIG. 29 . 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 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. 
         FIG. 30  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. 30 . In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in  FIG. 30 , including but not limited to, kynurenine, indole-3-aldehyde, indole-3-acetic acid, and/or indole-3 acetaldehyde. 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 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. 
         FIG. 31  depicts a schematic of the trypophan metabolic pathway. 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  FIG. 31 . In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in  FIG. 31 , including but not limited to, kynurenine, indole-3-aldehyde, indole-3-acetic acid, and/or indole-3 acetaldehyde. 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 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. 
         FIG. 32A  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. 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. 32A . In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes which produce one or more of the metabolites depicted in  FIG. 32A . 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 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.  FIG. 32B  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; I3C: Indole-3-carbinol; IAld: Indole-3-aldehyde; DIM: 3,3′-Diindolylmethane; ICZ: Indolo(3,2-b)carbazole. 
         FIG. 33  depicts a schematic showing an exemplary Kynurenine Degradation Circuit. Kynurenine is imported into the cell through expression of the aroP, tnaB or mtr transporter. Kynureninase is expressed to metabolize Kynurenine to Anthranilic acid in the cell. Both the transporter and kynureninase genes are optionally expressed from an inducible promoter, e.g., a FNR-inducible promoter. The bacteria may also include an auxotrophy, e.g., deletion of thyA (A thyA). 
         FIG. 34  depicts a schematic showing an exemplary Kynurenine Synthesis Circuit. Kynurenine and or Tryptophan is imported into the cell through expression of the aroP, tnaB or mtr transporter. Kynurenine biosynthetic cassette is expressed to produce Kynurenine. Both the transporter and Kynurenine biosynthetic cassette genes are optionally expressed from an inducible promoter, e.g., a FNR-inducible promoter. The bacteria may also include an auxotrophy, e.g., deletion of thyA (A thyA). 
         FIG. 35  depicts a schematic showing an exemplary Kynurenine Synthesis Circuit. Kynurenine and or Tryptophan is imported into the cell through expression of the aroP, tnaB or mtr transporter. Tryptophan is synthesized and then Kynurenine is synthesized from the synthesized tryptophan or from tryptophan imported into the cell. Both the transporter and kynureninase biosynthetic genes are optionally expressed from an inducible promoter, e.g., a FNR-inducible promoter. The bacteria may also include an auxotrophy, e.g., deletion of thyA (A thyA). 
         FIGS. 36A-36C  depicts a bar graphs showing the results of a checkerboard assay to establish the concentrations of kynurenine and 5-fluoro-L-tryptophan (ToxTrp) capable of sustaining growth of a trpE mutant of  E. coli  Nissle expressing pseudoKYNase. In  FIG. 36A , Bacteria were grown in the presence of different concentrations of KYNU and ToxTrp, and in the absence of Anhydrous Tetracycline (aTc). Growth was assessed at OD600. In  FIG. 36B , Bacteria were grown in the presence of different concentrations of KYNU and ToxTrp, and in the presence of Anhydrous Tetracycline (aTc). Growth was assessed at OD600.  FIG. 36C  depicts a bar graph showing the growth of the wild-type  E. coli  Nissle (SYN094) and trpE control strain in M9+KYNU, without ToxTrp. 
         FIGS. 37A-37H  depicts schematics of non-limiting examples of embodiments of the disclosure. In all embodiments, optil3 
       ally gene(s) which encode exporters may also be included.  FIG. 37A  depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce tryptamine from tryptophan. The circuits for tryptophan production are as depicted and described in  FIG. 27 . Alternatively, 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, under the control of an inducible promoter e.g., an FNR promoter.  FIG. 37B  depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-acetaldehyde and FICZ from tryptophan. The circuits for tryptophan production are as depicted and described in  FIG. 27 . Alternatively, 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 taal (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, under the control of an inducible promoter e.g., an FNR promoter.  FIG. 37C  depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-acetaldehyde and FICZ from tryptophan. The circuits for tryptophan production are as depicted and described in  FIG. 27 . Alternatively, 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 tynA (Monoamine oxidase, e.g., from  E. coli ), which converts tryptophan to indole-3-acetaldehyde and FICZ, under the control of an inducible promoter e.g., an FNR promoter.  FIG. 37D  depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole-3-acetonitrile from tryptophan. The circuits for tryptophan production are as depicted and described in  FIG. 27 . Alternatively, 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, under the control of an inducible promoter e.g., an FNR promoter.  FIG. 37E  depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce kynurenine from tryptophan. The circuits for tryptophan production are as depicted and described in  FIG. 27 . Alternatively, 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, under the control of an inducible promoter e.g., an FNR promoter.  FIG. 37F  depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce kynureninic acid from tryptophan. The circuits for tryptophan production are as depicted and described in  FIG. 27 . Alternatively, 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. 37G  depicts one embodiment of the disclosure, in which the genetically engineered bacteria produce indole from tryptophan. The circuits for tryptophan production are as depicted and described in  FIG. 27 . Alternatively, 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, under the control of an inducible promoter e.g., an FNR promoter.  FIG. 37H  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. 
         FIG. 38A  depicts schematics of exemplary embodiment of the disclosure, in which the genetically engineered bacteria convert tryptophan into indole-3-acetic acid. In  FIG. 38A , the circuits for tryptophan production are as depicted and described in  FIG. 27 . Alternatively, 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 taal (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, under the control of an inducible promoter e.g., an FNR promoter. 
       In  FIG. 38B  the circuits for tryptophan production are as depicted and described in  FIG. 27 . Alternatively, 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 ) 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 ), under the control of an inducible promoter e.g., an FNR promoter. In  FIG. 38C  the circuits for tryptophan production are as depicted and described in  FIG. 27 . Alternatively, 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 taal (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 ) under the control of an inducible promoter e.g., an FNR promoter. In  FIG. 38D  the circuits for tryptophan production are as depicted and described in  FIG. 27 . Alternatively, tryptophan can be imported through a transporter. In addition, the genetically engineered bacteria comprise a circuit comprising IaaM (Tryptophan 2-monooxygenase e.g., from  Pseudomonas savastanoi ) and iaaH (Indoleacetamide hydrolase, e.g., from  Pseudomonas savastanoi ), under the control of an inducible promoter e.g., an FNR promoter. 
       In  FIG. 38E  the circuits for tryptophan production are as depicted and described in  FIG. 27 . Alternatively, 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 Arabidopis  thaliana ) and nit1 (Nitrilase, e.g., from  Arabidopsis thaliana ) and iaaH (Indoleacetamide hydrolase, e.g., from  Pseudomonas savastanoi ), under the control of an inducible promoter e.g., an FNR promoter. 
         FIG. 39  depicts a schematic of an  E. coli  that is genetically engineered to express a butyrate cassette. 
         FIG. 40  depicts a schematic of an  E. coli  that is genetically engineered to express a a propionate biosynthestic cassette. 
         FIG. 41  depicts a schematic of an  E. coli  that is genetically engineered to express a GLP-1 and a secretion system as known in the art or described herein. 
         FIG. 42  depicts a schematic of an  E. coli  that is genetically engineered to express a butyrate and a propionate biosynthestic cassette. 
         FIG. 43  depicts a schematic of an  E. coli  that is genetically engineered to produce kynurenine, butyrate, and tryptophan (which can be converted to kynurenine or exported), under the control of a FNR-responsive promoter and further comprising a secretion system as known in the art or described herein. Export mechanism for kynurenine and/or tryptophan is also expressed or provided. 
         FIG. 44  depicts a schematic of an  E. coli  that is genetically engineered to produce kynurenine, butyrate, and tryptophan (which can be converted to kynurenine or exported), under the control of a FNR-responsive promoter and further comprising a secretion system as known in the art or described herein. A tryptophan transporter for import of tryptophan also expressed. Export mechanism for kynurenine is also expressed or provided. 
         FIG. 45  depicts a schematic of an  E. coli  that is genetically engineered to produce butyrate, tryptophan metabolites, and tryptophan (which can be converted to bioactive tryptophan metabolites or exported), under the control of a FNR-responsive promoter and further comprising a secretion system as known in the art or described herein. Export mechanism for tryptophan and/or tryptophan metabolites is also expressed or provided. 
         FIG. 46  depicts a schematic of an  E. coli  that is genetically engineered to produce butyrate, and propionate, kynurenine and/or other tryptophan metabolites, and GLP-1, under the control of a FNR-responsive promoter and further comprising a secretion system, e.g., for GLP-1 secretion as known in the art or described herein. Export mechanism for kynurenine/or tryptophan metabolites is also expressed or provided. 
         FIG. 47  depicts a map of exemplary integration sites within the  E. coli  1917 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. The malE/K site is circled. In some embodiments of the disclosure, FNR-ArgAfbr is inserted at the malEK locus. 
         FIG. 48  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. 49  depicts an exemplary schematic of the  E. coli  1917 Nissle chromosome comprising multiple mechanisms of action (MoAs). 
         FIG. 50  depicts an exemplary schematic of the  E. coli  1917 Nissle chromosome comprising multiple MoAs. In some embodiments, an Glp-1 expression circuit, a butyrate production circuit, a propionate production circuit, and a kynurenine biosynthetic cassette are inserted at four or more different chromosomal insertion sites 
         FIG. 51  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. 52  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. 53  depicts a schematic of a type I 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. 54  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. 55  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). 
         FIG. 56A  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 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. 56A  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. 56B  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. 56C  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. 57  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. 58  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. 59  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. 60  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. 61  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. 62  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 Table 2 (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. 
         FIG. 63A  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. 63B  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. 63C  depicts the growth of bacterial cell cultures expressing lacZ over time, both in the presence and absence of oxygen. 
         FIG. 64  depicts ATC ( FIG. 64A ) or nitric oxide-inducible ( FIG. 64B ) reporter constructs. 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. 64C  depicts a schematic of the constructs.  FIG. 64D  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. 65  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. 66  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. 66  depicts a bar graph of residence over time for streptomycin resistant Nissle. 
         FIG. 67  depicts a schematic diagram of a wild-type clbA construct (upper panel) and a schematic diagram of a clbA knockout construct (lower panel). 
         FIG. 68  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 animla disease model; 9. Assimilate into the microbiome; 10. Develop understanding of in vivo PK and dosing regimen. 
         FIGS. 69A , B, C, D, and E depict a schematic of non-limiting manufacturing processes for upstream and downstream production of the genetically engineered bacteria of the present disclosure.  FIG. 69A  depicts the parameters for starter culture 1 (SC1): loop full—glycerol stock, duration overnight, temperature 37° C., shaking at 250 rpm.  FIG. 69B  depicts the parameters for starter culture 2 (SC2): 1/100 dilution from SC1, duration 1.5 hours, temperature 37° C., shaking at 250 rpm.  FIG. 69C  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.  FIG. 69D  depicts the parameters for harvest: centrifugation at speed 4000 rpm and duration 30 minutes, wash 1×10% glycerol/PBS, centrifugation, re-suspension 10% glycerol/PBS.  FIG. 69E  depicts the parameters for vial fill/storage: 1-2 mL aliquots, −80° C. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     The invention includes genetically engineered bacteria, pharmaceutical compositions thereof, and methods of modulating and treating metabolic diseases. In some embodiments, the genetically engineered bacteria of the invention comprise a gene encoding a non-native metabolic and/or satiety effector molecule, or a gene cassette encoding a non-native biosynthetic pathway for producing a non-native metabolic and/or satiety effector molecule. The gene or gene cassette is further operably linked to a regulatory region that is controlled by a transcription factor that is capable of sensing low-oxygen conditions. The genetically engineered bacteria are capable of producing metabolic and/or satiety effector molecule in low-oxygen environments, e.g., the gut. Thus, the genetically engineered bacteria and pharmaceutical compositions comprising those bacteria may be used in order to treat and/or prevent conditions associated with metabolic diseases, including obesity and type 2 diabetes. 
     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, “metabolic diseases” include, but are not limited to, type 1 diabetes; type 2 diabetes; metabolic syndrome; Bardet-Biedel syndrome; Prader-Willi syndrome; non-alcoholic fatty liver disease; tuberous sclerosis; Albright hereditary osteodystrophy; brain-derived neurotrophic factor (BDNF) deficiency; Single-minded 1 (SIM1) deficiency; leptin deficiency; leptin receptor deficiency; pro-opiomelanocortin (POMC) defects; proprotein convertase subtilisin/kexin type 1 (PCSK1) deficiency; Src homology 2B1 (SH2B1) deficiency; pro-hormone convertase 1/3 deficiency; melanocortin-4-receptor (MC4R) deficiency; Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome; pseudohypoparathyroidism type 1A; Fragile X syndrome; Borjeson-Forsmann-Lehmann syndrome; Alstrom syndrome; Cohen syndrome; and ulnar-mammary syndrome. 
     Symptoms associated with the aforementioned diseases and conditions include, but are not limited to, one or more of weight gain, obesity, fatigue, hyperlipidemia, hyperphagia, hyperdipsia, polyphagia, polydipsia, polyuria, pain of the extremities, numbness of the extremities, blurry vision, nystagmus, hearing loss, cardiomyopathy, insulin resistance, light sensitivity, pulmonary disease, liver disease, liver cirrhosis, liver failure, kidney disease, kidney failure, seizures, hypogonadism, and infertility. 
     Metabolic diseases are associated with a variety of physiological changes, including but not limited to elevated glucose levels, elevated triglyceride levels, elevated cholesterol levels, insulin resistance, high blood pressure, hypogonadism, subfertility, infertility, abdominal obesity, pro-thrombotic conditions, and pro-inflammatory conditions. A metabolic effector is a molecule that is capable of minimizing any one or more of said physiological changes. For example, a metabolic effector molecule may enhance the body&#39;s sensitivity to insulin, thereby ameliorating insulin resistance. Insulin resistance is a physiological condition in which the body&#39;s insulin becomes less effective at lowering blood sugar. Excess blood sugar can cause adverse health effects such as type 2 diabetes. “Satiety” is used to refer to a homeostatic state in which a subject feels that hunger or food craving is minimized or satisfied. A satiety effector is a molecule that contributes to the minimization or satisfaction of said hunger or food craving. A molecule may be primarily a metabolic effector or primarily a satiety effector. A molecule may be both a metabolic and satiety effector, e.g., GLP-1. 
     “Metabolic effector molecules” and/or “satiety effector molecules” include, but are not limited to, n-acyl-phophatidylethanolamines (NAPEs), n-acyl-ethanolamines (NAEs), ghrelin receptor antagonists, peptide YY3-36, cholecystokinin (CCK) family molecules, CCK58, CCK33, CCK22, CCK8, bombesin family molecules, bombesin, gastrin releasing peptide (GRP), neuromedin B (P), glucagon, GLP-1, GLP-2, apolipoprotein A-IV, amylin, somatostatin, enterostatin, oxyntomodulin, pancreatic peptide, short-chain fatty acids, butyrate, propionate, acetate, serotonin receptor agonists, nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN), nucleotide riboside (NR), nicotinamide, and nicotinic acid (NA). Such molecules may also include compounds that inhibit a molecule that promotes metabolic disease, e.g., a single-chain variable fragment (scFv), antisense RNA, siRNA, or shRNA that inhibits dipeptidyl peptidase-4 (DPP4) or ghrelin receptor. A metabolic and/or satiety effector molecule may be encoded by a single gene, e.g., glucogon-like peptide 1 is encoded by the GLP-1 gene. Alternatively, a metabolic and/or satiety effector molecule may be synthesized by a biosynthetic pathway requiring multiple genes, e.g., propionate. These molecules may also be referred to as therapeutic molecules. 
     As used herein, the term “engineered bacterial cell” or “engineered bacteria” refers to a bacterial cell or bacteria that have been genetically modified from their native state. For instance, an engineered 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. Engineered bacterial cells disclosed herein may comprise exogenous nucleotide sequences on plasmids. Alternatively, engineered bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome. 
     A “programmed bacterial cell” or “programmed engineered bacterial cell” is an engineered bacterial cell 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, 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. “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 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, 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, and stem-loop structures. In one embodiment, the regulatory sequence comprises a promoter, e.g., an FNR responsive promoter. 
     As used herein, a “gene cassette” or “operon” encoding a biosynthetic pathway refers to the two or more genes that are required to produce a metabolic and/or satiety effector molecule, e.g., propionate. 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, cdC, 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, cdC, 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 a metabolic and/or satiety effector 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 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 “directly inducible promoter” refers to a regulatory region, wherein the regulatory region is operably linked to a gene or a gene cassette encoding a biosynthetic pathway for producing a metabolic and/or satiety effector molecule, e.g. propionate. In the presence of an inducer of said regulatory region, a metabolic and/or satiety effector molecule 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 gene encoding a first molecule, e.g., a transcription factor, which is capable of regulating a second regulatory region that is operably linked to a gene or a gene cassette encoding a biosynthetic pathway for producing a metabolic and/or satiety effector molecule, e.g. propionate. In the presence of an inducer of the first regulatory region, the second regulatory region may be activated or repressed, thereby activating or repressing production of propionate. Both a directly inducible promoter and an indirectly inducible promoter are encompassed by “inducible promoter.” 
     “Exogenous environmental condition(s)” refers to setting(s) or circumstance(s) under which the promoter described above is directly or indirectly induced. 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 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 gene or gene cassette for producing a therapeutic molecule is operably linked to an oxygen level-dependent promoter. 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. 
     In some embodiments, the gene or gene cassette for producing a metabolic and/or satiety effector molecule is operably linked to an oxygen level-dependent regulatory region such that the effector molecule is expressed in low-oxygen, microaerobic, or anaerobic conditions. For example, the oxygen level-dependent regulatory region is operably linked to a propionate gene cassette; in low oxygen conditions, the oxygen level-dependent regulatory region is activated by a corresponding oxygen level-sensing transcription factor, thereby driving expression of the propionate gene cassette. Examples of oxygen level-dependent transcription factors and corresponding promoters and/or regulatory regions include, but are not limited to, FNR, ANR, and DNR. Corresponding FNR-responsive promoters, ANR-responsive promoters, and DNR-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 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 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 
               
               
                   
                   
                 Table 4 
               
               
                   
                 ANR 
                 arcDABC 
               
               
                   
                 DNR 
                 norb, norC 
               
               
                   
                   
               
            
           
         
       
     
     As used herein, a “non-native” nucleic acid sequence refers to a nucleic acid sequence not normally present in a bacterium, 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 a sequence that is modified and/or mutated as compared to the unmodified sequence from bacteria 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 bacteria of the invention comprise a gene cassette that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene cassette in nature, e.g., a FNR-responsive promoter operably linked to a propionate gene cassette. 
     “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, BBa_J23100, a constitutive  Escherichia coli σ   S  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 σ   32  promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutive  Escherichia coli σ   70  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_M13110)), a constitutive  Bacillus subtilis  G A  promoter (e.g., promoter veg (BBa_K143013), promoter 43 (BBa_K143013), PliaG (BBa_K823000), P lepA  (BBa_K823002), P veg  (BBa_K823003)), a constitutive  Bacillus subtilis σ   B  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)). 
     “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. 
     “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, and protozoa. In some aspects, the microorganism is engineered (“engineered microorganism”) to produce one or more therapeutic molecules. In certain aspects, the microorganism is engineered to import and/or catabolize certain toxic metabolites, substrates, or other compounds from its environment, e.g., the gut. In certain aspects, the microorganism is engineered to synthesize certain beneficial metabolites, molecules, or other compounds (synthetic or naturally occurring) and release them into its environment. 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 are commensal bacteria, which are present in the indigenous microbiota of the gut. Examples of non-pathogenic bacteria include, but are not limited to  Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia, 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, 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). Naturally pathogenic bacteria may be genetically engineered to provide 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. Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic. Examples of probiotic bacteria include, but are not limited to, Bifidobacteria,  Escherichia, Lactobacillus , and  Saccharomyces , e.g.,  Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli, Escherichia coli  strain Nissle,  Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum , and  Saccharomyces boulardii  (Dinleyici et al., 2014; U.S. Pat. Nos. 5,589,168; 6,203,797; 6,835,376). 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, “stably maintained” or “stable” bacterium is used to refer to a bacterial host cell carrying non-native genetic material, e.g., a propionate gene cassette, 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/or 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 modified bacterium comprising a propionate gene cassette, in which the plasmid or chromosome carrying the propionate gene cassette is stably maintained in the host cell, such that the gene cassette can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro and/or in vivo. 
     As used herein, the term “treat” and its cognates refer to an amelioration of a disease or disorder, or at least one discernible symptom thereof. In another embodiment, “treat” refers to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient. In another embodiment, “treat” refers to inhibiting the progression of a disease or disorder, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. In another embodiment, “treat” refers to slowing the progression or reversing the progression of a disease or disorder. As used herein, “prevent” and its cognates refer to delaying the onset or reducing the risk of acquiring a given disease or disorder. 
     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 metabolic diseases may encompass reducing or eliminating associated symptoms, e.g., weight gain, and does not necessarily encompass the elimination of the underlying disease or disorder, e.g., congenital leptin deficiency. Treating the diseases described herein may encompass increasing levels of propionate, increasing levels of butyrate, and increasing GLP-1, and/or modulating levels of tryptophan and/or its metabolites (e.g., kynurenine), and does not necessarily encompass the elimination of the underlying disease. 
     As used herein a “pharmaceutical composition” refers to a preparation of genetically engineered bacteria of the invention 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 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, 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., obesity. 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 a metabolic disease. 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. 
     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. 
     Bacteria 
     The genetically engineered bacteria of the invention comprise a gene or gene cassette for producing a non-native metabolic and/or satiety effector molecule, 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 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, non-pathogenic bacteria are Gram-negative bacteria. In some embodiments, non-pathogenic bacteria are Gram-positive 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, 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, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis , and  Saccharomyces boulardii . In certain embodiments, the genetically engineered bacteria are selected from the group consisting of  Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Escherichia coli  Nissle,  Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri , and  Lactococcus lactis.    
     In some embodiments, the genetically engineered bacteria are  Escherichia coli  strain Nissle 1917 ( E. coli  Nissle), a Gram-positive 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 is 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 a recent study in non-human primates, Nissle was well tolerated by female cynomolgus monkeys after 28 days of daily NG dose administration at doses up to 1×1012 CFU/animal. No Nissle related mortality occurred and no Nissle related effects were identified upon clinical observation, body weight, and clinical pathology assessment (see, e.g., PCT/US16/34200). 
     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). 
     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). Thus the genetically engineered bacteria may require continued administration. Residence time in vivo may be calculated for the genetically engineered bacteria. 
     In certain embodiments, the payload(s) described below are expressed in one species, strain, or subtype of genetically engineered bacteria. In alternate embodiments, the payload is expressed in two or more species, strains, and/or subtypes of genetically engineered bacteria. 
     Metabolic Diseases 
     NASH 
     Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD), where excess fat accumulation in the liver results in chronic inflammation and damage. Nonalcoholic fatty liver disease is a component of metabolic syndrome and a spectrum of liver disorders ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). Simple liver steatosis is defined as a benign form of NAFLD with minimal risk of progression, in contrast to NASH, which tends to progress to cirrhosis in up to 20% of patients and can subsequently lead to liver failure or hepatocellular carcinoma. NASH affects approximately 3-5% of the population in America, especially in those identified as obese. NASH is characterized by such abnormalities as advanced lipotoxic metabolites, pro-inflammatory substrate, fibrosis, and increased hepatic lipid deposition. If left untreated, NASH can lead to cirrhosis, liver failure, and hepatocellular carcinoma. 
     Although patients diagnosed with alcoholic steatohepatitis demonstrate similar symptoms and liver damage, NASH develops in individuals who do not consume alcohol, and the underlying causes of NASH are unknown. Hepatic steatosis occurs when the amount of imported and synthesized lipids exceeds the export or catabolism in hepatocytes. An excess intake of fat or carbohydrate is the main cause of hepatic steatosis. NAFLD patients exhibit signs of liver inflammation and increased hepatic lipid accumulation. In addition, the development of NAFLD in obese individuals is closely associated with insulin resistance and other metabolic disorders and thus might be of clinical relevance). Therfore, Possible causative factors include insulin resistance, cytokine imbalance (specifically, an increase in the tumor necrosis factor-alpha (TNF-α)/adiponectin ratio), and oxidative stress resulting from mitochondrial abnormalities. 
     Currently, there is no accepted approach to treating NASH. Therapy generally involves treating known risk factors such as correction of obesity through diet and exercise, treating hyperglycemia through diet and insulin, avoiding alcohol consumption, and avoiding unnecessary medication. In animal models, administration of butyrate has been shown to reduce hepatic steatosis, inflammation, and fat deposition (see, for example, Jin et al., British J. Nutrition, 114(11):1745-1755, 2015 and Endo et al., PLoS One, 8(5):e63388, 2013). Colonic propionate delivery has also been shown to reduce intrahepatocellular lipid content in NASH patients, including improvements in weight gain and intra-abdominal fat deposition (see, for example, Chambers et al., Gut, gutjnl-2014), and GLP-1 administration has been shown to reduce the degree of lipotoxic metabolites and pro-inflammatory substrates, both of which have been shown to speed NASH development, as well as reduce hepatic lipid deposition (see, for example, Bernsmeier et al., PLoS One, 9(1):e87488, 2014 and Armstrong et al., J. Hepatol., 2015). 
     The liver has both an arterial and venous blood supply, with the majority of hepatic blood flow coming from the gut via the portal vein. In NASH the liver is exposed to potentially harmful substances derived from the gut (increased perability and reduced intestinal integrity), including translocated bacteria, LPS and endotoxins as well as secreted cytokines. Translocated microbial products might contribute to the pathogenesis of fatty liver disease by several mechanisms, including stimulating pro-inflammatory and profibrotic pathways via a range of cytokines. For example, butyrate and other SCFA, e.g., derived from the microbiota, are known to promote maintaining intestinal integrity. 
     The role of bile acids in the pathogenesis of NAFLD and NASH has been extensively studied (Leung et al., The Role Of The Gut Microbiota In NAFLD; Nature Reviews Gastroenterology &amp; Hepatology). For example, in one study study, manipulation of the gut microbiota changed intestinal bile acid composition leading to intestinal antagonism of FRX, the master regulator of bile acid metabolism. This FXR antagonism reduced ceramide synthesis and de novo lipogenesis in the liver (Jiang, C. et al. Intestinal farnesoid X receptor signaling promotes nonalcoholic fatty liver disease.  J. Clin. Invest.  125, 386-402 (2015)). 
     Studies have also suggested that rapid weight loss through bariatric surgery (e.g. gastric bypass) is effective in decreasing steatosis, hepatic inflammation, and fibrosis. Other treatments have involved using anti-diabetic medications such as metformin, rosiglitazone, and pioglitazone. Though inconclusive, the studies suggest that the medications stimulate insulin sensitivity in NASH patients, thus alleviating liver damage. In cases were NASH has resulted in advanced cirrhosis, the only treatment is a liver transplant. Regardless, no current treatments are wholly determinative or reliable for treating NASH. Therefore, a need exists for improved therapies and treatments of NASH. 
     In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of NAFLD and/or NASH. In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments the circuits stimulate insulin secretion and/or promote satiety. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate, and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of NAFLD and/or NASH. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including but not limited to bile salt hydrolase or bile salt transporter producing cassettes. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream indole tryptophan metabolites described herein, including, but not limited to those listed in Table 13 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more indole tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13, and elsewhere herein, in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. 
     In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios. 
     In certain embodiments, one or more of these circuits may be combined for the treatment of NASH and/or NAFLD. In a non-limiting example, butyrate producing, GLP-1 secreting, and ryptophan pathway modulating cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of NASH and/or NAFLD. 
     Diabetes 
     Diabetes mellitus type 1 (also known as type 1 diabetes) is a form of diabetes mellitus that results from the autoimmune destruction of the insulin-producing beta cells in the pancreas. The subsequent lack of insulin leads to increased glucose in blood and urine. The classical symptoms are frequent urination, increased thirst, increased hunger, and weight loss. In some embodiments the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of diabetes mellitus. 
     Diabetes mellitus type 2 is a long term metabolic disorder that is characterized by high blood sugar, insulin resistance, and relative lack of insulin. Common symptoms include increased thirst, frequent urination, and unexplained weight loss. Symptoms may also include increased hunger, feeling tired, and sores that do not heal. Often symptoms come on slowly. Long-term complications from high blood sugar include heart disease, strokes, diabetic retinopathy which can result in blindness, kidney failure, and poor blood flow in the limbs which may lead to amputations. 
     Insulin resistance (IR) is generally regarded as a pathological condition in which cells fail to respond to the normal actions of the hormone insulin. Normally insulin produced when glucose enters the circulation after a meal triggers glucose uptake into cells. Under conditions of insulin resistance, the cells in the body are resistant to the insulin produced after a meal, preventing glucose uptake and leading to high blood sugar. 
     The kynurenine hypothesis of diabetes is based on evidence of diabetogenic effects of the kynurenine metabolite Xanthurenic Acid (XA) and the realization that the KP is upregulated by low-grade inflammation and stress, two conditions involved in the pathogenesis of insulin resistance, and of diabetes type I and diabetes type II. Increased concentrations of KYNA and xanthurenic acid (3-Hydroxy KYNA, XA) were detected in the plasma of patients with type 2 diabetes, presumably due to chronic stress or the low-grade inflammation that are prominent risk factors for diabetes. The production of these kynurenine metabolites is a regulatory mechanism to attenuate damage by the inflammation-induced production of reactive oxygen species. 
     Experimental and clinical data have clearly established that besides fat, muscle and liver, pancreatic islet tissue itself is a site of inflammation during obesity and type 2 diabetes. It is therefore conceivable that in parallel to the high free fatty acids and glucose levels, pancreatic islet exposure to increased levels of cytokines may induce dysregulation of islet KP in a way resembling that seen in the brain in many neurodegenerative disorders. 
     In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of type 2 diabetes. In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments the circuits stimulate insulin secretion and/or promote satiety. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of type 2 diabetes. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including but not limited to bile salt hydrolase or bile salt transporter producing cassettes. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, not limited to those listed in Table 13 and elsewhere herein., in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. 
     In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios. 
     In certain embodiments, one or more of these circuits may be combined for the treatment of type 2 diabetes. In a non-limiting example, butyrate producing, GLP-1 secreting, and ryptophan pathway modulating cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of type 2 diabetes. 
     Obesity 
     Metabolic Syndrome affects approximately 20-30% of the middle-aged population, and represents an increased risk to cardiovascular disorders, the leading cause of death in the United States. Obesity, dyslipidemia, hypertension, and type 2 diabetes are described as metabolic syndrome. In some embodiments the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of metabolic syndrome and/or obesity. Several of the metabolites and polypeptides produced by the genetically engineered bacteria are useful for increasing insulin secretion and promoting satiety, e.g. GLP-1. 
     Obesity is a common, deadly, and costly disease in developed countries which impacts all age groups, race, and gender. Obesity can be classified as an inflammatory disease because it is associated with immune activation and a chronic, low-grade systemic inflammation. Endotoxemia, a process resulting from translocation of endotoxic compounds (lipopolysaccharides [LPS]), of gram-negative intestinal bacteria. In the last decade, it has become evident that insulin resistance and T2DM are characterized by low-grade inflammation. In this respect, LPS trigger a low-grade inflammatory response, and the process of endotoxemia can therefore result in the development of insulin resistance and other metabolic disorders. Several of the metabolites produced by the genetically engineered bacteria described herein are useful in the reduction of inflammation. For example, butyrate, contributes to maintaining intestinal integrity. Other anti-inflammatory metabolites as described herein may also be useful in the treatment of type 2 diaberes. 
     Over nutrition leads to an excess intake of tryptophan (TRP)—an essential amino acid, a precursor for serotonin (5-HT) and melatonin, and a key player in the caloric intake regulation. Yet, the circulating levels of TRP have been shown to be low in morbidly obese subjects (Brandacher G, Winkler C, Aigner F, et al. Bariatric surgery cannot prevent tryptophan depletion due to chronic immune activation in morbidly obese patients. Obes Surg 2006; 16:541-548). 
     Serotonin regulates carbohydrate and fat intake (Blundell J E, Lawton C L. Serotonin and dietary fat intake: effects of dexfenfluramine. Metabolism 1995; 44:33-37), relieves stress which is another caloric intake trigger (Buwalda B, Blom W A, Koolhaas J M, van Dijk G. Behavioral and physiological responses to stress are affected by high-fat feeding in male rats; Physiol Behav 2001; 73:371-377), and inhibits neuropeptide Y (NYP)—one of the most potent orexigenic peptides in the hypothalamus (Jia Y, El-Haddad M, Gendy A, Nguyen T, Ross M G. 
     In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of obesity. In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments the circuits stimulate insulin secretion and/or promote satiety. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1 and/or GLP-1 analog(s). In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of obesity. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including, but not limited, to bile salt hydrolase or bile salt transporter producing cassettes. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13 and elsewhere herein, in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13, and elsewhere herein, including but not limited to, Tryptamine, Indole-3-acetaldehyde, Indole-3-acetic acid, Indole, 6-formylindolo(3,2-b)carbazole, Kynurenic acid, Indole-3-aldehyde; 3,3′-Diindolylmethane. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein, in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and elsewhere herein. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein, in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. 
     In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios. 
     In certain embodiments, one or more of these circuits may be combined for the treatment of obesity. In a non-limiting example, butyrate producing, GLP-1 secreting, and ryptophan pathway modulating cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of obesity. 
     Prader Willi Syndrome 
     Prader-Willi syndrome (OMIM 176270) is a complex genetic neurodevelopmental disorder with manifested early in failure to thrive, feeding difficulties during infancy, hypogonadism/hypogenitalism, growth hormone deficiency, and typically a paternal 15q11-q13 chromosome deletion. In early childhood trough alduhood, food seeking behaviors and hyperphagia are noted along with a low metabolic rate and decreased physical activity leading to obesity which can be life-threatening, if not controlled. PWS is considered the most common syndromic cause of life threatening obesity in childhood (Buttler et al., Am J Med Genet A. 2015 March; 167A(3):563-71; Increased plasma chemokine levels in children with Prader-Willi syndrome). It has been reported that, when matched for body mass index (BMI), PWS adults had the same prevalence of metabolic syndrome (41.4%) and insulin resistance index as obese controls. 
     Prader-Willi syndrome (PWS) has no cure. PWS syndrome individuals present with obesity with hyperphagia and deficit of satiety, and in some cases insulin resistance, that persists thoughout youth and adulthood and remains a critical problem in PWS teenagers and adults because it leads to severe complications, such as limb edema, cardiac or respiratory failure, and physical disabilities. Severe obesity, and food seeking therfroe remains the larges problem with PWS. Access to food must be strictly supervised and limited. Therefore, agents which modulate satiety and orh insulin levels may be useful in the treatment of PWS. 
     In additiona, increased inflammatory markers and cytokine levels in the plasma have been observed in PWS individuals. These cytokines serve as chemoattractants for recruitment of immune cells and indicate an inflammatory component in PWS, which underlies certain aspects of the pathology (Buttler et al., Am J Med Genet A. 2015 March; 167A(3):563-71; Increased plasma chemokine levels in children with Prader-Willi syndrome). Therefore, anti-inflammatory agents may be useful in the treatment of certain aspects of PWS. 
     In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments the circuits stimulate insulin secretion and/or promote satiety. 
     In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of PWS. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of PWS. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including but not limited to bile salt hydrolase or bile salt transporter producing cassettes. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein, in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein, in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein, in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. 
     In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios. 
     In certain embodiments, one or more of these circuits may be combined for the treatment of PWS. In a non-limiting example, butyrate producing, GLP-1 secreting, and ryptophan pathway modulating cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of PWS. 
     Metabolic Syndrome 
     Metabolic syndrome is a clustering of at least three of five of the following medical conditions: abdominal (central) obesity, elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, and low high-density lipoprotein (HDL) levels. 
     In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of metabolic syndrome. In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments the circuits stimulate insulin secretion and/or promote satiety. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate, and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of metabolic syndrome. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including but not limited to bile salt hydrolase or bile salt transporter producing cassettes. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein, in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein, in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein, in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios. 
     In certain embodiments, one or more of these circuits may be combined for the treatment of metabolic syndrome. In a non-limiting example, butyrate producing, GLP-1 secreting, and ryptophan pathway modulating cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of metabolic syndrome. 
     Cardiovascular Disease 
     Metabolic syndrome is an important risk factor for cardiovascular disease incidence and mortality, as well as all-cause mortality. 
     Cardiovascular disease includes coronary artery diseases (CAD) such as angina and myocardial infarction, stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, and venous thrombosis. Coronary artery disease, stroke, and peripheral artery disease involve atherosclerosis, caused inter alia by high blood pressure, smoking, diabetes, lack of exercise, obesity, high blood cholesterol, poor diet, and excessive alcohol consumption, and the like. 
     The detection, prevention, and treatment of the underlying risk factors of the metabolic syndrome are a critical approach to lower the cardiovascular disease incidence in the general population. 
     Cellular adhesion molecules and oxidative stress play a role in the pathogenesis of atherosclerosis in patients with chronic kidney disease (CKD) and uremia. Uremia is condition that occurs when the kidneys no longer filter properly, and is likely to occur s in the final stage of chronic kidney disease. Several studies in CKD patients have shown that tryptophan metabolites along the kynurenine pathway are increased, possibly as consequence of inflammation. Therefore, anti-inflammatory agents may be useful in the treatment of cardiovascular disease, including CKD and artherosclerosis. In some embodiments the genetically engineered bacteria modulate the levels of one or more of tryptophan, kynurenine, kynurenine downstream metabolites, and other tryptophan metabolites and/or modulate one or more metabolite ratios. 
     Ischemic stroke, which results from cerebral arterial occlusion, is becoming a major cause of morbidity and mortality in today&#39;s society and affects millions of people every year. Currently, the only approved treatment for the acute phase of stroke is the recombinant thrombolytic tissue-type plasminogen activator. Identifying molecules that contribute to the ischemic damage may help to elucidate potential therapeutic targets. In some embodiments the genetically engineered bacteria described herein are useful in the treatment, prevention and/or management of ischemia and stroke. Inflammation and oxidative stress are also involved in brain damage following stroke, and tryptophan oxidation along the kynurenine pathway contributes to the modulation of oxidative stress. 
     In some embodiments, the genetically engineered bacteria are useful for the prevention, treatment, and/or management of cardiovascular disease, including but not limited to, one or more of coronary artery diseases, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, venous thrombosis, ischemic stroke, and/or chronic kidney disease. In some embodiments, the genetically engineered bacteria comprise circuits which reduce inflammation. In some embodiments the circuits stimulate insulin secretion and/or promote satiety. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate and/or acetate. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of GLP-1. In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the production of short-chain fatty acids, e.g., butyrate and/or propionate for the treatment of cardiovascular disease, including but not limited to, one or more of coronary artery diseases, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, venous thrombosis, ischemic stroke, and/or chronic kidney disease. 
     In some embodiments, the genetically engineered bacteria comprise one or more gene cassettes for the increase of bile salt catabolism, including but not limited to bile salt hydrolase or bile salt transporter producing cassettes. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein, in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which modulate the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which modulate the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein, in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which increase the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which increase the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease typtophan levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease kynurenine levels in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream kynurenine metabolites described herein in the patient, e.g., in the serum and/or in the gut. In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease levels of downstream tryptophan metabolites described herein, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein, in the patient, e.g., in the serum and/or in the gut. 
     In certain embodiments, the genetically engineered bacteria comprise one or more gene cassettes as described herein, which decrease the TRP/KYN ratio in the patient, e.g., in the serum and/or in the gut. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of tryptophan to one or more kynurenine downstream metabolites described herein, e.g., in  FIG. 29 . In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios of kynurenine to one or more downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between two downstream kynurenine metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. In some embodiments, the genetically engineered bacteria comprise gene cassettes which decrease the ratios between one or more tryptophan metabolites, including, but not limited to those listed in Table 13 and  FIG. 32  and elsewhere herein. 
     In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases serotonin and or melatonin levels. In some embodiments, the genetically engineered bacteria comprise a gene cassette which modulates the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which increases the tryptophan to serotonin and or melatonin ratios. In some embodiments, the genetically engineered bacteria comprise a gene cassette which decreases the tryptophan to serotonin and or melatonin ratios. 
     In certain embodiments, one or more of these circuits may be combined for the treatment of cardionvascular disorders. In a non-limiting example, butyrate producing, GLP-1 secreting, and ryptophan pathway modulating cassettes may be expressed in combination by the genetically engineered bacteria for the treatment of cardionvascular disorders. 
     Metabolic and Satiety Effector Molecules, and Modulators of Inflammation 
     The genetically engineered bacteria of the invention comprise a gene encoding a non-native metabolic and/or satiety effector molecule, or a gene cassette encoding a biosynthetic pathway capable of producing a metabolic and/or satiety effector molecule. In some embodiments, the metabolic and/or satiety effector molecule is selected from the group consisting of n-acyl-phophatidylethanolamines (NAPEs), n-acyl-ethanolamines (NAEs), ghrelin receptor antagonists, peptide YY3-36, cholecystokinin (CCK) family molecules, CCK58, CCK33, CCK22, CCK8, bombesin family molecules, bombesin, gastrin releasing peptide (GRP), neuromedin B (P), glucagon, GLP-1, GLP-2, apolipoprotein A-IV, amylin, somatostatin, enterostatin, oxyntomodulin, pancreatic peptide, short-chain fatty acids, butyrate, propionate, acetate, serotonin receptor agonists, nicotinamide adenine dinucleotide (NAD), nicotinamide mononucleotide (NMN), nucleotide riboside (NR), nicotinamide, and nicotinic acid (NA). A molecule may be primarily a metabolic effector, or primarily a satiety effector. Alternatively, a molecule may be both a metabolic and satiety effector. 
     In some embodiments, the genetically engineered bacteria of the invention comprise one or more gene(s) or gene cassette(s) which are capable of producing an effector, which can modulate the inflammatory status. Non-limiting examples include short shain fatty acides, and tryptophan and its metabolites, as described herein. 
     The effect of the genetically engineered bacteria on the inflammatory status can be measured by methods known in the art, e.g., plasma can be drawn before and after administration of the genetically engineered bacteria. The erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and plasma viscosity (PV) blood tests are commonly used to detect this increase n inflammation. In some embodiments the genetically engineered bacteria modulate, e.g. decrease or increase, levels of inflammatory markers, eg. C-reactive protein (CRP). 
     In some embodiments, the genetically engineered bacteria modulate, e.g. decrease, levels of inflammatory growth factors and cytokines, e.g., IL-10, IL-6, and/or TNF-α and proinflammatory signaling, e.g. NF-kappaB signaling. In some embodiments the genetically engineered bacteria modulate, e.g. increase, levels of anti-inflammatory growth factors and cytokines, e.g., IL4, IL-10, IL-13, IFN-alpha and/or transforming growth factor-beta. [0210] 
     In some embodiments, the genetically engineered bacteria produce effectors, which bind to and stimulate the aromatic hydrocarbon receptor. In some embodiments the genetically engineered bacteria stimulate AHR signaling in immune cell types, including T cells, B cells, NK cells, macrophages, and dendritic cells (DCs), and/or in epithelial cells. In some embodiments, the genetically engineered bacteria modulate, e.g., increase the levels of IL-22, e.g., through stimulation of AHR. 
     In some emobodiments, the genetically engineered bacteria may reduce gut permeability. In some embodiments, the the genetically engineered bacteria may reduce the amounts of LPS and in the circulation, which are increase in metabolic disease, e.g., in NASH. 
     The gene or gene cassette for producing the metabolic and/or satiety effector molecule and/or modulator of inflammation may be expressed under the control of a constitutive promoter, a promoter that is induced by exogenous environmental conditions, a promoter that is induced by exogenous environmental conditions, molecules, or metabolites specific to the gut of a mammal, and/or a promoter that is induced by low-oxygen or anaerobic conditions, such as the environment of the mammalian gut. 
     The gene or gene cassette for producing the metabolic and/or satiety effector and/or modulator of inflammation 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 metabolic and/or satiety effector molecule. In some embodiments, expression from the chromosome may be useful for increasing stability of expression of the metabolic and/or satiety effector molecule. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector molecule 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 propionate biosynthesis gene cassette may be integrated into the bacterial chromosome. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector molecule is expressed from a plasmid in the genetically engineered bacteria. In some embodiments, the gene or gene cassette for producing the metabolic and/or satiety effector molecule 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. 47 ). 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. In some embodiments, the genetically engineered bacteria of the invention are capable of expressing a metabolic and/or satiety effector molecule that is encoded by a single gene, e.g., the molecule is GLP-1 and encoded by the GLP-1 gene. 
     One of skill in the art would appreciate that additional genes and gene cassettes capable of producing metabolic and/or satiety effector molecules and/or modulator of inflammation 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 metabolic and/or satiety effector molecules and/or modulator of inflammation. In certain embodiments, the two or more molecules behave synergistically to ameliorate metabolic disease. In some embodiments, the genetically engineered bacteria express at least one metabolic effector molecule and at least one satiety effector molecule and at least one modulator of inflammation. 
     Short Chain Fatty Acids 
     Short-chain fatty acids (SCFAs), primarily acetate, propionate, and butyrate, are metabolites formed by gut microbiota from complex dietary carbohydrates. Butyrate and acetate were reported to protect against diet-induced obesity without causing hypophagia, while propionate was shown to reduce food intake. In rodent models of genetic or diet-induced obesity, supplementation of butyrate in diet, and oral administration of acetate was shown to suppress weight gain independent of food intake suppression; Propionate was reported to inhibit food intake in humans (see, e.g., Lin et al., Butyrate and Propionate Protect against Diet-Induced Obesity and Regulate Gut Hormones via Free Fatty Acid Receptor 3-Independent Mechanisms, and refernces therein). Therefore, the production of SCFAs is likely efficacious in the treatment of metabolic syndrome and related disorders, and/or diabetes type2, and/or obesity. 
     SCFAs represent a major constituent of the luminal contents of the colon. Among SCFAs butyrate is believed to play an important role for epithelial homeostasis. Acetate and propionate have anti-inflammatory properties, which are comparable to those of butyrate (Tedelind et al., World J Gastroenterol. 2007 May 28; 13(20): 2826-2832. Acetate and propionate, similar to butyrate, inhibit TNFα-mediated activation of the NF-KB pathway. These findings suggest that propionate and acetate, in addition to butyrate, could be efficacious in the treatment of inflammatory conditions. 
     Propionate 
     In alternate embodiments, the genetically engineered bacteria of the invention are capable of producing a metabolic and/or satiety effector 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 2). 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 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 2-4 lists the nucleic acid sequences of exemplary genes in the propionate biosynthesis gene cassette. Table 5 lists the polypeptide sequences expressed by exemplary propionate biosynthesis genes. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Propionate Cassette Sequences (Acrylate Pathway) 
               
            
           
           
               
               
            
               
                 Gene sequence 
                 Description 
               
               
                   
               
               
                 pct 
                 ATGCGCAAAGTGCCGATTATCACGGCTGACGAGGCCGCAAAACT 
               
               
                 SEQ ID NO: 1 
                 GATCAAGGACGGCGACACCGTGACAACTAGCGGCTTTGTGGGTA 
               
               
                   
                 ACGCGATCCCTGAGGCCCTTGACCGTGCAGTCGAAAAGCGTTTC 
               
               
                   
                 CTGGAAACGGGCGAACCGAAGAACATTACTTATGTATATTGCGG 
               
               
                   
                 CAGTCAGGGCAATCGCGACGGTCGTGGCGCAGAACATTTCGCGC 
               
               
                   
                 ATGAAGGCCTGCTGAAACGTTATATCGCTGGCCATTGGGCGACC 
               
               
                   
                 GTCCCGGCGTTAGGGAAAATGGCCATGGAGAATAAAATGGAGGC 
               
               
                   
                 CTACAATGTCTCTCAGGGCGCCTTGTGTCATCTCTTTCGCGATA 
               
               
                   
                 TTGCGAGCCATAAACCGGGTGTGTTCACGAAAGTAGGAATCGGC 
               
               
                   
                 ACCTTCATTGATCCACGTAACGGTGGTGGGAAGGTCAACGATAT 
               
               
                   
                 TACCAAGGAAGATATCGTAGAACTGGTGGAAATTAAAGGGCAGG 
               
               
                   
                 AATACCTGTTTTATCCGGCGTTCCCGATCCATGTCGCGCTGATT 
               
               
                   
                 CGTGGCACCTATGCGGACGAGAGTGGTAACATCACCTTTGAAAA 
               
               
                   
                 AGAGGTAGCGCCTTTGGAAGGGACTTCTGTCTGTCAAGCGGTGA 
               
               
                   
                 AGAACTCGGGTGGCATTGTCGTGGTTCAGGTTGAGCGTGTCGTC 
               
               
                   
                 AAAGCAGGCACGCTGGATCCGCGCCATGTGAAAGTTCCGGGTAT 
               
               
                   
                 CTATGTAGATTACGTAGTCGTCGCGGATCCGGAGGACCATCAAC 
               
               
                   
                 AGTCCCTTGACTGCGAATATGATCCTGCCCTTAGTGGAGAGCAC 
               
               
                   
                 CGTCGTCCGGAGGTGGTGGGTGAACCACTGCCTTTATCCGCGAA 
               
               
                   
                 GAAAGTCATCGGCCGCCGTGGCGCGATTGAGCTCGAGAAAGACG 
               
               
                   
                 TTGCAGTGAACCTTGGGGTAGGTGCACCTGAGTATGTGGCCTCC 
               
               
                   
                 GTGGCCGATGAAGAAGGCATTGTGGATTTTATGACTCTCACAGC 
               
               
                   
                 GGAGTCCGGCGCTATCGGTGGCGTTCCAGCCGGCGGTGTTCGCT 
               
               
                   
                 TTGGGGCGAGCTACAATGCTGACGCCTTGATCGACCAGGGCTAC 
               
               
                   
                 CAATTTGATTATTACGACGGTGGGGGTCTGGATCTTTGTTACCT 
               
               
                   
                 GGGTTTAGCTGAATGCGACGAAAAGGGTAATATCAATGTTAGCC 
               
               
                   
                 GCTTCGGTCCTCGTATCGCTGGGTGCGGCGGATTCATTAACATT 
               
               
                   
                 ACCCAAAACACGCCGAAAGTCTTCTTTTGTGGGACCTTTACAGC 
               
               
                   
                 CGGGGGGCTGAAAGTGAAAATTGAAGATGGTAAGGTGATTATCG 
               
               
                   
                 TTCAGGAAGGGAAACAGAAGAAATTCCTTAAGGCAGTGGAGCAA 
               
               
                   
                 ATCACCTTTAATGGAGACGTGGCCTTAGCGAACAAGCAACAAGT 
               
               
                   
                 TACCTACATCACGGAGCGTTGCGTCTTCCTCCTCAAAGAAGACG 
               
               
                   
                 GTTTACACCTTTCGGAAATCGCGCCAGGCATCGATCTGCAGACC 
               
               
                   
                 CAGATTTTGGATGTTATGGACTTTGCCCCGATCATTGATCGTGA 
               
               
                   
                 CGCAAACGGGCAGATTAAACTGATGGACGCGGCGTTATTCGCAG 
               
               
                   
                 AAGGGCTGATGGGCTTGAAAGAAATGAAGTCTTAA 
               
               
                   
               
               
                 IcdA 
                 ATGAGCTTAACCCAAGGCATGAAAGCTAAACAACTGTTAGCATA 
               
               
                 SEQ ID NO: 2 
                 CTTTCAGGGTAAAGCCGATCAGGATGCACGTGAAGCGAAAGCCC 
               
               
                   
                 GCGGTGAGCTGGTCTGCTGGTCGGCGTCAGTCGCGCCGCCGGAA 
               
               
                   
                 TTTTGCGTAACAATGGGCATTGCCATGATCTACCCGGAGACTCA 
               
               
                   
                 TGCAGCGGGCATCGGTGCCCGCAAAGGTGCGATGGACATGCTGG 
               
               
                   
                 AAGTTGCGGACCGCAAAGGCTACAACGTGGATTGTTGTTCCTAC 
               
               
                   
                 GGCCGTGTAAATATGGGTTACATGGAATGTTTAAAAGAAGCCGC 
               
               
                   
                 CATCACGGGCGTCAAGCCGGAAGTTTTGGTTAATTCCCCTGCTG 
               
               
                   
                 CTGACGTTCCGCTTCCCGATTTGGTGATTACGTGTAATAATATC 
               
               
                   
                 TGTAACACGCTGCTGAAATGGTACGAAAACTTAGCAGCAGAACT 
               
               
                   
                 CGATATTCCTTGCATCGTGATCGACGTACCGTTTAATCATACCA 
               
               
                   
                 TGCCGATTCCGGAATATGCCAAGGCCTACATCGCGGACCAGTTC 
               
               
                   
                 CGCAATGCAATTTCTCAGCTGGAAGTTATTTGTGGCCGTCCGTT 
               
               
                   
                 CGATTGGAAGAAATTTAAGGAGGTCAAAGATCAGACCCAGCGTA 
               
               
                   
                 GCGTATACCACTGGAACCGCATTGCCGAGATGGCGAAATACAAG 
               
               
                   
                 CCTAGCCCGCTGAACGGCTTCGATCTGTTCAATTACATGGCGTT 
               
               
                   
                 AATCGTGGCGTGCCGCAGCCTGGATTATGCAGAAATTACCTTTA 
               
               
                   
                 AAGCGTTCGCGGACGAATTAGAAGAGAATTTGAAGGCGGGTATC 
               
               
                   
                 TACGCCTTTAAAGGTGCGGAAAAAACGCGCTTTCAATGGGAAGG 
               
               
                   
                 TATCGCGGTGTGGCCACATTTAGGTCACACGTTTAAATCTATGA 
               
               
                   
                 AGAATCTGAATTCGATTATGACCGGTACGGCATACCCCGCCCTT 
               
               
                   
                 TGGGACCTGCACTATGACGCTAACGACGAATCTATGCACTCTAT 
               
               
                   
                 GGCTGAAGCGTACACCCGTATTTATATTAATACTTGTCTGCAGA 
               
               
                   
                 ACAAAGTAGAGGTCCTGCTTGGGATCATGGAAAAAGGCCAGGTG 
               
               
                   
                 GATGGTACCGTATATCATCTGAATCGCAGCTGCAAACTGATGAG 
               
               
                   
                 TTTCCTGAACGTGGAAACGGCTGAAATTATTAAAGAGAAGAACG 
               
               
                   
                 GTCTTCCTTACGTCTCCATTGATGGCGATCAGACCGATCCTCGC 
               
               
                   
                 GTTTTTTCTCCGGCCCAGTTTGATACCCGTGTTCAGGCCCTGGT 
               
               
                   
                 TGAGATGATGGAGGCCAATATGGCGGCAGCGGAATAA 
               
               
                   
               
               
                 IcdB 
                 ATGTCACGCGTGGAGGCAATCCTGTCGCAGCTGAAAGATGTCGC 
               
               
                 SEQ ID NO: 3 
                 CGCGAATCCGAAAAAAGCCATGGATGACTATAAAGCTGAAACAG 
               
               
                   
                 GTAAGGGCGCGGTTGGTATCATGCCGATCTACAGCCCCGAAGAA 
               
               
                   
                 ATGGTACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGCGC 
               
               
                   
                 CCAGGGCAAAACGATTAGTAAAGCGCGCACCTATCTGCCTGCTT 
               
               
                   
                 TTGCCTGCAGCGTAATGCAGCAGGTTATGGAATTACAGTGCGAG 
               
               
                   
                 GGCGCGTATGATGACCTGTCCGCAGTTATTTTTAGCGTACCGTG 
               
               
                   
                 CGACACTCTCAAATGTCTTAGCCAGAAATGGAAAGGTACGTCCC 
               
               
                   
                 CAGTGATTGTATTTACGCATCCGCAGAACCGCGGATTAGAAGCG 
               
               
                   
                 GCGAACCAATTCTTGGTTACCGAGTATGAACTGGTAAAAGCACA 
               
               
                   
                 ACTGGAATCAGTTCTGGGTGTGAAAATTTCAAACGCCGCCCTGG 
               
               
                   
                 AAAATTCGATTGCAATTTATAACGAGAATCGTGCCGTGATGCGT 
               
               
                   
                 GAGTTCGTGAAAGTGGCAGCGGACTATCCTCAAGTCATTGACGC 
               
               
                   
                 AGTGAGCCGCCACGCGGTTTTTAAAGCGCGCCAGTTTATGCTTA 
               
               
                   
                 AGGAAAAACATACCGCACTTGTGAAAGAACTGATCGCTGAGATT 
               
               
                   
                 AAAGCAACGCCAGTCCAGCCGTGGGACGGAAAAAAGGTTGTAGT 
               
               
                   
                 GACGGGCATTCTGTTGGAACCGAATGAGTTATTAGATATCTTTA 
               
               
                   
                 ATGAGTTTAAGATCGCGATTGTTGATGATGATTTAGCGCAGGAA 
               
               
                   
                 AGCCGTCAGATCCGTGTTGACGTTCTGGACGGAGAAGGCGGACC 
               
               
                   
                 GCTCTACCGTATGGCTAAAGCGTGGCAGCAAATGTATGGCTGCT 
               
               
                   
                 CGCTGGCAACCGACACCAAGAAGGGTCGCGGCCGTATGTTAATT 
               
               
                   
                 AACAAAACGATTCAGACCGGTGCGGACGCTATCGTAGTTGCAAT 
               
               
                   
                 GATGAAGTTTTGCGACCCAGAAGAATGGGATTATCCGGTAATGT 
               
               
                   
                 ACCGTGAATTTGAAGAAAAAGGGGTCAAATCACTTATGATTGAG 
               
               
                   
                 GTGGATCAGGAAGTATCGTCTTTCGAACAGATTAAAACCCGTCT 
               
               
                   
                 GCAGTCATTCGTCGAAATGCTTTAA 
               
               
                   
               
               
                 IcdC 
                 ATGTATACCTTGGGGATTGATGTCGGTTCTGCCTCTAGTAAAGC 
               
               
                 SEQ ID NO: 4 
                 GGTGATTCTGAAAGATGGAAAAGATATTGTCGCTGCCGAGGTTG 
               
               
                   
                 TCCAAGTCGGTACCGGCTCCTCGGGTCCCCAACGCGCACTGGAC 
               
               
                   
                 AAAGCCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACATCAGCTA 
               
               
                   
                 CACAGTAGCTACGGGCTATGGGCGCTTCAATTTTAGCGACGCGG 
               
               
                   
                 ATAAACAGATTTCGGAAATTAGCTGTCATGCCAAAGGCATTTAT 
               
               
                   
                 TTCTTAGTACCAACTGCGCGCACTATTATTGACATTGGCGGCCA 
               
               
                   
                 AGATGCGAAAGCCATCCGCCTGGACGACAAGGGGGGTATTAAGC 
               
               
                   
                 AATTCTTCATGAATGATAAATGCGCGGCGGGCACGGGGCGTTTC 
               
               
                   
                 CTGGAAGTCATGGCTCGCGTACTTGAAACCACCCTGGATGAAAT 
               
               
                   
                 GGCTGAACTGGATGAACAGGCGACTGACACCGCTCCCATTTCAA 
               
               
                   
                 GCACCTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAATTG 
               
               
                   
                 AGCAATGGTGTCTCACGCAACAACATCATTAAAGGTGTCCATCT 
               
               
                   
                 GAGCGTTGCGTCACGTGCGTGTGGTCTGGCGTATCGCGGCGGTT 
               
               
                   
                 TGGAGAAAGATGTTGTTATGACAGGTGGCGTGGCAAAAAATGCA 
               
               
                   
                 GGGGTGGTGCGCGCGGTGGCGGGCGTTCTGAAGACCGATGTTAT 
               
               
                   
                 CGTTGCTCCGAATCCTCAGACGACCGGTGCACTGGGGGCAGCGC 
               
               
                   
                 TGTATGCTTATGAGGCCGCCCAGAAGAAGTA 
               
               
                   
               
               
                 etfA 
                 ATGGCCTTCAATAGCGCAGATATTAATTCTTTCCGCGATATTTG 
               
               
                 SEQ ID NO: 5 
                 GGTGTTTTGTGAACAGCGTGAGGGCAAACTGATTAACACCGATT 
               
               
                   
                 TCGAATTAATTAGCGAAGGTCGTAAACTGGCTGACGAACGCGGA 
               
               
                   
                 AGCAAACTGGTTGGAATTTTGCTGGGGCACGAAGTTGAAGAAAT 
               
               
                   
                 CGCAAAAGAATTAGGCGGCTATGGTGCGGACAAGGTAATTGTGT 
               
               
                   
                 GCGATCATCCGGAACTTAAATTTTACACTACGGATGCTTATGCC 
               
               
                   
                 AAAGTTTTATGTGACGTCGTGATGGAAGAGAAACCGGAGGTAAT 
               
               
                   
                 TTTGATCGGTGCCACCAACATTGGCCGTGATCTCGGACCGCGTT 
               
               
                   
                 GTGCTGCACGCTTGCACACGGGGCTGACGGCTGATTGCACGCAC 
               
               
                   
                 CTGGATATTGATATGAATAAATATGTGGACTTTCTTAGCACCAG 
               
               
                   
                 TAGCACCTTGGATATCTCGTCGATGACTTTCCCTATGGAAGATA 
               
               
                   
                 CAAACCTTAAAATGACGCGCCCTGCATTTGGCGGACATCTGATG 
               
               
                   
                 GCAACGATCATTTGTCCACGCTTCCGTCCCTGTATGAGCACAGT 
               
               
                   
                 GCGCCCCGGAGTGATGAAGAAAGCGGAGTTCTCGCAGGAGATGG 
               
               
                   
                 CGCAAGCATGTCAAGTAGTGACCCGTCACGTAAATTTGTCGGAT 
               
               
                   
                 GAAGACCTTAAAACTAAAGTAATTAATATCGTGAAGGAAACGAA 
               
               
                   
                 AAAGATTGTGGATCTGATCGGCGCAGAAATTATTGTGTCAGTTG 
               
               
                   
                 GTCGTGGTATCTCGAAAGATGTCCAAGGTGGAATTGCACTGGCT 
               
               
                   
                 GAAAAACTTGCGGACGCATTTGGTAACGGTGTCGTGGGCGGCTC 
               
               
                   
                 GCGCGCAGTGATTGATTCCGGCTGGTTACCTGCGGATCATCAGG 
               
               
                   
                 TTGGACAAACCGGTAAGACCGTGCACCCGAAAGTCTACGTGGCG 
               
               
                   
                 CTGGGTATTAGTGGGGCTATCCAGCATAAGGCTGGGATGCAAGA 
               
               
                   
                 CTCTGAACTGATCATTGCCGTCAACAAAGACGAAACGGCGCCTA 
               
               
                   
                 TCTTCGACTGCGCCGATTATGGCATCACCGGTGATTTATTTAAA 
               
               
                   
                 ATCGTACCGATGATGATCGACGCGATCAAAGAGGGTAAAAACGC 
               
               
                   
                 ATGA 
               
               
                   
               
               
                 acrB 
                 ATGCGCATCTATGTGTGTGTGAAACAAGTCCCAGATACGAGCGG 
               
               
                 SEQ ID NO: 6 
                 CAAGGTGGCCGTTAACCCTGATGGGACCCTTAACCGTGCCTCAA 
               
               
                   
                 TGGCAGCGATTATTAACCCGGACGATATGTCCGCGATCGAACAG 
               
               
                   
                 GCATTAAAACTGAAAGATGAAACCGGATGCCAGGTTACGGCGCT 
               
               
                   
                 TACGATGGGTCCTCCTCCTGCCGAGGGCATGTTGCGCGAAATTA 
               
               
                   
                 TTGCAATGGGGGCCGACGATGGTGTGCTGATTTCGGCCCGTGAA 
               
               
                   
                 TTTGGGGGGTCCGATACCTTCGCAACCAGTCAAATTATTAGCGC 
               
               
                   
                 GGCAATCCATAAATTAGGCTTAAGCAATGAAGACATGATCTTTT 
               
               
                   
                 GCGGTCGTCAGGCCATTGACGGTGATACGGCCCAAGTCGGCCCT 
               
               
                   
                 CAAATTGCCGAAAAACTGAGCATCCCACAGGTAACCTATGGCGC 
               
               
                   
                 AGGAATCAAAAAATCTGGTGATTTAGTGCTGGTGAAGCGTATGT 
               
               
                   
                 TGGAGGATGGTTATATGATGATCGAAGTCGAAACTCCATGTCTG 
               
               
                   
                 ATTACCTGCATTCAGGATAAAGCGGTAAAACCACGTTACATGAC 
               
               
                   
                 TCTCAACGGTATTATGGAATGCTACTCCAAGCCGCTCCTCGTTC 
               
               
                   
                 TCGATTACGAAGCACTGAAAGATGAACCGCTGATCGAACTTGAT 
               
               
                   
                 ACCATTGGGCTTAAAGGCTCCCCGACGAATATCTTTAAATCGTT 
               
               
                   
                 TACGCCGCCTCAGAAAGGCGTTGGTGTCATGCTCCAAGGCACCG 
               
               
                   
                 ATAAGGAAAAAGTCGAGGATCTGGTGGATAAGCTGATGCAGAAA 
               
               
                   
                 CATGTCATCTAA 
               
               
                   
               
               
                 acrC 
                 ATGTTCTTACTGAAGATTAAAAAAGAACGTATGAAACGCATGGA 
               
               
                 SEQ ID NO: 7 
                 CTTTAGTTTAACGCGTGAACAGGAGATGTTAAAAAAACTGGCGC 
               
               
                   
                 GTCAGTTTGCTGAGATCGAGCTGGAACCGGTGGCCGAAGAGATT 
               
               
                   
                 GATCGTGAGCACGTTTTTCCTGCAGAAAACTTTAAGAAGATGGC 
               
               
                   
                 GGAAATTGGCTTAACCGGCATTGGTATCCCGAAAGAATTTGGTG 
               
               
                   
                 GCTCCGGTGGAGGCACCCTGGAGAAGGTCATTGCCGTGTCAGAA 
               
               
                   
                 TTCGGCAAAAAGTGTATGGCCTCAGCTTCCATTTTAAGCATTCA 
               
               
                   
                 TCTTATCGCGCCGCAGGCAATCTACAAATATGGGACCAAAGAAC 
               
               
                   
                 AGAAAGAGACGTACCTGCCGCGTCTTACCAAAGGTGGTGAACTG 
               
               
                   
                 GGCGCCTTTGCGCTGACAGAACCAAACGCCGGAAGCGATGCCGG 
               
               
                   
                 CGCGGTAAAAACGACCGCGATTCTGGACAGCCAGACAAACGAGT 
               
               
                   
                 ACGTGCTGAATGGCACCAAATGCTTTATCAGCGGGGGCGGGCGC 
               
               
                   
                 GCGGGTGTTCTTGTAATTTTTGCGCTTACTGAACCGAAAAAAGG 
               
               
                   
                 TCTGAAAGGGATGAGCGCGATTATCGTGGAGAAAGGGACCCCGG 
               
               
                   
                 GCTTCAGCATCGGCAAGGTGGAGAGCAAGATGGGGATCGCAGGT 
               
               
                   
                 TCGGAAACCGCGGAACTTATCTTCGAAGATTGTCGCGTTCCGGC 
               
               
                   
                 TGCCAACCTTTTAGGTAAAGAAGGCAAAGGCTTTAAAATTGCTA 
               
               
                   
                 TGGAAGCCCTGGATGGCGCCCGTATTGGCGTGGGCGCTCAAGCA 
               
               
                   
                 ATCGGAATTGCCGAGGGGGCGATCGACCTGAGTGTGAAGTACGT 
               
               
                   
                 TCACGAGCGCATTCAATTTGGTAAACCGATCGCGAATCTGCAGG 
               
               
                   
                 GAATTCAATGGTATATCGCGGATATGGCGACCAAAACCGCCGCG 
               
               
                   
                 GCACGCGCACTTGTTGAGTTTGCAGCGTATCTTGAAGACGCGGG 
               
               
                   
                 TAAACCGTTCACAAAGGAATCTGCTATGTGCAAGCTGAACGCCT 
               
               
                   
                 CCGAAAACGCGCGTTTTGTGACAAATTTAGCTCTGCAGATTCAC 
               
               
                   
                 GGGGGTTACGGTTATATGAAAGATTATCCGTTAGAGCGTATGTA 
               
               
                   
                 TCGCGATGCTAAGATTACGGAAATTTACGAGGGGACATCAGAAA 
               
               
                   
                 TCCATAAGGTGGTGATTGCGCGTGAAGTAATGAAACGCTAA 
               
               
                   
               
               
                 thrA fbr   
                 ATGCGAGTGTTGAAGTTCGGCGGTACATCAGTGGCAAATGCAGA 
               
               
                 SEQ ID NO: 8 
                 ACGTTTTCTGCGTGTTGCCGATATTCTGGAAAGCAATGCCAGGC 
               
               
                   
                 AGGGGCAGGTGGCCACCGTCCTCTCTGCCCCCGCCAAAATCACC 
               
               
                   
                 AACCACCTGGTGGCGATGATTGAAAAAACCATTAGCGGCCAGGA 
               
               
                   
                 TGCTTTACCCAATATCAGCGATGCCGAACGTATTTTTGCCGAAC 
               
               
                   
                 TTTTGACGGGACTCGCCGCCGCCCAGCCGGGGTTCCCGCTGGCG 
               
               
                   
                 CAATTGAAAACTTTCGTCGATCAGGAATTTGCCCAAATAAAACA 
               
               
                   
                 TGTCCTGCATGGCATTAGTTTGTTGGGGCAGTGCCCGGATAGCA 
               
               
                   
                 TCAACGCTGCGCTGATTTGCCGTGGCGAGAAAATGTCGATCGCC 
               
               
                   
                 ATTATGGCCGGCGTATTAGAAGCGCGCGGTCACAACGTTACTGT 
               
               
                   
                 TATCGATCCGGTCGAAAAACTGCTGGCAGTGGGGCATTACCTCG 
               
               
                   
                 AATCTACCGTCGATATTGCTGAGTCCACCCGCCGTATTGCGGCA 
               
               
                   
                 AGCCGCATTCCGGCTGATCACATGGTGCTGATGGCAGGTTTCAC 
               
               
                   
                 CGCCGGTAATGAAAAAGGCGAACTGGTGGTGCTTGGACGCAACG 
               
               
                   
                 GTTCCGACTACTCTGCTGCGGTGCTGGCTGCCTGTTTACGCGCC 
               
               
                   
                 GATTGTTGCGAGATTTGGACGGACGTTGACGGGGTCTATACCTG 
               
               
                   
                 CGACCCGCGTCAGGTGCCCGATGCGAGGTTGTTGAAGTCGATGT 
               
               
                   
                 CCTACCAGGAAGCGATGGAGCTTTCCTACTTCGGCGCTAAAGTT 
               
               
                   
                 CTTCACCCCCGCACCATTACCCCCATCGCCCAGTTCCAGATCCC 
               
               
                   
                 TTGCCTGATTAAAAATACCGGAAATCCTCAAGCACCAGGTACGC 
               
               
                   
                 TCATTGGTGCCAGCCGTGATGAAGACGAATTACCGGTCAAGGGC 
               
               
                   
                 ATTTCCAATCTGAATAACATGGCAATGTTCAGCGTTTCTGGTCC 
               
               
                   
                 GGGGATGAAAGGGATGGTCGGCATGGCGGCGCGCGTCTTTGCAG 
               
               
                   
                 CGATGTCACGCGCCCGTATTTCCGTGGTGCTGATTACGCAATCA 
               
               
                   
                 TCTTCCGAATACAGCATCAGTTTCTGCGTTCCACAAAGCGACTG 
               
               
                   
                 TGTGCGAGCTGAACGGGCAATGCAGGAAGAGTTCTACCTGGAAC 
               
               
                   
                 TGAAAGAAGGCTTACTGGAGCCGCTGGCAGTGACGGAACGGCTG 
               
               
                   
                 GCCATTATCTCGGTGGTAGGTGATGGTATGCGCACCTTGCGTGG 
               
               
                   
                 GATCTCGGCGAAATTCTTTGCCGCACTGGCCCGCGCCAATATCA 
               
               
                   
                 ACATTGTCGCCATTGCTCAGAGATCTTCTGAACGCTCAATCTCT 
               
               
                   
                 GTCGTGGTAAATAACGATGATGCGACCACTGGCGTGCGCGTTAC 
               
               
                   
                 TCATCAGATGCTGTTCAATACCGATCAGGTTATCGAAGTGTTTG 
               
               
                   
                 TGATTGGCGTCGGTGGCGTTGGCGGTGCGCTGCTGGAGCAACTG 
               
               
                   
                 AAGCGTCAGCAAAGCTGGCTGAAGAATAAACATATCGACTTACG 
               
               
                   
                 TGTCTGCGGTGTTGCCAACTCGAAGGCTCTGCTCACCAATGTAC 
               
               
                   
                 ATGGCCTTAATCTGGAAAACTGGCAGGAAGAACTGGCGCAAGCC 
               
               
                   
                 AAAGAGCCGTTTAATCTCGGGCGCTTAATTCGCCTCGTGAAAGA 
               
               
                   
                 ATATCATCTGCTGAACCCGGTCATTGTTGACTGCACTTCCAGCC 
               
               
                   
                 AGGCAGTGGCGGATCAATATGCCGACTTCCTGCGCGAAGGTTTC 
               
               
                   
                 CACGTTGTCACGCCGAACAAAAAGGCCAACACCTCGTCGATGGA 
               
               
                   
                 TTACTACCATCAGTTGCGTTATGCGGCGGAAAAATCGCGGCGTA 
               
               
                   
                 AATTCCTCTATGACACCAACGTTGGGGCTGGATTACCGGTTATT 
               
               
                   
                 GAGAACCTGCAAAATCTGCTCAATGCAGGTGATGAATTGATGAA 
               
               
                   
                 GTTCTCCGGCATTCTTTCTGGTTCGCTTTCTTATATCTTCGGCA 
               
               
                   
                 AGTTAGACGAAGGCATGAGTTTCTCCGAGGCGACCACGCTGGCG 
               
               
                   
                 CGGGAAATGGGTTATACCGAACCGGACCCGCGAGATGATCTTTC 
               
               
                   
                 TGGTATGGATGTGGCGCGTAAACTATTGATTCTCGCTCGTGAAA 
               
               
                   
                 CGGGACGTGAACTGGAGCTGGCGGATATTGAAATTGAACCTGTG 
               
               
                   
                 CTGCCCGCAGAGTTTAACGCCGAGGGTGATGTTGCCGCTTTTAT 
               
               
                   
                 GGCGAATCTGTCACAACTCGACGATCTCTTTGCCGCGCGCGTGG 
               
               
                   
                 CGAAGGCCCGTGATGAAGGAAAAGTTTTGCGCTATGTTGGCAAT 
               
               
                   
                 ATTGATGAAGATGGCGTCTGCCGCGTGAAGATTGCCGAAGTGGA 
               
               
                   
                 TGGTAATGATCCGCTGTTCAAAGTGAAAAATGGCGAAAACGCCC 
               
               
                   
                 TGGCCTTCTATAGCCACTATTATCAGCCGCTGCCGTTGGTACTG 
               
               
                   
                 CGCGGATATGGTGCGGGCAATGACGTTACAGCTGCCGGTGTCTT 
               
               
                   
                 TGCTGATCTGCTACGTACCCTCTCATGGAAGTTAGGAGTCTGA 
               
               
                   
               
               
                 thrB 
                 ATGGTTAAAGTTTATGCCCCGGCTTCCAGTGCCAATATGAGCGT 
               
               
                 SEQ ID NO: 9 
                 CGGGTTTGATGTGCTCGGGGCGGCGGTGACACCTGTTGATGGTG 
               
               
                   
                 CATTGCTCGGAGATGTAGTCACGGTTGAGGCGGCAGAGACATTC 
               
               
                   
                 AGTCTCAACAACCTCGGACGCTTTGCCGATAAGCTGCCGTCAGA 
               
               
                   
                 ACCACGGGAAAATATCGTTTATCAGTGCTGGGAGCGTTTTTGCC 
               
               
                   
                 AGGAACTGGGTAAGCAAATTCCAGTGGCGATGACCCTGGAAAAG 
               
               
                   
                 AATATGCCGATCGGTTCGGGCTTAGGCTCCAGTGCCTGTTCGGT 
               
               
                   
                 GGTCGCGGCGCTGATGGCGATGAATGAACACTGCGGCAAGCCGC 
               
               
                   
                 TTAATGACACTCGTTTGCTGGCTTTGATGGGCGAGCTGGAAGGC 
               
               
                   
                 CGTATCTCCGGCAGCATTCATTACGACAACGTGGCACCGTGTTT 
               
               
                   
                 TCTCGGTGGTATGCAGTTGATGATCGAAGAAAACGACATCATCA 
               
               
                   
                 GCCAGCAAGTGCCAGGGTTTGATGAGTGGCTGTGGGTGCTGGCG 
               
               
                   
                 TATCCGGGGATTAAAGTCTCGACGGCAGAAGCCAGGGCTATTTT 
               
               
                   
                 ACCGGCGCAGTATCGCCGCCAGGATTGCATTGCGCACGGGCGAC 
               
               
                   
                 ATCTGGCAGGCTTCATTCACGCCTGCTATTCCCGTCAGCCTGAG 
               
               
                   
                 CTTGCCGCGAAGCTGATGAAAGATGTTATCGCTGAACCCTACCG 
               
               
                   
                 TGAACGGTTACTGCCAGGCTTCCGGCAGGCGCGGCAGGCGGTCG 
               
               
                   
                 CGGAAATCGGCGCGGTAGCGAGCGGTATCTCCGGCTCCGGCCCG 
               
               
                   
                 ACCTTGTTCGCTCTGTGTGACAAGCCGGAAACCGCCCAGCGCGT 
               
               
                   
                 TGCCGACTGGTTGGGTAAGAACTACCTGCAAAATCAGGAAGGTT 
               
               
                   
                 TTGTTCATATTTGCCGGCTGGATACGGCGGGCGCACGAGTACTG 
               
               
                   
                 GAAAACTAA 
               
               
                   
               
               
                 thrC 
                 ATGAAACTCTACAATCTGAAAGATCACAACGAGCAGGTCAGCTT 
               
               
                 SEQ ID NO: 10 
                 TGCGCAAGCCGTAACCCAGGGGTTGGGCAAAAATCAGGGGCTGT 
               
               
                   
                 TTTTTCCGCACGACCTGCCGGAATTCAGCCTGACTGAAATTGAT 
               
               
                   
                 GAGATGCTGAAGCTGGATTTTGTCACCCGCAGTGCGAAGATCCT 
               
               
                   
                 CTCGGCGTTTATTGGTGATGAAATCCCACAGGAAATCCTGGAAG 
               
               
                   
                 AGCGCGTGCGCGCGGCGTTTGCCTTCCCGGCTCCGGTCGCCAAT 
               
               
                   
                 GTTGAAAGCGATGTCGGTTGTCTGGAATTGTTCCACGGGCCAAC 
               
               
                   
                 GCTGGCATTTAAAGATTTCGGCGGTCGCTTTATGGCACAAATGC 
               
               
                   
                 TGACCCATATTGCGGGTGATAAGCCAGTGACCATTCTGACCGCG 
               
               
                   
                 ACCTCCGGTGATACCGGAGCGGCAGTGGCTCATGCTTTCTACGG 
               
               
                   
                 TTTACCGAATGTGAAAGTGGTTATCCTCTATCCACGAGGCAAAA 
               
               
                   
                 TCAGTCCACTGCAAGAAAAACTGTTCTGTACATTGGGCGGCAAT 
               
               
                   
                 ATCGAAACTGTTGCCATCGACGGCGATTTCGATGCCTGTCAGGC 
               
               
                   
                 GCTGGTGAAGCAGGCGTTTGATGATGAAGAACTGAAAGTGGCGC 
               
               
                   
                 TAGGGTTAAACTCGGCTAACTCGATTAACATCAGCCGTTTGCTG 
               
               
                   
                 GCGCAGATTTGCTACTACTTTGAAGCTGTTGCGCAGCTGCCGCA 
               
               
                   
                 GGAGACGCGCAACCAGCTGGTTGTCTCGGTGCCAAGCGGAAACT 
               
               
                   
                 TCGGCGATTTGACGGCGGGTCTGCTGGCGAAGTCACTCGGTCTG 
               
               
                   
                 CCGGTGAAACGTTTTATTGCTGCGACCAACGTGAACGATACCGT 
               
               
                   
                 GCCACGTTTCCTGCACGACGGTCAGTGGTCACCCAAAGCGACTC 
               
               
                   
                 AGGCGACGTTATCCAACGCGATGGACGTGAGTCAGCCGAACAAC 
               
               
                   
                 TGGCCGCGTGTGGAAGAGTTGTTCCGCCGCAAAATCTGGCAACT 
               
               
                   
                 GAAAGAGCTGGGTTATGCAGCCGTGGATGATGAAACCACGCAAC 
               
               
                   
                 AGACAATGCGTGAGTTAAAAGAACTGGGCTACACTTCGGAGCCG 
               
               
                   
                 CACGCTGCCGTAGCTTATCGTGCGCTGCGTGATCAGTTGAATCC 
               
               
                   
                 AGGCGAATATGGCTTGTTCCTCGGCACCGCGCATCCGGCGAAAT 
               
               
                   
                 TTAAAGAGAGCGTGGAAGCGATTCTCGGTGAAACGTTGGATCTG 
               
               
                   
                 CCAAAAGAGCTGGCAGAACGTGCTGATTTACCCTTGCTTTCACA 
               
               
                   
                 TAATCTGCCCGCCGATTTTGCTGCGTTGCGTAAATTGATGATGA 
               
               
                   
                 ATCATCAGTAA 
               
               
                   
               
               
                 ilvA fbr   
                 ATGAGTGAAACATACGTGTCTGAGAAAAGTCCAGGAGTGATGG 
               
               
                 SEQ ID NO: 11 
                 TAGCGGAGCGGAGCTGATTCGTGCCGCCGACATTCAAACGGCGC 
               
               
                   
                 AGGCACGAATTTCCTCCGTCATTGCACCAACTCCATTGCAGTAT 
               
               
                   
                 TGCCCTCGTCTTTCTGAGGAAACCGGAGCGGAAATCTACCTTAA 
               
               
                   
                 GCGTGAGGATCTGCAGGATGTTCGTTCCTACAAGATCCGCGGTG 
               
               
                   
                 CGCTGAACTCTGGAGCGCAGCTCACCCAAGAGCAGCGCGATGCA 
               
               
                   
                 GGTATCGTTGCCGCATCTGCAGGTAACCATGCCCAGGGCGTGGC 
               
               
                   
                 CTATGTGTGCAAGTCCTTGGGCGTTCAGGGACGCATCTATGTTC 
               
               
                   
                 CTGTGCAGACTCCAAAGCAAAAGCGTGACCGCATCATGGTTCAC 
               
               
                   
                 GGCGGAGAGTTTGTCTCCTTGGTGGTCACTGGCAATAACTTCGA 
               
               
                   
                 CGAAGCATCGGCTGCAGCGCATGAAGATGCAGAGCGCACCGGCG 
               
               
                   
                 CAACGCTGATCGAGCCTTTCGATGCTCGCAACACCGTCATCGGT 
               
               
                   
                 CAGGGCACCGTGGCTGCTGAGATCTTGTCGCAGCTGACTTCCAT 
               
               
                   
                 GGGCAAGAGTGCAGATCACGTGATGGTTCCAGTCGGCGGTGGCG 
               
               
                   
                 GACTTCTTGCAGGTGTGGTCAGCTACATGGCTGATATGGCACCT 
               
               
                   
                 CGCACTGCGATCGTTGGTATCGAACCAGCGGGAGCAGCATCCAT 
               
               
                   
                 GCAGGCTGCATTGCACAATGGTGGACCAATCACTTTGGAGACTG 
               
               
                   
                 TTGATCCCTTTGTGGACGGCGCAGCAGTCAAACGTGTCGGAGAT 
               
               
                   
                 CTCAACTACACCATCGTGGAGAAGAACCAGGGTCGCGTGCACAT 
               
               
                   
                 GATGAGCGCGACCGAGGGCGCTGTGTGTACTGAGATGCTCGATC 
               
               
                   
                 TTTACCAAAACGAAGGCATCATCGCGGAGCCTGCTGGCGCGCTG 
               
               
                   
                 TCTATCGCTGGGTTGAAGGAAATGTCCTTTGCACCTGGTTCTGC 
               
               
                   
                 AGTGGTGTGCATCATCTCTGGTGGCAACAACGATGTGCTGCGTT 
               
               
                   
                 ATGCGGAAATCGCTGAGCGCTCCTTGGTGCACCGCGGTTTGAAG 
               
               
                   
                 CACTACTTCTTGGTGAACTTCCCGCAAAAGCCTGGTCAGTTGCG 
               
               
                   
                 TCACTTCCTGGAAGATATCCTGGGACCGGATGATGACATCACGC 
               
               
                   
                 TGTTTGAGTACCTCAAGCGCAACAACCGTGAGACCGGTACTGCG 
               
               
                   
                 TTGGTGGGTATTCACTTGAGTGAAGCATCAGGATTGGATTCTTT 
               
               
                   
                 GCTGGAACGTATGGAGGAATCGGCAATTGATTCCCGTCGCCTCG 
               
               
                   
                 AGCCGGGCACCCCTGAGTACGAATACTTGACCTAA 
               
               
                   
               
               
                 aceE 
                 ATGTCAGAACGTTTCCCAAATGACGTGGATCCGATCGAAACTCG 
               
               
                 SEQ ID NO: 12 
                 CGACTGGCTCCAGGCGATCGAATCGGTCATCCGTGAAGAAGGTG 
               
               
                   
                 TTGAGCGTGCTCAGTATCTGATCGACCAACTGCTTGCTGAAGCC 
               
               
                   
                 CGCAAAGGCGGTGTAAACGTAGCCGCAGGCACAGGTATCAGCAA 
               
               
                   
                 CTACATCAACACCATCCCCGTTGAAGAACAACCGGAGTATCCGG 
               
               
                   
                 GTAATCTGGAACTGGAACGCCGTATTCGTTCAGCTATCCGCTGG 
               
               
                   
                 AACGCCATCATGACGGTGCTGCGTGCGTCGAAAAAAGACCTCGA 
               
               
                   
                 ACTGGGCGGCCATATGGCGTCCTTCCAGTCTTCCGCAACCATTT 
               
               
                   
                 ATGATGTGTGCTTTAACCACTTCTTCCGTGCACGCAACGAGCAG 
               
               
                   
                 GATGGCGGCGACCTGGTTTACTTCCAGGGCCACATCTCCCCGGG 
               
               
                   
                 CGTGTACGCTCGTGCTTTCCTGGAAGGTCGTCTGACTCAGGAGC 
               
               
                   
                 AGCTGGATAACTTCCGTCAGGAAGTTCACGGCAATGGCCTCTCT 
               
               
                   
                 TCCTATCCGCACCCGAAACTGATGCCGGAATTCTGGCAGTTCCC 
               
               
                   
                 GACCGTATCTATGGGTCTGGGTCCGATTGGTGCTATTTACCAGG 
               
               
                   
                 CTAAATTCCTGAAATATCTGGAACACCGTGGCCTGAAAGATACC 
               
               
                   
                 TCTAAACAAACCGTTTACGCGTTCCTCGGTGACGGTGAAATGGA 
               
               
                   
                 CGAACCGGAATCCAAAGGTGCGATCACCATCGCTACCCGTGAAA 
               
               
                   
                 AACTGGATAACCTGGTCTTCGTTATCAACTGTAACCTGCAGCGT 
               
               
                   
                 CTTGACGGCCCGGTCACCGGTAACGGCAAGATCATCAACGAACT 
               
               
                   
                 GGAAGGCATCTTCGAAGGTGCTGGCTGGAACGTGATCAAAGTGA 
               
               
                   
                 TGTGGGGTAGCCGTTGGGATGAACTGCTGCGTAAGGATACCAGC 
               
               
                   
                 GGTAAACTGATCCAGCTGATGAACGAAACCGTTGACGGCGACTA 
               
               
                   
                 CCAGACCTTCAAATCGAAAGATGGTGCGTACGTTCGTGAACACT 
               
               
                   
                 TCTTCGGTAAATATCCTGAAACCGCAGCACTGGTTGCAGACTGG 
               
               
                   
                 ACTGACGAGCAGATCTGGGCACTGAACCGTGGTGGTCACGATCC 
               
               
                   
                 GAAGAAAATCTACGCTGCATTCAAGAAAGCGCAGGAAACCAAAG 
               
               
                   
                 GCAAAGCGACAGTAATCCTTGCTCATACCATTAAAGGTTACGGC 
               
               
                   
                 ATGGGCGACGCGGCTGAAGGTAAAAACATCGCGCACCAGGTTAA 
               
               
                   
                 GAAAATGAACATGGACGGTGTGCGTCATATCCGCGACCGTTTCA 
               
               
                   
                 ATGTGCCGGTGTCTGATGCAGATATCGAAAAACTGCCGTACATC 
               
               
                   
                 ACCTTCCCGGAAGGTTCTGAAGAGCATACCTATCTGCACGCTCA 
               
               
                   
                 GCGTCAGAAACTGCACGGTTATCTGCCAAGCCGTCAGCCGAACT 
               
               
                   
                 TCACCGAGAAGCTTGAGCTGCCGAGCCTGCAAGACTTCGGCGCG 
               
               
                   
                 CTGTTGGAAGAGCAGAGCAAAGAGATCTCTACCACTATCGCTTT 
               
               
                   
                 CGTTCGTGCTCTGAACGTGATGCTGAAGAACAAGTCGATCAAAG 
               
               
                   
                 ATCGTCTGGTACCGATCATCGCCGACGAAGCGCGTACTTTCGGT 
               
               
                   
                 ATGGAAGGTCTGTTCCGTCAGATTGGTATTTACAGCCCGAACGG 
               
               
                   
                 TCAGCAGTACACCCCGCAGGACCGCGAGCAGGTTGCTTACTATA 
               
               
                   
                 AAGAAGACGAGAAAGGTCAGATTCTGCAGGAAGGGATCAACGAG 
               
               
                   
                 CTGGGCGCAGGTTGTTCCTGGCTGGCAGCGGCGACCTCTTACAG 
               
               
                   
                 CACCAACAATCTGCCGATGATCCCGTTCTACATCTATTACTCGA 
               
               
                   
                 TGTTCGGCTTCCAGCGTATTGGCGATCTGTGCTGGGCGGCTGGC 
               
               
                   
                 GACCAGCAAGCGCGTGGCTTCCTGATCGGCGGTACTTCCGGTCG 
               
               
                   
                 TACCACCCTGAACGGCGAAGGTCTGCAGCACGAAGATGGTCACA 
               
               
                   
                 GCCACATTCAGTCGCTGACTATCCCGAACTGTATCTCTTACGAC 
               
               
                   
                 CCGGCTTACGCTTACGAAGTTGCTGTCATCATGCATGACGGTCT 
               
               
                   
                 GGAGCGTATGTACGGTGAAAAACAAGAGAACGTTTACTACTACA 
               
               
                   
                 TCACTACGCTGAACGAAAACTACCACATGCCGGCAATGCCGGAA 
               
               
                   
                 GGTGCTGAGGAAGGTATCCGTAAAGGTATCTACAAACTCGAAAC 
               
               
                   
                 TATTGAAGGTAGCAAAGGTAAAGTTCAGCTGCTCGGCTCCGGTT 
               
               
                   
                 CTATCCTGCGTCACGTCCGTGAAGCAGCTGAGATCCTGGCGAAA 
               
               
                   
                 GATTACGGCGTAGGTTCTGACGTTTATAGCGTGACCTCCTTCAC 
               
               
                   
                 CGAGCTGGCGCGTGATGGTCAGGATTGTGAACGCTGGAACATGC 
               
               
                   
                 TGCACCCGCTGGAAACTCCGCGCGTTCCGTATATCGCTCAGGTG 
               
               
                   
                 ATGAACGACGCTCCGGCAGTGGCATCTACCGACTATATGAAACT 
               
               
                   
                 GTTCGCTGAGCAGGTCCGTACTTACGTACCGGCTGACGACTACC 
               
               
                   
                 GCGTACTGGGTACTGATGGCTTCGGTCGTTCCGACAGCCGTGAG 
               
               
                   
                 AACCTGCGTCACCACTTCGAAGTTGATGCTTCTTATGTCGTGGT 
               
               
                   
                 TGCGGCGCTGGGCGAACTGGCTAAACGTGGCGAAATCGATAAGA 
               
               
                   
                 AAGTGGTTGCTGACGCAATCGCCAAATTCAACATCGATGCAGAT 
               
               
                   
                 AAAGTTAACCCGCGTCTGGCGTAA 
               
               
                   
               
               
                 aceF 
                 ATGGCTATCGAAATCAAAGTACCGGACATCGGGGCTGATGAAGT 
               
               
                 SEQ ID NO: 13 
                 TGAAATCACCGAGATCCTGGTCAAAGTGGGCGACAAAGTTGAAG 
               
               
                   
                 CCGAACAGTCGCTGATCACCGTAGAAGGCGACAAAGCCTCTATG 
               
               
                   
                 GAAGTTCCGTCTCCGCAGGCGGGTATCGTTAAAGAGATCAAAGT 
               
               
                   
                 CTCTGTTGGCGATAAAACCCAGACCGGCGCACTGATTATGATTT 
               
               
                   
                 TCGATTCCGCCGACGGTGCAGCAGACGCTGCACCTGCTCAGGCA 
               
               
                   
                 GAAGAGAAGAAAGAAGCAGCTCCGGCAGCAGCACCAGCGGCTGC 
               
               
                   
                 GGCGGCAAAAGACGTTAACGTTCCGGATATCGGCAGCGACGAAG 
               
               
                   
                 TTGAAGTGACCGAAATCCTGGTGAAAGTTGGCGATAAAGTTGAA 
               
               
                   
                 GCTGAACAGTCGCTGATCACCGTAGAAGGCGACAAGGCTTCTAT 
               
               
                   
                 GGAAGTTCCGGCTCCGTTTGCTGGCACCGTGAAAGAGATCAAAG 
               
               
                   
                 TGAACGTGGGTGACAAAGTGTCTACCGGCTCGCTGATTATGGTC 
               
               
                   
                 TTCGAAGTCGCGGGTGAAGCAGGCGCGGCAGCTCCGGCCGCTAA 
               
               
                   
                 ACAGGAAGCAGCTCCGGCAGCGGCCCCTGCACCAGCGGCTGGCG 
               
               
                   
                 TGAAAGAAGTTAACGTTCCGGATATCGGCGGTGACGAAGTTGAA 
               
               
                   
                 GTGACTGAAGTGATGGTGAAAGTGGGCGACAAAGTTGCCGCTGA 
               
               
                   
                 ACAGTCACTGATCACCGTAGAAGGCGACAAAGCTTCTATGGAAG 
               
               
                   
                 TTCCGGCGCCGTTTGCAGGCGTCGTGAAGGAACTGAAAGTCAAC 
               
               
                   
                 GTTGGCGATAAAGTGAAAACTGGCTCGCTGATTATGATCTTCGA 
               
               
                   
                 AGTTGAAGGCGCAGCGCCTGCGGCAGCTCCTGCGAAACAGGAAG 
               
               
                   
                 CGGCAGCGCCGGCACCGGCAGCAAAAGCTGAAGCCCCGGCAGCA 
               
               
                   
                 GCACCAGCTGCGAAAGCGGAAGGCAAATCTGAATTTGCTGAAAA 
               
               
                   
                 CGACGCTTATGTTCACGCGACTCCGCTGATCCGCCGTCTGGCAC 
               
               
                   
                 GCGAGTTTGGTGTTAACCTTGCGAAAGTGAAGGGCACTGGCCGT 
               
               
                   
                 AAAGGTCGTATCCTGCGCGAAGACGTTCAGGCTTACGTGAAAGA 
               
               
                   
                 AGCTATCAAACGTGCAGAAGCAGCTCCGGCAGCGACTGGCGGTG 
               
               
                   
                 GTATCCCTGGCATGCTGCCGTGGCCGAAGGTGGACTTCAGCAAG 
               
               
                   
                 TTTGGTGAAATCGAAGAAGTGGAACTGGGCCGCATCCAGAAAAT 
               
               
                   
                 CTCTGGTGCGAACCTGAGCCGTAACTGGGTAATGATCCCGCATG 
               
               
                   
                 TTACTCACTTCGACAAAACCGATATCACCGAGTTGGAAGCGTTC 
               
               
                   
                 CGTAAACAGCAGAACGAAGAAGCGGCGAAACGTAAGCTGGATGT 
               
               
                   
                 GAAGATCACCCCGGTTGTCTTCATCATGAAAGCCGTTGCTGCAG 
               
               
                   
                 CTCTTGAGCAGATGCCTCGCTTCAATAGTTCGCTGTCGGAAGAC 
               
               
                   
                 GGTCAGCGTCTGACCCTGAAGAAATACATCAACATCGGTGTGGC 
               
               
                   
                 GGTGGATACCCCGAACGGTCTGGTTGTTCCGGTATTCAAAGACG 
               
               
                   
                 TCAACAAGAAAGGCATCATCGAGCTGTCTCGCGAGCTGATGACT 
               
               
                   
                 ATTTCTAAGAAAGCGCGTGACGGTAAGCTGACTGCGGGCGAAAT 
               
               
                   
                 GCAGGGCGGTTGCTTCACCATCTCCAGCATCGGCGGCCTGGGTA 
               
               
                   
                 CTACCCACTTCGCGCCGATTGTGAACGCGCCGGAAGTGGCTATC 
               
               
                   
                 CTCGGCGTTTCCAAGTCCGCGATGGAGCCGGTGTGGAATGGTAA 
               
               
                   
                 AGAGTTCGTGCCGCGTCTGATGCTGCCGATTTCTCTCTCCTTCG 
               
               
                   
                 ACCACCGCGTGATCGACGGTGCTGATGGTGCCCGTTTCATTACC 
               
               
                   
                 ATCATTAACAACACGCTGTCTGACATTCGCCGTCTGGTGATGTA 
               
               
                   
                 A 
               
               
                   
               
               
                 lpd 
                 ATGAGTACTGAAATCAAAACTCAGGTCGTGGTACTTGGGGCAGG 
               
               
                 SEQ ID NO: 14 
                 CCCCGCAGGTTACTCCGCTGCCTTCCGTTGCGCTGATTTAGGTC 
               
               
                   
                 TGGAAACCGTAATCGTAGAACGTTACAACACCCTTGGCGGTGTT 
               
               
                   
                 TGCCTGAACGTCGGCTGTATCCCTTCTAAAGCACTGCTGCACGT 
               
               
                   
                 AGCAAAAGTTATCGAAGAAGCCAAAGCGCTGGCTGAACACGGTA 
               
               
                   
                 TCGTCTTCGGCGAACCGAAAACCGATATCGACAAGATTCGTACC 
               
               
                   
                 TGGAAAGAGAAAGTGATCAATCAGCTGACCGGTGGTCTGGCTGG 
               
               
                   
                 TATGGCGAAAGGCCGCAAAGTCAAAGTGGTCAACGGTCTGGGTA 
               
               
                   
                 AATTCACCGGGGCTAACACCCTGGAAGTTGAAGGTGAGAACGGC 
               
               
                   
                 AAAACCGTGATCAACTTCGACAACGCGATCATTGCAGCGGGTTC 
               
               
                   
                 TCGCCCGATCCAACTGCCGTTTATTCCGCATGAAGATCCGCGTA 
               
               
                   
                 TCTGGGACTCCACTGACGCGCTGGAACTGAAAGAAGTACCAGAA 
               
               
                   
                 CGCCTGCTGGTAATGGGTGGCGGTATCATCGGTCTGGAAATGGG 
               
               
                   
                 CACCGTTTACCACGCGCTGGGTTCACAGATTGACGTGGTTGAAA 
               
               
                   
                 TGTTCGACCAGGTTATCCCGGCAGCTGACAAAGACATCGTTAAA 
               
               
                   
                 GTCTTCACCAAGCGTATCAGCAAGAAATTCAACCTGATGCTGGA 
               
               
                   
                 AACCAAAGTTACCGCCGTTGAAGCGAAAGAAGACGGCATTTATG 
               
               
                   
                 TGACGATGGAAGGCAAAAAAGCACCCGCTGAACCGCAGCGTTAC 
               
               
                   
                 GACGCCGTGCTGGTAGCGATTGGTCGTGTGCCGAACGGTAAAAA 
               
               
                   
                 CCTCGACGCAGGCAAAGCAGGCGTGGAAGTTGACGACCGTGGTT 
               
               
                   
                 TCATCCGCGTTGACAAACAGCTGCGTACCAACGTACCGCACATC 
               
               
                   
                 TTTGCTATCGGCGATATCGTCGGTCAACCGATGCTGGCACACAA 
               
               
                   
                 AGGTGTTCACGAAGGTCACGTTGCCGCTGAAGTTATCGCCGGTA 
               
               
                   
                 AGAAACACTACTTCGATCCGAAAGTTATCCCGTCCATCGCCTAT 
               
               
                   
                 ACCAAACCAGAAGTTGCATGGGTGGGTCTGACTGAGAAAGAAGC 
               
               
                   
                 GAAAGAGAAAGGCATCAGCTATGAAACCGCCACCTTCCCGTGGG 
               
               
                   
                 CTGCTTCTGGTCGTGCTATCGCTTCCGACTGCGCAGACGGTATG 
               
               
                   
                 ACCAAGCTGATTTTCGACAAAGAATCTCACCGTGTGATCGGTGG 
               
               
                   
                 TGCGATTGTCGGTACTAACGGCGGCGAGCTGCTGGGTGAAATCG 
               
               
                   
                 GCCTGGCAATCGAAATGGGTTGTGATGCTGAAGACATCGCACTG 
               
               
                   
                 ACCATCCACGCGCACCCGACTCTGCACGAGTCTGTGGGCCTGGC 
               
               
                   
                 GGCAGAAGTGTTCGAAGGTAGCATTACCGACCTGCCGAACCCGA 
               
               
                   
                 AAGCGAAGAAGAAGTAA 
               
               
                   
               
               
                 tesB 
                 ATGAGTCAGGCGCTAAAAAATTTACTGACATTGTTAAATCTGGA 
               
               
                 SEQ ID NO: 15 
                 AAAAATTGAGGAAGGACTCTTTCGCGGCCAGAGTGAAGATTTAG 
               
               
                   
                 GTTTACGCCAGGTGTTTGGCGGCCAGGTCGTGGGTCAGGCCTTG 
               
               
                   
                 TATGCTGCAAAAGAGACCGTCCCTGAAGAGCGGCTGGTACATTC 
               
               
                   
                 GTTTCACAGCTACTTTCTTCGCCCTGGCGATAGTAAGAAGCCGA 
               
               
                   
                 TTATTTATGATGTCGAAACGCTGCGTGACGGTAACAGCTTCAGC 
               
               
                   
                 GCCCGCCGGGTTGCTGCTATTCAAAACGGCAAACCGATTTTTTA 
               
               
                   
                 TATGACTGCCTCTTTCCAGGCACCAGAAGCGGGTTTCGAACATC 
               
               
                   
                 AAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCCTCCCTTCG 
               
               
                   
                 GAAACGCAAATCGCCCAATCGCTGGCGCACCTGCTGCCGCCAGT 
               
               
                   
                 GCTGAAAGATAAATTCATCTGCGATCGTCCGCTGGAAGTCCGTC 
               
               
                   
                 CGGTGGAGTTTCATAACCCACTGAAAGGTCACGTCGCAGAACCA 
               
               
                   
                 CATCGTCAGGTGTGGATCCGCGCAAATGGTAGCGTGCCGGATGA 
               
               
                   
                 CCTGCGCGTTCATCAGTATCTGCTCGGTTACGCTTCTGATCTTA 
               
               
                   
                 ACTTCCTGCCGGTAGCTCTACAGCCGCACGGCATCGGTTTTCTC 
               
               
                   
                 GAACCGGGGATTCAGATTGCCACCATTGACCATTCCATGTGGTT 
               
               
                   
                 CCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATAGCGTGG 
               
               
                   
                 AGAGCACCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGAG 
               
               
                   
                 TTTTATACCCAAGACGGCGTACTGGTTGCCTCGACCGTTCAGGA 
               
               
                   
                 AGGGGTGATGCGTAATCACAATTAA 
               
               
                   
               
               
                 acuI 
                 ATGCGTGCGGTACTGATCGAGAAGTCCGATGATACACAGTCCGT 
               
               
                 SEQ ID NO: 16 
                 CTCTGTCACCGAACTGGCTGAAGATCAACTGCCGGAAGGCGACG 
               
               
                   
                 TTTTGGTAGATGTTGCTTATTCAACACTGAACTACAAAGACGCC 
               
               
                   
                 CTGGCAATTACCGGTAAAGCCCCCGTCGTTCGTCGTTTTCCGAT 
               
               
                   
                 GGTACCTGGAATCGACTTTACGGGTACCGTGGCCCAGTCTTCCC 
               
               
                   
                 ACGCCGACTTCAAGCCAGGTGATCGCGTAATCCTGAATGGTTGG 
               
               
                   
                 GGTGTGGGGGAAAAACATTGGGGCGGTTTAGCGGAGCGCGCTCG 
               
               
                   
                 CGTGCGCGGAGACTGGCTTGTTCCCTTGCCAGCCCCCCTGGACT 
               
               
                   
                 TACGCCAAGCGGCCATGATCGGTACAGCAGGATACACGGCGATG 
               
               
                   
                 TTGTGCGTTCTGGCGCTTGAACGTCACGGAGTGGTGCCGGGTAA 
               
               
                   
                 TGGGGAAATCGTGGTGTCCGGTGCAGCAGGCGGCGTCGGCTCCG 
               
               
                   
                 TTGCGACGACCCTTCTTGCCGCTAAGGGCTATGAGGTAGCGGCA 
               
               
                   
                 GTGACTGGACGTGCGTCCGAAGCAGAATATCTGCGCGGTTTGGG 
               
               
                   
                 GGCGGCGAGCGTAATTGATCGTAACGAATTAACGGGGAAGGTAC 
               
               
                   
                 GCCCGCTGGGTCAGGAGCGTTGGGCTGGCGGGATTGACGTGGCG 
               
               
                   
                 GGATCAACCGTGCTTGCGAACATGCTTTCTATGATGAAGTATCG 
               
               
                   
                 CGGGGTAGTCGCTGCGTGTGGCCTGGCCGCGGGCATGGATCTGC 
               
               
                   
                 CCGCGTCTGTCGCGCCCTTTATTCTTCGTGGGATGACGCTGGCA 
               
               
                   
                 GGGGTGGATAGCGTTATGTGCCCAAAGACAGATCGTTTAGCAGC 
               
               
                   
                 GTGGGCCCGTTTGGCGTCAGATCTTGACCCTGCCAAGCTGGAGG 
               
               
                   
                 AGATGACTACAGAGTTGCCGTTTAGTGAAGTAATCGAGACAGCA 
               
               
                   
                 CCCAAATTCTTGGACGGGACGGTTCGTGGCCGCATTGTTATCCC 
               
               
                   
                 CGTAACGCCCTAA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Propionate Cassette Sequences Sleeping 
               
               
                 Beauty Operon 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Sbm 
                 ATGTCTAACGTGCAGGAGTGGCAACAGCTTGCCAACAAGGAAT 
               
               
                 SEQ ID 
                 TGAGCCGTCGGGAGAAAACTGTCGACTCGCTGGTTCATCAAAC 
               
               
                 NO: 17 
                 CGCGGAAGGGATCGCCATCAAGCCGCTGTATACCGAAGCCGAT 
               
               
                   
                 CTCGATAATCTGGAGGTGACAGGTACCCTTCCTGGTTTGCCGC 
               
               
                   
                 CCTACGTTCGTGGCCCGCGTGCCACTATGTATACCGCCCAACC 
               
               
                   
                 GTGGACCATCCGTCAGTATGCTGGTTTTTCAACAGCAAAAGAG 
               
               
                   
                 TCCAACGCTTTTTATCGCCGTAACCTGGCCGCCGGGCAAAAAG 
               
               
                   
                 GTCTTTCCGTTGCGTTTGACCTTGCCACCCACCGTGGCTACGA 
               
               
                   
                 CTCCGATAACCCGCGCGTGGCGGGCGACGTCGGCAAAGCGGGC 
               
               
                   
                 GTCGCTATCGACACCGTGGAAGATATGAAAGTCCTGTTCGACC 
               
               
                   
                 AGATCCCGCTGGATAAAATGTCGGTTTCGATGACCATGAATGG 
               
               
                   
                 CGCAGTGCTACCAGTACTGGCGTTTTATATCGTCGCCGCAGAA 
               
               
                   
                 GAGCAAGGTGTTACACCTGATAAACTGACCGGCACCATTCAAA 
               
               
                   
                 ACGATATTCTCAAAGAGTACCTCTGCCGCAACACCTATATTTA 
               
               
                   
                 CCCACCAAAACCGTCAATGCGCATTATCGCCGACATCATCGCC 
               
               
                   
                 TGGTGTTCCGGCAACATGCCGCGATTTAATACCATCAGTATCA 
               
               
                   
                 GCGGTTACCACATGGGTGAAGCGGGTGCCAACTGCGTGCAGCA 
               
               
                   
                 GGTAGCATTTACGCTCGCTGATGGGATTGAGTACATCAAAGCA 
               
               
                   
                 GCAATCTCTGCCGGACTGAAAATTGATGACTTCGCTCCTCGCC 
               
               
                   
                 TGTCGTTCTTCTTCGGCATCGGCATGGATCTGTTTATGAACGT 
               
               
                   
                 CGCCATGTTGCGTGCGGCACGTTATTTATGGAGCGAAGCGGTC 
               
               
                   
                 AGTGGATTTGGCGCACAGGACCCGAAATCACTGGCGCTGCGTA 
               
               
                   
                 CCCACTGCCAGACCTCAGGCTGGAGCCTGACTGAACAGGATCC 
               
               
                   
                 GTATAACAACGTTATCCGCACCACCATTGAAGCGCTGGCTGCG 
               
               
                   
                 ACGCTGGGCGGTACTCAGTCACTGCATACCAACGCCTTTGACG 
               
               
                   
                 AAGCGCTTGGTTTGCCTACCGATTTCTCAGCACGCATTGCCCG 
               
               
                   
                 CAACACCCAGATCATCATCCAGGAAGAATCAGAACTCTGCCGC 
               
               
                   
                 ACCGTCGATCCACTGGCCGGATCCTATTACATTGAGTCGCTGA 
               
               
                   
                 CCGATCAAATCGTCAAACAAGCCAGAGCTATTATCCAACAGAT 
               
               
                   
                 CGACGAAGCCGGTGGCATGGCGAAAGCGATCGAAGCAGGTCTG 
               
               
                   
                 CCAAAACGAATGATCGAAGAGGCCTCAGCGCGCGAACAGTCGC 
               
               
                   
                 TGATCGACCAGGGCAAGCGTGTCATCGTTGGTGTCAACAAGTA 
               
               
                   
                 CAAACTGGATCACGAAGACGAAACCGATGTACTTGAGATCGAC 
               
               
                   
                 AACGTGATGGTGCGTAACGAGCAAATTGCTTCGCTGGAACGCA 
               
               
                   
                 TTCGCGCCACCCGTGATGATGCCGCCGTAACCGCCGCGTTGAA 
               
               
                   
                 CGCCCTGACTCACGCCGCACAGCATAACGAAAACCTGCTGGCT 
               
               
                   
                 GCCGCTGTTAATGCCGCTCGCGTTCGCGCCACCCTGGGTGAAA 
               
               
                   
                 TTTCCGATGCGCTGGAAGTCGCTTTCGACCGTTATCTGGTGCC 
               
               
                   
                 AAGCCAGTGTGTTACCGGCGTGATTGCGCAAAGCTATCATCAG 
               
               
                   
                 TCTGAGAAATCGGCCTCCGAGTTCGATGCCATTGTTGCGCAAA 
               
               
                   
                 CGGAGCAGTTCCTTGCCGACAATGGTCGTCGCCCGCGCATTCT 
               
               
                   
                 GATCGCTAAGATGGGCCAGGATGGACACGATCGCGGCGCGAAA 
               
               
                   
                 GTGATCGCCAGCGCCTATTCCGATCTCGGTTTCGACGTAGATT 
               
               
                   
                 TAAGCCCGATGTTCTCTACACCTGAAGAGATCGCCCGCCTGGC 
               
               
                   
                 CGTAGAAAACGACGTTCACGTAGTGGGCGCATCCTCACTGGCT 
               
               
                   
                 GCCGGTCATAAAACGCTGATCCCGGAACTGGTCGAAGCGCTGA 
               
               
                   
                 AAAAATGGGGACGCGAAGATATCTGCGTGGTCGCGGGTGGCGT 
               
               
                   
                 CATTCCGCCGCAGGATTACGCCTTCCTGCAAGAGCGCGGCGTG 
               
               
                   
                 GCGGCGATTTATGGTCCAGGTACACCTATGCTCGACAGTGTGC 
               
               
                   
                 GCGACGTACTGAATCTGATAAGCCAGCATCATGATTAA 
               
               
                   
               
               
                 ygfD 
                 ATGATTAATGAAGCCACGCTGGCAGAAAGTATTCGCCGCTTAC 
               
               
                 SEQ ID 
                 GTCAGGGTGAGCGTGCCACACTCGCCCAGGCCATGACGCTGGT 
               
               
                 NO: 18 
                 GGAAAGCCGTCACCCGCGTCATCAGGCACTAAGTACGCAGCTG 
               
               
                   
                 CTTGATGCCATTATGCCGTACTGCGGTAACACCCTGCGACTGG 
               
               
                   
                 GCGTTACCGGCACCCCCGGCGCGGGGAAAAGTACCTTTCTTGA 
               
               
                   
                 GGCCTTTGGCATGTTGTTGATTCGAGAGGGATTAAAGGTCGCG 
               
               
                   
                 GTTATTGCGGTCGATCCCAGCAGCCCGGTCACTGGCGGTAGCA 
               
               
                   
                 TTCTCGGGGATAAAACCCGCATGAATGACCTGGCGCGTGCCGA 
               
               
                   
                 AGCGGCGTTTATTCGCCCGGTACCATCCTCCGGTCATCTGGGC 
               
               
                   
                 GGTGCCAGTCAGCGAGCGCGGGAATTAATGCTGTTATGCGAAG 
               
               
                   
                 CAGCGGGTTATGACGTAGTGATTGTCGAAACGGTTGGCGTCGG 
               
               
                   
                 GCAGTCGGAAACAGAAGTCGCCCGCATGGTGGACTGTTTTATC 
               
               
                   
                 TCGTTGCAAATTGCCGGTGGCGGCGATGATCTGCAGGGCATTA 
               
               
                   
                 AAAAAGGGCTGATGGAAGTGGCTGATCTGATCGTTATCAACAA 
               
               
                   
                 AGACGATGGCGATAACCATACCAATGTCGCCATTGCCCGGCAT 
               
               
                   
                 ATGTACGAGAGTGCCCTGCATATTCTGCGACGTAAATACGACG 
               
               
                   
                 AATGGCAGCCACGGGTTCTGACTTGTAGCGCACTGGAAAAACG 
               
               
                   
                 TGGAATCGATGAGATCTGGCACGCCATCATCGACTTCAAAACC 
               
               
                   
                 GCGCTAACTGCCAGTGGTCGTTTACAACAAGTGCGGCAACAAC 
               
               
                   
                 AATCGGTGGAATGGCTGCGTAAGCAGACCGAAGAAGAAGTACT 
               
               
                   
                 GAATCACCTGTTCGCGAATGAAGATTTCGATCGCTATTACCGC 
               
               
                   
                 CAGACGCTTTTAGCGGTCAAAAACAATACGCTCTCACCGCGCA 
               
               
                   
                 CCGGCCTGCGGCAGCTCAGTGAATTTATCCAGACGCAATATTT 
               
               
                   
                 TGATTAA 
               
               
                   
               
               
                 ygfG 
                 ATGTCTTATCAGTATGTTAACGTTGTCACTATCAACAAAGTGG 
               
               
                 SEQ ID 
                 CGGTCATTGAGTTTAACTATGGCCGAAAACTTAATGCCTTAAG 
               
               
                 NO: 19 
                 TAAAGTCTTTATTGATGATCTTATGCAGGCGTTAAGCGATCTC 
               
               
                   
                 AACCGGCCGGAAATTCGCTGTATCATTTTGCGCGCACCGAGTG 
               
               
                   
                 GATCCAAAGTCTTCTCCGCAGGTCACGATATTCACGAACTGCC 
               
               
                   
                 GTCTGGCGGTCGCGATCCGCTCTCCTATGATGATCCATTGCGT 
               
               
                   
                 CAAATCACCCGCATGATCCAAAAATTCCCGAAACCGATCATTT 
               
               
                   
                 CGATGGTGGAAGGTAGTGTTTGGGGTGGCGCATTTGAAATGAT 
               
               
                   
                 CATGAGTTCCGATCTGATCATCGCCGCCAGTACCTCAACCTTC 
               
               
                   
                 TCAATGACGCCTGTAAACCTCGGCGTCCCGTATAACCTGGTCG 
               
               
                   
                 GCATTCACAACCTGACCCGCGACGCGGGCTTCCACATTGTCAA 
               
               
                   
                 AGAGCTGATTTTTACCGCTTCGCCAATCACCGCCCAGCGCGCG 
               
               
                   
                 CTGGCTGTCGGCATCCTCAACCATGTTGTGGAAGTGGAAGAAC 
               
               
                   
                 TGGAAGATTTCACCTTACAAATGGCGCACCACATCTCTGAGAA 
               
               
                   
                 AGCGCCGTTAGCCATTGCCGTTATCAAAGAAGAGCTGCGTGTA 
               
               
                   
                 CTGGGCGAAGCACACACCATGAACTCCGATGAATTTGAACGTA 
               
               
                   
                 TTCAGGGGATGCGCCGCGCGGTGTATGACAGCGAAGATTACCA 
               
               
                   
                 GGAAGGGATGAACGCTTTCCTCGAAAAACGTAAACCTAATTTC 
               
               
                   
                 GTTGGTCATTAA 
               
               
                   
               
               
                 ygfH 
                 ATGGAAACTCAGTGGACAAGGATGACCGCCAATGAAGCGGCAG 
               
               
                 SEQ ID 
                 AAATTATCCAGCATAACGACATGGTGGCATTTAGCGGCTTTAC 
               
               
                 NO: 20 
                 CCCGGCGGGTTCGCCGAAAGCCCTACCCACCGCGATTGCCCGC 
               
               
                   
                 AGAGCTAACGAACAGCATGAGGCCAAAAAGCCGTATCAAATTC 
               
               
                   
                 GCCTTCTGACGGGTGCGTCAATCAGCGCCGCCGCTGACGATGT 
               
               
                   
                 ACTTTCTGACGCCGATGCTGTTTCCTGGCGTGCGCCATATCAA 
               
               
                   
                 ACATCGTCCGGTTTACGTAAAAAGATCAATCAGGGCGCGGTGA 
               
               
                   
                 GTTTCGTTGACCTGCATTTGAGCGAAGTGGCGCAAATGGTCAA 
               
               
                   
                 TTACGGTTTCTTCGGCGACATTGATGTTGCCGTCATTGAAGCA 
               
               
                   
                 TCGGCACTGGCACCGGATGGTCGAGTCTGGTTAACCAGCGGGA 
               
               
                   
                 TCGGTAATGCGCCGACCTGGCTGCTGCGGGCGAAGAAAGTGAT 
               
               
                   
                 CATTGAACTCAATCACTATCACGATCCGCGCGTTGCAGAACTG 
               
               
                   
                 GCGGATATTGTGATTCCTGGCGCGCCACCGCGGCGCAATAGCG 
               
               
                   
                 TGTCGATCTTCCATGCAATGGATCGCGTCGGTACCCGCTATGT 
               
               
                   
                 GCAAATCGATCCGAAAAAGATTGTCGCCGTCGTGGAAACCAAC 
               
               
                   
                 TTGCCCGACGCCGGTAATATGCTGGATAAGCAAAATCCCATGT 
               
               
                   
                 GCCAGCAGATTGCCGATAACGTGGTCACGTTCTTATTGCAGGA 
               
               
                   
                 AATGGCGCATGGGCGTATTCCGCCGGAATTTCTGCCGCTGCAA 
               
               
                   
                 AGTGGCGTGGGCAATATCAATAATGCGGTAATGGCGCGTCTGG 
               
               
                   
                 GGGAAAACCCGGTAATTCCTCCGTTTATGATGTATTCGGAAGT 
               
               
                   
                 GCTACAGGAATCGGTGGTGCATTTACTGGAAACCGGCAAAATC 
               
               
                   
                 AGCGGGGCCAGCGCCTCCAGCCTGACAATCTCGGCCGATTCCC 
               
               
                   
                 TGCGCAAGATTTACGACAATATGGATTACTTTGCCAGCCGCAT TGTGTTGCGTCCGCAGGAGATTTCCAATAACCCGGAAATCATC CGTCGTCTGGGCGTCATCGCTCTGAACGTCGGCCTGGAGTTTG ATATTTACGGGCATGCCAACTCAACACACGTAGCCGGGGTCGA TCTGATGAACGGCATCGGCGGCAGCGGTGATTTTGAACGCAAC GCGTATCTGTCGATCTTTATGGCCCCGTCGATTGCTAAAGAAG GCAAGATCTCAACCGTCGTGCCAATGTGCAGCCATGTTGATCA CAGCGAACACAGCGTCAAAGTGATCATCACCGAACAAGGGATC GCCGATCTGCGCGGTCTTTCCCCGCTTCAACGCGCCCGCACTA TCATTGATAATTGTGCACATCCTATGTATCGGGATTATCTGCA TCGCTATCTGGAAAATGCGCCTGGCGGACATATTCACCACGAT CTTAGCCACGTCTTCGACTTACACCGTAATTTAATTGCAACCG GCTCGATGCTGGGTTAA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Sequences of Propionate Cassette from Propioni Bacteria 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 mutA 
                 ATGAGCAGCACGGATCAGGGGACCAACCCCGCCGACACTGACG 
               
               
                 SEQ ID NO: 21 
                 ACCTCACTCCCACCACACTCAGTCTGGCCGGGGATTTCCCCAA 
               
               
                   
                 GGCCACTGAGGAGCAGTGGGAGCGCGAAGTTGAGAAGGTATTC 
               
               
                   
                 AACCGTGGTCGTCCACCGGAGAAGCAGCTGACCTTCGCCGAGT 
               
               
                   
                 GTCTGAAGCGCCTGACGGTTCACACCGTCGATGGCATCGACAT 
               
               
                   
                 CGTGCCGATGTACCGTCCGAAGGACGCGCCGAAGAAGCTGGGT 
               
               
                   
                 TACCCCGGCGTCACCCCCTTCACCCGCGGCACCACGGTGCGCA 
               
               
                   
                 ACGGTGACATGGATGCCTGGGACGTGCGCGCCCTGCACGAGGA 
               
               
                   
                 TCCCGACGAGAAGTTCACCCGCAAGGCGATCCTTGAAGACCTG 
               
               
                   
                 GAGCGTGGCGTCACCTCCCTGTTGTTGCGCGTTGATCCCGACG 
               
               
                   
                 CGATCGCACCCGAGCACCTCGACGAGGTCCTCTCCGACGTCCT 
               
               
                   
                 GCTGGAAATGACCAAGGTGGAGGTCTTCAGCCGCTACGACCAG 
               
               
                   
                 GGTGCCGCCGCCGAGGCCTTGATGGGCGTCTACGAGCGCTCCG 
               
               
                   
                 ACAAGCCGGCGAAGGACCTGGCCCTGAACCTGGGCCTGGATCC 
               
               
                   
                 CATCGGCTTCGCGGCCCTGCAGGGCACCGAGCCGGATCTGACC 
               
               
                   
                 GTGCTCGGTGACTGGGTGCGCCGCCTGGCGAAGTTCTCACCGG 
               
               
                   
                 ACTCGCGCGCCGTCACGATCGACGCGAACGTCTACCACAACGC 
               
               
                   
                 CGGTGCCGGCGACGTGGCAGAGCTCGCTTGGGCACTGGCCACC 
               
               
                   
                 GGCGCGGAGTACGTGCGCGCCCTGGTCGAACAGGGCTTCAACG 
               
               
                   
                 CCACAGAGGCCTTCGACACGATCAACTTCCGTGTCACCGCCAC 
               
               
                   
                 CCACGACCAGTTCCTCACGATCGCCCGTCTTCGCGCCCTGCGC 
               
               
                   
                 GAGGCATGGGCCCGCATCGGCGAGGTCTTTGGCGTGGACGAGG 
               
               
                   
                 ACAAGCGCGGCGCTCGCCAGAATGCGATCACCAGTTGGCGTGA 
               
               
                   
                 GCTCACCCGCGAAGACCCCTATGTCAACATCCTTCGCGGTTCG 
               
               
                   
                 ATTGCCACCTTCTCCGCCTCCGTTGGCGGGGCCGAGTCGATCA 
               
               
                   
                 CGACGCTGCCCTTCACCCAGGCCCTCGGCCTGCCGGAGGACGA 
               
               
                   
                 CTTCCCGCTGCGCATCGCGCGCAACACGGGCATCGTGCTCGCC 
               
               
                   
                 GAAGAGGTGAACATCGGCCGCGTCAACGACCCGGCCGGTGGCT 
               
               
                   
                 CCTACTACGTCGAGTCGCTCACTCGCACCCTGGCCGACGCTGC 
               
               
                   
                 CTGGAAGGAATTCCAGGAGGTCGAGAAGCTCGGTGGCATGTCG 
               
               
                   
                 AAGGCGGTCATGACCGAGCACGTCACCAAGGTGCTCGACGCCT 
               
               
                   
                 GCAATGCCGAGCGCGCCAAGCGCCTGGCCAACCGCAAGCAGCC 
               
               
                   
                 GATCACCGCGGTCAGCGAGTTCCCGATGATCGGGGCCCGCAGC 
               
               
                   
                 ATCGAGACCAAGCCGTTCCCAACCGCTCCGGCGCGCAAGGGCC 
               
               
                   
                 TGGCCTGGCATCGCGATTCCGAGGTGTTCGAGCAGCTGATGGA 
               
               
                   
                 TCGCTCCACCAGCGTCTCCGAGCGCCCCAAGGTGTTCCTTGCC 
               
               
                   
                 TGCCTGGGCACCCGTCGCGACTTCGGTGGCCGCGAGGGCTTCT 
               
               
                   
                 CCAGCCCGGTATGGCACATCGCCGGTATCGACACCCCGCAGGT 
               
               
                   
                 CGAAGGCGGCACCACCGCCGAGATCGTCGAGGCGTTCAAGAAG 
               
               
                   
                 TCGGGCGCCCAGGTGGCCGATCTCTGCTCGTCCGCCAAGATCT 
               
               
                   
                 ACGCGCAGCAGGGACTTGAGGTTGCCAAGGCGCTCAAGGCCGC 
               
               
                   
                 CGGCGCGAAGGCCCTGTATCTGTCGGGCGCCTTCAAGGAGTTC 
               
               
                   
                 GGCGATGACGCCGCCGAGGCCGAGAAGCTGATCGACGGACGCC 
               
               
                   
                 TGTACATGGGCATGGATGTCGTCGACACCCTGTCCTCCACCCT 
               
               
                   
                 TGATATCTTGGGAGTCGCGAAGTGA 
               
               
                   
               
               
                 mutB 
                 GTGAGCACTCTGCCCCGTTTTGATTCAGTTGACCTGGGCAATG 
               
               
                 SEQ ID NO: 22 
                 CCCCGGTTCCTGCTGATGCCGCACAGCGCTTCGAGGAGTTGGC 
               
               
                   
                 CGCCAAGGCCGGCACCGAAGAGGCGTGGGAGACGGCTGAGCAG 
               
               
                   
                 ATTCCGGTTGGCACCCTGTTCAACGAAGACGTCTACAAGGACA 
               
               
                   
                 TGGACTGGCTGGACACCTACGCCGGTATCCCGCCGTTCGTCCA 
               
               
                   
                 CGGCCCATATGCAACCATGTACGCGTTCCGTCCCTGGACGATT 
               
               
                   
                 CGCCAGTACGCCGGCTTCTCCACGGCCAAGGAGTCCAACGCCT 
               
               
                   
                 TCTACCGCCGCAACCTTGCGGCGGGCCAGAAGGGCCTGTCGGT 
               
               
                   
                 TGCCTTCGACCTGCCCACCCACCGCGGCTACGACTCGGACAAT 
               
               
                   
                 CCCCGCGTCGCCGGTGACGTCGGCATGGCCGGGGTGGCCATCG 
               
               
                   
                 ACTCCATCTATGACATGCGCGAGCTGTTCGCCGGCATTCCGCT 
               
               
                   
                 GGACCAGATGAGCGTGTCGATGACCATGAACGGCGCCGTGCTG 
               
               
                   
                 CCGATCCTGGCCCTCTATGTGGTGACCGCCGAGGAGCAGGGCG 
               
               
                   
                 TCAAGCCCGAGCAGCTCGCCGGGACGATCCAGAACGACATCCT 
               
               
                   
                 CAAGGAGTTCATGGTTCGTAACACCTATATCTACCCGCCGCAG 
               
               
                   
                 CCGAGTATGCGAATCATCTCCGAGATCTTCGCCTACACGAGTG 
               
               
                   
                 CCAATATGCCGAAGTGGAATTCGATTTCCATTTCCGGCTACCA 
               
               
                   
                 CATGCAGGAAGCCGGCGCCACGGCCGACATCGAGATGGCCTAC 
               
               
                   
                 ACCCTGGCCGACGGTGTCGACTACATCCGCGCCGGCGAGTCGG 
               
               
                   
                 TGGGCCTCAATGTCGACCAGTTCGCGCCGCGTCTGTCCTTCTT 
               
               
                   
                 CTGGGGCATCGGCATGAACTTCTTCATGGAGGTTGCCAAGCTG 
               
               
                   
                 CGTGCCGCACGTATGTTGTGGGCCAAGCTGGTGCATCAGTTCG 
               
               
                   
                 GGCCGAAGAATCCGAAGTCGATGAGCCTGCGCACCCACTCGCA 
               
               
                   
                 GACCTCCGGTTGGTCGCTGACCGCCCAGGACGTCTACAACAAC 
               
               
                   
                 GTCGTGCGTACCTGCATCGAGGCCATGGCCGCCACCCAGGGCC 
               
               
                   
                 ATACCCAGTCGCTGCACACGAACTCGCTCGACGAGGCCATTGC 
               
               
                   
                 CCTACCGACCGATTTCAGCGCCCGCATCGCCCGTAACACCCAG 
               
               
                   
                 CTGTTCCTGCAGCAGGAATCGGGCACGACGCGCGTGATCGACC 
               
               
                   
                 CGTGGAGCGGCTCGGCATACGTCGAGGAGCTCACCTGGGACCT 
               
               
                   
                 GGCCCGCAAGGCATGGGGCCACATCCAGGAGGTCGAGAAGGTC 
               
               
                   
                 GGCGGCATGGCCAAGGCCATCGAAAAGGGCATCCCCAAGATGC 
               
               
                   
                 GCATTGAGGAAGCCGCCGCCCGCACCCAGGCACGCATCGACTC 
               
               
                   
                 CGGCCGTCAGCCGCTGATCGGCGTGAACAAGTACCGCCTGGAG 
               
               
                   
                 CACGAGCCGCCGCTCGATGTGCTCAAGGTTGACAACTCCACGG 
               
               
                   
                 TGCTCGCCGAGCAGAAGGCCAAGCTGGTCAAGCTGCGCGCCGA 
               
               
                   
                 GCGCGATCCCGAGAAGGTCAAGGCCGCCCTCGACAAGATCACC 
               
               
                   
                 TGGGCTGCCGCCAACCCCGACGACAAGGATCCGGATCGCAACC 
               
               
                   
                 TGCTGAAGCTGTGCATCGACGCTGGCCGCGCCATGGCGACGGT 
               
               
                   
                 CGGCGAGATGAGCGACGCGCTCGAGAAGGTCTTCGGACGCTAC 
               
               
                   
                 ACCGCCCAGATTCGCACCATCTCCGGTGTGTACTCGAAGGAAG 
               
               
                   
                 TGAAGAACACGCCTGAGGTTGAGGAAGCACGCGAGCTCGTTGA 
               
               
                   
                 GGAATTCGAGCAGGCCGAGGGCCGTCGTCCTCGCATCCTGCTG 
               
               
                   
                 GCCAAGATGGGCCAGGACGGTCACGACCGTGGCCAGAAGGTCA 
               
               
                   
                 TCGCCACCGCCTATGCCGACCTCGGTTTCGACGTCGACGTGGG 
               
               
                   
                 CCCGCTGTTCCAGACCCCGGAGGAGACCGCACGTCAGGCCGTC 
               
               
                   
                 GAGGCCGATGTGCACGTGGTGGGCGTTTCGTCGCTCGCCGGCG 
               
               
                   
                 GGCATCTGACGCTGGTTCCGGCCCTGCGCAAGGAGCTGGACAA 
               
               
                   
                 GCTCGGACGTCCCGACATCCTCATCACCGTGGGCGGCGTGATC 
               
               
                   
                 CCTGAGCAGGACTTCGACGAGCTGCGTAAGGACGGCGCCGTGG 
               
               
                   
                 AGATCTACACCCCCGGCACCGTCATTCCGGAGTCGGCGATCTC 
               
               
                   
                 GCTGGTCAAGAAACTGCGGGCTTCGCTCGATGCCTAG 
               
               
                   
               
               
                 GI: 18042134 
                 ATGAGTAATGAGGATCTTTTCATCTGTATCGATCACGTGGCAT 
               
               
                 SEQ ID NO: 23 
                 ATGCGTGCCCCGACGCCGACGAGGCTTCCAAGTACTACCAGGA 
               
               
                   
                 GACCTTCGGCTGGCATGAGCTCCACCGCGAGGAGAACCCGGAG 
               
               
                   
                 CAGGGAGTCGTCGAGATCATGATGGCCCCGGCTGCGAAGCTGA 
               
               
                   
                 CCGAGCACATGACCCAGGTTCAGGTCATGGCCCCGCTCAACGA 
               
               
                   
                 CGAGTCGACCGTTGCCAAGTGGCTTGCCAAGCACAATGGTCGC 
               
               
                   
                 GCCGGACTGCACCACATGGCATGGCGTGTCGATGACATCGACG 
               
               
                   
                 CCGTCAGCGCCACCCTGCGCGAGCGCGGCGTGCAGCTGCTGTA 
               
               
                   
                 TGACGAGCCCAAGCTCGGCACCGGCGGCAACCGCATCAACTTC 
               
               
                   
                 ATGCATCCCAAGTCGGGCAAGGGCGTGCTCATCGAGCTCACCC 
               
               
                   
                 AGTACCCGAAGAACTGA 
               
               
                   
               
               
                 mmdA 
                 ATGGCTGAAAACAACAATTTGAAGCTCGCCAGCACCATGGAAG 
               
               
                 SEQ ID NO: 24 
                 GTCGCGTGGAGCAGCTCGCAGAGCAGCGCCAGGTGATCGAAGC 
               
               
                   
                 CGGTGGCGGCGAACGTCGCGTCGAGAAGCAACATTCCCAGGGT 
               
               
                   
                 AAGCAGACCGCTCGTGAGCGCCTGAACAACCTGCTCGATCCCC 
               
               
                   
                 ATTCGTTCGACGAGGTCGGCGCTTTCCGCAAGCACCGCACCAC 
               
               
                   
                 GTTGTTCGGCATGGACAAGGCCGTCGTCCCGGCAGATGGCGTG 
               
               
                   
                 GTCACCGGCCGTGGCACCATCCTTGGTCGTCCCGTGCACGCCG 
               
               
                   
                 CGTCCCAGGACTTCACGGTCATGGGTGGTTCGGCTGGCGAGAC 
               
               
                   
                 GCAGTCCACGAAGGTCGTCGAGACGATGGAACAGGCGCTGCTC 
               
               
                   
                 ACCGGCACGCCCTTCCTGTTCTTCTACGATTCGGGCGGCGCCC 
               
               
                   
                 GGATCCAGGAGGGCATCGACTCGCTGAGCGGTTACGGCAAGAT 
               
               
                   
                 GTTCTTCGCCAACGTGAAGCTGTCGGGCGTCGTGCCGCAGATC 
               
               
                   
                 GCCATCATTGCCGGCCCCTGTGCCGGTGGCGCCTCGTATTCGC 
               
               
                   
                 CGGCACTGACTGACTTCATCATCATGACCAAGAAGGCCCATAT 
               
               
                   
                 GTTCATCACGGGCCCCCAGGTCATCAAGTCGGTCACCGGCGAG 
               
               
                   
                 GATGTCACCGCTGACGAACTCGGTGGCGCTGAGGCCCATATGG 
               
               
                   
                 CCATCTCGGGCAATATCCACTTCGTGGCCGAGGACGACGACGC 
               
               
                   
                 CGCGGAGCTCATTGCCAAGAAGCTGCTGAGCTTCCTTCCGCAG 
               
               
                   
                 AACAACACTGAGGAAGCATCCTTCGTCAACCCGAACAATGACG 
               
               
                   
                 TCAGCCCCAATACCGAGCTGCGCGACATCGTTCCGATTGACGG 
               
               
                   
                 CAAGAAGGGCTATGACGTGCGCGATGTCATTGCCAAGATCGTC 
               
               
                   
                 GACTGGGGTGACTACCTCGAGGTCAAGGCCGGCTATGCCACCA 
               
               
                   
                 ACCTCGTGACCGCCTTCGCCCGGGTCAATGGTCGTTCGGTGGG 
               
               
                   
                 CATCGTGGCCAATCAGCCGTCGGTGATGTCGGGTTGCCTCGAC 
               
               
                   
                 ATCAACGCCTCTGACAAGGCCGCCGAATTCGTGAATTTCTGCG 
               
               
                   
                 ATTCGTTCAACATCCCGCTGGTGCAGCTGGTCGACGTGCCGGG 
               
               
                   
                 CTTCCTGCCCGGCGTGCAGCAGGAGTACGGCGGCATCATTCGC 
               
               
                   
                 CATGGCGCGAAGATGCTGTACGCCTACTCCGAGGCCACCGTGC 
               
               
                   
                 CGAAGATCACCGTGGTGCTCCGCAAGGCCTACGGCGGCTCCTA 
               
               
                   
                 CCTGGCCATGTGCAACCGTGACCTTGGTGCCGACGCCGTGTAC 
               
               
                   
                 GCCTGGCCCAGCGCCGAGATTGCGGTGATGGGCGCCGAGGGTG 
               
               
                   
                 CGGCAAATGTGATCTTCCGCAAGGAGATCAAGGCTGCCGACGA 
               
               
                   
                 TCCCGACGCCATGCGCGCCGAGAAGATCGAGGAGTACCAGAAC 
               
               
                   
                 GCGTTCAACACGCCGTACGTGGCCGCCGCCCGCGGTCAGGTCG 
               
               
                   
                 ACGACGTGATTGACCCGGCTGATACCCGTCGAAAGATTGCTTC 
               
               
                   
                 CGCCCTGGAGATGTACGCCACCAAGCGTCAGACCCGCCCGGCG 
               
               
                   
                 AAGAAGCATGGAAACTTCCCCTGCTGA 
               
               
                   
               
               
                 PFREUD_18870 
                 ATGAGTCCGCGAGAAATTGAGGTTTCCGAGCCGCGCGAGGTTG 
               
               
                 SEQ ID NO: 25 
                 GTATCACCGAGCTCGTGCTGCGCGATGCCCATCAGAGCCTGAT 
               
               
                   
                 GGCCACACGAATGGCAATGGAAGACATGGTCGGCGCCTGTGCA 
               
               
                   
                 GACATTGATGCTGCCGGGTACTGGTCAGTGGAGTGTTGGGGTG 
               
               
                   
                 GTGCCACGTATGACTCGTGTATCCGCTTCCTCAACGAGGATCC 
               
               
                   
                 TTGGGAGCGTCTGCGCACGTTCCGCAAGCTGATGCCCAACAGC 
               
               
                   
                 CGTCTCCAGATGCTGCTGCGTGGCCAGAACCTGCTGGGTTACC 
               
               
                   
                 GCCACTACAACGACGAGGTCGTCGATCGCTTCGTCGACAAGTC 
               
               
                   
                 CGCTGAGAACGGCATGGACGTGTTCCGTGTCTTCGACGCCATG 
               
               
                   
                 AATGATCCCCGCAACATGGCGCACGCCATGGCTGCCGTCAAGA 
               
               
                   
                 AGGCCGGCAAGCACGCGCAGGGCACCATTTGCTACACGATCAG 
               
               
                   
                 CCCGGTCCACACCGTTGAGGGCTATGTCAAGCTTGCTGGTCAG 
               
               
                   
                 CTGCTCGACATGGGTGCTGATTCCATCGCCCTGAAGGACATGG 
               
               
                   
                 CCGCCCTGCTCAAGCCGCAGCCGGCCTACGACATCATCAAGGC 
               
               
                   
                 CATCAAGGACACCTACGGCCAGAAGACGCAGATCAACCTGCAC 
               
               
                   
                 TGCCACTCCACCACGGGTGTCACCGAGGTCTCCCTCATGAAGG 
               
               
                   
                 CCATCGAGGCCGGCGTCGACGTCGTCGACACCGCCATCTCGTC 
               
               
                   
                 CATGTCGCTCGGCCCGGGCCACAACCCCACCGAGTCGGTTGCC 
               
               
                   
                 GAGATGCTCGAGGGCACCGGGTACACCACCAACCTTGACTACG 
               
               
                   
                 ATCGCCTGCACAAGATCCGCGATCACTTCAAGGCCATCCGCCC 
               
               
                   
                 GAAGTACAAGAAGTTCGAGTCGAAGACGCTTGTCGACACCTCG 
               
               
                   
                 ATCTTCAAGTCGCAGATCCCCGGCGGCATGCTCTCCAACATGG 
               
               
                   
                 AGTCGCAGCTGCGCGCCCAGGGCGCCGAGGACAAGATGGACGA 
               
               
                   
                 GGTCATGGCAGAGGTGCCGCGCGTCCGCAAGGCCGCCGGCTTC 
               
               
                   
                 CCGCCCCTGGTCACCCCGTCCAGCCAGATCGTCGGCACGCAGG 
               
               
                   
                 CCGTGTTCAACGTGATGATGGGCGAGTACAAGAGGATGACCGG 
               
               
                   
                 CGAGTTCGCCGACATCATGCTCGGCTACTACGGCGCCAGCCCG 
               
               
                   
                 GCCGATCGCGATCCGAAGGTGGTCAAGTTGGCCGAGGAGCAGT 
               
               
                   
                 CCGGCAAGAAGCCGATCACCCAGCGCCCGGCCGATCTGCTGCC 
               
               
                   
                 CCCCGAGTGGGAGGAGCAGTCCAAGGAGGCCGCGGCCCTCAAG 
               
               
                   
                 GGCTTCAACGGCACCGACGAGGACGTGCTCACCTATGCACTGT 
               
               
                   
                 TCCCGCAGGTCGCTCCGGTCTTCTTCGAGCATCGCGCCGAGGG 
               
               
                   
                 CCCGCACAGCGTGGCTCTCACCGATGCCCAGCTGAAGGCCGAG 
               
               
                   
                 GCCGAGGGCGACGAGAAGTCGCTCGCCGTGGCCGGTCCCGTCA 
               
               
                   
                 CCTACAACGTGAACGTGGGCGGAACCGTCCGCGAAGTCACCGT 
               
               
                   
                 TCAGCAGGCGTGA 
               
               
                   
               
               
                 Bccp 
                 ATGAAACTGAAGGTAACAGTCAACGGCACTGCGTATGACGTTG 
               
               
                 SEQ ID NO: 26 
                 ACGTTGACGTCGACAAGTCACACGAAAACCCGATGGGCACCAT 
               
               
                   
                 CCTGTTCGGCGGCGGCACCGGCGGCGCGCCGGCACCGCGCGCA 
               
               
                   
                 GCAGGTGGCGCAGGCGCCGGTAAGGCCGGAGAGGGCGAGATTC 
               
               
                   
                 CCGCTCCGCTGGCCGGCACCGTCTCCAAGATCCTCGTGAAGGA 
               
               
                   
                 GGGTGACACGGTCAAGGCTGGTCAGACCGTGCTCGTTCTCGAG 
               
               
                   
                 GCCATGAAGATGGAGACCGAGATCAACGCTCCCACCGACGGCA 
               
               
                   
                 AGGTCGAGAAGGTCCTTGTCAAGGAGCGTGACGCCGTGCAGGG 
               
               
                   
                 CGGTCAGGGTCTCATCAAGATCGGCTGA 
               
               
                   
               
            
           
         
       
     
     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: 26) 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: 26) 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: 26) 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: 26) or a functional fragment thereof. 
     Table 5 lists exemplary polypeptide sequences, which may be encoded by the propionate production gene(s) or cattette(s) of the genetically engineered bacteria. 
     
       
         
           
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 Polypeptide Sequences for Propionate Synthesis 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Pct 
                 MRKVPIITADEAAKLIKDGDTVTTSGFVGNAIPEALDRAVEKRFLET 
               
               
                 SEQ ID 
                 GEPKNITYVYCGSQGNRDGRGAEHFAHEGLLKRYIAGHWATVPALGK 
               
               
                 NO: 27 
                 MAMENKMEAYNVSQGALCHLFRDIASHKPGVFTKVGIGTFIDPRNGG 
               
               
                   
                 GKVNDITKEDIVELVEIKGQEYLFYPAFPIHVALIRGTYADESGNIT 
               
               
                   
                 FEKEVAPLEGTSVCQAVKNSGGIVVVQVERVVKAGTLDPRHVKVPGI 
               
               
                   
                 YVDYVVVADPEDHQQSLDCEYDPALSGEHRRPEVVGEPLPLSAKKVI 
               
               
                   
                 GRRGAIELEKDVAVNLGVGAPEYVASVADEEGIVDFMTLTAESGAIG 
               
               
                   
                 GVPAGGVRFGASYNADALIDQGYQFDYYDGGGLDLCYLGLAECDEKG 
               
               
                   
                 NINVSRFGPRIAGCGGFINITQNTPKVFFCGTFTAGGLKVKIEDGKV 
               
               
                   
                 IIVQEGKQKKFLKAVEQITFNGDVALANKQQVTYITERCVFLLKEDG 
               
               
                   
                 LHLSEIAPGIDLQTQILDVMDFAPIIDRDANGQIKLMDAALFAEGLM 
               
               
                   
                 GLKEMKS* 
               
               
                   
               
               
                 lcdA 
                 MSLTQGMKAKQLLAYFQGKADQDAREAKARGELVCWSASVAPPEFCV 
               
               
                 SEQ ID 
                 TMGIAMIYPETHAAGIGARKGAMDMLEVADRKGYNVDCCSYGRVNMG 
               
               
                 NO: 28 
                 YMECLKEAAITGVKPEVLVNSPAADVPLPDLVITCNNICNTLLKWYE 
               
               
                   
                 NLAAELDIPCIVIDVPFNHTMPIPEYAKAYIADQFRNAISQLEVICG 
               
               
                   
                 RPFDWKKFKEVKDQTQRSVYHWNRIAEMAKYKPSPLNGFDLFNYMAL 
               
               
                   
                 IVACRSLDYAEITFKAFADELEENLKAGIYAFKGAEKTRFQWEGIAV 
               
               
                   
                 WPHLGHTFKSMKNLNSIMTGTAYPALWDLHYDANDESMHSMAEAYTR 
               
               
                   
                 IYINTCLQNKVEVLLGIMEKGQVDGTVYHLNRSCKLMSFLNVETAEI 
               
               
                   
                 IKEKNGLPYVSIDGDQTDPRVFSPAQFDTRVQALVEMMEANMAAAE* 
               
               
                   
               
               
                 lcdB 
                 MSRVEAILSQLKDVAANPKKAMDDYKAETGKGAVGIMPIYSPEEMVH 
               
               
                 SEQ ID 
                 AAGYLPMGIWGAQGKTISKARTYLPAFACSVMQQVMELQCEGAYDDL 
               
               
                 NO: 29 
                 SAVIFSVPCDTLKCLSQKWKGTSPVIVFTHPQNRGLEAANQFLVTEY 
               
               
                   
                 ELVKAQLESVLGVKISNAALENSIAIYNENRAVMREFVKVAADYPQV 
               
               
                   
                 IDAVSRHAVFKARQFMLKEKHTALVKELIAEIKATPVQPWDGKKVVV 
               
               
                   
                 TGILLEPNELLDIFNEFKIAIVDDDLAQESRQIRVDVLDGEGGPLYR 
               
               
                   
                 MAKAWQQMYGCSLATDTKKGRGRMLINKTIQTGADAIVVAMMKFCDP 
               
               
                   
                 EEWDYPVMYREFEEKGVKSLMIEVDQEVSSFEQIKTRLQSFVEML* 
               
               
                   
               
               
                 lcdC 
                 MYTLGIDVGSASSKAVILKDGKDIVAAEVVQVGTGSSGPQRALDKAFE 
               
               
                 SEQ ID 
                 VSGLKKEDISYTVATGYGRFNFSDADKQISEISCHAKGIYFLVPTART 
               
               
                 NO: 30 
                 IIDIGGQDAKAIRLDDKGGIKQFFMNDKCAAGTGRFLEVMARVLETTL 
               
               
                   
                 DEMAELDEQATDTAPISSTCTVFAESEVISQLSNGVSRNNIIKGVHLS 
               
               
                   
                 VASRACGLAYRGGLEKDVVMTGGVAKNAGVVRAVAGVLKTDVIVAPNP 
               
               
                   
                 QTTGALGAALYAYEAAQKKX 
               
               
                   
               
               
                 etfA 
                 MAFNSADINSFRDIWVFCEQREGKLINTDFELISEGRKLADERGSKL 
               
               
                 SEQ ID 
                 VGILLGHEVEEIAKELGGYGADKVIVCDHPELKFYTTDAYAKVLCDV 
               
               
                 NO: 31 
                 VMEEKPEVILIGATNIGRDLGPRCAARLHTGLTADCTHLDIDMNKYV 
               
               
                   
                 DFLSTSSTLDISSMTFPMEDTNLKMTRPAFGGHLMATIICPRFRPCM 
               
               
                   
                 STVRPGVMKKAEFSQEMAQACQVVTRHVNLSDEDLKTKVINIVKETK 
               
               
                   
                 KIVDLIGAEIIVSVGRGISKDVQGGIALAEKLADAFGNGVVGGSRAV 
               
               
                   
                 IDSGWLPADHQVGQTGKTVHPKVYVALGISGAIQHKAGMQDSELIIA 
               
               
                   
                 VNKDETAPIFDCADYGITGDLFKIVPMMIDAIKEGKNA* 
               
               
                   
               
               
                 acrB 
                 MRIYVCVKQVPDTSGKVAVNPDGTLNRASMAAIINPDDMSAIEQALK 
               
               
                 SEQ ID 
                 LKDETGCQVTALTMGPPPAEGMLREIIAMGADDGVLISAREFGGSDT 
               
               
                 NO: 32 
                 FATSQIISAAIHKLGLSNEDMIFCGRQAIDGDTAQVGPQIAEKLSIP 
               
               
                   
                 QVTYGAGIKKSGDLVLVKRMLEDGYMMIEVETPCLITCIQDKAVKPR 
               
               
                   
                 YMTLNGIMECYSKPLLVLDYEALKDEPLIELDTIGLKGSPTNIFKSF 
               
               
                   
                 TPPQKGVGVMLQGTDKEKVEDLVDKLMQKHVI* 
               
               
                   
               
               
                 acrC 
                 MFLLKIKKERMKRMDFSLTREQEMLKKLARQFAEIELEPVAEEIDRE 
               
               
                 SEQ ID 
                 HVFPAENFKKMAEIGLTGIGIPKEFGGSGGGTLEKVIAVSEFGKKCM 
               
               
                 NO: 33 
                 ASASILSIHLIAPQAIYKYGTKEQKETYLPRLTKGGELGAFALTEPN 
               
               
                   
                 AGSDAGAVKTTAILDSQTNEYVLNGTKCFISGGGRAGVLVIFALTEP 
               
               
                   
                 KKGLKGMSAIIVEKGTPGFSIGKVESKMGIAGSETAELIFEDCRVPA 
               
               
                   
                 ANLLGKEGKGFKIAMEALDGARIGVGAQAIGIAEGAIDLSVKYVHER 
               
               
                   
                 IQFGKPIANLQGIQWYIADMATKTAAARALVEFAAYLEDAGKPFTKE 
               
               
                   
                 SAMCKLNASENARFVTNLALQIHGGYGYMKDYPLERMYRDAKITEIY 
               
               
                   
                 EGTSEIHKWIAREVMKR* 
               
               
                   
               
               
                 thrAfbr 
                 MRVLKFGGTSVANAERFLRVADILESNARQGQVATVLSAPAKITNHL 
               
               
                 SEQ ID 
                 VAMIEKTISGQDALPNISDAERIFAELLTGLAAAQPGFPLAQLKTFV 
               
               
                 NO: 34 
                 DQEFAQIKHVLHGISLLGQCPDSINAALICRGEKMSIAIMAGVLEAR 
               
               
                   
                 GHNVTVIDPVEKLLAVGHYLESTVDIAESTRRIAASRIPADHMVLMA 
               
               
                   
                 GFTAGNEKGELVVLGRNGSDYSAAVLAACLRADCCEIWTDVDGVYTC 
               
               
                   
                 DPRQVPDARLLKSMSYQEAMELSYFGAKVLHPRTITPIAQFQIPCLI 
               
               
                   
                 KNTGNPQAPGTLIGASRDEDELPVKGISNLNNMAMFSVSGPGMKGMV 
               
               
                   
                 GMAARVFAAMSRARISVVLITQSSSEYSISFCVPQSDCVRAERAMQE 
               
               
                   
                 EFYLELKEGLLEPLAVTERLAIISVVGDGMRTLRGISAKFFAALARA 
               
               
                   
                 NINIVAIAQRSSERSISVVVNNDDATTGVRVTHQMLFNTDQVIEVFV 
               
               
                   
                 IGVGGVGGALLEQLKRQQSWLKNKHTDLRVCGVANSKALLTNVHGLN 
               
               
                   
                 LENWQEELAQAKEPFNLGRLIRLVKEYHLLNPVIVDCTSSQAVADQY 
               
               
                   
                 ADFLREGFHVVTPNKKANTSSMDYYHQLRYAAEKSRRKFLYDTNVGA 
               
               
                   
                 GLPVIENLQNLLNAGDELMKFSGILSGSLSYIFGKLDEGMSFSEATT 
               
               
                   
                 LAREMGYTEPDPRDDLSGMDVARKLLILARETGRELELADIEIEPVL 
               
               
                   
                 PAEFNAEGDVAAFMANLSQLDDLFAARVAKARDEGKVLRYVGNIDED 
               
               
                   
                 GVCRVKIAEVDGNDPLFKVKNGENALAFYSHYYQPLPLVLRGYGAGN 
               
               
                   
                 DVTAAGVFADLLRTLSWKLGV* 
               
               
                   
               
               
                 thrB 
                 MVKVYAPASSANMSVGFDVLGAAVTPVDGALLGDVVTVEAAETFSLN 
               
               
                 SEQ ID 
                 NLGRFADKLPSEPRENIVYQCWERFCQELGKQIPVAMTLEKNMPIGS 
               
               
                 NO: 35 
                 GLGSSACSVVAALMAMNEHCGKPLNDTRLLALMGELEGRISGSIHYD 
               
               
                   
                 NVAPCFLGGMQLMIEENDIISQQVPGFDEWLWVLAYPGIKVSTAEAR 
               
               
                   
                 AILPAQYRRQDCIAHGRHLAGFIHACYSRQPELAAKLMKDVIAEPYR 
               
               
                   
                 ERLLPGFRQARQAVAEIGAVASGISGSGPTLFALCDKPETAQRVADW 
               
               
                   
                 LGKNYLQNQEGFVHICRLDTAGARVLEN* 
               
               
                   
               
               
                 thrC 
                 MKLYNLKDHNEQVSFAQAVTQGLGKNQGLFFPHDLPEFSLTEIDEML 
               
               
                 SEQ ID 
                 KLDFVTRSAKILSAFIGDEIPQEILEERVRAAFAFPAPVANVESDVG 
               
               
                 NO: 36 
                 CLELFHGPTLAFKDFGGRFMAQMLTHIAGDKPVTILTATSGDTGAAV 
               
               
                   
                 AHAFYGLPNVKVVILYPRGKISPLQEKLFCTLGGNIETVAIDGDFDA 
               
               
                   
                 CQALVKQAFDDEELKVALGLNSANSINISRLLAQICYYFEAVAQLPQ 
               
               
                   
                 ETRNQLVVSVPSGNFGDLTAGLLAKSLGLPVKRFIAATNVNDTVPRF 
               
               
                   
                 LHDGQWSPKATQATLSNAMDVSQPNNWPRVEELFRRKIWQLKELGYA 
               
               
                   
                 AVDDETTQQTMRELKELGYTSEPHAAVAYRALRDQLNPGEYGLFLGT 
               
               
                   
                 AHPAKFKESVEAILGETLDLPKELAERADLPLLSHNLPADFAALRKL 
               
               
                   
                 MMNHQ* 
               
               
                   
               
               
                 ilvA fbr   
                 MSETYVSEKSPGVMASGAELIRAADIQTAQARISSVIAPTPLQYCPR 
               
               
                 SEQ ID 
                 LSEETGAEIYLKREDLQDVRSYKIRGALNSGAQLTQEQRDAGIVAAS 
               
               
                 NO: 37 
                 AGNHAQGVAYVCKSLGVQGRIYVPVQTPKQKRDRIMVHGGEFVSLVV 
               
               
                   
                 TGNNFDEASAAAHEDAERTGATLIEPFDARNTVIGQGTVAAEILSQL 
               
               
                   
                 TSMGKSADHVMVPVGGGGLLAGVVSYMADMAPRTAIVGIEPAGAASM 
               
               
                   
                 QAALHNGGPITLETVDPFVDGAAVKRVGDLNYTIVEKNQGRVHMMSA 
               
               
                   
                 TEGAVCTEMLDLYQNEGIIAEPAGALSIAGLKEMSFAPGSAVVCIIS 
               
               
                   
                 GGNNDVLRYAEIAERSLVHRGLKHYFLVNFPQKPGQLRHFLEDILGP 
               
               
                   
                 DDDITLFEYLKRNNRETGTALVGIHLSEASGLDSLLERMEESAIDSR 
               
               
                   
                 RLEPGTPEYEYLT* 
               
               
                   
               
               
                 ace 
                 MSERFPNDVDPIETRDWLQAIESVIREEGVERAQYLIDQLLAEARKG 
               
               
                 SEQ ID 
                 GVNVAAGTGISNYINTIPVEEQPEYPGNLELERRIRSAIRWNAIMTV 
               
               
                 NO: 38 
                 LRASKKDLELGGHMASFQSSATIYDVCFNHFFRARNEQDGGDLVYFQ 
               
               
                   
                 GHISPGVYARAFLEGRLTQEQLDNFRQEVHGNGLSSYPHPKLMPEFW 
               
               
                   
                 QFPTVSMGLGPIGAIYQAKFLKYLEHRGLKDTSKQTVYAFLGDGEMD 
               
               
                   
                 EPESKGAITIATREKLDNLVFVINCNLQRLDGPVTGNGKIINELEGI 
               
               
                   
                 FEGAGWNVIKVMWGSRWDELLRKDTSGKLIQLMNETVDGDYQTFKSK 
               
               
                   
                 DGAYVREHFFGKYPETAALVADWTDEQIWALNRGGHDPKKIYAAFKK 
               
               
                   
                 AQETKGKATVILAHTIKGYGMGDAAEGKNIAHQVKKMNMDGVRHIRD 
               
               
                   
                 RFNVPVSDADIEKLPYITFPEGSEEHTYLHAQRQKLHGYLPSRQPNF 
               
               
                   
                 TEKLELPSLQDFGALLEEQSKEISTTIAFVRALNVMLKNKSIKDRLV 
               
               
                   
                 PIIADEARTFGMEGLFRQIGIYSPNGQQYTPQDREQVAYYKEDEKGQ 
               
               
                   
                 ILQEGINELGAGCSWLAAATSYSTNNLPMIPFYIYYSMFGFQRIGDL 
               
               
                   
                 CWAAGDQQARGFLIGGTSGRTTLNGEGLQHEDGHSHIQSLTIPNCIS 
               
               
                   
                 YDPAYAYEVAVIMHDGLERMYGEKQENVYYYITTLNENYHMPAMPEG 
               
               
                   
                 AEEGIRKGIYKLETIEGSKGKVQLLGSGSILRHVREAAEILAKDYGV 
               
               
                   
                 GSDVYSVTSFTELARDGQDCERWNMLHPLETPRVPYIAQVMNDAPAV 
               
               
                   
                 ASTDYMKLFAEQVRTYVPADDYRVLGTDGFGRSDSRENLRHHFEVDA 
               
               
                   
                 SYVVVAALGELAKRGEIDKKVVADAIAKFNIDADKVNPRLA* 
               
               
                   
               
               
                 aceF 
                 MAIEIKVPDIGADEVEITEILVKVGDKVEAEQSLITVEGDKASMEVP 
               
               
                 SEQ ID 
                 SPQAGIVKEIKVSVGDKTQTGALIMIFDSADGAADAAPAQAEEKKEA 
               
               
                 NO: 39 
                 APAAAPAAAAAKDVNVPDIGSDEVEVTEILVKVGDKVEAEQSLITVE 
               
               
                   
                 GDKASMEVPAPFAGTVKEIKVNVGDKVSTGSLIMVFEVAGEAGAAAP 
               
               
                   
                 AAKQEAAPAAAPAPAAGVKEVNVPDIGGDEVEVTEVMVKVGDKVAAE 
               
               
                   
                 QSLITVEGDKASMEVPAPFAGVVKELKVNVGDKVKTGSLIMIFEVEG 
               
               
                   
                 AAPAAAPAKQEAAAPAPAAKAEAPAAAPAAKAEGKSEFAENDAYVHA 
               
               
                   
                 TPLTRRLAREFGVNLAKVKGTGRKGRILREDVQAYVKEAIKRAEAAP 
               
               
                   
                 AATGGGIPGMLPWPKVDFSKFGEIEEVELGRIQKISGANLSRNWVMI 
               
               
                   
                 PHVTHFDKTDITELEAFRKQQNEEAAKRKLDVKITPVVFIMKAVAAA 
               
               
                   
                 LEQMPRFNSSLSEDGQRLTLKKYINIGVAVDTPNGLVVPVFKDVNKK 
               
               
                   
                 GIIELSRELMTISKKARDGKLTAGEMQGGCFTISSIGGLGTTHFAPI 
               
               
                   
                 VNAPEVAILGVSKSAMEPVWNGKEFVPRLMLPISLSFDHRVIDGADG 
               
               
                   
                 ARFITIINNTLSDIRRLVM* 
               
               
                   
               
               
                 Lpd 
                 MSTEIKTQVVVLGAGPAGYSAAFRCADLGLETVIVERYNTLGGVCLN 
               
               
                 SEQ ID 
                 VGCIPSKALLHVAKVIEEAKALAEHGIVFGEPKTDIDKIRTWKEKVI 
               
               
                 NO: 40 
                 NQLTGGLAGMAKGRKVKVVNGLGKFTGANTLEVEGENGKTVINFDNA 
               
               
                   
                 IIAAGSRPIQLPFIPHEDPRIWDSTDALELKEVPERLLVMGGGIIGL 
               
               
                   
                 EMGTVYHALGSQIDVVEMFDQVIPAADKDIVKVFTKRISKKFNLMLE 
               
               
                   
                 TKVTAVEAKEDGIYVTMEGKKAPAEPQRYDAVLVAIGRVPNGKNLDA 
               
               
                   
                 GKAGVEVDDRGFIRVDKQLRTNVPHIFAIGDIVGQPMLAHKGVHEGH 
               
               
                   
                 VAAEVIAGKKHYFDPKVIPSIAYTKPEVAWVGLTEKEAKEKGISYET 
               
               
                   
                 ATFPWAASGRAIASDCADGMTKLIFDKESHRVIGGAIVGTNGGELLG 
               
               
                   
                 EIGLAIEMGCDAEDIALTIHAHPTLHESVGLAAEVFEGSITDLPNPK 
               
               
                   
                 AKKK* 
               
               
                   
               
               
                 tesB 
                 MSQALKNLLTLLNLEKIEEGLFRGQSEDLGLRQVFGGQVVGQALYAA 
               
               
                 SEQ ID 
                 KETVPEERLVHSFHSYFLRPGDSKKPIIYDVETLRDGNSFSARRVAA 
               
               
                 NO: 41 
                 IQNGKPIFYMTASFQAPEAGFEHQKTMPSAPAPDGLPSETQIAQSLA 
               
               
                   
                 HLLPPVLKDKFICDRPLEVRPVEFHNPLKGHVAEPHRQVWIRANGSV 
               
               
                   
                 PDDLRVHQYLLGYASDLNFLPVALQPHGIGFLEPGIQIATIDHSMWF 
               
               
                   
                 HRPFNLNEWLLYSVESTSASSARGFVRGEFYTQDGVLVASTVQEGVM 
               
               
                   
                 RNHN* 
               
               
                   
               
               
                 acuI 
                 MRAVLIEKSDDTQSVSVTELAEDQLPEGDVLVDVAYSTLNYKDALAI 
               
               
                 SEQ ID 
                 TGKAPVVRRFPMVPGIDFTGTVAQSSHADFKPGDRVILNGWGVGEKH 
               
               
                 NO: 42 
                 WGGLAERARVRGDWLVPLPAPLDLRQAAMIGTAGYTAMLCVLALERH 
               
               
                   
                 GVVPGNGEIVVSGAAGGVGSVATTLLAAKGYEVAAVTGRASEAEYLR 
               
               
                   
                 GLGAASVIDRNELTGKVRPLGQERWAGGIDVAGSTVLANMLSMMKYR 
               
               
                   
                 GVVAACGLAAGMDLPASVAPFILRGMTLAGVDSVMCPKTDRLAAWAR 
               
               
                   
                 LASDLDPAKLEEMTTELPFSEVIETAPKFLDGTVRGRIVIPVTP* 
               
               
                   
               
               
                 Sbm 
                 MSNVQEWQQLANKELSRREKTVDSLVHQTAEGIAIKPLYTEADLDNL 
               
               
                 SEQ ID 
                 EVTGTLPGLPPYVRGPRATMYTAQPWTIRQYAGFSTAKESNAFYRRN 
               
               
                 NO: 43 
                 LAAGQKGLSVAFDLATHRGYDSDNPRVAGDVGKAGVAIDTVEDMKVL 
               
               
                   
                 FDQIPLDKMSVSMTMNGAVLPVLAFYIVAAEEQGVTPDKLTGTIQND 
               
               
                   
                 ILKEYLCRNTYIYPPKPSMRIIADIIAWCSGNMPRFNTISISGYHMG 
               
               
                   
                 EAGANCVQQVAFTLADGIEYIKAAISAGLKIDDFAPRLSFFFGIGMD 
               
               
                   
                 LFMNVAMLRAARYLWSEAVSGFGAQDPKSLALRTHCQTSGWSLTEQD 
               
               
                   
                 PYNNVIRTTIEALAATLGGTQSLHTNAFDEALGLPTDFSARIARNTQ 
               
               
                   
                 IIIQEESELCRTVDPLAGSYYIESLTDQIVKQARAIIQQIDEAGGMA 
               
               
                   
                 KAIEAGLPKRMIEEASAREQSLIDQGKRVIVGVNKYKLDHEDETDVL 
               
               
                   
                 EIDNVMVRNEQIASLERIRATRDDAAVTAALNALTHAAQHNENLLAA 
               
               
                   
                 AVNAARVRATLGEISDALEVAFDRYLVPSQCVTGVIAQSYHQSEKSA 
               
               
                   
                 SEFDAIVAQTEQFLADNGRRPRILIAKMGQDGHDRGAKVIASAYSDL 
               
               
                   
                 GFDVDLSPMFSTPEEIARLAVENDVHVVGASSLAAGHKTLIPELVEA 
               
               
                   
                 LKKWGREDICVVAGGVIPPQDYAFLQERGVAAIYGPGTPMLDSVRDV 
               
               
                   
                 LNLISQHHD* 
               
               
                   
               
               
                 ygfD 
                 MINEATLAESIRRLRQGERATLAQAMTLVESRHPRHQALSTQLLDAI 
               
               
                 SEQ ID 
                 MPYCGNTLRLGVTGTPGAGKSTFLEAFGMLLIREGLKVAVIAVDPSS 
               
               
                 NO: 44 
                 PVTGGSILGDKTRMNDLARAEAAFIRPVPSSGHLGGASQRARELMLL 
               
               
                   
                 CEAAGYDVVIVETVGVGQSETEVARMVDCFISLQIAGGGDDLQGIKK 
               
               
                   
                 GLMEVADLIVINKDDGDNHTNVAIARHMYESALHILRRKYDEWQPRV 
               
               
                   
                 LTCSALEKRGIDEIWHAIIDFKTALTASGRLQQVRQQQSVEWLRKQT 
               
               
                   
                 EEEVLNHLFANEDFDRYYRQTLLAVKNNTLSPRTGLRQLSEFIQTQY 
               
               
                   
                 FD* 
               
               
                   
               
               
                 ygfG 
                 MSYQYVNVVTINKVAVIEFNYGRKLNALSKVFIDDLMQALSDLNRPE 
               
               
                 SEQ ID 
                 IRCIILRAPSGSKVFSAGHDIHELPSGGRDPLSYDDPLRQITRMTQK 
               
               
                 NO: 45 
                 FPKPIISMVEGSVWGGAFEMIMSSDLIIAASTSTFSMTPVNLGVPYN 
               
               
                   
                 LVGIHNLTRDAGFHIVKELIFTASPITAQRALAVGILNHVVEVEELE 
               
               
                   
                 DFTLQMAHHISEKAPLAIAVIKEELRVLGEAHTMNSDEFERIQGMRR 
               
               
                   
                 AVYDSEDYQEGMNAFLEKRKPNFVGH* 
               
               
                   
               
               
                 ygfH 
                 METQWTRMTANEAAEIIQHNDMVAFSGFTPAGSPKALPTAIARRANE 
               
               
                 SEQ ID 
                 QHEAKKPYQIRLLTGASISAAADDVLSDADAVSWRAPYQTSSGLRKK 
               
               
                 NO: 46 
                 INQGAVSFVDLHLSEVAQMVNYGFFGDIDVAVIEASALAPDGRVWLT 
               
               
                   
                 SGIGNAPTWLLRAKKVIIELNHYHDPRVAELADIVIPGAPPRRNSVS 
               
               
                   
                 IFHAMDRVGTRYVQIDPKKIVAVVETNLPDAGNMLDKQNPMCQQIAD 
               
               
                   
                 NVVTFLLQEMAHGRIPPEFLPLQSGVGNINNAVMARLGENPVIPPFM 
               
               
                   
                 MYSEVLQESVVHLLETGKISGASASSLTISADSLRKIYDNMDYFASR 
               
               
                   
                 IVLRPQEISNNPEIIRRLGVIALNVGLEFDIYGHANSTHVAGVDLMN 
               
               
                   
                 GIGGSGDFERNAYLSIFMAPSIAKEGKISTVVPMCSHVDHSEHSVKV 
               
               
                   
                 IITEQGIADLRGLSPLQRARTIIDNCAHPMYRDYLHRYLENAPGGHI 
               
               
                   
                 HHDLSHVFDLHRNLIATGSMLG* 
               
               
                   
               
               
                 mutA 
                 MSSTDQGTNPADTDDLTPTTLSLAGDFPKATEEQWEREVEKVFNRGRP 
               
               
                 SEQ ID 
                 PEKQLTFAECLKRLTVHTVDGIDIVPMYRPKDAPKKLGYPGVTPFTRG 
               
               
                 NO: 47 
                 TTVRNGDMDAWDVRALHEDPDEKFTRKAILEDLERGVTSLLLRVDPDA 
               
               
                   
                 IAPEHLDEVLSDVLLEMTKVEVFSRYDQGAAAEALMGVYERSDKPAKD 
               
               
                   
                 LALNLGLDPIGFAALQGTEPDLTVLGDWVRRLAKFSPDSRAVTIDANV 
               
               
                   
                 YHNAGAGDVAELAWALATGAEYVRALVEQGFNATEAFDTINFRVTATH 
               
               
                   
                 DQFLTIARLRALREAWARIGEVFGVDEDKRGARQNAITSWRELTREDP 
               
               
                   
                 YVNILRGSIATFSASVGGAESITTLPFTQALGLPEDDFPLRIARNTGI 
               
               
                   
                 VLAEEVNIGRVNDPAGGSYYVESLTRTLADAAWKEFQEVEKLGGMSKA 
               
               
                   
                 VMTEHVTKVLDACNAERAKRLANRKQPITAVSEFPMIGARSIETKPFP 
               
               
                   
                 TAPARKGLAWHRDSEVFEQLMDRSTSVSERPKVFLACLGTRRDFGGRE 
               
               
                   
                 GFSSPVWHIAGIDTPQVEGGTTAEIVEAFKKSGAQVADLCSSAKIYAQ 
               
               
                   
                 QGLEVAKALKAAGAKALYLSGAFKEFGDDAAEAEKLIDGRLYMGMDVV 
               
               
                   
                 DTLSSTLDILGVAK 
               
               
                   
               
               
                 mutB 
                 VSTLPRFDSVDLGNAPVPADAAQRFEELAAKAGTEEAWETAEQIPVGT 
               
               
                 SEQ ID 
                 LFNEDVYKDMDWLDTYAGIPPFVHGPYATMYAFRPWTIRQYAGFSTAK 
               
               
                 NO: 48 
                 ESNAFYRRNLAAGQKGLSVAFDLPTHRGYDSDNPRVAGDVGMAGVAID 
               
               
                   
                 SIYDMRELFAGIPLDQMSVSMTMNGAVLPILALYVVTAEEQGVKPEQL 
               
               
                   
                 AGTIQNDILKEFMVRNTYIYPPQPSMRIISEIFAYTSANMPKWNSISI 
               
               
                   
                 SGYHMQEAGATADIEMAYTLADGVDYIRAGESVGLNVDQFAPRLSFFW 
               
               
                   
                 GIGMNFFMEVAKLRAARMLWAKLVHQFGPKNPKSMSLRTHSQTSGWSL 
               
               
                   
                 TAQDVYNNVVRTCIEAMAATQGHTQSLHTNSLDEAIALPTDFSARIAR 
               
               
                   
                 NTQLFLQQESGTTRVIDPWSGSAYVEELTWDLARKAWGHIQEVEKVGG 
               
               
                   
                 MAKAIEKGIPKMRIEEAAARTQARIDSGRQPLIGVNKYRLEHEPPLDV 
               
               
                   
                 LKVDNSTVLAEQKAKLVKLRAERDPEKVKAALDKITWAAANPDDKDPD 
               
               
                   
                 RNLLKLCIDAGRAMATVGEMSDALEKVFGRYTAQIRTISGVYSKEVKN 
               
               
                   
                 TPEVEEARELVEEFEQAEGRRPRILLAKMGQDGHDRGQKVIATAYADL 
               
               
                   
                 GFDVDVGPLFQTPEETARQAVEADVHVVGVSSLAGGHLTLVPALRKEL 
               
               
                   
                 DKLGRPDILITVGGVIPEQDFDELRKDGAVEIYTPGTVIPESAISLVK 
               
               
                   
                 KLRASLDA 
               
               
                   
               
               
                 GI: 18042134 
                 MSNEDLFICIDHVAYACPDADEASKYYQETFGWHELHREENPEQGVVE 
               
               
                 SEQ ID 
                 IMMAPAAKLTEHMTQVQVMAPLNDESTVAKWLAKHNGRAGLHHMAWRV 
               
               
                 NO: 49 
                 DDIDAVSATLRERGVQLLYDEPKLGTGGNRINFMHPKSGKGVLIELTQ 
               
               
                   
                 YPKN 
               
               
                   
               
               
                 mmdA 
                 MAENNNLKLASTMEGRVEQLAEQRQVIEAGGGERRVEKQHSQGKQTAR 
               
               
                 SEQ ID 
                 ERLNNLLDPHSFDEVGAFRKHRTTLFGMDKAVVPADGVVTGRGTILGR 
               
               
                 NO: 50 
                 PVHAASQDFTVMGGSAGETQSTKVVETMEQALLTGTPFLFFYDSGGAR 
               
               
                   
                 IQEGIDSLSGYGKMFFANVKLSGVVPQIAIIAGPCAGGASYSPALTDF 
               
               
                   
                 IIMTKKAHMFITGPQVIKSVTGEDVTADELGGAEAHMAISGNIHFVAE 
               
               
                   
                 DDDAAELIAKKLLSFLPQNNTEEASFVNPNNDVSPNTELRDIVPIDGK 
               
               
                   
                 KGYDVRDVIAKIVDWGDYLEVKAGYATNLVTAFARVNGRSVGIVANQP 
               
               
                   
                 SVMSGCLDINASDKAAEFVNFCDSFNIPLVQLVDVPGFLPGVQQEYGG 
               
               
                   
                 IIRHGAKMLYAYSEATVPKITVVLRKAYGGSYLAMCNRDLGADAVYAW 
               
               
                   
                 PSAEIAVMGAEGAANVIFRKEIKAADDPDAMRAEKIEEYQNAFNTPYV 
               
               
                   
                 AAARGQVDDVIDPADTRRKIASALEMYATKRQTRPAKKHGNFPC 
               
               
                   
               
               
                 PFREUD_188870 
                 MSPREIEVSEPREVGITELVLRDAHQSLMATRMAMEDMVGACADIDAA 
               
               
                 SEQ ID 
                 GYWSVECWGGATYDSCIRFLNEDPWERLRTFRKLMPNSRLQMLLRGQN 
               
               
                 NO: 51 
                 LLGYRHYNDEVVDRFVDKSAENGMDVFRVFDAMNDPRNMAHAMAAVKK 
               
               
                   
                 AGKHAQGTICYTISPVHTVEGYVKLAGQLLDMGADSIALKDMAALLKP 
               
               
                   
                 QPAYDIIKAIKDTYGQKTQINLHCHSTTGVTEVSLMKAIEAGVDVVDT 
               
               
                   
                 AISSMSLGPGHNPTESVAEMLEGTGYTTNLDYDRLHKIRDHFKAIRPK 
               
               
                   
                 YKKFESKTLVDTSIFKSQIPGGMLSNMESQLRAQGAEDKMDEVMAEVP 
               
               
                   
                 RVRKAAGFPPLVTPSSQIVGTQAVFNVMMGEYKRMTGEFADIMLGYYG 
               
               
                   
                 ASPADRDPKVVKLAEEQSGKKPITQRPADLLPPEWEEQSKEAAALKGF 
               
               
                   
                 NGTDEDVLTYALFPQVAPVFFEHRAEGPHSVALTDAQLKAEAEGDEKS 
               
               
                   
                 LAVAGPVTYNVNVGGTVREVTVQQA 
               
               
                   
               
               
                 Bccp 
                 MKLKVTVNGTAYDVDVDVDKSHENPMGTILFGGGTGGAPAPRAAGGAG 
               
               
                 SEQ ID 
                 AGKAGEGEIPAPLAGTVSKILVKEGDTVKAGQTVLVLEAMKMETEINA 
               
               
                 NO: 52 
                 PTDGKVEKVLVKERDAVQGGQGLIKIG 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the genetically engineered bacteria encode one or more polypeptide sequences of Table 5 (SEQ ID NO: 27-SEQ ID NO: 52) 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 5 (SEQ ID NO: 27-SEQ ID NO: 52) or a functional fragment thereof. 
     In one embodiment, the bacterial cell comprises a heterologous propionate gene cassette. In some embodiments, the disclosure provides a bacterial cell that comprises a 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 liver disease, such as nonalcoholic steatohepatitis (NASH). 
     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, Gln, 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, 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: 1. In another embodiment, the pct gene has at least about 85% identity with SEQ ID NO: 1. In one embodiment, the pct gene has at least about 90% identity with SEQ ID NO: 1. In one embodiment, the pct gene has at least about 95% identity with SEQ ID NO: 1. In another embodiment, the pct gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. Accordingly, in one embodiment, the pct 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 pct gene comprises the sequence of SEQ ID NO: 1. In yet another embodiment the pct gene consists of the sequence of SEQ ID NO: 1. 
     In one embodiment, the lcdA gene has at least about 80% identity with SEQ ID NO: 2. In another embodiment, the lcdA gene has at least about 85% identity with SEQ ID NO: 2. In one embodiment, the lcdA gene has at least about 90% identity with SEQ ID NO: 2. In one embodiment, the lcdA gene has at least about 95% identity with SEQ ID NO: 2. In another embodiment, the lcdA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2. Accordingly, in one embodiment, the lcdA 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 lcdA gene comprises the sequence of SEQ ID NO: 2. In yet another embodiment the lcdA gene consists of the sequence of SEQ ID NO: 2. 
     In one embodiment, the lcdB gene has at least about 80% identity with SEQ ID NO: 3. In another embodiment, the lcdB gene has at least about 85% identity with SEQ ID NO: 3. In one embodiment, the lcdB gene has at least about 90% identity with SEQ ID NO: 3. In one embodiment, the IcdB gene has at least about 95% identity with SEQ ID NO: 3. In another embodiment, the IcdB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3. Accordingly, in one embodiment, the IcdB 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 IcdB gene comprises the sequence of SEQ ID NO: 3. In yet another embodiment the IcdB gene consists of the sequence of SEQ ID NO: 3. 
     In one embodiment, the IcdC gene has at least about 80% identity with SEQ ID NO: 4. In another embodiment, the IcdC gene has at least about 85% identity with SEQ ID NO: 4. In one embodiment, the IcdC gene has at least about 90% identity with SEQ ID NO: 4. In one embodiment, the IcdC gene has at least about 95% identity with SEQ ID NO: 4. In another embodiment, the IcdC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. Accordingly, in one embodiment, the WcdA 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 IcdC gene comprises the sequence of SEQ ID NO: 4. In yet another embodiment the IcdC gene consists of the sequence of SEQ ID NO: 4. 
     In one embodiment, the etfA gene has at least about 80% identity with SEQ ID NO: 5. In another embodiment, the etfA gene has at least about 85% identity with SEQ ID NO: 5. In one embodiment, the etfA gene has at least about 90% identity with SEQ ID NO: 5. In one embodiment, the etfA gene has at least about 95% identity with SEQ ID NO: 5. In another embodiment, the etfA gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5. Accordingly, in one embodiment, the etfA 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 etfA gene comprises the sequence of SEQ ID NO: 5. In yet another embodiment the etfA gene consists of the sequence of SEQ ID NO: 5. 
     In one embodiment, the acrB gene has at least about 80% identity with SEQ ID NO: 6. In another embodiment, the acrB gene has at least about 85% identity with SEQ ID NO: 6. In one embodiment, the acrB gene has at least about 90% identity with SEQ ID NO: 6. In one embodiment, the acrB gene has at least about 95% identity with SEQ ID NO: 6. In another embodiment, the acrB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6. Accordingly, in one embodiment, the acrB 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 acrB gene comprises the sequence of SEQ ID NO: 6. In yet another embodiment the acrB gene consists of the sequence of SEQ ID NO: 6. 
     In one embodiment, the acrC gene has at least about 80% identity with SEQ ID NO: 7. In another embodiment, the acrC gene has at least about 85% identity with SEQ ID NO: 7. In one embodiment, the acrC gene has at least about 90% identity with SEQ ID NO: 7. In one embodiment, the acrC gene has at least about 95% identity with SEQ ID NO: 7. In another embodiment, the acrC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 7. Accordingly, in one embodiment, the acrC 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 acrC gene comprises the sequence of SEQ ID NO: 7. In yet another embodiment the acrC gene consists of the sequence of SEQ ID NO: 7. 
     In one embodiment, the thrA fbr  gene has at least about 80% identity with SEQ ID NO: 8. In another embodiment, the thrA fbr  gene has at least about 85% identity with SEQ ID NO: 8. In one embodiment, the thrA fbr  gene has at least about 90% identity with SEQ ID NO: 8. In one embodiment, the thrA fbr  gene has at least about 95% identity with SEQ ID NO: 8. In another embodiment, the thrA fbr  gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 8. Accordingly, in one embodiment, the thrA 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: 8. In another embodiment, the thrA fbr  gene comprises the sequence of SEQ ID NO: 8. In yet another embodiment the thrA fbr  gene consists of the sequence of SEQ ID NO: 8. 
     In one embodiment, the thrB gene has at least about 80% identity with SEQ ID NO: 9. In another embodiment, the thrB gene has at least about 85% identity with SEQ ID NO: 9. In one embodiment, the thrB gene has at least about 90% identity with SEQ ID NO: 9. In one embodiment, the thrB gene has at least about 95% identity with SEQ ID NO: 9. In another embodiment, the thrB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9. Accordingly, in one embodiment, the thrB 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 thrB gene comprises the sequence of SEQ ID NO: 9. In yet another embodiment the thrB gene consists of the sequence of SEQ ID NO: 9. 
     In one embodiment, the thrC gene has at least about 80% identity with SEQ ID NO: 10. In another embodiment, the thrC gene has at least about 85% identity with SEQ ID NO: 10. In one embodiment, the thrC gene has at least about 90% identity with SEQ ID NO: 10. In one embodiment, the thrC gene has at least about 95% identity with SEQ ID NO: 10. In another embodiment, the thrC gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. 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: 10. In another embodiment, the thrC gene comprises the sequence of SEQ ID NO: 10. In yet another embodiment the thrC gene consists of the sequence of SEQ ID NO: 10. 
     In one embodiment, the ilvA fbr  gene has at least about 80% identity with SEQ ID NO: 11. In another embodiment, the ilvA fbr  gene has at least about 85% identity with SEQ ID NO: 11. In one embodiment, the ilvA fbr  gene has at least about 90% identity with SEQ ID NO: 11. In one embodiment, the ilvA fbr  gene has at least about 95% identity with SEQ ID NO: 11. In another embodiment, the ilvA fbr  gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 11. 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: 11. In another embodiment, the ilvA fbr  gene comprises the sequence of SEQ ID NO: 11. In yet another embodiment the ilvA fbr  gene consists of the sequence of SEQ ID NO: 11. 
     In one embodiment, the aceE gene has at least about 80% identity with SEQ ID NO: 12. In another embodiment, the aceE gene has at least about 85% identity with SEQ ID NO: 12. In one embodiment, the aceE gene has at least about 90% identity with SEQ ID NO: 12. In one embodiment, the aceE gene has at least about 95% identity with SEQ ID NO: 12. In another embodiment, the aceE gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 12. 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: 12. In another embodiment, the aceE gene comprises the sequence of SEQ ID NO: 12. In yet another embodiment the aceE gene consists of the sequence of SEQ ID NO: 12. 
     In one embodiment, the aceF gene has at least about 80% identity with SEQ ID NO: 13. In another embodiment, the aceF gene has at least about 85% identity with SEQ ID NO: 13. In one embodiment, the aceF gene has at least about 90% identity with SEQ ID NO: 13. In one embodiment, the aceF gene has at least about 95% identity with SEQ ID NO: 13. In another embodiment, the aceF gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 13. 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: 13. In another embodiment, the aceF gene comprises the sequence of SEQ ID NO: 13. In yet another embodiment the aceF gene consists of the sequence of SEQ ID NO: 13. 
     In one embodiment, the lpd gene has at least about 80% identity with SEQ ID NO: 14. In another embodiment, the lpd gene has at least about 85% identity with SEQ ID NO: 14. In one embodiment, the lpd gene has at least about 90% identity with SEQ ID NO: 14. In one embodiment, the lpd gene has at least about 95% identity with SEQ ID NO: 14. In another embodiment, the lpd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 14. 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: 14. In another embodiment, the lpd gene comprises the sequence of SEQ ID NO: 14. In yet another embodiment the lpd gene consists of the sequence of SEQ ID NO: 14. 
     In one embodiment, the tesB gene has at least about 80% identity with SEQ ID NO: 15. In another embodiment, the tesB gene has at least about 85% identity with SEQ ID NO: 15. In one embodiment, the tesB gene has at least about 90% identity with SEQ ID NO: 15. In one embodiment, the tesB gene has at least about 95% identity with SEQ ID NO: 15. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15. 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: 15. In another embodiment, the tesB gene comprises the sequence of SEQ ID NO: 15. In yet another embodiment the tesB gene consists of the sequence of SEQ ID NO: 15. 
     In one embodiment, the acuI gene has at least about 80% identity with SEQ ID NO: 16. In another embodiment, the acuI gene has at least about 85% identity with SEQ ID NO: 16. In one embodiment, the acuI gene has at least about 90% identity with SEQ ID NO: 16. In one embodiment, the acuI gene has at least about 95% identity with SEQ ID NO: 16. In another embodiment, the acuI gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 16. 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: 16. In another embodiment, the acuI gene comprises the sequence of SEQ ID NO: 16. In yet another embodiment the acuI gene consists of the sequence of SEQ ID NO: 16. 
     In one embodiment, the sbm gene has at least about 80% identity with SEQ ID NO: 17. In another embodiment, the sbm gene has at least about 85% identity with SEQ ID NO: 17. In one embodiment, the sbm gene has at least about 90% identity with SEQ ID NO: 17. In one embodiment, the sbm gene has at least about 95% identity with SEQ ID NO: 17. In another embodiment, the sbm gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 17.0. 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: 17. In another embodiment, the sbm gene comprises the sequence of SEQ ID NO: 17. In yet another embodiment the sbm gene consists of the sequence of SEQ ID NO: 17. 
     In one embodiment, the ygfD gene has at least about 80% identity with SEQ ID NO: 18. In another embodiment, the ygfD gene has at least about 85% identity with SEQ ID NO: 18. In one embodiment, the ygfD gene has at least about 90% identity with SEQ ID NO: 18. In one embodiment, the ygfD gene has at least about 95% identity with SEQ ID NO: 18. In another embodiment, the ygfD gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 18. 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: 18. In another embodiment, the ygfD gene comprises the sequence of SEQ ID NO: 18. In yet another embodiment the ygfD gene consists of the sequence of SEQ ID NO: 18. 
     In one embodiment, the ygfG gene has at least about 80% identity with SEQ ID NO: 19. In another embodiment, the ygfG gene has at least about 85% identity with SEQ ID NO: 19. In one embodiment, the ygfG gene has at least about 90% identity with SEQ ID NO: 19. In one embodiment, the ygfG gene has at least about 95% identity with SEQ ID NO: 19. In another embodiment, the ygfG gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 19. 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: 19. In another embodiment, the ygfG gene comprises the sequence of SEQ ID NO: 19. In yet another embodiment the ygfG gene consists of the sequence of SEQ ID NO: 19. 
     In one embodiment, the ygfH gene has at least about 80% identity with SEQ ID NO: 20. In another embodiment, the ygfH gene has at least about 85% identity with SEQ ID NO: 20. In one embodiment, the ygfH gene has at least about 90% identity with SEQ ID NO: 20. In one embodiment, the ygfH gene has at least about 95% identity with SEQ ID NO: 20. In another embodiment, the ygfH gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 20. 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: 20. In another embodiment, the ygfH gene comprises the sequence of SEQ ID NO: 20. In yet another embodiment the ygfH gene consists of the sequence of SEQ ID NO: 20. 
     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: 27 through SEQ ID NO: 52. 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: 27 through SEQ ID NO: 52. 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: 27 through SEQ ID NO: 52. 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: 27 through SEQ ID NO: 52. 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: 27 through SEQ ID NO: 52. 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: 27 through SEQ ID NO: 52. 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: 27 through SEQ ID NO: 52. 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: 27 through SEQ ID NO: 52. 
     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. 
     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 ameliorates metabolic disease (Lin et al., 2012). 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 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 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. 
     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 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: bcd2, 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 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. 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 6 depicts the nucleic acid sequences of exemplary genes in exemplary butyrate biosynthesis gene cassettes. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Exemplary Butyrate Cassette Sequences 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 bcd2 
                 ATGGATTTAAATTCTAAAAAATATCAGATGCTTAAAGAGCTATA 
               
               
                 SEQ ID NO: 53 
                 TGTAAGCTTCGCTGAAAATGAAGTTAAACCTTTAGCAACAGAAC 
               
               
                   
                 TTGATGAAGAAGAAAGATTTCCTTATGAAACAGTGGAAAAAATG 
               
               
                   
                 GCAAAAGCAGGAATGATGGGTATACCATATCCAAAAGAATATGG 
               
               
                   
                 TGGAGAAGGTGGAGACACTGTAGGATATATAATGGCAGTTGAAG 
               
               
                   
                 AATTGTCTAGAGTTTGTGGTACTACAGGAGTTATATTATCAGCT 
               
               
                   
                 CATACATCTCTTGGCTCATGGCCTATATATCAATATGGTAATGA 
               
               
                   
                 AGAACAAAAACAAAAATTCTTAAGACCACTAGCAAGTGGAGAAA 
               
               
                   
                 AATTAGGAGCATTTGGTCTTACTGAGCCTAATGCTGGTACAGAT 
               
               
                   
                 GCGTCTGGCCAACAAACAACTGCTGTTTTAGACGGGGATGAATA 
               
               
                   
                 CATACTTAATGGCTCAAAAATATTTATAACAAACGCAATAGCTG 
               
               
                   
                 GTGACATATATGTAGTAATGGCAATGACTGATAAATCTAAGGGG 
               
               
                   
                 AACAAAGGAATATCAGCATTTATAGTTGAAAAAGGAACTCCTGG 
               
               
                   
                 GTTTAGCTTTGGAGTTAAAGAAAAGAAAATGGGTATAAGAGGTT 
               
               
                   
                 CAGCTACGAGTGAATTAATATTTGAGGATTGCAGAATACCTAAA 
               
               
                   
                 GAAAATTTACTTGGAAAAGAAGGTCAAGGATTTAAGATAGCAAT 
               
               
                   
                 GTCTACTCTTGATGGTGGTAGAATTGGTATAGCTGCACAAGCTT 
               
               
                   
                 TAGGTTTAGCACAAGGTGCTCTTGATGAAACTGTTAAATATGTA 
               
               
                   
                 AAAGAAAGAGTACAATTTGGTAGACCATTATCAAAATTCCAAAA 
               
               
                   
                 TACACAATTCCAATTAGCTGATATGGAAGTTAAGGTACAAGCGG 
               
               
                   
                 CTAGACACCTTGTATATCAAGCAGCTATAAATAAAGACTTAGGA 
               
               
                   
                 AAACCTTATGGAGTAGAAGCAGCAATGGCAAAATTATTTGCAGC 
               
               
                   
                 TGAAACAGCTATGGAAGTTACTACAAAAGCTGTACAACTTCATG 
               
               
                   
                 GAGGATATGGATACACTCGTGACTATCCAGTAGAAAGAATGATG 
               
               
                   
                 AGAGATGCTAAGATAACTGAAATATATGAAGGAACTAGTGAAGT 
               
               
                   
                 TCAAAGAATGGTTATTTCAGGAAAACTATTAAAATAG 
               
               
                   
               
               
                 etfB3 
                 ATGAATATAGTCGTTTGTATAAAACAAGTTCCAGATACAACAGA 
               
               
                 SEQ ID NO: 54 
                 AGTTAAACTAGATCCTAATACAGGTACTTTAATTAGAGATGGAG 
               
               
                   
                 TACCAAGTATAATAAACCCTGATGATAAAGCAGGTTTAGAAGAA 
               
               
                   
                 GCTATAAAATTAAAAGAAGAAATGGGTGCTCATGTAACTGTTAT 
               
               
                   
                 AACAATGGGACCTCCTCAAGCAGATATGGCTTTAAAAGAAGCTT 
               
               
                   
                 TAGCAATGGGTGCAGATAGAGGTATATTATTAACAGATAGAGCA 
               
               
                   
                 TTTGCGGGTGCTGATACTTGGGCAACTTCATCAGCATTAGCAGG 
               
               
                   
                 AGCATTAAAAAATATAGATTTTGATATTATAATAGCTGGAAGAC 
               
               
                   
                 AGGCGATAGATGGAGATACTGCACAAGTTGGACCTCAAATAGCT 
               
               
                   
                 GAACATTTAAATCTTCCATCAATAACATATGCTGAAGAAATAAA 
               
               
                   
                 AACTGAAGGTGAATATGTATTAGTAAAAAGACAATTTGAAGATT 
               
               
                   
                 GTTGCCATGACTTAAAAGTTAAAATGCCATGCCTTATAACAACT 
               
               
                   
                 CTTAAAGATATGAACACACCAAGATACATGAAAGTTGGAAGAAT 
               
               
                   
                 ATATGATGCTTTCGAAAATGATGTAGTAGAAACATGGACTGTAA 
               
               
                   
                 AAGATATAGAAGTTGACCCTTCTAATTTAGGTCTTAAAGGTTCT 
               
               
                   
                 CCAACTAGTGTATTTAAATCATTTACAAAATCAGTTAAACCAGC 
               
               
                   
                 TGGTACAATATACAATGAAGATGCGAAAACATCAGCTGGAATTA 
               
               
                   
                 TCATAGATAAATTAAAAGAGAAGTATATCATATAA 
               
               
                   
               
               
                 etfA3 
                 ATGGGTAACGTTTTAGTAGTAATAGAACAAAGAGAAAATGTAAT 
               
               
                 SEQ ID NO: 55 
                 TCAAACTGTTTCTTTAGAATTACTAGGAAAGGCTACAGAAATAG 
               
               
                   
                 CAAAAGATTATGATACAAAAGTTTCTGCATTACTTTTAGGTAGT 
               
               
                   
                 AAGGTAGAAGGTTTAATAGATACATTAGCACACTATGGTGCAGA 
               
               
                   
                 TGAGGTAATAGTAGTAGATGATGAAGCTTTAGCAGTGTATACAA 
               
               
                   
                 CTGAACCATATACAAAAGCAGCTTATGAAGCAATAAAAGCAGCT 
               
               
                   
                 GACCCTATAGTTGTATTATTTGGTGCAACTTCAATAGGTAGAGA 
               
               
                   
                 TTTAGCGCCTAGAGTTTCTGCTAGAATACATACAGGTCTTACTG 
               
               
                   
                 CTGACTGTACAGGTCTTGCAGTAGCTGAAGATACAAAATTATTA 
               
               
                   
                 TTAATGACAAGACCTGCCTTTGGTGGAAATATAATGGCAACAAT 
               
               
                   
                 AGTTTGTAAAGATTTCAGACCTCAAATGTCTACAGTTAGACCAG 
               
               
                   
                 GGGTTATGAAGAAAAATGAACCTGATGAAACTAAAGAAGCTGTA 
               
               
                   
                 ATTAACCGTTTCAAGGTAGAATTTAATGATGCTGATAAATTAGT 
               
               
                   
                 TCAAGTTGTACAAGTAATAAAAGAAGCTAAAAAACAAGTTAAAA 
               
               
                   
                 TAGAAGATGCTAAGATATTAGTTTCTGCTGGACGTGGAATGGGT 
               
               
                   
                 GGAAAAGAAAACTTAGACATACTTTATGAATTAGCTGAAATTAT 
               
               
                   
                 AGGTGGAGAAGTTTCTGGTTCTCGTGCCACTATAGATGCAGGTT 
               
               
                   
                 GGTTAGATAAAGCAAGACAAGTTGGTCAAACTGGTAAAACTGTA 
               
               
                   
                 AGACCAGACCTTTATATAGCATGTGGTATATCTGGAGCAATACA 
               
               
                   
                 ACATATAGCTGGTATGGAAGATGCTGAGTTTATAGTTGCTATAA 
               
               
                   
                 ATAAAAATCCAGAAGCTCCAATATTTAAATATGCTGATGTTGGT 
               
               
                   
                 ATAGTTGGAGATGTTCATAAAGTGCTTCCAGAACTTATCAGTCA 
               
               
                   
                 GTTAAGTGTTGCAAAAGAAAAAGGTGAAGTTTTAGCTAACTAA 
               
               
                   
               
               
                 thiA1 
                 ATGAGAGAAGTAGTAATTGCCAGTGCAGCTAGAACAGCAGTAGG 
               
               
                 SEQ ID NO: 56 
                 AAGTTTTGGAGGAGCATTTAAATCAGTTTCAGCGGTAGAGTTAG 
               
               
                   
                 GGGTAACAGCAGCTAAAGAAGCTATAAAAAGAGCTAACATAACT 
               
               
                   
                 CCAGATATGATAGATGAATCTCTTTTAGGGGGAGTACTTACAGC 
               
               
                   
                 AGGTCTTGGACAAAATATAGCAAGACAAATAGCATTAGGAGCAG 
               
               
                   
                 GAATACCAGTAGAAAAACCAGCTATGACTATAAATATAGTTTGT 
               
               
                   
                 GGTTCTGGATTAAGATCTGTTTCAATGGCATCTCAACTTATAGC 
               
               
                   
                 ATTAGGTGATGCTGATATAATGTTAGTTGGTGGAGCTGAAAACA 
               
               
                   
                 TGAGTATGTCTCCTTATTTAGTACCAAGTGCGAGATATGGTGCA 
               
               
                   
                 AGAATGGGTGATGCTGCTTTTGTTGATTCAATGATAAAAGATGG 
               
               
                   
                 ATTATCAGACATATTTAATAACTATCACATGGGTATTACTGCTG 
               
               
                   
                 AAAACATAGCAGAGCAATGGAATATAACTAGAGAAGAACAAGAT 
               
               
                   
                 GAATTAGCTCTTGCAAGTCAAAATAAAGCTGAAAAAGCTCAAGC 
               
               
                   
                 TGAAGGAAAATTTGATGAAGAAATAGTTCCTGTTGTTATAAAAG 
               
               
                   
                 GAAGAAAAGGTGACACTGTAGTAGATAAAGATGAATATATTAAG 
               
               
                   
                 CCTGGCACTACAATGGAGAAACTTGCTAAGTTAAGACCTGCATT 
               
               
                   
                 TAAAAAAGATGGAACAGTTACTGCTGGTAATGCATCAGGAATAA 
               
               
                   
                 ATGATGGTGCTGCTATGTTAGTAGTAATGGCTAAAGAAAAAGCT 
               
               
                   
                 GAAGAACTAGGAATAGAGCCTCTTGCAACTATAGTTTCTTATGG 
               
               
                   
                 AACAGCTGGTGTTGACCCTAAAATAATGGGATATGGACCAGTTC 
               
               
                   
                 CAGCAACTAAAAAAGCTTTAGAAGCTGCTAATATGACTATTGAA 
               
               
                   
                 GATATAGATTTAGTTGAAGCTAATGAGGCATTTGCTGCCCAATC 
               
               
                   
                 TGTAGCTGTAATAAGAGACTTAAATATAGATATGAATAAAGTTA 
               
               
                   
                 ATGTTAATGGTGGAGCAATAGCTATAGGACATCCAATAGGATGC 
               
               
                   
                 TCAGGAGCAAGAATACTTACTACACTTTTATATGAAATGAAGAG 
               
               
                   
                 AAGAGATGCTAAAACTGGTCTTGCTACACTTTGTATAGGCGGTG 
               
               
                   
                 GAATGGGAACTACTTTAATAGTTAAGAGATAG 
               
               
                   
               
               
                 hbd 
                 ATGAAATTAGCTGTAATAGGTAGTGGAACTATGGGAAGTGGTAT 
               
               
                 SEQ ID NO: 57 
                 TGTACAAACTTTTGCAAGTTGTGGACATGATGTATGTTTAAAGA 
               
               
                   
                 GTAGAACTCAAGGTGCTATAGATAAATGTTTAGCTTTATTAGAT 
               
               
                   
                 AAAAATTTAACTAAGTTAGTTACTAAGGGAAAAATGGATGAAGC 
               
               
                   
                 TACAAAAGCAGAAATATTAAGTCATGTTAGTTCAACTACTAATT 
               
               
                   
                 ATGAAGATTTAAAAGATATGGATTTAATAATAGAAGCATCTGTA 
               
               
                   
                 GAAGACATGAATATAAAGAAAGATGTTTTCAAGTTACTAGATGA 
               
               
                   
                 ATTATGTAAAGAAGATACTATCTTGGCAACAAATACTTCATCAT 
               
               
                   
                 TATCTATAACAGAAATAGCTTCTTCTACTAAGCGCCCAGATAAA 
               
               
                   
                 GTTATAGGAATGCATTTCTTTAATCCAGTTCCTATGATGAAATT 
               
               
                   
                 AGTTGAAGTTATAAGTGGTCAGTTAACATCAAAAGTTACTTTTG 
               
               
                   
                 ATACAGTATTTGAATTATCTAAGAGTATCAATAAAGTACCAGTA 
               
               
                   
                 GATGTATCTGAATCTCCTGGATTTGTAGTAAATAGAATACTTAT 
               
               
                   
                 ACCTATGATAAATGAAGCTGTTGGTATATATGCAGATGGTGTTG 
               
               
                   
                 CAAGTAAAGAAGAAATAGATGAAGCTATGAAATTAGGAGCAAAC 
               
               
                   
                 CATCCAATGGGACCACTAGCATTAGGTGATTTAATCGGATTAGA 
               
               
                   
                 TGTTGTTTTAGCTATAATGAACGTTTTATATACTGAATTTGGAG 
               
               
                   
                 ATACTAAATATAGACCTCATCCACTTTTAGCTAAAATGGTTAGA 
               
               
                   
                 GCTAATCAATTAGGAAGAAAAACTAAGATAGGATTCTATGATTA 
               
               
                   
                 TAATAAATAA 
               
               
                   
               
               
                 crt2 
                 ATGAGTACAAGTGATGTTAAAGTTTATGAGAATGTAGCTGTTGA 
               
               
                 SEQ ID NO: 58 
                 AGTAGATGGAAATATATGTACAGTGAAAATGAATAGACCTAAAG 
               
               
                   
                 CCCTTAATGCAATAAATTCAAAGACTTTAGAAGAACTTTATGAA 
               
               
                   
                 GTATTTGTAGATATTAATAATGATGAAACTATTGATGTTGTAAT 
               
               
                   
                 ATTGACAGGGGAAGGAAAGGCATTTGTAGCTGGAGCAGATATTG 
               
               
                   
                 CATACATGAAAGATTTAGATGCTGTAGCTGCTAAAGATTTTAGT 
               
               
                   
                 ATCTTAGGAGCAAAAGCTTTTGGAGAAATAGAAAATAGTAAAAA 
               
               
                   
                 AGTAGTGATAGCTGCTGTAAACGGATTTGCTTTAGGTGGAGGAT 
               
               
                   
                 GTGAACTTGCAATGGCATGTGATATAAGAATTGCATCTGCTAAA 
               
               
                   
                 GCTAAATTTGGTCAGCCAGAAGTAACTCTTGGAATAACTCCAGG 
               
               
                   
                 ATATGGAGGAACTCAAAGGCTTACAAGATTGGTTGGAATGGCAA 
               
               
                   
                 AAGCAAAAGAATTAATCTTTACAGGTCAAGTTATAAAAGCTGAT 
               
               
                   
                 GAAGCTGAAAAAATAGGGCTAGTAAATAGAGTCGTTGAGCCAGA 
               
               
                   
                 CATTTTAATAGAAGAAGTTGAGAAATTAGCTAAGATAATAGCTA 
               
               
                   
                 AAAATGCTCAGCTTGCAGTTAGATACTCTAAAGAAGCAATACAA 
               
               
                   
                 CTTGGTGCTCAAACTGATATAAATACTGGAATAGATATAGAATC 
               
               
                   
                 TAATTTATTTGGTCTTTGTTTTTCAACTAAAGACCAAAAAGAAG 
               
               
                   
                 GAATGTCAGCTTTCGTTGAAAAGAGAGAAGCTAACTTTATAAAA 
               
               
                   
                 GGGTAA 
               
               
                   
               
               
                 pbt 
                 ATGAGAAGTTTTGAAGAAGTAATTAAGTTTGCAAAAGAAAGAGG 
               
               
                 SEQ ID NO: 59 
                 ACCTAAAACTATATCAGTAGCATGTTGCCAAGATAAAGAAGTTT 
               
               
                   
                 TAATGGCAGTTGAAATGGCTAGAAAAGAAAAAATAGCAAATGCC 
               
               
                   
                 ATTTTAGTAGGAGATATAGAAAAGACTAAAGAAATTGCAAAAAG 
               
               
                   
                 CATAGACATGGATATCGAAAATTATGAACTGATAGATATAAAAG 
               
               
                   
                 ATTTAGCAGAAGCATCTCTAAAATCTGTTGAATTAGTTTCACAA 
               
               
                   
                 GGAAAAGCCGACATGGTAATGAAAGGCTTAGTAGACACATCAAT 
               
               
                   
                 AATACTAAAAGCAGTTTTAAATAAAGAAGTAGGTCTTAGAACTG 
               
               
                   
                 GAAATGTATTAAGTCACGTAGCAGTATTTGATGTAGAGGGATAT 
               
               
                   
                 GATAGATTATTTTTCGTAACTGACGCAGCTATGAACTTAGCTCC 
               
               
                   
                 TGATACAAATACTAAAAAGCAAATCATAGAAAATGCTTGCACAG 
               
               
                   
                 TAGCACATTCATTAGATATAAGTGAACCAAAAGTTGCTGCAATA 
               
               
                   
                 TGCGCAAAAGAAAAAGTAAATCCAAAAATGAAAGATACAGTTGA 
               
               
                   
                 AGCTAAAGAACTAGAAGAAATGTATGAAAGAGGAGAAATCAAAG 
               
               
                   
                 GTTGTATGGTTGGTGGGCCTTTTGCAATTGATAATGCAGTATCT 
               
               
                   
                 TTAGAAGCAGCTAAACATAAAGGTATAAATCATCCTGTAGCAGG 
               
               
                   
                 ACGAGCTGATATATTATTAGCCCCAGATATTGAAGGTGGTAACA 
               
               
                   
                 TATTATATAAAGCTTTGGTATTCTTCTCAAAATCAAAAAATGCA 
               
               
                   
                 GGAGTTATAGTTGGGGCTAAAGCACCAATAATATTAACTTCTAG 
               
               
                   
                 AGCAGACAGTGAAGAAACTAAACTAAACTCAATAGCTTTAGGTG 
               
               
                   
                 TTTTAATGGCAGCAAAGGCATAA 
               
               
                   
               
               
                 buk 
                 ATGAGCAAAATATTTAAAATCTTAACAATAAATCCTGGTTCGAC 
               
               
                 SEQ ID NO: 60 
                 ATCAACTAAAATAGCTGTATTTGATAATGAGGATTTAGTATTTG 
               
               
                   
                 AAAAAACTTTAAGACATTCTTCAGAAGAAATAGGAAAATATGAG 
               
               
                   
                 AAGGTGTCTGACCAATTTGAATTTCGTAAACAAGTAATAGAAGA 
               
               
                   
                 AGCTCTAAAAGAAGGTGGAGTAAAAACATCTGAATTAGATGCTG 
               
               
                   
                 TAGTAGGTAGAGGAGGACTTCTTAAACCTATAAAAGGTGGTACT 
               
               
                   
                 TATTCAGTAAGTGCTGCTATGATTGAAGATTTAAAAGTGGGAGT 
               
               
                   
                 TTTAGGAGAACACGCTTCAAACCTAGGTGGAATAATAGCAAAAC 
               
               
                   
                 AAATAGGTGAAGAAGTAAATGTTCCTTCATACATAGTAGACCCT 
               
               
                   
                 GTTGTTGTAGATGAATTAGAAGATGTTGCTAGAATTTCTGGTAT 
               
               
                   
                 GCCTGAAATAAGTAGAGCAAGTGTAGTACATGCTTTAAATCAAA 
               
               
                   
                 AGGCAATAGCAAGAAGATATGCTAGAGAAATAAACAAGAAATAT 
               
               
                   
                 GAAGATATAAATCTTATAGTTGCACACATGGGTGGAGGAGTTTC 
               
               
                   
                 TGTTGGAGCTCATAAAAATGGTAAAATAGTAGATGTTGCAAACG 
               
               
                   
                 CATTAGATGGAGAAGGACCTTTCTCTCCAGAAAGAAGTGGTGGA 
               
               
                   
                 CTACCAGTAGGTGCATTAGTAAAAATGTGCTTTAGTGGAAAATA 
               
               
                   
                 TACTCAAGATGAAATTAAAAAGAAAATAAAAGGTAATGGCGGAC 
               
               
                   
                 TAGTTGCATACTTAAACACTAATGATGCTAGAGAAGTTGAAGAA 
               
               
                   
                 AGAATTGAAGCTGGTGATGAAAAAGCTAAATTAGTATATGAAGC 
               
               
                   
                 TATGGCATATCAAATCTCTAAAGAAATAGGAGCTAGTGCTGCAG 
               
               
                   
                 TTCTTAAGGGAGATGTAAAAGCAATATTATTAACTGGTGGAATC 
               
               
                   
                 GCATATTCAAAAATGTTTACAGAAATGATTGCAGATAGAGTTAA 
               
               
                   
                 ATTTATAGCAGATGTAAAAGTTTATCCAGGTGAAGATGAAATGA 
               
               
                   
                 TTGCATTAGCTCAAGGTGGACTTAGAGTTTTAACTGGTGAAGAA 
               
               
                   
                 GAGGCTCAAGTTTATGATAACTAA 
               
               
                   
               
               
                 ter 
                 ATGATCGTAAAACCTATGGTACGCAACAATATCTGCCTGAACGC 
               
               
                 SEQ ID NO: 61 
                 CCATCCTCAGGGCTGCAAGAAGGGAGTGGAAGATCAGATTGAAT 
               
               
                   
                 ATACCAAGAAACGCATTACCGCAGAAGTCAAAGCTGGCGCAAAA 
               
               
                   
                 GCTCCAAAAAACGTTCTGGTGCTTGGCTGCTCAAATGGTTACGG 
               
               
                   
                 CCTGGCGAGCCGCATTACTGCTGCGTTCGGATACGGGGCTGCGA 
               
               
                   
                 CCATCGGCGTGTCCTTTGAAAAAGCGGGTTCAGAAACCAAATAT 
               
               
                   
                 GGTACACCGGGATGGTACAATAATTTGGCATTTGATGAAGCGGC 
               
               
                   
                 AAAACGCGAGGGTCTTTATAGCGTGACGATCGACGGCGATGCGT 
               
               
                   
                 TTTCAGACGAGATCAAGGCCCAGGTAATTGAGGAAGCCAAAAAA 
               
               
                   
                 AAAGGTATCAAATTTGATCTGATCGTATACAGCTTGGCCAGCCC 
               
               
                   
                 AGTACGTACTGATCCTGATACAGGTATCATGCACAAAAGCGTTT 
               
               
                   
                 TGAAACCCTTTGGAAAAACGTTCACAGGCAAAACAGTAGATCCG 
               
               
                   
                 TTTACTGGCGAGCTGAAGGAAATCTCCGCGGAACCAGCAAATGA 
               
               
                   
                 CGAGGAAGCAGCCGCCACTGTTAAAGTTATGGGGGGTGAAGATT 
               
               
                   
                 GGGAACGTTGGATTAAGCAGCTGTCGAAGGAAGGCCTCTTAGAA 
               
               
                   
                 GAAGGCTGTATTACCTTGGCCTATAGTTATATTGGCCCTGAAGC 
               
               
                   
                 TACCCAAGCTTTGTACCGTAAAGGCACAATCGGCAAGGCCAAAG 
               
               
                   
                 AACACCTGGAGGCCACAGCACACCGTCTCAACAAAGAGAACCCG 
               
               
                   
                 TCAATCCGTGCCTTCGTGAGCGTGAATAAAGGCCTGGTAACCCG 
               
               
                   
                 CGCAAGCGCCGTAATCCCGGTAATCCCTCTGTATCTCGCCAGCT 
               
               
                   
                 TGTTCAAAGTAATGAAAGAGAAGGGCAATCATGAAGGTTGTATT 
               
               
                   
                 GAACAGATCACGCGTCTGTACGCCGAGCGCCTGTACCGTAAAGA 
               
               
                   
                 TGGTACAATTCCAGTTGATGAGGAAAATCGCATTCGCATTGATG 
               
               
                   
                 ATTGGGAGTTAGAAGAAGACGTCCAGAAAGCGGTATCCGCGTTG 
               
               
                   
                 ATGGAGAAAGTCACGGGTGAAAACGCAGAATCTCTCACTGACTT 
               
               
                   
                 AGCGGGGTACCGCCATGATTTCTTAGCTAGTAACGGCTTTGATG 
               
               
                   
                 TAGAAGGTATTAATTATGAAGCGGAAGTTGAACGCTTCGACCGT 
               
               
                   
                 ATCTGA 
               
               
                   
               
               
                 tesB 
                 ATGAGTCAGGCGCTAAAAAATTTACTGACATTGTTAAATCTGGA 
               
               
                 SEQ ID NO: 15 
                 AAAAATTGAGGAAGGACTCTTTCGCGGCCAGAGTGAAGATTTAG 
               
               
                   
                 GTTTACGCCAGGTGTTTGGCGGCCAGGTCGTGGGTCAGGCCTTG 
               
               
                   
                 TATGCTGCAAAAGAGACCGTCCCTGAAGAGCGGCTGGTACATTC 
               
               
                   
                 GTTTCACAGCTACTTTCTTCGCCCTGGCGATAGTAAGAAGCCGA 
               
               
                   
                 TTATTTATGATGTCGAAACGCTGCGTGACGGTAACAGCTTCAGC 
               
               
                   
                 GCCCGCCGGGTTGCTGCTATTCAAAACGGCAAACCGATTTTTTA 
               
               
                   
                 TATGACTGCCTCTTTCCAGGCACCAGAAGCGGGTTTCGAACATC 
               
               
                   
                 AAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCCTCCCTTCG 
               
               
                   
                 GAAACGCAAATCGCCCAATCGCTGGCGCACCTGCTGCCGCCAGT 
               
               
                   
                 GCTGAAAGATAAATTCATCTGCGATCGTCCGCTGGAAGTCCGTC 
               
               
                   
                 CGGTGGAGTTTCATAACCCACTGAAAGGTCACGTCGCAGAACCA 
               
               
                   
                 CATCGTCAGGTGTGGATCCGCGCAAATGGTAGCGTGCCGGATGA 
               
               
                   
                 CCTGCGCGTTCATCAGTATCTGCTCGGTTACGCTTCTGATCTTA 
               
               
                   
                 ACTTCCTGCCGGTAGCTCTACAGCCGCACGGCATCGGTTTTCTC 
               
               
                   
                 GAACCGGGGATTCAGATTGCCACCATTGACCATTCCATGTGGTT 
               
               
                   
                 CCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATAGCGTGG 
               
               
                   
                 AGAGCACCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGAG 
               
               
                   
                 TTTTATACCCAAGACGGCGTACTGGTTGCCTCGACCGTTCAGGA 
               
               
                   
                 AGGGGTGATGCGTAATCACAATTAA 
               
               
                   
               
            
           
         
       
     
     Exemplary polypeptide sequences for the production of butyrate by the genetically engineered bacteria are provided in Table 7. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Exemplary Polypeptide Sequences for 
               
               
                 Butyrate Production 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 Bcd2 
                 MDLNSKKYQMLKELYVSFAENEVKPLATELDEEERF 
               
               
                 SEQ ID NO: 62 
                 PYETVEKMAKAGMMGIPYPKEYGGEGGDTVGYIMAV 
               
               
                   
                 EELSRVCGTTGVILSAHTSLGSWPIYQYGNEEQKQK 
               
               
                   
                 FLRPLASGEKLGAFGLTEPNAGTDASGQQTTAVLDG 
               
               
                   
                 DEYILNGSKIFITNAIAGDIYVVMAMTDKSKGNKGI 
               
               
                   
                 SAFIVEKGTPGFSFGVKEKKMGIRGSATSELIFEDC 
               
               
                   
                 RIPKENLLGKEGQGFKIAMSTLDGGRIGIAAQALGL 
               
               
                   
                 AQGALDETVKYVKERVQFGRPLSKFQNTQFQLADME 
               
               
                   
                 VKVQAARHLVYQAAINKDLGKPYGVEAAMAKLFAAE 
               
               
                   
                 TAMEVTTKAVQLHGGYGYTRDYPVERMMRDAKITEI 
               
               
                   
                 YEGTSEVQRMVISGKLLK 
               
               
                   
               
               
                 etfB3 
                 MNIVVCIKQVPDTTEVKLDPNTGTLIRDGVPSIINP 
               
               
                 SEQ ID NO: 63 
                 DDKAGLEEAIKLKEEMGAHVTVITMGPPQADMALKE 
               
               
                   
                 ALAMGADRGILLTDRAFAGADTWATSSALAGALKNI 
               
               
                   
                 DFDIIIAGRQAIDGDTAQVGPQIAEHLNLPSITYAE 
               
               
                   
                 EIKTEGEYVLVKRQFEDCCHDLKVKMPCLITTLKDM 
               
               
                   
                 NTPRYMKVGRIYDAFENDVVETWTVKDIEVDPSNLG 
               
               
                   
                 LKGSPTSVFKSFTKSVKPAGTIYNEDAKTSAGIIID 
               
               
                   
                 KLKEKYII 
               
               
                   
               
               
                 etfA3 
                 MGNVLVVIEQRENVIQTVSLELLGKATEIAKDYDTK 
               
               
                 SEQ ID NO: 64 
                 VSALLLGSKVEGLIDTLAHYGADEVIVVDDEALAVY 
               
               
                   
                 TTEPYTKAAYEAIKAADPIVVLFGATSIGRDLAPRV 
               
               
                   
                 SARIHTGLTADCTGLAVAEDTKLLLMTRPAFGGNIM 
               
               
                   
                 ATIVCKDFRPQMSTVRPGVMKKNEPDETKEAVINRF 
               
               
                   
                 KVEFNDADKLVQVVQVIKEAKKQVKIEDAKILVSAG 
               
               
                   
                 RGMGGKENLDILYELAEIIGGEVSGSRATIDAGWLD 
               
               
                   
                 KARQVGQTGKTVRPDLYIACGISGAIQHIAGMEDAE 
               
               
                   
                 FIVAINKNPEAPIFKYADVGIVGDVHKVLPELISQL 
               
               
                   
                 SVAKEKGEVLAN 
               
               
                   
               
               
                 Ter 
                 MIVKPMVRNNICLNAHPQGCKKGVEDQIEYTKKRIT 
               
               
                 SEQ ID NO: 65 
                 AEVKAGAKAPKNVLVLGCSNGYGLASRITAAFGYGA 
               
               
                   
                 ATIGVSFEKAGSETKYGTPGWYNNLAFDEAAKREGL 
               
               
                   
                 YSVTIDGDAFSDEIKAQVIEEAKKKGIKFDLIVYSL 
               
               
                   
                 ASPVRTDPDTGIMHKSVLKPFGKTFTGKTVDPFTGE 
               
               
                   
                 LKEISAEPANDEEAAATVKVMGGEDWERWIKQLSKE 
               
               
                   
                 GLLEEGCITLAYSYIGPEATQALYRKGTIGKAKEHL 
               
               
                   
                 EATAHRLNKENPSIRAFVSVNKGLVTRASAVIPVIP 
               
               
                   
                 LYLASLFKVMKEKGNHEGCIEQITRLYAERLYRKDG 
               
               
                   
                 TIPVDEENRIRIDDWELEEDVQKAVSALMEKVTGEN 
               
               
                   
                 AESLTDLAGYRHDFLASNGFDVEGINYEAEVERFDR 
               
               
                   
                 I 
               
               
                   
               
               
                 ThiA 
                 MREVVIASAARTAVGSFGGAFKSVSAVELGVTAAKE 
               
               
                 SEQ ID NO: 66 
                 AIKRANITPDMIDESLLGGVLTAGLGQNIARQIALG 
               
               
                   
                 AGIPVEKPAMTINIVCGSGLRSVSMASQLIALGDAD 
               
               
                   
                 IMLVGGAENMSMSPYLVPSARYGARMGDAAFVDSMI 
               
               
                   
                 KDGLSDIFNNYHMGITAENIAEQWNITREEQDELAL 
               
               
                   
                 ASQNKAEKAQAEGKFDEEIVPVVIKGRKGDTVVDKD 
               
               
                   
                 EYIKPGTTMEKLAKLRPAFKKDGTVTAGNASGINDG 
               
               
                   
                 AAMLVVMAKEKAEELGIEPLATIVSYGTAGVDPKIM 
               
               
                   
                 GYGPVPATKKALEAANMTIEDIDLVEANEAFAAQSV 
               
               
                   
                 AVIRDLNIDMNKVNVNGGAIAIGHPIGCSGARILTT 
               
               
                   
                 LLYEMKRRDAKTGLATLCIGGGMGTTLIVKR 
               
               
                   
               
               
                 Hbd 
                 MKLAVIGSGTMGSGIVQTFASCGHDVCLKSRTQGAI 
               
               
                 SEQ ID NO: 67 
                 DKCLALLDKNLTKLVTKGKMDEATKAEILSHVSSTT 
               
               
                   
                 NYEDLKDMDLIIEASVEDMNIKKDVFKLLDELCKED 
               
               
                   
                 TILATNTSSLSITEIASSTKRPDKVIGMHFFNPVPM 
               
               
                   
                 MKLVEVISGQLTSKVTFDTVFELSKSINKVPVDVSE 
               
               
                   
                 SPGFVVNRILIPMINEAVGIYADGVASKEEIDEAMK 
               
               
                   
                 LGANHPMGPLALGDLIGLDVVLAIMNVLYTEFGDTK 
               
               
                   
                 YRPHPLLAKMVRANQLGRKTKIGFYDYNK 
               
               
                   
               
               
                 Crt2 
                 MSTSDVKVYENVAVEVDGNICTVKMNRPKALNAINS 
               
               
                 SEQ ID NO: 68 
                 KTLEELYEVFVDINNDETIDVVILTGEGKAFVAGAD 
               
               
                   
                 IAYMKDLDAVAAKDFSILGAKAFGEIENSKKVVIAA 
               
               
                   
                 VNGFALGGGCELAMACDIRIASAKAKFGQPEVTLGI 
               
               
                   
                 TPGYGGTQRLTRLVGMAKAKELIFTGQVIKADEAEK 
               
               
                   
                 IGLVNRVVEPDILIEEVEKLAKIIAKNAQLAVRYSK 
               
               
                   
                 EAIQLGAQTDINTGIDIESNLFGLCFSTKDQKEGMS 
               
               
                   
                 AFVEKREANFIKG 
               
               
                   
               
               
                 Pbt 
                 MRSFEEVIKFAKERGPKTISVACCQDKEVLMAVEMA 
               
               
                 SEQ ID NO: 69 
                 RKEKIANAILVGDIEKTKEIAKSIDMDIENYELIDI 
               
               
                   
                 KDLAEASLKSVELVSQGKADMVMKGLVDTSIILKAV 
               
               
                   
                 LNKEVGLRTGNVLSHVAVFDVEGYDRLFFVTDAAMN 
               
               
                   
                 LAPDTNTKKQIIENACTVAHSLDISEPKVAAICAKE 
               
               
                   
                 KVNPKMKDTVEAKELEEMYERGEIKGCMVGGPFAID 
               
               
                   
                 NAVSLEAAKHKGINHPVAGRADILLAPDIEGGNILY 
               
               
                   
                 KALVFFSKSKNAGVIVGAKAPIILTSRADSEETKLN 
               
               
                   
                 SIALGVLMAAKA 
               
               
                   
               
               
                 Buk 
                 MSKIFKILTINPGSTSTKIAVFDNEDLVFEKTLRHS 
               
               
                 SEQ ID NO: 70 
                 SEEIGKYEKVSDQFEFRKQVIEEALKEGGVKTSELD 
               
               
                   
                 AVVGRGGLLKPIKGGTYSVSAAMIEDLKVGVLGEHA 
               
               
                   
                 SNLGGIIAKQIGEEVNVPSYIVDPVVVDELEDVARI 
               
               
                   
                 SGMPEISRASVVHALNQKAIARRYAREINKKYEDIN 
               
               
                   
                 LIVAHMGGGVSVGAHKNGKIVDVANALDGEGPFSPE 
               
               
                   
                 RSGGLPVGALVKMCFSGKYTQDEIKKKIKGNGGLVA 
               
               
                   
                 YLNTNDAREVEERIEAGDEKAKLVYEAMAYQISKEI 
               
               
                   
                 GASAAVLKGDVKAILLTGGIAYSKMFTEMIADRVKF 
               
               
                   
                 IADVKVYPGEDEMIALAQGGLRVLTGEEEAQVYDN 
               
               
                   
               
               
                 TesB 
                 MSQALKNLLTLLNLEKIEEGLFRGQSEDLGLRQVFG 
               
               
                 SEQ ID NO: 41 
                 GQVVGQALYAAKETVPEERLVHSFHSYFLRPGDSKK 
               
               
                   
                 PIIYDVETLRDGNSFSARRVAAIQNGKPIFYMTASF 
               
               
                   
                 QAPEAGFEHQKTMPSAPAPDGLPSETQIAQSLAHLL 
               
               
                   
                 PPVLKDKFICDRPLEVRPVEFHNPLKGHVAEPHRQV 
               
               
                   
                 WIRANGSVPDDLRVHQYLLGYASDLNFLPVALQPHG 
               
               
                   
                 IGFLEPGIQTATIDHSMWFHRPFNLNEWLLYSVEST 
               
               
                   
                 SASSARGFVRGEFYTQDGVLVASTVQEGVMRNHN* 
               
               
                   
               
            
           
         
       
     
     The gene products of the bcd2, etfA3, 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, erfB3, and erfA3 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 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, 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 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, 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 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. 
     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 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 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 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, 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 hunger, appetite, craving, obesity, metablic syndrome, insulin resistance, liver damage, or other 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, the local production of butyrate protects against diet-induced obesity (Lin et al., 2012). In some embodiments, the local production of butyrate protects against diet-induced obesity without causing decreased food intake (Lin et al., 2012). In some embodiments, local butyrate production reduces gut inflammation, a symptom of metabolic disease. 
     In one embodiment, the bcd2 gene has at least about 80% identity with SEQ ID NO: 53. In another embodiment, the bcd2 gene has at least about 85% identity with SEQ ID NO: 53. In one embodiment, the bcd2 gene has at least about 90% identity with SEQ ID NO: 53. In one embodiment, the bcd2 gene has at least about 95% identity with SEQ ID NO: 53. In another embodiment, the bcd2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 53. 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: 53. In another embodiment, the bcd2 gene comprises the sequence of SEQ ID NO: 53. In yet another embodiment the bcd2 gene consists of the sequence of SEQ ID NO: 53. 
     In one embodiment, the etB3 gene has at least about 80% identity with SEQ ID NO: 54. In another embodiment, the etfB3 gene has at least about 85% identity with SEQ ID NO: 54. In one embodiment, the etB3 gene has at least about 90% identity with SEQ ID NO: 54. In one embodiment, the etfB3 gene has at least about 95% identity with SEQ ID NO: 54. In another embodiment, the etB3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 54. 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: 54. In another embodiment, the etB3 gene comprises the sequence of SEQ ID NO: 54. In yet another embodiment the etfB3 gene consists of the sequence of SEQ ID NO: 54. 
     In one embodiment, the etfA3 gene has at least about 80% identity with SEQ ID NO: 55. In another embodiment, the etfA3 gene has at least about 85% identity with SEQ ID NO: 55. In one embodiment, the etfA3 gene has at least about 90% identity with SEQ ID NO: 55. In one embodiment, the etfA3 gene has at least about 95% identity with SEQ ID NO: 55. In another embodiment, the etfA3 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 55. 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: 55. In another embodiment, the etfA3 gene comprises the sequence of SEQ ID NO: 55. In yet another embodiment the etfA3 gene consists of the sequence of SEQ ID NO: 55. 
     In one embodiment, the thiA1 gene has at least about 80% identity with SEQ ID NO: 56. In another embodiment, the thiA1 gene has at least about 85% identity with SEQ ID NO: 56. In one embodiment, the thiA1 gene has at least about 90% identity with SEQ ID NO: 56. In one embodiment, the thiA1 gene has at least about 95% identity with SEQ ID NO: 56. In another embodiment, the thiA1 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 56. 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: 56. In another embodiment, the thiA1 gene comprises the sequence of SEQ ID NO: 56. In yet another embodiment the thiA1 gene consists of the sequence of SEQ ID NO: 56. 
     In one embodiment, the hbd gene has at least about 80% identity with SEQ ID NO: 57. In another embodiment, the hbd gene has at least about 85% identity with SEQ ID NO: 57. In one embodiment, the hbd gene has at least about 90% identity with SEQ ID NO: 57. In one embodiment, the hbd gene has at least about 95% identity with SEQ ID NO: 57. In another embodiment, the hbd gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 57. 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: 57. In another embodiment, the hbd gene comprises the sequence of SEQ ID NO: 57. In yet another embodiment the hbd gene consists of the sequence of SEQ ID NO: 57. 
     In one embodiment, the crt2 gene has at least about 80% identity with SEQ ID NO: 58. In another embodiment, the crt2 gene has at least about 85% identity with SEQ ID NO: 58. In one embodiment, the crt2 gene has at least about 90% identity with SEQ ID NO: 58. In one embodiment, the crt2 gene has at least about 95% identity with SEQ ID NO: 58. In another embodiment, the crt2 gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 58. 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: 58. In another embodiment, the crt2 gene comprises the sequence of SEQ ID NO: 58. In yet another embodiment the crt2 gene consists of the sequence of SEQ ID NO: 58. 
     In one embodiment, the pbt gene has at least about 80% identity with SEQ ID NO: 59. In another embodiment, the pbt gene has at least about 85% identity with SEQ ID NO: 59. In one embodiment, the pbt gene has at least about 90% identity with SEQ ID NO: 59. In one embodiment, the pbt gene has at least about 95% identity with SEQ ID NO: 59. In another embodiment, the pbt gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 59. 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: 59. In another embodiment, the pbt gene comprises the sequence of SEQ ID NO: 59. In yet another embodiment the pbt gene consists of the sequence of SEQ ID NO: 59. 
     In one embodiment, the buk gene has at least about 80% identity with SEQ ID NO: 60. In another embodiment, the buk gene has at least about 85% identity with SEQ ID NO: 60. In one embodiment, the buk gene has at least about 90% identity with SEQ ID NO: 60. In one embodiment, the buk gene has at least about 95% identity with SEQ ID NO: 60. In another embodiment, the buk gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 60. 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: 60. In another embodiment, the buk gene comprises the sequence of SEQ ID NO: 60. In yet another embodiment the buk gene consists of the sequence of SEQ ID NO: 60. 
     In one embodiment, the ter gene has at least about 80% identity with SEQ ID NO: 61. In another embodiment, the ter gene has at least about 85% identity with SEQ ID NO: 61. In one embodiment, the ter gene has at least about 90% identity with SEQ ID NO: 61. In one embodiment, the ter gene has at least about 95% identity with SEQ ID NO: 61. In another embodiment, the ter gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 61. 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: 61. In another embodiment, the ter gene comprises the sequence of SEQ ID NO: 61. In yet another embodiment the ter gene consists of the sequence of SEQ ID NO: 61. 
     In one embodiment, the tesB gene has at least about 80% identity with SEQ ID NO: 15. In another embodiment, the tesB gene has at least about 85% identity with SEQ ID NO: 15. In one embodiment, the tesB gene has at least about 90% identity with SEQ ID NO: 15. In one embodiment, the tesB gene has at least about 95% identity with SEQ ID NO: 15. In another embodiment, the tesB gene has at least about 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15. 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: 15. In another embodiment, the tesB gene comprises the sequence of SEQ ID NO: 15. In yet another embodiment the tesB gene consists of the sequence of SEQ ID NO: 15. 
     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: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. 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 with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. 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 with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. 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 with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. 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 with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. 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 with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. 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: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. 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: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. 
     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. 
     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 metabolic disease (Lin et al., 2012). 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 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 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. 
     Acetate 
     In some embodiments, the genetically engineered bacteria of the invention comprise an acetate gene cassette and produce acetate under particular exogenous environmental conditions. The genetically engineered bacteria may include any suitable set of acetate biosynthesis genes. 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 et al., 2008). 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 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, the genetically engineered bacteria are capable of producing an alternate short-chain fatty acid. 
     GLP-1 
     In some embodiments, the genetically engineered bacteria of the invention are capable of producing GLP-1 or proglucagon. GLP-1 and several other insulin and satiety regulating peptides result from cleaved of preproglucagon. Preproglucagon is proteolytically cleaved in a tissue-specific manner. Post-translational processing in the gut and brain by prohormone convertases results in the secretion of GLP-1 and GLP-2, while the glucagon sequence remains in a larger peptide, glicentin or glicentin-related pancreatic peptide (GRPP) and oxyntomodulin. Glucagon-like peptide 1 (GLP-1) is produced by intestinal cells, e.g., ileal L cells, and is capable of stimulating insulin secretion and the differentiation of insulin-secreting cells and inhibiting glucagon secretion. GLP-1 is capable of restoring glucose sensitivity and increasing satiety. 
     Glucagon-like peptide 1 (GLP-1) is also used to treat those suffering from non-alcoholic steatohepatitis by reducing the degree of lipotoxic metabolites, pro-inflammatory substrate, and hepatic lipid deposition. Glucagon-like peptide 1 is well known to those of skill in the art. For example, glucagon-like peptide 1 has been used to stimulate insulin secretion in the treatment of type-two diabetes and non-alcoholic steatohepatitis (NASH). See, for example, Armstrong, et al., J. of Hepatology, 64:399-408 (2016); Bernsmeier, et al., PLOS One, 9(1): e87488 (2014); Kjems, et al., Diabetes, 52:380-386 (2003); Knudsen et al., J. Med. Chem., 43:1664-1669 (2000); MacDonald, et al., Diabetes, 51(supp. 3):5434-5442 (2002); Werner, et al., Regulatory Peptides, 164:58-34 (2010); Drucker and Nauck, Lancet, 368:1696-1705 (2006); Jiminez-Solem, et al., Cur. Opinion in Mol. Therap., 12(6):760-797 (2010); Schnabel, et al., Vasc. Health and Risk Mgmt., 2(1):69-77 (2006); and WO1995/017510, the entire contents of each of which are expressly incorporated herein by reference. 
     Proteolytic cleavage of proglucagon produces GLP-1 and GLP-2. GLP-1 adminstration has therapeutic potential in treating type 2 diabetes (Gallwitz et al., 2000). The genetically engineered bacteria may comprise any suitable gene encoding GLP-1 or proglucagon, e.g., human GLP-1 or proglucagon. In some embodiments, a protease inhibitor, e.g., an inhibitor of dipeptidyl peptidase, is also administered to decrease GLP-1 degradation. In some embodiments, the genetically engineered bacteria express a degradation resistant GLP-1 analog (see, e.g., Gallwitz et al., 2000). In some embodiments, the gene encoding GLP-1 or proglucagon is modified and/or mutated, e.g., to enhance stability, increase GLP-1 production, and/or increase metabolic disease attenuation potency. In some embodiments, the local production of GLP-1 induces insulin secretion and/or differentiation of insulin-secreting cells. In some embodiments, the local production of GLP-1 produces satiety in a subject and ameliorates obesity. In some embodiments, the genetically engineered bacteria are capable of expressing GLP-1 or proglucagon 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. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 GLP-1 Polynucleotide Sequences 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 GLP-1 (1-37), with 
                 ATGGACGAGTTCGAACGCCACG 
               
               
                 initiation 
                 CGGAGGGAACTTTCACTTCTGA 
               
               
                 met codon; codon 
                 TGTTTCTAGCTATTTGGAGGGC 
               
               
                 optimized for 
                 CAGGCTGCGAAAGAGTTTATTG 
               
               
                 expression in  E .  coli . 
                 CTTGGCTGGTTAAAGGTCGTGG 
               
               
                 SEQ ID NO: 71 
                 TTAA 
               
               
                   
               
               
                 GLP1 (1-37) codon 
                 GACGAGTTCGAACGCCACGCGG 
               
               
                 optimized for 
                 AGGGAACTTTCACTTCTGATGT 
               
               
                 expression in  E .  coli . 
                 TTCTAGCTATTTGGAGGGCCAG 
               
               
                 SEQ ID NO: 72 
                 GCTGCGAAAGAGTTTATTGCTT 
               
               
                   
                 GGCTGGTTAAAGGTCGTGGTTA 
               
               
                   
                 A 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 GLP-1 Polypeptide Sequences 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 GLP-1 (1-37) 
                 HDEFERHAEGTFTSDVSSYLEGQAAKEFIAW 
               
               
                 SEQ ID NO: 73 
                 LVKGRG 
               
               
                   
               
               
                 GLP-1 (1-37) H-&gt;M substitution 
                 MDEFERHAEGTFTSDVSSYLEGQAAKEFIAW 
               
               
                 SEQ ID NO: 74 
                 LVKGRG 
               
               
                   
               
               
                 GLP-1-(7-37) 
                 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 
               
               
                 SEQ ID NO: 75 
                   
               
               
                   
               
               
                 GLP-1-(7-36)NH2 
                 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR 
               
               
                 SEQ ID NO: 76 
                   
               
               
                   
               
               
                 glucagon preproprotein 
                 MKSIYFVAGLFVMLVQGSWQRSLQDTEEKSR 
               
               
                 (NP_002045.1) 1-20 is signal 
                 SFSASQADPLSDPDQMNEDKRHSQGTFTSDY 
               
               
                 peptide 
                 SKYLDSRRAQDFVQWLMNTKRNRNNIAKRHD 
               
               
                 SEQ ID NO: 77 
                 EFERHAEGTFTSDVSSYLEGQAAKEFIAWLV 
               
               
                   
                 KGRGRRDFPEEVAIVEELGRRHADGSFSDEM 
               
               
                   
                 NTILDNLAARDFINWLIQTKITDRK 
               
               
                   
               
               
                 Proglucagon (Signal peptide 1 - 
                 RSLQDTEEKSRSFSASQADPLSDPDQMNEDK 
               
               
                 20; Glucagon-like peptide 1 (92- 
                 RHSQGTFTSDYSKYLDSRRAQDFVQWLMNTK 
               
               
                 128); Glucagon-like peptide 2 146- 
                 RNRNNIAKRHDEFERHAEGTFTSDVSSYLEG 
               
               
                 178 
                 QAAKEFIAWLVKGRGRRDFPEEVAIVEELGR 
               
               
                 SEQ ID NO: 78 
                 RHADGSFSDEMNTILDNLAARDFINWLIQTK 
               
               
                   
                 ITDRK 
               
               
                   
               
               
                 Glucagon 
                 HSQGTFTSDYSKYLDSRRAQDFVQWLMNT 
               
               
                 SEQ ID NO: 79 
                   
               
               
                   
               
               
                 Glicentin 
                 RSLQDTEEKSRSFSASQADPLSDPDQMNEDK 
               
               
                 SEQ ID NO: 80 
                 RHSQGTFTSDYSKYLDSRRAQDFVQWLMNTK 
               
               
                   
                 RNRNNIA 
               
               
                   
               
               
                 Glicentin related peptide 
                 RSLQDTEEKSRSFSASQADPLSDPDQMNED 
               
               
                 SEQ ID NO: 81 
                   
               
               
                   
               
               
                 Oxyntomodulin 
                 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKR 
               
               
                 SEQ ID NO: 82 
                 NRNNIA 
               
               
                   
               
            
           
         
       
     
     The circulating active form of GLP-1 is GLP-1(7-37), which has a very short biological half-life of the order of just a few minutes in blood. The relatively low stability of GLP-1 (3-5 min) has significantly limited its clinical utility because of the rapid degradation catalyzed by the enzyme dipeptidyl peptidase IV (DPP-JV), but also other enzyrnes such as neutral endopeptidase (NEP), plasma lkailikrein or plasmin. One strategy to prolong in vivo half-life is stabilization towards degradation by DPPJV, which preferably cleaves N-terminal Xaa-Pro or Xaa-Ala dipeptide sequences. Alteration of that N-terminal sequence, especially the second amino acid, has proven to reduce degradation by DPPJV (e.g., reviewed in Lorenz et al., Recent progress and future options in the development of GLP-1 receptor agonists for the treatment of diabesity; Bioorganic &amp; Medicinal Chemistry Letters, 23 (14);4011-4018). In some embodiments, the genetically engineered bacteria comprise a cassette encoding GLP-1 fragment or variant, in which the DPP-JV is mutated, such that it can no longer be cleaved by the enzyme. 
     GLP-1 is released in a tissue specific manner, though post-translational processing of pre-pro-glucagon, from the neuroendocrine L-cells predominantly in two forms, GLP-1 (7-36) amide, which constitutes approximately 80% of circulating GLP-1, and GLP-1 (7-37) amide. GLP-1 (1-36 amide) is predominantly secreted in the pancreas, whereas GLP-1 (1-37) is secreted in the ileum and hypothalamus. 
     In addition, full length GLP-1-(1-37) is produced in much smaller amounts. This full-length form of GLP-1(1-37), was previously thought to be inactive, but was found to stimulate rat intestinal epithelial cells to become glucose-responsive insulin-secreting cells, i.e., full length GLP-1 could convert intestinal epithelial progenitors in the small intestine into insulin-producing cells (Suzuki et al., Glucagon-like peptide 1 (1-37) converts intestinal epithelial cells into insulin-producing cells; Proc Natl Acad Sci USA. 2003 Apr. 29; 100(9): 5034-5039). While the amounts of GLP-1 (1-37) produced endogenously likely are not sufficient for these effects, secretion of large amounts of GLP-1, e.g., by the genetically engineered bacteria, are likely sufficient to alter a balance in the developmental environment of the intestinal epithelia, leading to the induction of insulin-producing cells from intestinal epithelial progenitors. As such, secretion of full-length GLP-1 by the genetically engineered bacteria of the disclosure is a novel therapeutic strategy for the treatment of a number of diseases related to dysregulation of insulin production and/or secretion, including diabetes. 
     GLP-1 analogs, which exhibit extended stability in serum, have become important in the clinic. Exendin-4, a peptide produced in the salivary glands of the Gila monster (Heloderma suspectum), possesses similar glucose regulatory function to the human GLP-1 peptide. In exendin-4, the second amino acid is a Gly rendering it resistant to DPPIV mediated degradation. Furthermore, the Leu21-Ser39 span of exendin-4 forms a compact tertiary fold (the Trp-cage) which shields the side chain of Trp25 from solvent exposure, leading to enhanced helicity and stability of the peptide (see Lorenz et al. for review). Exenatide BID is a synthetic version of exendin-4, represents the first GLP-1 RA approved in 2005 as antidiabetic therapy for the treatment of T2DM. Following the FDA approval of exendin-4, liraglutide and albiglutide, which are long-acting GLP-1 analogs using palmitic acid conjugation and albumin fusion, respectively, were approved. Many other strategies have also been employed to achieve long-acting activity of GLP-1, including dimerization, intra-molecular conjugation, and additional variant positive charged amino acids on the N terminus. Table 10 lists non-limiting examples of GLP-iR agonists. In some embodiments, the genetically engineered bacteria comprise a gene encoding Exenatide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Liraglutide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Lixisenatide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Albiglutide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Dulaglutide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Taspoglutide. In some embodiments, the genetically engineered bacteria comprise a gene encoding Semaglutide. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Non-limiting examples of GLP-1R agonists 
               
            
           
           
               
               
               
            
               
                 Name and SEQ ID NO 
                 Sequence 
                 Short description 
               
               
                   
               
               
                 Exenatide 
                 HGEGTFTSDLSKQMEE 
                 Second amino acid is a Gly 
               
               
                 SEQ ID NO: 83 
                 EAVRLFIEWLKNGGPS 
                 rendering it resistant to 
               
               
                   
                 SGAPPPS 
                 DPPIV mediated 
               
               
                   
                   
                 degradation. Furthermore, 
               
               
                   
                   
                 the Leu21-Ser39 span of 
               
               
                   
                   
                 exendin-4 forms a compact 
               
               
                   
                   
                 tertiary fold (the Trp-cage) 
               
               
                   
                   
                 which shields the side chain 
               
               
                   
                   
                 of Trp25 from solvent 
               
               
                   
                   
                 exposure, leading to 
               
               
                   
                   
                 enhanced helicity and 
               
               
                   
                   
                 stability of the peptide 
               
               
                   
               
               
                 Liraglutide 
                 HAEGTFTSDVSSYLEG 
                 a close structural homolog to 
               
               
                 SEQ ID NO: 84 
                 QAAKEEFIIAWLVKGR 
                 GLP-1(7-37) with 97% 
               
               
                   
                 G 
                 sequence identity to the 
               
               
                   
                   
                 native hormone. Lys in 
               
               
                   
                   
                 position 34 is substituted by 
               
               
                   
                   
                 Arg and a palmitic acid is 
               
               
                   
                   
                 conjugated to Lys in position 
               
               
                   
                   
                 26 via a glutamate spacer 
               
               
                   
               
               
                 Lixisenatide 
                 HGEGTFTSDLSKQMEE 
                 synthetic analog of exendin- 
               
               
                 SEQ ID NO: 85 
                 EAVRLFIEWLKNGGPS 
                 4. Compared to exendin-4, 
               
               
                   
                 SGAPPSKKKKKK 
                 six Lys residues have been 
               
               
                   
                   
                 added to the C-terminus (also 
               
               
                   
                   
                 amidated), while one Pro in 
               
               
                   
                   
                 the C-terminal region has 
               
               
                   
                   
                 been deleted. 
               
               
                   
               
               
                 Albiglutide 
                 HGEGTFTSDVSSYLEG 
                 two copies of GLP-1 are 
               
               
                 SEQ ID NO: 86 
                 QAAKEFIAWLVKGRH 
                 fused as tandem repeat to the 
               
               
                   
                 GEGTFTSDVSSYLEGQ 
                 N-terminus of albumin. 
               
               
                   
                 AAKEFIAWLVKGRDA 
                 DPPIV-resistance is 
               
               
                   
                 HKSEVAHRFKDLGEEN 
                 achieved by a single 
               
               
                   
                 FKALVLIAFAQYLQQC 
                 substitution, Ala for Gly, at 
               
               
                   
                 PFEDHVKLVNEVTEFA 
                 the DPPIV cleavage site. 
               
               
                   
                 KTCVADESAENCDKSL 
                   
               
               
                   
                 HTLFGDKLCTVATLRE TYGEMADCCAKQEPE 
                   
               
               
                   
                 RNECFLQHKDDNPNLP 
                   
               
               
                   
                 RLVRPEVDVMCTAFH 
                   
               
               
                   
                 DNEETFLKKYLYEIAR 
                   
               
               
                   
                 RHPYFYAPELLFFAKR 
                   
               
               
                   
                 YKAAFTECCQAADKA 
                   
               
               
                   
                 ACLLPKLDELRDEGKA 
                   
               
               
                   
                 SSAKQRLKCASLQKFG 
                   
               
               
                   
                 ERAFKAWAVARLSQR 
                   
               
               
                   
                 FPKAEFAEVSKLVTDL 
                   
               
               
                   
                 TKVHTECCHGDLLECA 
                   
               
               
                   
                 DDRADLAKYICENQDS 
                   
               
               
                   
                 ISSKLKECCEKPLLEKS 
                   
               
               
                   
                 HCIAEVENDEMPADLP 
                   
               
               
                   
                 SLAADFVESKDVCKN 
                   
               
               
                   
                 YAEAKDVFLGMFLYE 
                   
               
               
                   
                 YARRHPDYSVVLLLRL 
                   
               
               
                   
                 AKTYETTLEKCCAAA 
                   
               
               
                   
                 DPHECYAKVFDEFKPL 
                   
               
               
                   
                 VEEPQNLIKQNCELFE 
                   
               
               
                   
                 QLGEYKFQNALLVRY 
                   
               
               
                   
                 TKKVPQVSTPTLVEVS 
                   
               
               
                   
                 RNLGKVGSKCCKHPE 
                   
               
               
                   
                 AKRMPCAEDYLSVVL 
                   
               
               
                   
                 NQLCVLHEKTPVSDRV 
                   
               
               
                   
                 TKCCTESLVNRRPCFS 
                   
               
               
                   
                 ALEVDETYVPKEFNAE 
                   
               
               
                   
                 TFTFHADICTLSEKERQ 
                   
               
               
                   
                 IKKQTALVELVKHKPK 
                   
               
               
                   
                 ATKEQLKAVMDDFAA 
                   
               
               
                   
                 FVEKCCKADDKETCFA 
                   
               
               
                   
                 EEGKKLVAASQAALG 
                   
               
               
                   
                 L 
                   
               
               
                   
               
               
                 Dulaglutide 
                 HGEGTFTSDVSSYLEE 
                 A recombinant fusion 
               
               
                 SEQ ID NO: 87 
                 QAAKEFIAWLVKGGG 
                 protein, which consists of 
               
               
                   
                 GGGGSGGGGSGGGGS 
                 two GLP-1 peptides 
               
               
                   
                 AESKYGPPCPPCPAPE 
                 covalently linked by a small 
               
               
                   
                 AAGGPSVFLFPPKPKD 
                 peptide [tetraglycyl-1- 
               
               
                   
                 TLMISRTPEVTCVVVD 
                 seryltetraglycyl-1- 
               
               
                   
                 VSQEDPEVQFNWYVD 
                 seryltetraglycyl-l-seryl-1- 
               
               
                   
                 GVEVHNAKTKPREEQF 
                 alanyl] to a human IgG4-Fc 
               
               
                   
                 NSTYRVVSVLTVLHQD 
                 heavy chain variant. 
               
               
                   
                 WLNGKEYKCKVSNKG 
                 Compared to natural GLP-1, 
               
               
                   
                 LPSSIEKTISKAKGQPR 
                 the GLP-1 moieties contain 
               
               
                   
                 EPQVYTLPPSQEEMTK 
                 amino acid substitutions 
               
               
                   
                 NQVSLTCLVKGFYPSD 
                 (Ala8→Gly, Gly26→Glu, 
               
               
                   
                 IAVEWESNGQPENNYK 
                 Arg36→Gly) to ensure 
               
               
                   
                 TTPPVLDSDGSFFLYSR 
                 protection from DPPIV 
               
               
                   
                 LTVDKSRWQEGNVFS 
                 cleavage as well as 
               
               
                   
                 CSVMHEALHNHYTQK 
                 maintenance of the potency 
               
               
                   
                 SLSLSLG 
                 of the construct. 
               
               
                   
               
               
                 Taspoglutide 
                 His-Aib-Glu-Gly-Thr- 
                 a close analog of natural 
               
               
                 SEQ ID NO: 88 
                 Phe-Thr-Ser Asp-Val-Ser- 
                 GLP-1(7-36) in which the 
               
               
                   
                 Ser-Tyr-Leu-Glu-Gly- 
                 unnatural amino acid 
               
               
                   
                 Gln-Ala-Ala-Lys-Glu- 
                 aminoisobutyric acid (Aib) 
               
               
                   
                 Phe-Ile-Ala-Trp-Leu-Val- 
                 has been introduced in 
               
               
                   
                 Lys-Aib-Arg-NH 2   
                 position 8 and 35 in order to 
               
               
                   
                   
                 avoid degradation by DPPIV, 
               
               
                   
                   
                 but also by other serine 
               
               
                   
                   
                 proteases such as plasma 
               
               
                   
                   
                 kallikrein and plasmin. 
               
               
                   
               
               
                 Semaglutide 
                 MAGAPGPLRLALLLLG 
                   
               
               
                 SEQ ID NO: 89 
                 MVGRAGPRPQGATVS 
                   
               
               
                   
                 LWETVQKWREYRRQC 
                   
               
               
                   
                 QRSLTEDPPPATDLFC 
                   
               
               
                   
                 NRTFDEYACWPDGEP 
                   
               
               
                   
                 GSFVNVSCPWYLPWA 
                   
               
               
                   
                 SSVPQGHVYRFCTAEG 
                   
               
               
                   
                 LWLQKDNSSLPWRDL 
                   
               
               
                   
                 SECEESKRGERSSPEEQ 
                   
               
               
                   
                 LLFLYIIYTVGYALSFS 
                   
               
               
                   
                 ALVIASAILLGFRHLHC 
                   
               
               
                   
                 TRNYIHLNLFASFILRA 
                   
               
               
                   
                 LSVFIKDAALKWMYST 
                   
               
               
                   
                 AAQQHQWDGLLSYQD 
                   
               
               
                   
                 SLSCRLVFLLMQYCVA 
                   
               
               
                   
                 ANYYWLLVEGVYLYT 
                   
               
               
                   
                 LLAFSVLSEQWIFRLY 
                   
               
               
                   
                 VSIGWGVPLLFVVPWG 
                   
               
               
                   
                 IVKYLYEDEGCWTRNS 
                   
               
               
                   
                 NMNYWLIIRLPILFAIG 
                   
               
               
                   
                 VNFLIFVRVICIVVSKL 
                   
               
               
                   
                 KANLMCKTDIKCRLA 
                   
               
               
                   
                 KSTLTLIPLLGTHEVIF 
                   
               
               
                   
                 AFVMDEHARGTLRFIK 
                   
               
               
                   
                 LFTELSFTSFQGLMVAI 
                   
               
               
                   
                 LYCFVNNEVQLEFRKS 
                   
               
               
                   
                 WERWRLEHLHIQRDSS 
                   
               
               
                   
                 MKPLKCPTSSLSSGAT 
                   
               
               
                   
                 AGSSMYTATCQASCS 
               
               
                   
               
            
           
         
       
     
     In one embodiment, GLP-1 and/or a GLP-1R agonist of Table 10 stimulates the rate of insulin secretion in the body. In one embodiment, GLP-1 and/or a GLP-1R agonist of Table 10 inhibits and lowers plasma glucose produced in the body. In one embodiment, GLP-1 and/or a GLP-1R agonist of Table 10 decreases the level of lipotoxic metabolites in the body. In one embodiment, GLP-1 and/or a GLP-1R agonist of Table 10 decreases the degree of pro-inflammatory substrate in the body. In one embodiment, GLP-1 decreases the level of insulin resistance (IR) in the body. In one embodiment, GLP-1 and/or a GLP-1R agonist of Table 10 decreases the level of hepatic lipid deposition in the body. Methods for measuring the insulin secretion rates and glucose levels are well known to one of ordinary skill in the art. For example, blood samples taken periodically, and standard statistical analysis methods may be used to determine the insulin secretion rates and plasma glucose levels in a subject. 
     GLP-1 and/or a GLP-1R agonist of Table 10 may be expressed or modified in bacteria of this disclosure in order to enhance insulin stimulation and reduce plasma glucose levels in subjects having liver disease, such as NASH. Specifically, when GLP-1 and/or a GLP-1R agonist of Table 10 is expressed in the engineered bacterial cells of the disclosure, the expressed GLP-1 and/or a GLP-1R agonist of Table 10 will reduce the degree of lipotoxic metabolites, pro-inflammatory substrate, and hepatic lipid deposition in the subject. 
     GLP-1 and/or a GLP-1R agonist of Table 10 may be expressed or modified in bacteria of this disclosure in order to enhance insulin stimulation and reduce plasma glucose levels in subjects having type two diabetes, obesity, and/or metabolic syndrome, or metabolic syndrome related disorders, including cardiovascular disorders, and obesity in a subject. 
     In one embodiment, the bacterial cell comprises one or more genes encoding a GLP-1 and/or a GLP-1R agonist of Table 10. In some embodiments, the disclosure provides a bacterial cell that comprises a heterologous gene encoding a glucagon-like peptide 1 operably linked to a first promoter. In one embodiment, the first promoter is an inducible promoter. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of a gene encoding a glucagon-like peptide 1. In one embodiment, the bacterial cell comprises multiple copies of a gene or genes encoding a glucagon-like peptide 1. 
     Multiple distinct embodiments of GLP-1 and/or a GLP-1R agonist of Table 10 are known in the art. In some embodiments, the glucagon-like peptide 1 is encoded by a gene derived from a bacterial species. In some embodiments, a glucagon-like peptide 1 is encoded by a gene derived from a non-bacterial species. In some embodiments, a glucagon-like peptide 1 is encoded by a gene derived from a eukaryotic species, e.g.  Homo sapiens . In one embodiment, the gene encoding the glucagon-like peptide 1 is expressed in an organism of the genus or species that includes, but is not limited to,  Lactobacillus  spp., such as  Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus brevis , or  Lactobacillus  gasseri;  Bifidobacterium  spp., such as  Bifidobacterium longum; Bacillus  spp., such as  Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus ; and  Streptomyces  spp., such as  Streptomyces lividans.    
     In one embodiment, the gene encoding the GLP-1 and/or a GLP-1R agonist of Table 10 has been codon-optimized for use in the engineered bacterial cell. In one embodiment, the gene encoding the glucagon-like peptide 1 has been codon-optimized for use in  Escherichia coli . In another embodiment, the gene encoding the glucagon-like peptide 1 has been codon-optimized for use in  Lactococcus . When the gene encoding the GLP-1 and/or a GLP-1R agonist of Table 10 is expressed in the engineered bacterial cells, the bacterial cells express more GLP-1 and/or a GLP-1R agonist of Table 10 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 gene encoding a GLP-1 and/or a GLP-1R agonist of Table 10 may be used to express more GLP-1 and/or a GLP-1R agonist of Table 10 to treat liver disease, such as nonalcoholic steatohepatitis, type two diabetes, metabolic syndrome, and metabolic syndrome related disorders, including cardiovascular disorders and obesity in a subject. 
     Assays for testing the activity of a GLP-1 and/or a GLP-1R agonist of Table 10 or a glucagon-like peptide 1 receptor are well known to one of ordinary skill in the art. For example, glucose and insulin levels can be assessed by drawing plasma samples from subjects previously administered intravenous infusions of the glucagon-like peptide 1 as described in Kjems, et al., Diabetes, 52:380-386 (2003), the entire contents of which are expressly incorporated herein by reference. Briefly, plasma samples from a subject are treated with heparin and sodium fluoride, centrifuged, and plasma glucose levels measured by a glucose oxidase technique. Likewise, the plasma insulin concentrations are measured by a two-site insulin enzyme linked immunosorbent method. Alternatively, baby hamster kidney cells can be used to assay structure-activity relationships of glucagon-like peptide 1 derivatives (see, for example, Knudsen et al., J. Med. Chem., 43:1664-1669 (2000), the entire contents of which are expressly incorporated herein by reference). The present disclosure encompasses genes encoding a GLP-1 and/or a GLP-1R agonist of Table 10 comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein. 
     In some embodiments, the gene encoding a GLP-1 and/or a GLP-1R agonist of Table 10 is mutagenized; mutants exhibiting increased activity are selected; and the mutagenized gene encoding the GLP-1 and/or a GLP-1R agonist of Table 10 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 gene encoding the glucagon-like peptide 1 is from  Homo sapiens . In one embodiment, the gene encoding the glucagon-like peptide 1 is from  Lactobacillus  spp. In one embodiment, the Lacotbacillus spp. is  Lactobacillus plantarum  WCFS1,  Lactobacillus plantarum  80,  Lactobacillus johnsonii  NCC533,  Lactobacillus johnsonii  100-100,  Lactobacillus acidophilus  NCFM ATCC700396,  Lactobacillus brevis  ATCC 367,  Lactobacillus  gasseri ATCC 33323, or  Lactobacillus acidophilus . In another embodiment, the gene encoding the glucagon-like peptide 1 is from a  Bifidobacterium  spp. In one embodiment, the  Bifidobacterium  spp. is  Bifidobacterium longum  NCC2705,  Bifidobacterium longum  DJO10A,  Bifidobacterium longum  BB536, or  Bifidobacterium longum  SBT2928. In another embodiment, the gene encoding the glucagon-like peptide 1 is from  Bacillus  spp. In one embodiment, the  Bacillus  spp is  Bacillus subtilis , or  Bacillus licheniformis , or  Bacillus lentus , or  Bacillus brevis , or  Bacillus stearothermophilus , or  Bacillus alkalophilus , or  Bacillus amyloliquefaciens , or  Bacillus coagulans , or  Bacillus circulans , or  Bacillus lautus . In another embodiment, the gene encoding the glucagon-like peptide 1 is from  Streptomyces  spp. In one embodiment, the  Streptomyces  spp. is  Streptomyces lividans . Other genes encoding glucagon-like peptide 1 are well-known to one of ordinary skill in the art and described in, for example, MacDonald, et al., Diabetes, 51(supp. 3):S434-S442 (2002) and WO1995/017510. 
     In one embodiment, the gene encoding the glucagon-like peptide 1 has at least about 80% identity with a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. In another embodiment, the gene encoding the glucagon-like peptide 1 has at least about 85% identity with a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. In one embodiment, the gene encoding the glucagon-like peptide 1 has at least about 90% identity with a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. In one embodiment, the gene encoding the glucagon-like peptide 1 has at least about 95% identity with a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. In another embodiment, the gene encoding the glucagon-like peptide 1 has at least about 96%, 97%, 98%, or 99% identity with a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. Accordingly, in one embodiment, the gene encoding the glucagon-like peptide 1 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 a nucleic acid sequence encoding SEQ ID NO:40. In another embodiment, the gene encoding the glucagon-like peptide 1 comprises a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. In yet another embodiment the gene encoding the glucagon-like peptide 1 consists of a nucleic acid sequence encoding SEQ ID NO: 71 or SEQ ID NO: 72. 
     In one embodiment, the gene encoding the glucagon-like peptide 1 is directly operably linked to a first promoter. In another embodiment, the gene encoding the glucagon-like peptide 1 is indirectly operably linked to a first promoter. In one embodiment, the promoter is not operably linked with the gene encoding the glucagon-like peptide 1 in nature. 
     In some embodiments, the gene encoding the glucagon-like peptide 1 is expressed under the control of a constitutive promoter. In another embodiment, the gene encoding the glucagon-like peptide 1 is expressed under the control of an inducible promoter. In some embodiments, the gene encoding the glucagon-like peptide 1 is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the gene encoding the glucagon-like peptide 1 is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the gene encoding the glucagon-like peptide 1 is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. In one embodiment, the gene encoding the glucagon-like peptide 1 is expressed under the control of a promoter that is directly or indirectly induced 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. Inducible promoters are described in more detail infra. 
     The gene encoding the glucagon-like peptide 1 may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the gene encoding the glucagon-like peptide 1 is located on a plasmid in the bacterial cell. In another embodiment, the gene encoding the glucagon-like peptide 1 is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the gene encoding the glucagon-like peptide 1 is located in the chromosome of the bacterial cell, and a second gene encoding a second glucagon-like peptide 1 is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding the glucagon-like peptide 1 is located on a plasmid in the bacterial cell, and a second gene encoding a second glucagon-like peptide 1 is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding the glucagon-like peptide 1 is located in the chromosome of the bacterial cell, and a second gene encoding a second glucagon-like peptide 1 is located in the chromosome of the bacterial cell. 
     In some embodiments, the gene encoding the glucagon-like peptide 1 is expressed on a low-copy plasmid. In some embodiments, the gene encoding the glucagon-like peptide 1 is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the glucagon-like peptide 1, thereby reducing the degree of lipotoxic metabolites, pro-inflammatory substrate, and hepatic lipid deposition prevalent to those suffering from non-alcoholic steatohepatitis. 
     In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding GLP-1 (1-37), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 73. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding GLP-1 (1-37) H-&gt;M substitution), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 74. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding GLP-1-(7-37), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 75. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding GLP-1-(7-36), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 76. 
     In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding glucagon preproprotein (NP_002045.1), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Proglucagon, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 78. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Glucagon, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 79. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Glicentin), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 80 In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Glicentin related peptide), or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 81. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Oxyntomodulin. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 82. 
     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: 73 through SEQ ID NO: 82. 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 with one or more of SEQ ID NO: 73 through SEQ ID NO: 82. 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 with one or more of SEQ ID NO: 73 through SEQ ID NO: 82. 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 with one or more of SEQ ID NO: 73 through SEQ ID NO: 82. 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 with one or more of SEQ ID NO: 73 through SEQ ID NO: 82. 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 with one or more of SEQ ID NO: 62 through SEQ ID NO: 70, and SEQ ID NO: 41. 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: 73 through SEQ ID NO: 82. 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: 73 through SEQ ID NO: 82. 
     In embodiments, the pro-glucagon derived polypeptides, GLP-1 polypeptides, GLP-1 analogs described herein, and functional variants or fragments thereof are secreted. In some embodiments, the genetically engineered bacteria comprise one or more cassettes encoding pro-glucagon derived polypeptides, GLP-1 polypeptides, GLP-1 analogs, and/or functional variants or fragments and a secretion gene cassette and/or mutations generating a leaky phenotype. In some embodiments, a flagellar type III secretion pathway is used to secrete pro-glucagon derived polypeptides, GLP-1 polypeptides, and/or GLP-1 analogs described herein. In some embodiments, a Type V Autotransporter Secretion System is used to secrete pro-glucagon derived polypeptides, GLP-1 polypeptides, and/or GLP-1 analogs described herein. In some embodiments, a Hemolysin-based Secretion System is used to secrete the pro-glucagon derived polypeptides, GLP-1 polypeptides, and/or GLP-1 analogs described herein. In alternate embodiments, the genetically engineered bacteria expressing the pro-glucagon derived polypeptides, GLP-1 polypeptides, and/or GLP-1 analogs described herein further comprise a non-native single membrane-spanning secretion system. As described herein. In some embodiments, the engineered bacteria expressing the pro-glucagon derived polypeptides, GLP-1 polypeptides, and/or GLP-1 analogs described herein. have one or more deleted or mutated membrane genes to generate a leaky phenotype as described herein. 
     In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Exenatide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 83. 
     In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Liraglutide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 84. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Lixisenatide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 85. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Albiglutide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 86. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Dulaglutide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 87. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Taspoglutide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 88. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding Semaglutide, or a functional fragment or variant thereof. In one embodiment, the genetically engineered bacteria comprise a gene cassette encoding SEQ ID NO: 89. 
     In one embodiment, one or more polypeptides encoded by the and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. 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: 83 through SEQ ID NO: 89. 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 with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. 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 with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. 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 with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. 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 with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. 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 with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. In yet another embodiment one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria consist of the sequence of with one or more of SEQ ID NO: 83 through SEQ ID NO: 89. 
     GLP-2 
     In some embodiments, the genetically engineered bacteria of the invention 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 (Yazbeck et al., 2009). Obesity is associated with systemic inflammation and intestinal permeability, and commensal bacteria that produce GLP-2 may ameliorate those symptoms of the metabolic disease (Musso et al., 2010). 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. In some embodiments, the genetically engineered bacteria are capable of expressing GLP-2 or proglucagon 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. 
     Bile Salts 
     Bile salts (also called conjugated bile acids) are cholesterol derivatives synthesized in the liver which comprise a steroid ring component conjugated with either taurine (taurocholic acid; TCA) or glycine (glycochenodeoxycholic acid; GCDCA). Bile salts act as signaling molecules to regulate systemic endocrine functions, including triglyceride, cholesterol, and glucose homeostasis (Houten et al.,  EMBO J.,  25:1419-1425 (2006) and Watanabe et al.,  Nature,  439:484-489 (2006)). Specifically, bile acids trigger cellular farnesoid X receptor (FXR)- and G-protein coupled receptor (TGR4)-mediated host responses. Additionally, bile salts have been shown to facilitate lipid absorption and repress bacterial cell growth in the small intestine, thereby influencing both host metabolic pathways and the microflora present in the gut (Jones et al.,  PNAS,  105(36):13580-13585 (2008) and Ridlon et al.,  J. Lipid Research,  47(2):241-259 (2006)). 
     Bile salts are stored in the gallbladder and then subsequently released into the duodenum via the common bile duct. In the small intestine, microbial bile salt hydrolase (BSH) enzymes remove the glycine or taurine molecules, a process referred to as deconjugation, to produce the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA). In the gut, bile acids are reabsorbed within the terminal ileum, while non-reabsorbed bile acids enter the large intestine. Once in the large intestine, bile acids are amenable to further modification by microbial 7α-dehydroxylase enzymes to yield secondary bile acids, such as deoxycholic acid (DCA) and lithocholic acid (LCA) (Joyce et al.,  Gut Microbes,  5(5):669-674 (2014); Bhowmik et al., Accepted Article, doi:10.1002/prot.24971 (2015); see also  FIG. 1 ). 
     It has been shown that bile salt metabolism is involved in host physiology (Ridlon et al.,  Current Opinion Gastroenterol.,  30(3):332 (2014) and Jones et al., 2008). For example, it is known that the expression of bile salt hydrolase enzymes functionally regulate host lipid metabolism and play a role in cholesterol metabolism and transport, circadian rhythm, gut homeostasis/barrier function, weight gain, adiposity, and possibly gastrointestinal cancers in the host (Joyce et al.,  PNAS,  111(20):7421-7426 (2014);  Zhou and Hylemon, Steroids,  86:62-68, (2014); Mitchell et al.,  Expert Opinion Biolog. Therapy,  13(5):631-642 (2013); and WO14/198857, the entire contents of each of which are expressly incorporated herein by reference). Specifically, potential effects of bile salt hydrolase-expressing bacteria on cholesterol metabolic pathways have been shown to upregulate the ATP binding cassette A1 (ABCA1), the ATP binding cassette G1 (ABCG1), the ATP binding cassette G5/G8 (ABCG5/G8), cholesterol 7 alpha-hydroxylase (CYP7A1), and liver X receptor (LXR), and to downregulate farnesoid X receptor (FXR), Niemann-Pick C1-like 1 (NPC1L1), and small heterodimer partner (SHP), which impacts cholesterol efflux, plasma HDL-C levels, biliary excretion, cholesterol catabolism, bile acid synthesis, cholesterol levels, and decreased intestinal cholesterol absorption, among other effects (Mitchel et al. (2014) and Zhou and Hylemon (2014)). Additionally, bile salt hydrolase activity has been shown to impact bile detoxification, gastrointestinal persistence, nutrition, membrane alterations, altered digestive functions (lipid malabsorption, weight loss), cholesterol lowering, cancer, and formation of gallstones (see Begley et al.,  Applied and Environmental Microbiology,  72(3):1729-1738 (2006)). Moreover, a  Clostridium scindens  bacterium expressing a 7α-dehydroxylase enzyme has been shown to produce resistance to  C. difficile  infection in hosts (Buffie et al.,  Nature,  517:205-208 (2015), and bile salt metabolism has been shown to play a role in both regulating the microbiome as well as in cirrhosis (Ridlon et al.,  Gut Microbes,  4(5):382-387 (2013) and Kakiyama et al.,  J. Hepatol.,  58(5):949-955 (2013)). Thus, a need exists for treatments which address the metabolism of bile salts in subjects in order to treat and prevent diseases and disorders in which bile salts play a role, such as cardiovascular disease, metabolic disease, cirrhosis, gastrointestinal cancer, and  C. difficile  infection. 
     As used herein, the term “bile salt” or “conjugated bile acid” refers to a cholesterol derivative that is synthesized in the liver and consists of a steroid ring component that is conjugated with either glycine (glycochenodeoxycholic acid; GCDCA) or taurine (taurocholic acid; TCA). Bile salts are stored in the gallbladder and then subsequently released into the duodenum. Bile salts act as signaling molecules to regulate systemic endocrine functions including triglyceride, cholesterol, and glucose homeostasis, and also facilitate lipid absorption. In the small intestine, microbial bile salt hydrolase (BSH) enzymes remove the glycine or taurine molecules to produce bile acids. 
     As used herein, the term “bile acid” or “unconjugated bile acid” refers to cholic acid (CA) or chenodeoxycholic acid (CDCA). In the gut, bile acids are reabsorbed within the terminal ileum, while non-reabsorbed bile acids enter the large intestine. In the large intestine, bile acids are amenable to further modification by microbial 7α-dehydroxylase enzymes to yield secondary bile acids, such as deoxycholic acid (DCA) and lithocholic acid (LCA). As used herein, the term “catabolism” refers to the processing, breakdown and/or degradation of a metabolite or a complex molecule, such as tryptophan or a bile salt, into compounds that are non-toxic or which can be utilized by the bacterial cell or can be exported inot the extracellular environment, where these compounds may function as effectors. 
     In one embodiment, the term “bile salt catabolism” refers to the processing, breakdown, and/or degradation of bile salts into unconjugated bile acid(s). In one embodiment, “abnormal catabolism” refers to any condition(s), disorder(s), disease(s), predisposition(s), and/or genetic mutations(s) that result in increased levels of bile salts. In one embodiment, “abnormal catabolism” refers to an inability and/or decreased capacity of a cell, organ, and/or system to process, degrade, and/or secrete bile salts. In healthy adult humans, 600 mg of bile salts are secreted daily. In one embodiment, said inability or decreased capacity of a cell, organ, and/or system to process and/or degrade bile salts is caused by the decreased endogenous deconjugation of bile salts, e.g., decreased endogenous deconjugation of bile salts into bile acids by the intestinal microbiota in the gut. In one embodiment, the inability or decreased capacity of a cell, organ, and/or system to process and/or degrade bile salts results from a decrease in the number of or activity of intestinal bile salt hydrolase (BSH)-producing microorganisms. 
     In one embodiment, a “disease associated with bile salts” or a “disorder associated with bile salts” is a disease or disorder involving the abnormal, e.g., increased, levels of bile salts in a subject. Alternatively, a disease or disorder associated with bile salts is a disease or disorder wherein a subject exhibits normal levels of bile salts, but wherein the subject would benefit from decreased levels of bile salts. Bile salts function to solubilize dietary fat and enable its absorption into host circulation, and healthy adult humans secrete about 600 mg of bile salts daily through the stool. Thus, decreasing increased levels of bile salts, or normal levels of bile salts, in a subject would result in less uptake of dietary fat, causing the subject&#39;s liver to pull cholesterol from systemic circulation as it attempts to synthesize more. Thus, in one embodiment, a subject having a disease or disorder associated with bile salts secretes about 600 mg of bile salts in their stool daily. In another embodiment, a subject having a disease or disorder associated with bile salts secretes more than 600 mg, 700 mg, 800 mg, 900 mg, or 1 g of bile salts in their stool daily. In one embodiment, a disease or disorder associated with bile salts is a cardiovascular disease. In another embodiment, a disease or disorder associated with bile salts is a metabolic disease. In another embodiment, a disease or disorder associated with bile salts is a liver disease, such as cirrhosis, nonalcoholic steatohepatitis (NASH), or progressive familialintrahepatic cholestasis type 2 (PFIC2). 
     As used herein, the terms “cardiovascular disease” or “cardiovascular disorder” are terms used to classify numerous conditions affecting the heart, heart valves, and vasculature (e.g., veins and arteries) of the body, and encompasses diseases and conditions including, but not limited to hypercholesterolemia, diabetic dyslipidemia, hypertension, arteriosclerosis, atherosclerosis, myocardial infarction, acute coronary syndrome, angina, congestive heart failure, aortic aneurysm, aortic dissection, iliac or femoral aneurysm, pulmonary embolism, primary hypertension, atrial fibrillation, stroke, transient ischemic attack, systolic dysfunction, diastolic dysfunction, myocarditis, atrial tachycardia, ventricular fibrillation, endocarditis, arteriopathy, vasculitis, atherosclerotic plaque, vulnerable plaque, acute coronary syndrome, acute ischemic attack, sudden cardiac death, peripheral vascular disease, coronary artery disease (CAD), peripheral artery disease (PAD), and cerebrovascular disease. As used herein, a subject having “hypercholesterolemia” may have a total cholesterol of greater than 4 mmol/L, and a low-density lipoprotein cholesterol (LDL) of greater than 3 mmol/L. 
     As used herein, the terms “metabolic disease” or “metabolic disorder” refer to diseases caused by lipid and cholesterol metabolic pathways that are regulated by or affected by bile salts and bile acids. For example, cholesterol metabolic diseases and disorders include diabetes (including Type 1 diabetes, Type 2 diabetes, and maturity onset diabetes of the young (MODY)), obesity, weight gain, gallstones, hypertriglyceridemia, hyperfattyacidemia, and hyperinsulinemia. 
     As used herein, the term “bile salt hydrolase” enzyme refers to an enzyme involved in the cleavage of the amino acid sidechain of glycol- or tauro-conjugated bile acids to generate unconjugated bile acids ( FIG. 2 ). Bile salt hydrolase (BSH) enzymes are well known to those of skill in the art. For example, bile salt hydrolase activity has been detected in  Lactobacillus  spp.,  Bifidobacterium  spp.,  Enterococcus  spp.,  Clostridum  spp.,  Bacteroides  spp.,  Methanobrevibacter  spp., and  Listeria  spp. See, for example, Begley et al.,  Applied and Environmental Microbiology,  72(3):1729-1738 (2006); Jones et al.,  Proc. Natl. Acad. Sci.,  105(36):13580-13585 (2008); Ridlon et al.,  J. Lipid Res.,  47(2):241-259 (2006); and WO2014/198857, the entire contents of each of which are expressly incorporated herein by reference. 
     Bile Salt Hydrolases 
     The bacterial cells described herein comprise a heterologous gene encoding a bile salt hydrolase enzyme and are capable of deconjugating bile salts into unconjugated bile acids (see  FIGS. 1 and 2 ). 
     In one embodiment, the bile salt hydrolase enzyme increases the rate of bile salt catabolism in the cell. In one embodiment, the bile salt hydrolase enzyme decreases the level of bile salts in the cell or in the subject. In one embodiment, the bile salt hydrolase enzyme decreases the level of taurocholic acid (TCA) in the cell or in the subject. In one embodiment, the bile salt hydrolase enzyme decreases the level of glycochenodeoxycholic acid (GCDCA) in the cell or in the subject. Methods for measuring the rate of bile salt catabolism and the level of bile salts and bile acids are well known to one of ordinary skill in the art. For example, bile salts and acids may be extracted from a sample, and standard LC/MS methods may be used to determine the rate of bile salt catabolism and/or level of bile salts and bile acids. 
     In another embodiment, the bile salt hydrolase enzyme increases the level of bile acids in the cell or in the subject as compared to the level of bile salts in the cell or in the subject. In another embodiment, the bile salt hydrolase enzyme increases the level of cholic acid (CA) in the cell. In another embodiment, the bile salt hydrolase enzyme increases the level of chenodeoxycholic acid (CDCA) in the cell. 
     Enzymes involved in the catabolism of bile salts may be expressed or modified in the bacteria of the disclosure in order to enhance catabolism of bile salts. Specifically, when a bile salt hydrolase enzyme is expressed in the recombinant bacterial cells of the disclosure, the bacterial cells convert more bile salts into unconjugated bile acids when the bile salt hydrolase enzyme is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. In another embodiment, when a bile salt hydrolase enzyme is expressed in the recombinant bacterial cells of the disclosure, the bacterial cells convert more bile salts, such as TCA or GCDCA, into CA and CDCA when the bile salt hydrolase enzyme 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 bile salt hydrolase enzyme can catabolize bile salts to treat disorders associated with bile salts, including cardiovascular diseases, metabolic diseases, liver disease, such as cirrhosis or NASH, gastrointestinal cancers, and  C. difficile  infection. 
     In one embodiment, the bacterial cell comprises a heterologous gene encoding a bile salt hydrolase enzyme. In some embodiments, the disclosure provides a bacterial cell that comprises a heterologous gene encoding a bile salt hydrolase enzyme operably linked to a first promoter. In one embodiment, the first promoter is an inducible promoter. In one embodiment, the bacterial cell comprises a gene encoding a bile salt hydrolase enzyme 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 bile salt hydrolase enzyme. In yet another embodiment, the bacterial cell comprises at least one native gene encoding a bile salt hydrolase enzyme, as well as at least one copy of a gene encoding a bile salt hydrolase enzyme 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 bile salt hydrolase enzyme. In one embodiment, the bacterial cell comprises multiple copies of a gene or genes encoding a bile salt hydrolase enzyme. 
     Multiple distinct bile salt hydrolase enzymes are known in the art. In some embodiments, bile salt hydrolase enzyme is encoded by a gene encoding a bile salt hydrolase enzyme derived from a bacterial species. In some embodiments, a bile salt hydrolase enzyme is encoded by a gene encoding a bile salt hydrolase enzyme derived from a non-bacterial species. In some embodiments, a bile salt hydrolase enzyme is encoded by a gene derived from a eukaryotic species, e.g., a fungi. In one embodiment, the gene encoding the bile salt hydrolase enzyme is derived from an organism of the genus or species that includes, but is not limited to,  Lactobacillus  spp., such as  Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus brevis , or  Lactobacillus  gasseri;  Bifidobacterium  spp., such as  Bifidobacterium longum, Bifidobacterium bifidum , or  Bifidobacterium adolescentis; Bacteroides  spp., such as  Bacteroides fragilis  or  Bacteroides vlugatus; Clostridium  spp., such as  Clostridium perfringens; Listeria  spp., such as  Listeria monocytogenes, Enterococcus  spp., such as  Enterococcus faecium  or  Enterococcus faecalis; Brucella  spp., such as  Brucella abortus; Methanobrevibacter  spp., such as  Methanobrevibacter smithii, Staphylococcus  spp., such as  Staphylococcus aureus, Mycobacterium  spp., such as  Mycobacterium tuberculosis; Salmonella  spp., such as  Salmonella enterica; Listeria  spp., such as  Listeria monocytogenes.    
     In one embodiment, the gene encoding the bile salt hydrolase enzyme has been codon-optimized for use in the recombinant bacterial cell. In one embodiment, the gene encoding the bile salt hydrolase enzyme has been codon-optimized for use in  Escherichia coli . In another embodiment, the gene encoding the bile salt hydrolase enzyme has been codon-optimized for use in  Lactococcus . When the gene encoding the bile salt hydrolase enzyme is expressed in the recombinant bacterial cells, the bacterial cells catabolize more bile salt 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 gene encoding a bile salt hydrolase enzyme may be used to catabolize excess bile salts to treat a disorder associated with bile salts, such as cardiovascular disease, metabolic disease, liver disease, such as cirrhosis or NASH. 
     The present disclosure further comprises genes encoding functional fragments of a bile salt hydrolase enzyme or functional variants of a bile salt hydrolase enzyme. As used herein, the term “functional fragment thereof” or “functional variant thereof” of a bile salt hydrolase enzyme relates to an element having qualitative biological activity in common with the wild-type bile salt hydrolase enzyme from which the fragment or variant was derived. For example, a functional fragment or a functional variant of a mutated bile salt hydrolase enzyme is one which retains essentially the same ability to catabolize bile salts as the bile salt hydrolase enzyme from which the functional fragment or functional variant was derived. For example, a polypeptide having bile salt hydrolase enzyme activity may be truncated at the N-terminus or C-terminus and the retention of bile salt hydrolase enzyme activity assessed using assays known to those of skill in the art, including the exemplary assays provided herein. In one embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a bile salt hydrolase enzyme functional variant. In another embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a bile salt hydrolase enzyme functional fragment. 
     Assays for testing the activity of a bile salt hydrolase enzyme, a bile salt hydrolase enzyme functional variant, or a bile salt hydrolase enzyme functional fragment are well known to one of ordinary skill in the art. For example, bile salt catabolism can be assessed by expressing the protein, functional variant, or fragment thereof, in a recombinant bacterial cell that lacks endogenous bile salt hydrolase enzyme activity. Bile salt hydrolase activity can be assessed using a plate assay as described in Dashkevicz and Feighner,  Applied Environ. Microbiol.,  55:11-16 (1989) and Christiaens et al.,  Appl. Environ. Microbiol.,  58:3792-3798 (1992), the entire contents of each of which are expressly incorporated herein by reference. Briefly, bacterial cultures that are grown overnight can be spotted onto LB bile agar supplemented with either 0.5% (wt/vol) TDCA, 0.5% (wt/vol) GDCA, or 3% (vol/vol) human bile. BSH activity can be indicated by halos of precipitated deconjugated bile acids (see, also, Jones et al.,  PNAS,  105(36):13580-13585 (2008), the entire contents of which are expressly incorporated herein by reference). A ninhydrine assay for free taurine has also been described (see, for example, Clarke et al.,  Gut Microbes,  3(3):186-202 (2012), the entire contents of which are expressly incorporated herein by reference. Alternatively, a mouse model can be used to assay bile salt and bile acid signatures in vivo (see, for example, Joyce et al.,  PNAS,  111(20):7421-7426 (2014), the entire contents of which are expressly incorporated herein by reference). The present disclosure encompasses genes encoding a bile salt hydrolase enzyme comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein. 
     In some embodiments, the gene encoding a bile salt hydrolase enzyme is mutagenized; mutants exhibiting increased activity are selected; and the mutagenized gene encoding the bile salt hydrolase 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 gene encoding the bile salt hydrolase enzyme is from  Lactobacillus  spp. In one embodiment, the Lacotbacillus spp. is  Lactobacillus plantarum  WCFS1,  Lactobacillus plantarum  80,  Lactobacillus johnsonii  NCC533,  Lactobacillus johnsonii  100-100,  Lactobacillus acidophilus  NCFM ATCC700396,  Lactobacillus brevis  ATCC 367,  Lactobacillus gasseri  ATCC 33323, or  Lactobacillus acidophilus . In another embodiment, the gene encoding the bile salt hydrolase enzyme is from a  Bifidobacterium  spp. In one embodiment, the  Bifidobacterium  spp. is  Bifidobacterium longum  NCC2705,  Bifidobacterium longum  DJO10A,  Bifidobacterium longum  BB536,  Bifidobacterium longum  SBT2928,  Bifidobacterium bifidum  ATCC 11863, or  Bifidobacterium adolescentis . In another embodiment, the gene encoding the bile salt hydrolase enzyme is from  Bacteroides  spp. In one embodiment, the  Bacteroides  spp. is  Bacteroides fragilis  or  Bacteroides vlugatus . In another embodiment, the gene encoding the bile salt hydrolase enzyme is from  Clostridium  spp. In one embodiment, the  Clostridum  spp. is  Clostridum perfringens  MCV 185 or  Clostridum perfringens  13. In another embodiment, the gene encoding the bile salt hydrolase enzyme is from  Listeria  spp. In one embodiment, the  Listeria  spp. is  Listeria monocytogenes . In one embodiment, the gene encoding the bile salt hydrolase enzyme is from  Methanobrevibacter  spp. In one embodiment, the  Methanobrevibacter  spp. is  Methanobrevibacter smithii . Other genes encoding bile salt hydrolase enzymes are well-known to one of ordinary skill in the art and described in, for example, Jones et al.,  PNAS,  105(36):13580-13585 (2008) and WO2014/198857. Table 11 lists non-limiting examples of bile salt hydrolases. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Bile Salt Hydrolases 
               
            
           
           
               
               
            
               
                 Gene or Operon 
                 Sequence 
               
               
                   
               
               
                 Bile salt hydrolase 
                 ATGTGTACTGCCATAACTTATCAATCTTATAATAATTACTTC 
               
               
                 from  Lactobacillus   
                 GGTAGAAATTTCGATTATGAAATTTCATACAATGAAATGGTT 
               
               
                 
                   plantarum 
                 
                 ACGATTACGCCTAGAAAATATCCACTAGTATTTCGTAAGGTG 
               
               
                 SEQ ID NO: 90 
                 GAGAACTTAGATCACCATTATGCAATAATTGGAATTACTGCT 
               
               
                   
                 GATGTAGAAAGCTATCCACTTTACTACGATGCGATGAATGAA 
               
               
                   
                 AAAGGCTTGTGTATTGCGGGATTAAATTTTGCAGGTTATGCT 
               
               
                   
                 GATTATAAAAAATATGATGCTGATAAAGTTAATATCACACCA 
               
               
                   
                 TTTGAATTAATTCCTTGGTTATTGGGACAATTTTCAAGTGTT 
               
               
                   
                 AGAGAAGTGAAAAAGAACATACAAAAACTAAACTTGGTTAAT 
               
               
                   
                 ATTAATTTTAGTGAACAATTACCATTATCACCGCTACATTGG 
               
               
                   
                 TTGGTTGCTGATAAACAGGAATCGATAGTTATTGAAAGTGTC 
               
               
                   
                 AAAGAAGGACTAAAAATTTACGACAATCCAGTAGGTGTGTTA 
               
               
                   
                 ACAAACAATCCTAATTTTGACTACCAATTATTTAATTTGAAC 
               
               
                   
                 AACTATCGTGCCTTATCAAATAGCACACCCCAAAATAGTTTT 
               
               
                   
                 TCGGAAAAAGTGGATTTAGATAGTTATAGTAGAGGAATGGGC 
               
               
                   
                 GGACTAGGATTACCTGGAGACTTGTCCTCAATGTCTAGATTT 
               
               
                   
                 GTCAGAGCCGCTTTTACTAAATTAAACTCGTTGTCGATGCAG 
               
               
                   
                 ACAGAGAGTGGCAGTGTTAGTCAGTTTTTCCATATACTAGGG 
               
               
                   
                 TCTGTAGAACAACAAAAAGGGCTATGTGAAGTTACTGACGGA 
               
               
                   
                 AAGTACGAATATACAATCTATTCTTCTTGTTGTGATATGGAC 
               
               
                   
                 AAAGGAGTTTATTACTATAGAACTTATGACAATAGTCAAATT 
               
               
                   
                 AACAGTGTCAGTTTAAACCATGAGCACTTGGATACGACTGAA 
               
               
                   
                 TTAATTTCTTATCCATTACGATCAGAAGCACAATACTATGCA 
               
               
                   
                 GTTAACTAA 
               
               
                   
               
               
                 Bile salt hydrolase 
                 MCTAITYQSYNNYFGRNFDYEISYNEMVTITPRKYPLVFRKV 
               
               
                 protein from 
                 ENLDHHYAIIGITADVESYPLYYDAMNEKGLCIAGLNFAGYA 
               
               
                 
                   Lactobacillus 
                 
                 DYKKYDADKVNITPFELIPWLLGQFSSVREVKKNIQKLNLVN 
               
               
                 
                   plantarum 
                 
                 INFSEQLPLSPLHWLVADKQESIVIESVKEGLKIYDNPVGVL 
               
               
                 SEQ ID NO: 91 
                 TNNPNFDYQLFNLNNYRALSNSTPQNSFSEKVDLDSYSRGMG 
               
               
                   
                 GLGLPGDLSSMSRFVRAAFTKLNSLSMQTESGSVSQFFHILG 
               
               
                   
                 SVEQQKGLCEVTDGKYEYTIYSSCCDMDKGVYYYRTYDNSQI 
               
               
                   
                 NSVSLNHEHLDTTELISYPLRSEAQYYAVN 
               
               
                   
               
               
                 Bile salt hydrolase 
                 ATGTGTACTGCTGCAAATTATTTAACAAAATGCCATTATTTT 
               
               
                 from 
                 GGCCGTAATTTTGACTATGAAATTTCATATAATGAAAGAGTA 
               
               
                 
                   Methanobrevibacter 
                 
                 ACGATAACTCCTAGAAACTATCCTTTAATATTCAGGGATACT 
               
               
                   smithii  3142 
                 GAGGACATTGAAAATCATTATGGGATTATTGGCATAGCTGCA 
               
               
                 SEQ ID NO: 92 
                 GGTATTGATGAATATCCTTTGTATTATGATGCATGTAATGAG 
               
               
                   
                 AAAGGATTAGCTATGGGGGGATTAAACTTTCCGGATTACTGT 
               
               
                   
                 GACTACAAACCACTAGATAAATCTAAAGTTAACATAGCTTCT 
               
               
                   
                 TTTGAGATTATTCCATATATATTATCTCAAGCAAAAACCATC 
               
               
                   
                 AGTGATGCCGAAAGGTTATTGGAAAACTTAAATATTTCAGAT 
               
               
                   
                 GAGAAATTTTCCGCCCAGTTGCCTCCATCTCCACTTCATTGG 
               
               
                   
                 ATTATTTCAGATAGGAATGCTTCAATTGTTGTAGAGGTTGTA 
               
               
                   
                 GAGGAAGGACTGGATATTTATGATAATCCTGTAGGAGTTTTA 
               
               
                   
                 ACAAACAACCCTCCTTTTGATAAACAGCTATTTAATTTAAAT 
               
               
                   
                 AATTATATGGCATTATCAAACAGAACGCCTGAAAATACCTTT 
               
               
                   
                 GGAGGCAATTTGGATTTGGCAACTTATAGTCGGGGAATGGGT 
               
               
                   
                 TCAATTGGTCTTCCGGGGGATGTTTCTTCACAGTCCCGTTTT 
               
               
                   
                 GTAAAAGCAGCTTTTGTTAAAGAAAATTCCGTTTCCGGAGAT 
               
               
                   
                 TCTGAAAAAGAAAGTGTGTCTCAGTTTTTCCATATTCTGGCA 
               
               
                   
                 TCTGTTGAACAGCAAAAAGGATGTACGTTAGTGGAAGAACCT 
               
               
                   
                 GATAAATTTGAGTATACTATTTATTCAGACTGTTACAATACA 
               
               
                   
                 GATAAGGGAATATTGTATTATAAAACATATGATGGTCCTCAA 
               
               
                   
                 ACATCTGTTAATATACATGATGAGGATTTGGAAACCAATCAG 
               
               
                   
                 TTAATTAATTTTGAGTTGGTTGATTAA 
               
               
                   
               
               
                 Bile salt hydrolase 
                 MCTAANYLTKCHYFGRNFDYEISYNERVTITPRNYPLIFRDT 
               
               
                 protein from 
                 EDIENHYGIIGIAAGIDEYPLYYDACNEKGLAMGGLNFPDYC 
               
               
                 
                   Methanobrevibacter 
                 
                 DYKPLDKSKVNIASFEIIPYILSQAKTISDAERLLENLNISD 
               
               
                   smithii  3142 
                 EKFSAQLPPSPLHWIISDRNASIVVEVVEEGLDIYDNPVGVL 
               
               
                 SEQ ID NO: 93 
                 TNNPPFDKQLFNLNNYMALSNRTPENTFGGNLDLATYSRGMG 
               
               
                   
                 SIGLPGDVSSQSRFVKAAFVKENSVSGDSEKESVSQFFHILA 
               
               
                   
                 SVEQQKGCTLVEEPDKFEYTIYSDCYNTDKGILYYKTYDGPQ 
               
               
                   
                 TSVNIHDEDLETNQLINFELVD 
               
               
                   
               
               
                 Bile salt hydrolase 
                 ATGGTTATGAAAAAGATTTTGATAGCTTTGGCCTTATTGCTG 
               
               
                 from  Bacteroides   
                 ACAGGCATTGCAAGCGGATCGGCATGTACCGGTATTTCATTC 
               
               
                 
                   vulgatus 
                 
                 CTCGCTGAAGATGGCGGATATGTGCAGGCACGTACTATAGAG 
               
               
                 SEQ ID NO: 94 
                 TGGGGGAACAGTTATCTTCCGAGTGAATATGTTATTGTTCCC 
               
               
                   
                 AGAGGACAGGATTTGGTATCTTATACTCCAACGGGTGTAAAT 
               
               
                   
                 GGCTTGAGATTTCGGGCTAAATATGGTCTGGTAGGACTGGCT 
               
               
                   
                 ATCATTCAGAAAGAGTTTGTGGCTGAAGGACTGAATGAAGTA 
               
               
                   
                 GGGCTTTCGGCTGGATTGTTTTATTTTCCCCATTATGGGAAG 
               
               
                   
                 TATGAAGAATATGATGAGGCTCAAAATGCAATTACTTTGTCG 
               
               
                   
                 GATTTGCAGGTGGTGAACTGGATGCTTTCCCAATTTGCTACT 
               
               
                   
                 ATAGACGAAGTGAGAGAAGCTATAGAAGGGGTGAAGGTGGTG 
               
               
                   
                 TCTCTTGATAAACCTGGTAAAAGTTCTACGGTACATTGGCGC 
               
               
                   
                 ATTGGCGATGCTAAAGGAAATCAAATGGTGTTGGAATTTGTA 
               
               
                   
                 GGTGGTGTTCCTTATTTTTATGAAAATAAAGTAGGAGTACTC 
               
               
                   
                 ACCAATTCTCCCGATTTTCCATGGCAGGTGATTAACTTGAAT 
               
               
                   
                 AATTATGTAAATCTATATCCGGGAGCTGTCACTCCACAGCAA 
               
               
                   
                 TGGGGTGGGGTGACTATTTTCCCTTTTGGCGCAGGTGCCGGA 
               
               
                   
                 TTTCATGGTATTCCGGGGGATGTAACTCCTCCATCCCGTTTT 
               
               
                   
                 GTTCGTGTAGCGTTTTATAAGGCAACAGCTCCGGTGTGTCCT 
               
               
                   
                 ACAGCGTATGACGCTATATTACAAAGCTTTCATATCCTGAAT 
               
               
                   
                 AATTTTGATATTCCTATTGGTATAGAATATGCGTTAGGGAAA 
               
               
                   
                 GCACCTGATATTCCTAGTGCCACACAATGGACTTCGGCTATT 
               
               
                   
                 GATTTGACAAACAGGAAAGTGTATTATAAAACAGCATACAAT 
               
               
                   
                 AACAATATTCGTTGTATTAGTATGAAGAAGATTGATTTTGAT 
               
               
                   
                 AAAGTGAAGTATCAGTCGTATCCATTGGATAAGGAGTTGAAA 
               
               
                   
                 CAGCCTGTAGAAGAGATTATTGTGAAATAG 
               
               
                   
               
               
                 Bile salt hydrolase 
                 MVMKKILIALALLLTGIASGSACTGISFLAEDGGYVQARTIE 
               
               
                 protein from 
                 WGNSYLPSEYVIVPRGQDLVSYTPTGVNGLRFRAKYGLVGLA 
               
               
                 
                   Bacteroides 
                 
                 IIQKEFVAEGLNEVGLSAGLFYFPHYGKYEEYDEAQNAITLS 
               
               
                 
                   vulgatus 
                 
                 DLQVVNWMLSQFATIDEVREAIEGVKVVSLDKPGKSSTVHWR 
               
               
                 SEQ ID NO: 95 
                 IGDAKGNQMVLEFVGGVPYFYENKVGVLTNSPDFPWQVINLN 
               
               
                   
                 NYVNLYPGAVTPQQWGGVTIFPFGAGAGFHGIPGDVTPPSRF 
               
               
                   
                 VRVAFYKATAPVCPTAYDAILQSFHILNNFDIPIGIEYALGK 
               
               
                   
                 APDIPSATQWTSAIDLTNRKVYYKTAYNNNIRCISMKKIDFD 
               
               
                   
                 KVKYQSYPLDKELKQPVEEIIVK 
               
               
                   
               
               
                 Bile salt hydrolase 
                 ATGTGCACTGGTGTCCGTTTCTCCGATGATGAGGGCAACAC 
               
               
                 from 
                 CTATTTCGGCCGTAATCTCGACTGGAGTTTCTCATATGGGG 
               
               
                 
                   Bifidobacterium 
                 
                 AGACCATCCTGGTTACTCCGCGCGGCTACCACTATGACACG 
               
               
                 
                   longum 
                 
                 GTGTTTGGTGCGGGCGGCAAGGCGAAGCCGAACGCGGTGAT 
               
               
                 SEQ ID NO: 96 
                 CGGCGTGGGTGTGGTCATGGCCGATAGGCCGATGTATTTCG 
               
               
                   
                 ACTGCGCCAATGAACATGGTCTGGCCATCGCCGGCTTGAAT 
               
               
                   
                 TTCCCCGGCTACGCCTCGTTCGTCCACGAACCGGTCGAAGG 
               
               
                   
                 CACGGAAAACGTCGCCACGTTCGAATTTCCGCTGTGGGTGG 
               
               
                   
                 CGCGTAATTTCGACTCCGTCGACGAGGTCGAGGAGGCGCTC 
               
               
                   
                 AGGAACGTGACGCTCGTCTCCCAGATCGTGCCGGGACAGCA 
               
               
                   
                 GGAGTCTCTGCTGCACTGGTTCATCGGCGACGGCAAGCGCA 
               
               
                   
                 GCATCGTCGTCGAGCAGATGGCCGATGGCATGCACGTGCAT 
               
               
                   
                 CATGATGACGTCGATGTGCTGACCAATCAGCCGACGTTCGA 
               
               
                   
                 CTTCCATATGGAAAACCTGCGCAACTACATGTGCGTCAGCA 
               
               
                   
                 ACGAGATGGCCGAACCGACTTCATGGGGCAAGGCCTCCTTG 
               
               
                   
                 ACCGCCTGGGGTGCGGGTGTGGGCATGCATGGCATCCCGGG 
               
               
                   
                 CGACGTGAGTTCCCCGTCGCGCTTCGTTCGTGTGGCCTACA 
               
               
                   
                 CCAACGCGCATTACCCGCAGCAGAACGATGAAGCCGCCAAT 
               
               
                   
                 GTGTCGCGCCTGTTCCACACCCTCGGCTCCGTGCAGATGGT 
               
               
                   
                 GGACGGCATGGCGAAGATGGGCGACGGCCAGTTCGAACGCA 
               
               
                   
                 CGCTGTTCACCAGCGGATATTCGTCCAAGACCAACACCTAT 
               
               
                   
                 TACATGAACACCTATGATGACCCCGCCATCCGTTCCTACGC 
               
               
                   
                 CATGGCCGATTACGATATGGATTCCTCGGAGCTCATCAGCG 
               
               
                   
                 TCGCCCGATGA 
               
               
                   
               
               
                 Bile salt hydrolase 
                 MCTGVRFSDDEGNTYFGRNLDWSFSYGETILVTPRGYHYDTV 
               
               
                 protein from 
                 FGAGGKAKPNAVIGVGVVMADRPMYFDCANEHGLAIAGLNFP 
               
               
                 
                   Bifidobacterium 
                 
                 GYASFVHEPVEGTENVATFEFPLWVARNFDSVDEVEEALRNV 
               
               
                 
                   longum 
                 
                 TLVSQIVPGQQESLLHWFIGDGKRSIVVEQMADGMHVHHDDV 
               
               
                 SEQ ID NO: 97 
                 DVLTNQPTFDFHMENLRNYMCVSNEMAEPTSWGKASLTAWGA 
               
               
                   
                 GVGMHGIPGDVSSPSRFVRVAYTNAHYPQQNDEAANVSRLFH 
               
               
                   
                 TLGSVQMVDGMAKMGDGQFERTLFTSGYSSKTNTYYMNTYDD 
               
               
                   
                 PAIRSYAMADYDMDSSELISVAR 
               
               
                   
               
               
                 Bile salt hydrolase 
                 ATGTGTACGTCAATAACTTATACAACGAAGGATCACTATTT 
               
               
                 from  Listeria   
                 TGGAAGGAATTTCGATTATGAACTTTCTTACAAAGAAGTTG 
               
               
                 
                   monocytogenes 
                 
                 TGGTTGTTACGCCGAAAAATTACCCGTTCCATTTTCGCAAG 
               
               
                 SEQ ID NO: 98 
                 GTAGAGGATATAGAGAAGCATTATGCACTTATTGGTATTGC 
               
               
                   
                 TGCTGTGATGGAAAACTACCCGTTGTATTACGATGCTACCA 
               
               
                   
                 ATGAAAAAGGCCTTAGTATGGCAGGACTCAATTTCTCAGGA 
               
               
                   
                 AATGCGGATTACAAGGATTTTGCAGAAGGTAAGGACAATGT 
               
               
                   
                 GACCCCCTTTGAATTTATTCCGTGGATTCTTGGTCAATGCG 
               
               
                   
                 CTACTGTAAAAGAAGCAAGAAGATTACTTCAGAGAATCAAT 
               
               
                   
                 CTCGTGAATATTAGTTTTAGTGAAAATTTACCGCTGTCTCC 
               
               
                   
                 ATTACATTGGTTGATGGCTGATCAAACAGAATCTATTGTAG 
               
               
                   
                 TGGAATGTGTGAAAGATGGACTTCACATTTATGATAATCCT 
               
               
                   
                 GTTGGCGTGTTAACAAATAATCCAACATTTGATTACCAACT 
               
               
                   
                 ATTTAATTTAAACAATTATCGCGTTCTTTCGAGTGAAACCC 
               
               
                   
                 CAGAAAATAATTTTTCCAAAGAGATTGATTTGGATGCTTAT 
               
               
                   
                 AGTCGTGGGATGGGCGGAATTGGCTTACCTGGTGATTTATC 
               
               
                   
                 TTCTATGTCTCGTTTTGTGAAAGCAACTTTTACCAAATTGA 
               
               
                   
                 ATTCTGTTTCAGGTGATTCTGAATCAGAAAGTATTAGCCAA 
               
               
                   
                 TTTTTCCATATTTTAGGCTCGGTGGAACAACAAAAAGGTCT 
               
               
                   
                 TTGTGATGTTGGTGGGGGAAAATACGAGCATACTATTTATT 
               
               
                   
                 CCTCGTGTTGCAATATCGATAAAGGAATTTATTATTATAGA 
               
               
                   
                 ACATACGGAAACAGTCAAATTACTGGTGTGGATATGCACCA 
               
               
                   
                 AGAGGATTTAGAGAGCAAAGAACTAGCTATTTATCCACTCG 
               
               
                   
                 TCAATGAGCAACGACTAAACATTGTTAACAAATAA 
               
               
                   
               
               
                 Bile salt hydrolase 
                 MCTSITYTTKDHYFGRNFDYELSYKEVVVVTPKNYPFHFRKV 
               
               
                 protein from 
                 EDIEKHYALIGIAAVMENYPLYYDATNEKGLSMAGLNFSGNA 
               
               
                 
                   Listeria 
                 
                 DYKDFAEGKDNVTPFEFIPWILGQCATVKEARRLLQRINLVN 
               
               
                 
                   monocytogenes 
                 
                 ISFSENLPLSPLHWLMADQTESIVVECVKDGLHIYDNPVGVL 
               
               
                 SEQ ID NO: 99 
                 TNNPTFDYQLFNLNNYRVLSSETPENNFSKEIDLDAYSRGMG 
               
               
                   
                 GIGLPGDLSSMSRFVKATFTKLNSVSGDSESESISQFFHILG 
               
               
                   
                 SVEQQKGLCDVGGGKYEHTIYSSCCNIDKGIYYYRTYGNSQI 
               
               
                   
                 TGVDMHQEDLESKELAIYPLVNEQRLNIVNK 
               
               
                   
               
               
                 Bile salt hydrolase 
                 ATGTGTACAGGATTAGCCTTAGAAACAAAAGATGGATTACAT 
               
               
                 from  Clostridium   
                 TTGTTTGGAAGAAATATGGATATTGAATATTCATTTAATCAA 
               
               
                 
                   perfringens 
                 
                 TCTATTATATTTATTCCTAGGAATTTTAAATGTGTAAACAAA 
               
               
                 SEQ ID NO: 100 
                 TCAAACAAAAAAGAATTAACAACAAAATATGCTGTTCTTGGA 
               
               
                   
                 ATGGGAACTATTTTTGATGATTATCCTACCTTTGCAGATGGT 
               
               
                   
                 ATGAATGAAAAGGGATTAGGGTGTGCTGGCTTAAATTTCCCT 
               
               
                   
                 GTTTATGTTAGCTATTCTAAAGAAGATATAGAAGGTAAAACT 
               
               
                   
                 AATATTCCAGTATATAATTTCTTATTATGGGTTTTAGCTAAT 
               
               
                   
                 TTTAGCTCAGTAGAAGAGGTAAAGGAAGCATTAAAAAATGCT 
               
               
                   
                 AATATAGTGGATATACCTATTAGCGAAAATATTCCTAATACA 
               
               
                   
                 ACTCTTCATTGGATGATAAGCGATATAACAGGAAAGTCTATT 
               
               
                   
                 GTGGTTGAACAAACAAAGGAAAAATTAAATGTATTTGATAAT 
               
               
                   
                 AATATTGGAGTATTAACTAATTCACCTACTTTTGATTGGCAT 
               
               
                   
                 GTAGCAAATTTAAATCAATATGTAGGTTTGAGATATAATCAA 
               
               
                   
                 GTTCCAGAATTTAAGTTAGGAGATCAATCTTTAACTGCTTTA 
               
               
                   
                 GGTCAAGGAACTGGTTTAGTAGGATTACCAGGGGACTTTACA 
               
               
                   
                 CCTGCATCTAGATTTATAAGAGTAGCATTTTTAAGAGATGCA 
               
               
                   
                 ATGATAAAAAATGATAAAGATTCAATAGACTTAATTGAATTT 
               
               
                   
                 TTCCATATATTAAATAATGTTGCTATGGTAAGAGGATCAACT 
               
               
                   
                 AGAACTGTAGAAGAAAAAAGTGATCTTACTCAATATACAAGT 
               
               
                   
                 TGCATGTGTTTAGAAAAAGGAATTTATTATTATAATACCTAT 
               
               
                   
                 GAAAATAATCAAATTAATGCAATAGACATGAATAAAGAAAAC 
               
               
                   
                 TTAGATGGAAATGAAATTAAAACATATAAATACAACAAAACT 
               
               
                   
                 TTAAGTATTAATCATGTAAATTAG 
               
               
                   
               
               
                 Bile salt hydrolase 
                 MCTGLALETKDGLHLFGRNMDIEYSFNQSIIFIPRNFKCVNK 
               
               
                 protein from 
                 SNKKELTTKYAVLGMGTIFDDYPTFADGMNEKGLGCAGLNFP 
               
               
                 
                   Clostridium 
                 
                 VYVSYSKEDIEGKTNIPVYNFLLWVLANFSSVEEVKEALKNA 
               
               
                 
                   perfringens 
                 
                 NTVDIPISENIPNTTLHWMISDITGKSIVVEQTKEKLNVFDN 
               
               
                 SEQ ID NO: 101 
                 NIGVLTNSPTFDWHVANLNQYVGLRYNQVPEFKLGDQSLTAL 
               
               
                   
                 GQGTGLVGLPGDFTPASRFIRVAFLRDAMIKNDKDSIDLIEF 
               
               
                   
                 FHILNNVAMVRGSTRTVEEKSDLTQYTSCMCLEKGIYYYNTY 
               
               
                   
                 ENNQINAIDMNKENLDGNEIKTYKYNKTLSINHVN 
               
               
                   
               
               
                 Bile salt hydrolase 
                 ATGTGTACGTCTATTACTTATGTAACAAGTGATCATTATTTT 
               
               
                 from  Enterococcus   
                 GGAAGGAATTTTGATTATGAAATATCTTACAATGAAGTAGTT 
               
               
                 
                   faecium 
                 
                 ACTGTTACTCCAAGAAATTATAAGTTGAATTTTCGAAAGGTA 
               
               
                 SEQ ID NO: 102 
                 AATGATTTGGATACTCATTATGCAATGATTGGTATTGCCGCT 
               
               
                   
                 GGTATAGCTGACTACCCTCTTTATTACGATGCGACAAATGAA 
               
               
                   
                 AAAGGATTGAGTATGGCTGGGCTAAATTTTTCTGGGTATGCT 
               
               
                   
                 GATTATAAAGAAATACAAGAAGGGAAAGACAATGTATCTCCT 
               
               
                   
                 TTTGAATTTATTCCTTGGATTTTAGGACAATGCTCAACAGTA 
               
               
                   
                 GGAGAAGCTAAAAAATTGTTAAAAAATATCAATTTAGCAAAT 
               
               
                   
                 ATAAATTATAGTGACGAACTTCCTTTATCCCCTTTACATTGG 
               
               
                   
                 CTATTAGCTGATAAAGAAAAATCAATTGTCATTGAAAGTATG 
               
               
                   
                 AAAGATGGACTTCATATATATGATAACCCTGTGGGCGTTCTT 
               
               
                   
                 ACCAATAATCCTTCATTTGACTATCAATTATTTAATTTAAAC 
               
               
                   
                 AATTATCGTGTCTTATCGAGTGAAACTCCTAAAAATAATTTT 
               
               
                   
                 TCAAATCAAATAAGTTTGAATGCCTATAGCCGCGGTATGGGA 
               
               
                   
                 GGGATAGGCTTGCCTGGAGATTTATCCTCAGTATCTCGTTTT 
               
               
                   
                 GTTAAAGCGACTTTTACGAAGCTGAATTCTGTATCTGGAGAT 
               
               
                   
                 TCAGAGTCAGAAAGTATTAGTCAATTTTTCCATATCTTAGGT 
               
               
                   
                 TCAGTAGAACAACAAAAAGGTTTGTGTGATGTAGGTGATGGA 
               
               
                   
                 AAATATGAATATACAATTTATTCTTCTTGTTGCAATGTTGAC 
               
               
                   
                 AAAGGAATCTATTATTATCGAACATATGAAGACAGTCAAATT 
               
               
                   
                 ACTGCAATTGATATGAATAAAGAAGACTTAGATAGTCATAAG 
               
               
                   
                 TTAATTAGTTATCCAATTATAGAAAAACAACAAATTAAATAT 
               
               
                   
                 ATAAATTAG 
               
               
                   
               
               
                 Bile salt hydrolase 
                 MCTSITYVTSDHYFGRNFDYEISYNEVVTVTPRNYKLNFRKV 
               
               
                 protein from 
                 NDLDTHYAMIGIAAGIADYPLYYDATNEKGLSMAGLNFSGYA 
               
               
                 
                   Enterococcus 
                 
                 DYKEIQEGKDNVSPFEFIPWILGQCSTVGEAKKLLKNINLAN 
               
               
                 
                   faecium 
                 
                 INYSDELPLSPLHWLLADKEKSIVIESMKDGLHIYDNPVGVL 
               
               
                 SEQ ID NO: 103 
                 TNNPSFDYQLFNLNNYRVLSSETPKNNFSNQISLNAYSRGMG 
               
               
                   
                 GIGLPGDLSSVSRFVKATFTKLNSVSGDSESESISQFFHILG 
               
               
                   
                 SVEQQKGLCDVGDGKYEYTIYSSCCNVDKGIYYYRTYEDSQI 
               
               
                   
                 TAIDMNKEDLDSHKLISYPIIEKQQIKYIN 
               
               
                   
               
               
                 Bile salt hydrolase 
                 AAGAGAAAAATATGTGTACATCAATTATATTCAGTCCCAAAG 
               
               
                 A from 
                 ATCATTACTTTGGTCGTAACCTTGATTTAGAAATTACTTTTG 
               
               
                 
                   Lacotbacillus 
                 
                 GTCAACAAGTTGTTATTACGCCACGCAATTACACTTTTAAAT 
               
               
                 
                   acidophilus 
                 
                 TCCGTAAGATGCCCAGTTTAAAAAAGCACTATGCAATGATTG 
               
               
                 SEQ ID NO: 104 
                 GTATCTCATTAGATATGGATGATTATCCCCTATATTTCGACG 
               
               
                   
                 CTACAAATGAAAAAGGTTTAGGTATGGCCGGACTCAACTATC 
               
               
                   
                 CAGGAAATGCTACATATTATGAAGAAAAAGAAAATAAAGATA 
               
               
                   
                 ATATTGCTTCCTTTGAATTCATCCCTTGGATTTTAGGACAGT 
               
               
                   
                 GTAGCACTATTAGCGAAGTAAAGGATTTACTTAGCAGAATCA 
               
               
                   
                 ACATCGCCGATTTAAATTTCAGCGAAAAAATGCAAGCCTCCT 
               
               
                   
                 CTCTTCACTGGCTTATTGCAGATAAAACAGGTACATCATTAG 
               
               
                   
                 TTGTTGAAACAGACAAAGATGGAATGCATATTTATGATAATC 
               
               
                   
                 CAGTTGGCTGCTTAACTAATAATCCACAATTTCCAAAGCAAT 
               
               
                   
                 TATTCAATTTAAATAACTATGCTGACGTATCTCCAAAAATGC 
               
               
                   
                 CTAAAAATAACTTCTCAGATAAAGTAAATATGGCTGGCTACA 
               
               
                   
                 GCCGTGGATTAGGGTCTCACAACTTACCAGGTGGAATGGATT 
               
               
                   
                 CTGAATCACGTTTTGTCAGAGTAGCTTTCAATAAATTTAATG 
               
               
                   
                 CTCCAATTGCTGAAACCGAAGAAGAAAATATTGATACTTACT 
               
               
                   
                 TCCACATTTTACATTCGGTTGAACAACAAAAGGGACTGGATG 
               
               
                   
                 AAGTTGGTCCAAACTCATTTGAATATACAATTTATTCTGATG 
               
               
                   
                 GAACTAACTTAGACAAAGGTATTTTCTACTACACCACTTATT 
               
               
                   
                 CAAACAAACAAATTAACGTTGTTGATATGAATAAAGAAGATC 
               
               
                   
                 TAGATAGCAGCAATTTGATCACTTATGATATGCTTGATAAAA 
               
               
                   
                 CTAAATTTAACCATCAAAACTAA 
               
               
                   
               
               
                 Bile salt hydrolase 
                 MCTSIIFSPKDHYFGRNLDLEITFGQQVVITPRNYTFKFRKM 
               
               
                 A protein from 
                 PSLKKHYAMIGISLDMDDYPLYFDATNEKGLGMAGLNYPGNA 
               
               
                 
                   Lacotbacillus 
                 
                 TYYEEKENKDNIASFEFIPWILGQCSTISEVKDLLSRINIAD 
               
               
                 
                   acidophilus 
                 
                 LNFSEKMQASSLHWLIADKTGTSLVVETDKDGMHIYDNPVGC 
               
               
                 SEQ ID NO: 105 
                 LTNNPQFPKQLFNLNNYADVSPKMPKNNFSDKVNMAGYSRGL 
               
               
                   
                 GSHNLPGGMDSESRFVRVAFNKFNAPIAETEEENIDTYPHIL 
               
               
                   
                 HSVEQQKGLDEVGPNSFEYTIYSDGTNLDKGIFYYTTYSNKQ 
               
               
                   
                 INVVDMNKEDLDSSNLITYDMLDKTKFNHQN 
               
               
                   
               
               
                 Bile salt hydrolase 
                 AGAAAGCGTGCAGTAAATGTGTACATCAATTTGTTATAATC 
               
               
                 B from 
                 CTAACGATCATTATTTTGGTAGAAATCTTGACTATGAAATT 
               
               
                 
                   Lacotbacillus 
                 
                 GCTTATGGTCAAAAAGTAGTCATTGTACCAAGAAACTACGA 
               
               
                 
                   acidophilus 
                 
                 ATTTAAGTATAGAGAAATGCCCTCTCAAAAGATGCATTATG 
               
               
                 SEQ ID NO: 106 
                 CTTTTATCGGAGTATCTGTAGTTAATGATGATTATCCATTA 
               
               
                   
                 TTATGTGATGCAATTAATGAAAAGGGGCTTGGTATTGCAGG 
               
               
                   
                 ATTAAATTTTCAAGGTCCTAATCATTACTTTCCTAAAATCG 
               
               
                   
                 AAGGTAAGAAGAATATTGCTTCTTTTGAATTAATGCCATAC 
               
               
                   
                 TTATTAAGTAATTGTGAAAATACTGACGATGTTAAAGAAAT 
               
               
                   
                 CTTAGATAATGCAAATATTTTAAATATTAGCTTTTCAGCAA 
               
               
                   
                 ATTATCCTGCAGCTGATTTACATTGGATTTTAAGTGATAAA 
               
               
                   
                 GCTGGTAAGAGTATCGTAGTTGAATCAACCAATTCAGGTTT 
               
               
                   
                 ACATATTTATGATAATCCAGTGAATGTCTTAACTAACAATC 
               
               
                   
                 CTGAATTTCCGGATCAATTAATTAAATTAAGTGACTACGCC 
               
               
                   
                 GACGTTACTCCACATAATCCTAAGAATACATTGGTTCCTAA 
               
               
                   
                 TGTTGATCTTAATCTATATAGTAGAGGCTTAGGTACTCACC 
               
               
                   
                 ACTTACCTGGTGGAATGGATTCTAGCTCTCGATTTGTTAAG 
               
               
                   
                 GTAGCTTTTGTCTTGGCACACACTCCACAAGGAAAAAATGA 
               
               
                   
                 AGTGGAAAATGTTACTAATTATTTCCATATTCTGCATTCAG 
               
               
                   
                 TAGAACAACCTGATGGTTTAGATGAAGTAGAAGATAATCGC 
               
               
                   
                 TATGAATATACTATGTATACAGATTGTATGAACTTAGATAA 
               
               
                   
                 AGGTATTTTGTACTTTACTACTTATGACAATAATCGGATTA 
               
               
                   
                 ATGCAGTAGATATGCATAAAGCAGATTTAGATTCAGAAGAT 
               
               
                   
                 TTAATCTGCTACGATTTGTTTAAGAAACAAGATATTGAATA 
               
               
                   
                 TATGAATTAA 
               
               
                   
               
               
                 Bile salt hydrolase 
                 MCTSICYNPNDHYFGRNLDYEIAYGQKVVIVPRNYEFKYREM 
               
               
                 B protein from 
                 PSQKMHYAFIGVSVVNDDYPLLCDAINEKGLGIAGLNFQGPN 
               
               
                 
                   Lacotbacillus 
                 
                 HYFPKIEGKKNIASFELMPYLLSNCENTDDVKEILDNANILN 
               
               
                 
                   acidophilus 
                 
                 ISFSANYPAADLHWILSDKAGKSIVVESTNSGLHIYDNPVNV 
               
               
                 SEQ ID NO: 107 
                 LTNNPEFPDQLIKLSDYADVTPHNPKNTLVPNVDLNLYSRGL 
               
               
                   
                 GTHHLPGGMDSSSRFVKVAFVLAHTPQGKNEVENVTNYFHIL 
               
               
                   
                 HSVEQPDGLDEVEDNRYEYTMYTDCMNLDKGILYFTTYDNNR 
               
               
                   
                 INAVDMHKADLDSEDLICYDLFKKQDIEYMN 
               
               
                   
               
               
                 Bile salt hydrolase 
                 ATGGAAACGAAAAGCTCTCTCTGGAAATCATCGCGCCGCGT 
               
               
                 from  Brucella   
                 GCTTGCACATGGGGCTGCAACTGTTCTGGTCGCGGCGGGCC 
               
               
                 
                   abortus 
                 
                 TTATCGTTCCCCAGGCGGCTATGGCTTGCACGAGCTTCGTT 
               
               
                 SEQ ID NO: 108 
                 CTGCCGACGAGCGACGGTGGTATGGTCTATGGTCGCACGAT 
               
               
                   
                 GGAATTCGGGTTCAATCTCAAATCCGACATGATTGCCATTC 
               
               
                   
                 CGCGCAATTACACCATCACGGCAAGCGGGCCGGACGGTGCT 
               
               
                   
                 GCGGGCAAGAAATGGAAGGGCAAATATGCCACGATCGGCAT 
               
               
                   
                 GAATGCTTTTGGTATCGTCGCTCTCACCGACGGTATGAACG 
               
               
                   
                 AGAAGGGGCTTGCAGGCGGGCTTCTCTATTTCCCGGAATAT 
               
               
                   
                 GCCAAGTATCAGGACCCATCCACGGCGAAGCCGGAAGACAG 
               
               
                   
                 CCTCGCTCCGTGGGATTTCCTGACCTGGGCGCTGGCCAATT 
               
               
                   
                 TTTCGACAGTGGCCGAAGTCAAGGATGCTTTGAGCACCATT 
               
               
                   
                 TCCATCGTCGATGTGAAACAAAAGGACCTGGGATTTACCCC 
               
               
                   
                 GCCCGCTCACTACACGCTGCATGATGCGACCGGCGCATCCA 
               
               
                   
                 TCGTGATCGAACCGATCGACGGCAAGCTCAAGGTTTACGAC 
               
               
                   
                 AACAAGCTCGGTGTCATGACCAATTCGCCGTCTTTCGACTG 
               
               
                   
                 GCACATGACCAATCTGCGCAACTATGTCTATCTCTCGCGTG 
               
               
                   
                 AAAATCCGAAGCCGTTGCAGATCCTTGGCGAGACGATCCAG 
               
               
                   
                 TCATTCGGGCAAGGCGCCGGTATGCATGGTATTCCGGGCGA 
               
               
                   
                 CACCACGCCGCCATCGCGTTTCGTGCGTGCAAGCGCCTACG 
               
               
                   
                 TCCTTTCCGCCAAGAAGGTGCCGAGCGGCCTTGAAAGCGTG 
               
               
                   
                 CGGCTGGCCGAGCATATTGCCAATAACTTCGACATTCCAAA 
               
               
                   
                 GGGATGGAGCGAAGAGCAGAATATGTTTGAATATACCCAGT 
               
               
                   
                 GGACCGCCTTTGCGGACATGAAGAACGATGTCTATTACATC 
               
               
                   
                 AAGACCTATGACGATCAGGTTCTGCGCAGCTTCAGCTTCAA 
               
               
                   
                 GGATTTTGATGTCGATAGCAAAGATATTCTAACGATCAAGT 
               
               
                   
                 TCGAGCCAAAACTGGACGCGCCGTCACTGAAAAAGTAA 
               
               
                   
               
               
                 Bile salt hydrolase 
                 METKSSLWKSSRRVLAHGAATVLVAAGLIVPQAAMACTSFVL 
               
               
                 protein from 
                 PTSDGGMVYGRTMEFGFNLKSDMIAIPRNYTITASGPDGAAG 
               
               
                 
                   Brucella abortus 
                 
                 KKWKGKYATIGMNAFGIVALTDGMNEKGLAGGLLYFPEYAKY 
               
               
                 SEQ ID NO: 109 
                 QDPSTAKPEDSLAPWDFLTWALANFSTVAEVKDALSTISIVD 
               
               
                   
                 VKQKDLGFTPPAHYTLHDATGASIVIEPIDGKLKVYDNKLGV 
               
               
                   
                 MTNSPSFDWHMTNLRNYVYLSRENPKPLQILGETIQSFGQGA 
               
               
                   
                 GMHGIPGDTTPPSRFVRASAYVLSAKKVPSGLESVRLAEHIA 
               
               
                   
                 NNFDIPKGWSEEQNMFEYTQWTAFADMKNDVYYIKTYDDQVL 
               
               
                   
                 RSFSFKDFDVDSKDILTIKFEPKLDAPSLKK 
               
               
                   
               
            
           
         
       
     
     In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 90. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 90. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 90. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 90. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 90. Accordingly, in one embodiment, the bile salt hydrolase 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 the entire sequence of SEQ ID NO: 90. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 90. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 90. 
     In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 92. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 92. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 92. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 92. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 92. Accordingly, in one embodiment, the bile salt hydrolase 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 the entire sequence of SEQ ID NO: 92. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 92. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 92. 
     In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 94 In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 94. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 93. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 94. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 94. Accordingly, in one embodiment, the bile salt hydrolase 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 the entire sequence of SEQ ID NO: 94. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 94. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 94. 
     In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 96 In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 96. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 96. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 96. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 96. Accordingly, in one embodiment, the bile salt hydrolase 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 the entire sequence of SEQ ID NO: 96. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 96. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 96. 
     In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 98. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 98. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 98. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 98. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 98. Accordingly, in one embodiment, the bile salt hydrolase 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 the entire sequence of SEQ ID NO: 98. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 98. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 98. 
     In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 100. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 100. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 100. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 100. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 100. Accordingly, in one embodiment, the bile salt hydrolase 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 the entire sequence of SEQ ID NO: 100. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 100. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 100. 
     In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 102. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 102. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 102. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 102. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 102. Accordingly, in one embodiment, the bile salt hydrolase 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 the entire sequence of SEQ ID NO: 102. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 102. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 102. 
     In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 104. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 104. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 104. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 104. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 104. Accordingly, in one embodiment, the bile salt hydrolase 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 the entire sequence of SEQ ID NO: 104. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 104. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 104. 
     In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 106. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 106. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 106. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 106. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 106. Accordingly, in one embodiment, the bile salt hydrolase 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 the entire sequence of SEQ ID NO: 106. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 106. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 106. 
     In one embodiment, the bile salt hydrolase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 108. In another embodiment, the bile salt hydrolase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 108. In one embodiment, the bile salt hydrolase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 108. In one embodiment, the bile salt hydrolase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 108. In another embodiment, the bile salt hydrolase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 108. Accordingly, in one embodiment, the bile salt hydrolase 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 the entire sequence of SEQ ID NO: 108. In another embodiment, the bile salt hydrolase gene comprises the sequence of SEQ ID NO: 108. In yet another embodiment the bile salt hydrolase gene consists of the sequence of SEQ ID NO: 108. 
     In one embodiment, one or more polypeptides encoded by the and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. 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: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. 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 with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. 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 with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. 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 with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. 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 with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. 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 with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. In yet another embodiment one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria consist of the sequence of with one or more of SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109. 
     In one embodiment, the gene encoding the bile salt hydrolase enzyme is directly operably linked to a first promoter. In another embodiment, the gene encoding the bile salt hydrolase enzyme is indirectly operably linked to a first promoter. In one embodiment, the promoter is not operably linked with the gene encoding the bile salt hydrolase enzyme in nature. 
     In some embodiments, the gene encoding the bile salt hydrolase enzyme is expressed under the control of a constitutive promoter. In another embodiment, the gene encoding the bile salt hydrolase enzyme is expressed under the control of an inducible promoter. In some embodiments, the gene encoding the bile salt hydrolase enzyme is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the gene encoding the bile salt hydrolase enzyme is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the gene encoding the bile salt hydrolase enzyme is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. Inducible promoters are described in more detail infra. 
     In some embodiments, the genetically engineered bacteria are capable of expressing bile sale hydrolase under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of expressing bile sale hydrolase in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, 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 encoding the bile salt hydrolase enzyme may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the gene encoding the bile salt hydrolase enzyme is located on a plasmid in the bacterial cell. In another embodiment, the gene encoding the bile salt hydrolase is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the gene encoding the bile salt hydrolase enzyme is located in the chromosome of the bacterial cell, and a gene encoding a bile salt hydrolase enzyme from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding the bile salt hydrolase enzyme is located on a plasmid in the bacterial cell, and a gene encoding the bile salt hydrolase enzyme from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding the bile salt hydrolase enzyme is located in the chromosome of the bacterial cell, and a gene encoding the bile salt hydrolase enzyme from a different species of bacteria is located in the chromosome of the bacterial cell. For example,  E. coli  comprises a native bile salt hydrolase gene. 
     In some embodiments, the gene encoding the bile salt hydrolase enzyme is expressed on a low-copy plasmid. In some embodiments, the gene encoding the bile salt hydrolase enzyme is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the bile salt hydrolase enzyme, thereby increasing the catabolism of bile salts. 
     Transporters of Bile Salts and Bile Acids 
     The uptake of bile salts into the  Lactobacillus  and  Bifidobacterium  has been found to occur via the bile salt transporters CbsT1 and CbsT2 (see, e.g., Elkins et al., Microbiology, 147(Pt. 12):3403-3412 (2001), the entire contents of which are expressly incorporated herein by reference). The uptake of bile acids into the  Neisseria meningitides  has been found to occur via the bile acid sodium symporter ASBT (see, e.g., Hu et al.,  Nature,  478(7369):408-411 (2011), the contents of which are expressly incorporated herein by reference. Other proteins that mediate the import of bile salts or acids into cells are well known to those of skill in the art. For the purposes of this invention, a bile salt transporter includes bile salt importers and bile acid symporters. 
     Bile salt transporters, e.g., bile salt importers or bile acid symporters, may be expressed or modified in the bacteria in order to enhance bile salt or acid transport into the cell. Specifically, when the transporter of bile salts is expressed in the recombinant bacterial cells, the bacterial cells import more bile salts 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 transporter of bile salts may be used to import bile salts into the bacteria so that any gene encoding a bile salt hydrolase (BSH) enzyme expressed in the organism can be used to treat disorders associated with bile salts, such as cardiac disease, metabolic disease, liver disease, cancer, and  C. difficile  infection. In one embodiment, the bacterial cell comprises a heterologous gene encoding a transporter of a bile salt. In one embodiment, the bacterial cell comprises a heterologous gene encoding a transporter of a bile salt and a heterologous gene encoding a bile salt hydrolase (BSH) enzyme. 
     Thus, in some embodiments, the disclosure provides a bacterial cell that comprises a heterologous gene encoding a bile salt hydrolase enzyme operably linked to a first promoter and a heterologous gene encoding a transporter of a bile salt. In some embodiments, the disclosure provides a bacterial cell that comprises a heterologous gene encoding a transporter of a bile salt operably linked to the first promoter. In another embodiment, the disclosure provides a bacterial cell that comprises a heterologous gene encoding at least one bile salt hydrolase enzyme operably linked to a first promoter and a heterologous gene encoding transporter of a bile salt operably linked to a second promoter. In one embodiment, the first promoter and the second promoter are separate copies of the same promoter. In another embodiment, the first promoter and the second promoter are different promoters. 
     In one embodiment, the bacterial cell comprises a gene encoding a transporter of a bile salt from a different organism, e.g., a different species of bacteria. In one embodiment, the bacterial cell comprises at least one native gene encoding transporter of a bile salt. In some embodiments, the at least one native gene encoding atransporter of a bile salt is not modified. In another embodiment, the bacterial cell comprises more than one copy of at least one native gene encoding a transporter of a bile salt. In yet another embodiment, the bacterial cell comprises a copy of a gene encoding a native transporter of a bile salt, as well as at least one copy of a heterologous gene encoding a transporter of a bile salt from a different bacterial species. In one embodiment, the bacterial cell comprises at least one, two, three, four, five, or six copies of the heterologous gene encoding a tarnsporter of a bile salt. In one embodiment, the bacterial cell comprises multiple copies of the heterologous gene encoding a transporter of a bile salt. 
     In some embodiments, the transporterof a bile salt is encoded by a transporter of a bile salt gene derived from a bacterial genus or species, including but not limited to,  Lactobacillus . In some embodiments, the transporterof a bile salt gene is derived from a bacteria of the species  Lactobacillus  johnsonni strain 100-100. 
     The present disclosure further comprises genes encoding functional fragments of a transporter of a bile salt or functional variants of a transporter of a bile salt. As used herein, the term “functional fragment thereof” or “functional variant thereof” of a transporter of a bile salt relates to an element having qualitative biological activity in common with the wild-type transporter of a bile salt from which the fragment or variant was derived. For example, a functional fragment or a functional variant of a mutated transporter of bile salt protein is one which retains essentially the same ability to import the bile salt into the bacterial cell as does the transporter protein from which the functional fragment or functional variant was derived. In one embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a functional fragment of a transporter of a bile salt. In another embodiment, the recombinant bacterial cell comprises a heterologous gene encoding a functional variant of a transporter of a bile salt. 
     Assays for testing the activity of a transporter of a bile salt, a functional variant of a transporter of a bile salt, or a functional fragment of a transporter of a bile salt are well known to one of ordinary skill in the art. For example, bile salt import can be assessed as described in Elkins et al.,  Microbiology,  147:3403-3412 (2001), the entire contents of which are expressly incorporated herein by reference. 
     In one embodiment the gene(s) encoding the transporter of a bile salt have been codon-optimized for use in the host organism. In one embodiment, the genes encoding the transporter of a bile salt have been codon-optimized for use in  Escherichia coli.    
     The present disclosure also encompasses genes encoding a transporter of a bile salt 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. 
     In some embodiments, the gene encoding a transporter of a bile salt is mutagenized; mutants exhibiting increased bile salt transport are selected; and the mutagenized a gene encoding a transporter of a bile salt is isolated and inserted into the bacterial cell. In some embodiments, the gene encoding a transporter of a bile salt is mutagenized; mutants exhibiting decreased bile salt transport are selected; and the mutagenized a gene encoding a transporter of the bile salt is isolated and inserted into the bacterial cell. The transporter modifications described herein may be present on a plasmid or chromosome. Non-limiting examples of bile salt transporters, which are encoded in the genetically engineered bacteria, are in Table 12. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Bile Salt Transport and Export Sequences 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 cbsT1 from 
                 ATGTCGACCACACCGACACAGCCATCATCACGAAAACAG 
               
               
                 
                   Lactobacillus 
                 
                 GCTGTTTACCCGTACTTGATCGTGCTGTCGGGCATCGTCT 
               
               
                 
                   johnsonii 
                 
                 TCACGGCCATCCCGGTATCGCTGGTCTGCAGTTGCGCAGG 
               
               
                 SEQ ID NO: 110 
                 TATCTTCTTCACGCCTGTCAGCAGCTACTTCCATGTTCCCA 
               
               
                   
                 AGGCCGCATTCACCGGATATTTCAGCATATTCAGCATCAC 
               
               
                   
                 CATGGTCGCCTTCCTGCCGGTGGCCGGATGGCTGATGCAC 
               
               
                   
                 CGCTACGATCTGCGCATCGTACTGACCGCAAGCACCGTCC 
               
               
                   
                 TGGCTGGACTGGGCTGCCTGGGTATGTCCCGATCATCCGC 
               
               
                   
                 CATGTGGCAGTTCTATCTATGCGGAGTGGTTCTGGGAATC 
               
               
                   
                 GGCATGCCGGCCGTCCTCTATCTGTCAGTGCCAACACTCA 
               
               
                   
                 TCAACGCCTGGTTCCGCAAGCGGGTCGGGTTCTTCATCGG 
               
               
                   
                 CCTGTGCATGGCCTTCACCGGCATAGGCGGCGTGATCTTC 
               
               
                   
                 AACCAGATAGGCACCATGATCATCAGATCCGCCCCTGAT 
               
               
                   
                 GGATGGAGGCGGGGATATCTGGTTTTCGCTATTCTCATCC 
               
               
                   
                 TGGTGATCACCCTGCCCTTCACCATTTTCGTCATTCGCAG 
               
               
                   
                 CACACCCGAACAGATGGGTCTGCATCCCTACGGCGCCGA 
               
               
                   
                 CCAGGAGCCTGATGCAGCTGAGACGGCCACCAATAGTGC 
               
               
                   
                 AGGCACCGGGAGCAAAGACCAAAAGAGTCCTGAGCCTGC 
               
               
                   
                 AGCGTCAACCGTAGGCATGACTGCCTCCCAGGCCTTGCGC 
               
               
                   
                 TCCCCTGCCTTCTGGGCGCTGGCGCTCTTCTGCGGTCTGA 
               
               
                   
                 TCACCATGAATCAGACCATTTACCAGTTCCTGCCCTCCTA 
               
               
                   
                 CGCGGCATCCCTGCCATCCATGGCAGCCTACACGGGACT 
               
               
                   
                 GATCGCCTCCTCCTGCATGGCCGGCCAGGCCATCGGCAA 
               
               
                   
                 GATCATCCTGGGCATGGTCAACGACGGCAGCATCGTAGG 
               
               
                   
                 CGGTCTCTGTCTGGGCATCGGCGGCGGCATTCTCGGCGTC 
               
               
                   
                 TGCCTCATGGTCGCCTTCCCCGGATTGCCCGTGCTCCTCCT 
               
               
                   
                 GCTGGGAGCCTTTGCCTTCGGCCTTGTCTACGCCTGCACT 
               
               
                   
                 ACTGTGCAGACACCAATCCTGGTTACAGCGGTCTTCGGCT 
               
               
                   
                 CGCGCGACTACACCAACATCTATGCACGTATCCAGATGGT 
               
               
                   
                 TGGGTCCCTAGCCTCGGCCTTCGCAGCTCTCTTCTGGGGC 
               
               
                   
                 GCCATCGCTGACCAGCCCCACGGCTACATCATCATGTTCG 
               
               
                   
                 GTCTGAGCATCCTGATCATGGTTGTGGCCTTGTTCCTAGG 
               
               
                   
                 CATTATCCCTCTGAAAGGTACGCGCAAGTTGACCGATCAG 
               
               
                   
                 ATCGCCTGA 
               
               
                   
               
               
                 CbsT1 protein 
                 MSTTPTQPSSRKQAVYPYLIVLSGIVFTAIPVSLVCSCAGIFFT 
               
               
                 from 
                 PVSSYFHVPKAAFTGYFSIFSITMVAFLPVAGWLMHRYDLRI 
               
               
                 
                   Lactobacillus 
                 
                 VLTASTVLAGLGCLGMSRSSAMWQFYLCGVVLGIGMPAVL 
               
               
                 
                   johnsonii 
                 
                 YLSVPTLINAWFRKRVGFFIGLCMAFTGIGGVIFNQIGTMIIR 
               
               
                 SEQ ID NO: 111 
                 SAPDGWRRGYLVFAILILVITLPFTIFVIRSTPEQMGLHPYGA 
               
               
                   
                 DQEPDAAETATNSAGTGSKDQKSPEPAASTVGMTASQALRS 
               
               
                   
                 PAFWALALFCGLITMNQTIYQFLPSYAASLPSMAAYTGLIAS 
               
               
                   
                 SCMAGQAIGKIILGMVNDGSIVGGLCLGIGGGILGVCLMVAF 
               
               
                   
                 PGLPVLLLLGAFAFGLVYACTTVQTPILVTAVFGSRDYTNIY 
               
               
                   
                 ARIQMVGSLASAFAALFWGAIADQPHGYIIMFGLSILIMVVA 
               
               
                   
                 LFLGIIPLKGTRKLTDQIA 
               
               
                   
               
               
                 cbsT2 from 
                 ATGTCTACTGATGCCGCTACTAAAGATAAAGTAGTAAGC 
               
               
                 
                   Lactobacillus 
                 
                 AAGGGCTATAAATACTTCATGGTTTTCCTTTGTATGTTAA 
               
               
                 
                   johnsonii 
                 
                 CCCAAGCTATTCCTTATGGAATTGCTCAAAACATTCAGCC 
               
               
                 SEQ ID NO: 112 
                 TTTGTTTATCCACCCTTTAGTTAATACTTTCCACTTTACCT 
               
               
                   
                 TAGCATCGTACACATTAATTTTTACGTTTGGTGCGGTTTTT 
               
               
                   
                 GCTTCAGTTGCTTCTCCATTTATTGGTAAGGCATTAGAAA 
               
               
                   
                 AAGTTAACTTCCGACTAATGTATTTAATTGGTATTGGTCT 
               
               
                   
                 TTCTGCTATTGCCTACGTAATTTTTGGAATTAGTACAAAA 
               
               
                   
                 CTACCCGGTTTCTATATTGCCGCTATCATTTGTATGGTTGG 
               
               
                   
                 TTCAACCTTTTACTCCGGCCAAGGTGTTCCCTGGGTTATT 
               
               
                   
                 AACCACTGGTTCCCAGCAAAGGGACGTGGGGCTGCCTTA 
               
               
                   
                 GGAATTGCCTTCTGCGGTGGTTCTATTGGTAATATCTTTTT 
               
               
                   
                 ACAACCAGCAACCCAAGCTATTTTAAAACACTACATGAC 
               
               
                   
                 AGGTAATACTAAGACCGGTCATTTAACCTCTATGGCACCA 
               
               
                   
                 TTCTTTATCTTTGCCGTAGCTTTATTAGTAATCGGTGTAAT 
               
               
                   
                 TATCGCCTGCTTCATTAGAACCCCTAAGAAAGACGAAATT 
               
               
                   
                 GTTGTTTCTGATGCAGAACTAGCTGAAAGCAAGAAAGCT 
               
               
                   
                 GAAGCCGCAGCCAAAGCTAAAGAGTTTAAAGGCTGGACT 
               
               
                   
                 AGTAAACAAGTGTTACAAATGAAATGGTTCTGGATTTTCA 
               
               
                   
                 GCCTTGGTTTCTTAATCATTGGTTTAGGCTTAGCTTCTTTA 
               
               
                   
                 AATGAAGACTATGCCGCCTTCCTTGATACTAAGCTTTCTT 
               
               
                   
                 TAACCGATGTTGGTTTAGTTGGGTCAATGTACGGTGTTGG 
               
               
                   
                 TTGTTTAATCGGAAATATTTCTGGTGGTTTCTTATTTGATA 
               
               
                   
                 AATTTGGTACAGCAAAATCAATGACCTATGCTGGTTGTAT 
               
               
                   
                 GTATATTTTATCTATTCTGATGATGATCTTTATTAGCTTCC 
               
               
                   
                 AGCCATATGGTTCATCTATTAGTAAGGCTGCTGGCATTGG 
               
               
                   
                 CTATGCTATCTTTTGCGGCTTAGCTGTATTTAGTTACATGT 
               
               
                   
                 CAGGCCCAGCCTTCATGGCAAAAGACCTCTTTGGTTCAAG 
               
               
                   
                 AGATCAAGGTGTCATGCTTGGATACGTTGGTTTAGCTTAT 
               
               
                   
                 GCAATTGGCTATGCCATTGGTGCTCCACTATTTGGGATTA 
               
               
                   
                 TTAAGGGAGCGGCAAGCTTTACAGTTGCTTGGTACTTTAT 
               
               
                   
                 GATTGCCTTTGTTGCAATTGGTTTTATCATTTTAGTATTTG 
               
               
                   
                 CCGTTATCCAAATTAAGAGATACCAAAAGAAATACATTG 
               
               
                   
                 CAGAGCAAGCAGCAAAAGCTAATGCTAAATAA 
               
               
                   
               
               
                 CbsT2 protein 
                 MSTDAATKDKVVSKGYKYFMVFLCMLTQAIPYGIAQNIQPL 
               
               
                 from 
                 FIHPLVNTFHFTLASYTLIFTFGAVFASVASPFIGKALEKVNF 
               
               
                 
                   Lactobacillus 
                 
                 RLMYLIGIGLSAIAYVIFGISTKLPGFYIAAIICMVGSTFYSGQ 
               
               
                 
                   johnsonii 
                 
                 GVPWVINHWFPAKGRGAALGIAFCGGSIGNIFLQPATQAILK 
               
               
                 SEQ ID NO: 113 
                 HYMTGNTKTGHLTSMAPFFIFAVALLVIGVIIACFIRTPKKDE 
               
               
                   
                 IVVSDAELAESKKAEAAAKAKEFKGWTSKQVLQMKWFWIF 
               
               
                   
                 SLGFLIIGLGLASLNEDYAAFLDTKLSLTDVGLVGSMYGVG 
               
               
                   
                 CLIGNISGGFLFDKFGTAKSMTYAGCMYILSILMMIFISFQPY 
               
               
                   
                 GSSISKAAGIGYAIFCGLAVFSYMSGPAFMAKDLFGSRDQG 
               
               
                   
                 VMLGYVGLAYAIGYAIGAPLFGIIKGAASFTVAWYFMIAFV 
               
               
                   
                 AIGFIILVFAVIQIKRYQKKYIAEQAAKANAK 
               
               
                   
               
               
                 ABCB11 bile 
                 GAATGATGAAAACCGAGGTTGGAAAAGGTTGTGAAACCT 
               
               
                 salt exporter 
                 TTTAACTCTCCACAGTGGAGTCCATTATTTCCTCTGGCTTC 
               
               
                 
                   Homo sapiens 
                 
                 CTCAAATTCATATTCACAGGGTCGTTGGCTGTGGGTTGCA 
               
               
                 SEQ ID NO: 114 
                 ATTACCATGTCTGACTCAGTAATTCTTCGAAGTATAAAGA 
               
               
                   
                 AATTTGGAGAGGAGAATGATGGTTTTGAGTCAGATAAAT 
               
               
                   
                 CATATAATAATGATAAGAAATCAAGGTTACAAGATGAGA 
               
               
                   
                 AGAAAGGTGATGGCGTTAGAGTTGGCTTCTTTCAATTGTT 
               
               
                   
                 TCGGTTTTCTTCATCAACTGACATTTGGCTGATGTTTGTGG 
               
               
                   
                 GAAGTTTGTGTGCATTTCTCCATGGAATAGCCCAGCCAGG 
               
               
                   
                 CGTGCTACTCATTTTTGGCACAATGACAGATGTTTTTATT 
               
               
                   
                 GACTACGACGTTGAGTTACAAGAACTCCAGATTCCAGGA 
               
               
                   
                 AAAGCATGTGTGAATAACACCATTGTATGGACTAACAGT 
               
               
                   
                 TCCCTCAACCAGAACATGACAAATGGAACACGTTGTGGG 
               
               
                   
                 TTGCTGAACATCGAGAGCGAAATGATCAAATTTGCCAGTT 
               
               
                   
                 ACTATGCTGGAATTGCTGTCGCAGTACTTATCACAGGATA 
               
               
                   
                 TATTCAAATATGCTTTTGGGTCATTGCCGCAGCTCGTCAG 
               
               
                   
                 ATACAGAAAATGAGAAAATTTTACTTTAGGAGAATAATG 
               
               
                   
                 AGAATGGAAATAGGGTGGTTTGACTGCAATTCAGTGGGG 
               
               
                   
                 GAGCTGAATACAAGATTCTCTGATGATATTAATAAAATCA 
               
               
                   
                 ATGATGCCATAGCTGACCAAATGGCCCTTTTCATTCAGCG 
               
               
                   
                 CATGACCTCGACCATCTGTGGTTTCCTGTTGGGATTTTTCA 
               
               
                   
                 GGGGTTGGAAACTGACCTTGGTTATTATTTCTGTCAGCCC 
               
               
                   
                 TCTCATTGGGATTGGAGCAGCCACCATTGGTCTGAGTGTG 
               
               
                   
                 TCCAAGTTTACGGACTATGAGCTGAAGGCCTATGCCAAA 
               
               
                   
                 GCAGGGGTGGTGGCTGATGAAGTCATTTCATCAATGAGA 
               
               
                   
                 ACAGTGGCTGCTTTTGGTGGTGAGAAAAGAGAGGTTGAA 
               
               
                   
                 AGGTATGAGAAAAATCTTGTGTTCGCCCAGCGTTGGGGA 
               
               
                   
                 ATTAGAAAAGGAATAGTGATGGGATTCTTTACTGGATTCG 
               
               
                   
                 TGTGGTGTCTCATCTTTTTGTGTTATGCACTGGCCTTCTGG 
               
               
                   
                 TACGGCTCCACACTTGTCCTGGATGAAGGAGAATATACA 
               
               
                   
                 CCAGGAACCCTTGTCCAGATTTTCCTCAGTGTCATAGTAG 
               
               
                   
                 GAGCTTTAAATCTTGGCAATGCCTCTCCTTGTTTGGAAGC 
               
               
                   
                 CTTTGCAACTGGACGTGCAGCAGCCACCAGCATTTTTGAG 
               
               
                   
                 ACAATAGACAGGAAACCCATCATTGACTGCATGTCAGAA 
               
               
                   
                 GATGGTTACAAGTTGGATCGAATCAAGGGTGAAATTGAA 
               
               
                   
                 TTCCATAATGTGACCTTCCATTATCCTTCCAGACCAGAGG 
               
               
                   
                 TGAAGATTCTAAATGACCTCAACATGGTCATTAAACCAG 
               
               
                   
                 GGGAAATGACAGCTCTGGTAGGACCCAGTGGAGCTGGAA 
               
               
                   
                 AAAGTACAGCACTGCAACTCATTCAGCGATTCTATGACCC 
               
               
                   
                 CTGTGAAGGAATGGTGACCGTGGATGGCCATGACATTCG 
               
               
                   
                 CTCTCTTAACATTCAGTGGCTTAGAGATCAGATTGGGATA 
               
               
                   
                 GTGGAGCAAGAGCCAGTTCTGTTCTCTACCACCATTGCAG 
               
               
                   
                 AAAATATTCGCTATGGCAGAGAAGATGCAACAATGGAAG 
               
               
                   
                 ACATAGTCCAAGCTGCCAAGGAGGCCAATGCCTACAACT 
               
               
                   
                 TCATCATGGACCTGCCACAGCAATTTGACACCCTTGTTGG 
               
               
                   
                 AGAAGGAGGAGGCCAGATGAGTGGTGGCCAGAAACAAA 
               
               
                   
                 GGGTAGCTATCGCCAGAGCCCTCATCCGAAATCCCAAGA 
               
               
                   
                 TTCTGCTTTTGGACATGGCCACCTCAGCTCTGGACAATGA 
               
               
                   
                 GAGTGAAGCCATGGTGCAAGAAGTGCTGAGTAAGATTCA 
               
               
                   
                 GCATGGGCACACAATCATTTCAGTTGCTCATCGCTTGTCT 
               
               
                   
                 ACGGTCAGAGCTGCAGATACCATCATTGGTTTTGAACATG 
               
               
                   
                 GCACTGCAGTGGAAAGAGGGACCCATGAAGAATTACTGG 
               
               
                   
                 AAAGGAAAGGTGTTTACTTCACTCTAGTGACTTTGCAAAG 
               
               
                   
                 CCAGGGAAATCAAGCTCTTAATGAAGAGGACATAAAGGA 
               
               
                   
                 TGCAACTGAAGATGACATGCTTGCGAGGACCTTTAGCAG 
               
               
                   
                 AGGGAGCTACCAGGATAGTTTAAGGGCTTCCATCCGGCA 
               
               
                   
                 ACGCTCCAAGTCTCAGCTTTCTTACCTGGTGCACGAACCT 
               
               
                   
                 CCATTAGCTGTTGTAGATCATAAGTCTACCTATGAAGAAG 
               
               
                   
                 ATAGAAAGGACAAGGACATTCCTGTGCAGGAAGAAGTTG 
               
               
                   
                 AACCTGCCCCAGTTAGGAGGATTCTGAAATTCAGTGCTCC 
               
               
                   
                 AGAATGGCCCTACATGCTGGTAGGGTCTGTGGGTGCAGC 
               
               
                   
                 TGTGAACGGGACAGTCACACCCTTGTATGCCTTTTTATTC 
               
               
                   
                 AGCCAGATTCTTGGGACTTTTTCAATTCCTGATAAAGAGG 
               
               
                   
                 AACAAAGGTCACAGATCAATGGTGTGTGCCTACTTTTTGT 
               
               
                   
                 AGCAATGGGCTGTGTATCTCTTTTCACCCAATTTCTACAG 
               
               
                   
                 GGATATGCCTTTGCTAAATCTGGGGAGCTCCTAACAAAA 
               
               
                   
                 AGGCTACGTAAATTTGGTTTCAGGGCAATGCTGGGGCAA 
               
               
                   
                 GATATTGCCTGGTTTGATGACCTCAGAAATAGCCCTGGAG 
               
               
                   
                 CATTGACAACAAGACTTGCTACAGATGCTTCCCAAGTTCA 
               
               
                   
                 AGGGGCTGCCGGCTCTCAGATCGGGATGATAGTCAATTC 
               
               
                   
                 CTTCACTAACGTCACTGTGGCCATGATCATTGCCTTCTCCT 
               
               
                   
                 TTAGCTGGAAGCTGAGCCTGGTCATCTTGTGCTTCTTCCC 
               
               
                   
                 CTTCTTGGCTTTATCAGGAGCCACACAGACCAGGATGTTG 
               
               
                   
                 ACAGGATTTGCCTCTCGAGATAAGCAGGCCCTGGAGATG 
               
               
                   
                 GTGGGACAGATTACAAATGAAGCCCTCAGTAACATCCGC 
               
               
                   
                 ACTGTTGCTGGAATTGGAAAGGAGAGGCGGTTCATTGAA 
               
               
                   
                 GCACTTGAGACTGAGCTGGAGAAGCCCTTCAAGACAGCC 
               
               
                   
                 ATTCAGAAAGCCAATATTTACGGATTCTGCTTTGCCTTTG 
               
               
                   
                 CCCAGTGCATCATGTTTATTGCGAATTCTGCTTCCTACAG 
               
               
                   
                 ATATGGAGGTTACTTAATCTCCAATGAGGGGCTCCATTTC 
               
               
                   
                 AGCTATGTGTTCAGGGTGATCTCTGCAGTTGTACTGAGTG 
               
               
                   
                 CAACAGCTCTTGGAAGAGCCTTCTCTTACACCCCAAGTTA 
               
               
                   
                 TGCAAAAGCTAAAATATCAGCTGCACGCTTTTTTCAACTG 
               
               
                   
                 CTGGACCGACAACCCCCAATCAGTGTATACAATACTGCA 
               
               
                   
                 GGTGAAAAATGGGACAACTTCCAGGGGAAGATTGATTTT 
               
               
                   
                 GTTGATTGTAAATTTACATATCCTTCTCGACCTGACTCGC 
               
               
                   
                 AAGTTCTGAATGGTCTCTCAGTGTCGATTAGTCCAGGGCA 
               
               
                   
                 GACACTGGCGTTTGTTGGGAGCAGTGGATGTGGCAAAAG 
               
               
                   
                 CACTAGCATTCAGCTGTTGGAACGTTTCTATGATCCTGAT 
               
               
                   
                 CAAGGGAAGGTGATGATAGATGGTCATGACAGCAAAAAA 
               
               
                   
                 GTAAATGTCCAGTTCCTCCGCTCAAACATTGGAATTGTTT 
               
               
                   
                 CCCAGGAACCAGTGTTGTTTGCCTGTAGCATAATGGACAA 
               
               
                   
                 TATCAAGTATGGAGACAACACCAAAGAAATTCCCATGGA 
               
               
                   
                 AAGAGTCATAGCAGCTGCAAAACAGGCTCAGCTGCATGA 
               
               
                   
                 TTTTGTCATGTCACTCCCAGAGAAATATGAAACTAACGTT 
               
               
                   
                 GGGTCCCAGGGGTCTCAACTCTCTAGAGGGGAGAAACAA 
               
               
                   
                 CGCATTGCTATTGCTCGGGCCATTGTACGAGATCCTAAAA 
               
               
                   
                 TCTTGCTACTAGATGAAGCCACTTCTGCCTTAGACACAGA 
               
               
                   
                 AAGTGAAAAGACGGTGCAGGTTGCTCTAGACAAAGCCAG 
               
               
                   
                 AGAGGGTCGGACCTGCATTGTCATTGCCCATCGCTTGTCC 
               
               
                   
                 ACCATCCAGAACGCGGATATCATTGCTGTCATGGCACAG 
               
               
                   
                 GGGGTGGTGATTGAAAAGGGGACCCATGAAGAACTGATG 
               
               
                   
                 GCCCAAAAAGGAGCCTACTACAAACTAGTCACCACTGGA 
               
               
                   
                 TCCCCCATCAGTTGACCCAATGCAAGAATCTCAGACACAC 
               
               
                   
                 ATGACGCACCAGTTACAGGGGTTGTTTTTAAAGAAAAAA 
               
               
                   
                 ACAATCCCAGCAGGAGGGATTGCTGGGATTGTTTTTTCTT 
               
               
                   
                 TAAAGAAGAATGTTAATATTTTACTTTTACAGTCATTTTC 
               
               
                   
                 CTACATCGGAATCCAAGCTAATTTCTAATGGCCTTCCATA 
               
               
                   
                 ATAATTCTGCTTTAGATGTGTATACAGAAAATGAAAGAA 
               
               
                   
                 ACTAGGGTCCATATGAGGGAAAACCCAATGTCAAGTGGC 
               
               
                   
                 AGCTCAGCCACCACTCAGTGCTTCTCTGTGCAGGAGCCAG 
               
               
                   
                 TCCTGATTAATATGTGGGAATTAGTGAGACATCAGGGAG 
               
               
                   
                 TAAGTGACACTTTGAACTCCTCAAGGGCAGAGAACTGTCT 
               
               
                   
                 TTCATTTTTGAACCCTCGGTGTACACAGAGGCGGGTCTAT 
               
               
                   
                 AACAGGCAATCAACAAACGTTTCTTGAGCTAGACCAAGG 
               
               
                   
                 TCAGATTTGAAAAGAACAGAAGGACTGAAGACCAGCTGT 
               
               
                   
                 GTTTCTTAACTAAATTTGTCTTTCAAGTGAAACCAGCTTC 
               
               
                   
                 CTTCATCTCTAAGGCTAAGGATAGGGAAAGGGTGGATGC 
               
               
                   
                 TCTCAGGCTGAGGGAGGCAGAAAGGGAAAGTATTAGCAT 
               
               
                   
                 GAGCTTTCCAGTTAGGGCTGTTGATTTATGCTTTAACTTC 
               
               
                   
                 AGAGTGAGTGTAGGGGTGGTGATGCT 
               
               
                   
               
               
                 ABCB11 bile 
                 MSDSVILRSIKKFGEENDGFESDKSYNNDKKSRLQDEKKGD 
               
               
                 salt exporter 
                 GVRVGFFQLFRFSSSTDIWLMFVGSLCAFLHGIAQPGVLLIF 
               
               
                 protein  Homo   
                 GTMTDVFIDYDVELQELQIPGKACVNNTIVWTNSSLNQNMT 
               
               
                 
                   sapiens 
                 
                 NGTRCGLLNIESEMIKFASYYAGIAVAVLITGYIQICFWVIAA 
               
               
                 SEQ ID NO: 115 
                 ARQIQKMRKFYFRRIMRMEIGWFDCNSVGELNTRFSDDINKI 
               
               
                   
                 NDAIADQMALFIQRMTSTICGFLLGFFRGWKLTLVIISVSPLI 
               
               
                   
                 GIGAATIGLSVSKFTDYELKAYAKAGVVADEVISSMRTVAA 
               
               
                   
                 FGGEKREVERYEKNLVFAQRWGIRKGIVMGFFTGFVWCLIF 
               
               
                   
                 LCYALAFWYGSTLVLDEGEYTPGTLVQIFLSVIVGALNLGN 
               
               
                   
                 ASPCLEAFATGRAAATSIFETIDRKPIIDCMSEDGYKLDRIKG 
               
               
                   
                 EIEFHNVTFHYPSRPEVKILNDLNMVIKPGEMTALVGPSGAG 
               
               
                   
                 KSTALQLIQRFYDPCEGMVTVDGHDIRSLNIQWLRDQIGIVE 
               
               
                   
                 QEPVLFSTTIAENIRYGREDATMEDIVQAAKEANAYNFIMDL 
               
               
                   
                 PQQFDTLVGEGGGQMSGGQKQRVAIARALIRNPKILLLDMA 
               
               
                   
                 TSALDNESEAMVQEVLSKIQHGHTIISVAHRLSTVRAADTIIG 
               
               
                   
                 FEHGTAVERGTHEELLERKGVYFTLVTLQSQGNQALNEEDI 
               
               
                   
                 KDATEDDMLARTFSRGSYQDSLRASIRQRSKSQLSYLVHEPP 
               
               
                   
                 LAVVDHKSTYEEDRKDKDIPVQEEVEPAPVRRILKFSAPEWP 
               
               
                   
                 YMLVGSVGAAVNGTVTPLYAFLFSQILGTFSIPDKEEQRSQI 
               
               
                   
                 NGVCLLFVAMGCVSLFTQFLQGYAFAKSGELLTKRLRKFGF 
               
               
                   
                 RAMLGQDIAWFDDLRNSPGALTTRLATDASQVQGAAGSQI 
               
               
                   
                 GMIVNSFTNVTVAMIIAFSFSWKLSLVILCFFPFLALSGATQT 
               
               
                   
                 RMLTGFASRDKQALEMVGQITNEALSNIRTVAGIGKERRFIE 
               
               
                   
                 ALETELEKPFKTAIQKANIYGFCFAFAQCIMFIANSASYRYG 
               
               
                   
                 GYLISNEGLHFSYVFRVISAVVLSATALGRAFSYTPSYAKAK 
               
               
                   
                 ISAARFFQLLDRQPPISVYNTAGEKWDNFQGKIDFVDCKFTY 
               
               
                   
                 PSRPDSQVLNGLSVSISPGQTLAFVGSSGCGKSTSIQLLERFY 
               
               
                   
                 DPDQGKVMIDGHDSKKVNVQFLRSNIGIVSQEPVLFACSIM 
               
               
                   
                 DNIKYGDNTKEIPMERVIAAAKQAQLHDFVMSLPEKYETNV 
               
               
                   
                 GSQGSQLSRGEKQRIAIARAIVRDPKILLLDEATSALDTESEK 
               
               
                   
                 TVQVALDKAREGRTCIVIAHRLSTIQNADIIAVMAQGVVIEK 
               
               
                   
                 GTHEELMAQKGAYYKLVTTGSPIS 
               
               
                   
               
               
                 
                   Streptococcus 
                 
                 MEGRTVFVIAHRLSTIVNSDVILVMDHGRIIKRGDHDTLMEQ 
               
               
                 
                   thermophilus 
                 
                 GGTYYRLYTGSLEID 
               
               
                 Msba subfamily 
                   
               
               
                 ABC transporter 
                   
               
               
                 ATP-binding 
                   
               
               
                 protein 
                   
               
               
                 STH8232_1633 
                   
               
               
                 SEQ ID NO: 116 
                   
               
               
                   
               
               
                   Nostoc  spp. 
                 ATGTGGGGGAAACAAAGACAAAGAATCGCCATTGCACGA 
               
               
                 Asl1293 ABC 
                 GGGGGTTTTAAGAATTTGCAGGTTTTGATTTTAGATAAAG 
               
               
                 transporter 
                 CAACCTCGGCATTGGATAATAAAACAGAAGCAGCTATTG 
               
               
                 gene 
                 AGCGATCGCTGGTGTTGACTGTTGACCAATGA 
               
               
                 SEQ ID NO: 117 
                   
               
               
                   
               
               
                   Nostoc  spp. 
                 MWGKQRQRIAIARGGFKNLQVLILDKATSALDNKTEAAIER 
               
               
                 Asl1293 ABC 
                 SLVLTVDQ 
               
               
                 transporter 
                   
               
               
                 protein 
                   
               
               
                 SEQ ID NO: 118 
                   
               
               
                   
               
               
                 
                   Neisseria 
                 
                 ATGAATATCCTCAGTAAAATCAGCAGCTTTATCGGAAAA 
               
               
                 
                   meningitides 
                 
                 ACATTTTCCCTCTGGGCCGCGCTCTTTGCCGCCGCCGCTTT 
               
               
                 (MC58) 
                 TTTCGCGCCCGACACCTTCAAATGGGCGGGGCCTTATATT 
               
               
                 ASBT NM  bile 
                 CCTTGGCTGTTGGGCATTATTATGTTCGGTATGGGTTTGA 
               
               
                 acid sodium 
                 CGCTCAAACCTTCCGACTTCGATATTTTGTTCAAACATCC 
               
               
                 symporter 
                 CAAAGTCGTCATCATCGGCGTAATCGCACAATTCGCCATT 
               
               
                 (NMB0705) 
                 ATGCCGGCAACCGCCTGGCTGCTGTCCAAACTGTTGAACC 
               
               
                 SEQ ID NO: 119 
                 TGCCTGCCGAAATCGCGGTCGGCGTGATTTTGGTCGGCTG 
               
               
                   
                 CTGCCCGGGCGGTACGGCTTCCAATGTGATGACCTATCTG 
               
               
                   
                 GCGCGTGGCAATGTGGCTTTGTCGGTTGCCGTTACGTCTG 
               
               
                   
                 TTTCCACCCTGATTTCCCCATTGCTGACTCCCGCCATCTTC 
               
               
                   
                 CTGATGCTTGCCGGCGAAATGCTGGAAATCCAAGCGGCC 
               
               
                   
                 GGTATGTTGATGTCCATCGTCAAAATGGTTTTGCTCCCCA 
               
               
                   
                 TTGTTTTGGGTTTGATTGTCCATAAGGTTTTGGGCAGTAA 
               
               
                   
                 AACCGAAAAGCTGACCGATGCGCTGCCGCTGGTTTCCGTT 
               
               
                   
                 GCCGCCATCGTGCTGATTATCGGCGCGGTTGTTGGGGCAA 
               
               
                   
                 GCAAAGGCAAGATTATGGAAAGCGGCCTGCTGATTTTTG 
               
               
                   
                 CGGTTGTCGTACTCCACAACGGCATCGGCTACCTGCTCGG 
               
               
                   
                 CTTCTTTGCCGCCAAATGGACCGGCCTGCCTTATGATGCA 
               
               
                   
                 CAAAAAACGCTGACCATCGAAGTCGGTATGCAAAACTCG 
               
               
                   
                 GGCCTGGCCGCCGCGCTTGCCGCCGCACACTTTGCCGCCG 
               
               
                   
                 CGCCGGTCGTTGCCGTTCCGGGCGCATTGTTCAGCGTGTG 
               
               
                   
                 GCACAATATCTCCGGCTCGCTGCTGGCAACTTATTGGGCG 
               
               
                   
                 GCCAAAGCCGGTAAACATAAAAAACCCTAA 
               
               
                   
               
               
                 
                   Neisseria 
                 
                 MNILSKISSFIGKTFSLWAALFAAAAFFAPDTFKWAGPYIPW 
               
               
                 
                   meningitides 
                 
                 LLGIIMFGMGLTLKPSDFDILFKHPKVVIIGVIAQFAIMPATA 
               
               
                 (MC58) 
                 WLLSKLLNLPAEIAVGVILVGCCPGGTASNVMTYLARGNVA 
               
               
                 ASBT NM  bile 
                 LSVAVTSVSTLISPLLTPAIFLMLAGEMLEIQAAGMLMSIVK 
               
               
                 acid sodium 
                 MVLLPIVLGLIVHKVLGSKTEKLTDALPLVSVAAIVLIIGAVV 
               
               
                 symporter 
                 GASKGKIMESGLLIFAVVVLHNGIGYLLGFFAAKWTGLPYD 
               
               
                 protein 
                 AQKTLTIEVGMQNSGLAAALAAAHFAAAPVVAVPGALFSV 
               
               
                 SEQ ID NO: 120 
                 WHNISGSLLATYWAAKAGKHKKPGSENLYFQ 
               
               
                   
               
            
           
         
       
     
     In one embodiment, the bile salt transporter is the bile salt importer CbsT1. In one embodiment, the cbsT1 gene has at least about 80% identity to SEQ ID NO: 110. Accordingly, in one embodiment, the cbsT1 gene has at least about 90% identity to SEQ ID NO: 110. Accordingly, in one embodiment, the cbsT1 gene has at least about 95% identity to SEQ ID NO: 110. Accordingly, in one embodiment, the cbsT1 gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 110. In another embodiment, the cbsT1 gene comprises the sequence of SEQ ID NO: 110. In yet another embodiment the cbsT1 gene consists of the sequence of SEQ ID NO: 110. 
     In one embodiment, the bile salt transporter is the bile salt importer CbsT2. In one embodiment, the cbsT2 gene has at least about 80% identity to SEQ ID NO: 112. Accordingly, in one embodiment, the cbsT2 gene has at least about 90% identity to SEQ ID NO: 112. Accordingly, in one embodiment, the cbsT2 gene has at least about 95% identity to SEQ ID NO: 112. Accordingly, in one embodiment, the cbsT2 gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 112. In another embodiment, the cbsT2 gene comprises the sequence of SEQ ID NO: 112. In yet another embodiment the cbsT2 gene consists of the sequence of SEQ ID NO: 112. 
     In one embodiment, the bile acid transporter is the bile acid sodium symporter ASBT NM . In one embodiment, the NMB0705 gene of  Neisseria meningitides  has at least about 80% identity to SEQ ID NO: 117. Accordingly, in one embodiment, the NMB0705 gene has at least about 90% identity to SEQ ID NO: 117. Accordingly, in one embodiment, the NMB0705 gene has at least about 95% identity to SEQ ID NO: 117. Accordingly, in one embodiment, the NMB0705 gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 117. In another embodiment, the NMB0705 gene comprises the sequence of SEQ ID NO: 117. In yet another embodiment the NMB0705 gene consists of the sequence of SEQ ID NO: 117. 
     In one embodiment, one or more polypeptides encoded by the and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. 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: 111, 113, 115, 116, 118 and 120. 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 with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. 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 with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. 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 with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. 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 with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. 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 with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. In yet another embodiment one or more polypeptides encoded by the propionate circuits and expressed by the genetically engineered bacteria consist of the sequence of with one or more of SEQ ID NO: 111, 113, 115, 116, 118 and 120. 
     In some embodiments, the bacterial cell comprises a heterologous gene encoding a bile salt hydrolase enzyme operably linked to a first promoter and a heterologous gene encoding a transporter of a bile salt. In some embodiments, the heterologous gene encoding a transporter of the bile salt is operably linked to the first promoter. In other embodiments, the heterologous gene encoding a transporter of the bile salt is operably linked to a second promoter. In one embodiment, the gene encoding a transporter of the bile salt is directly operably linked to the second promoter. In another embodiment, the gene encoding a transporter of the bile salt is indirectly operably linked to the second promoter. 
     In some embodiments, expression of a gene encoding a transporter of a bile salt is controlled by a different promoter than the promoter that controls expression of the gene encoding the bile salt hydrolase enzyme. In some embodiments, expression of the gene encoding a transporter of a bile salt is controlled by the same promoter that controls expression of the bile salt hydrolase enzyme. In some embodiments, a gene encoding a transporter of a bile salt and the bile salt hydrolase enzyme are divergently transcribed from a promoter region. In some embodiments, expression of each of genes encoding the gene encoding a transporter of a bile salt and the gene encoding the bile salt hydrolase enzyme is controlled by different promoters. 
     In one embodiment, the the gene encoding a transporter of a bile salt is not operably linked with its natural promoter. In some embodiments, the gene encoding the transporter of the bile salt is controlled by its native promoter. In some embodiments, the gene encoding the transporter of the bile salt is controlled by an inducible promoter. In some embodiments, the gene encoding the transporter of the bile salt is controlled by a promoter that is stronger than its native promoter. In some embodiments, the gene encoding the transporter of the bile salt is controlled by a constitutive promoter. 
     In another embodiment, the promoter is an inducible promoter. Inducible promoters are described in more detail infra. 
     In one embodiment, the gene encoding a transporter of a bile salt is located on a plasmid in the bacterial cell. In another embodiment, the gene encoding a transporter of a bile salt is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the gene encoding a transporter of a bile salt is located in the chromosome of the bacterial cell, and a copy of a gene encoding a transporter of a bile salt from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding a transporter of a bile salt is located on a plasmid in the bacterial cell, and a copy of a gene encoding a transporter of a bile salt from a different species of bacteria is located on a plasmid in the bacterial cell. In yet another embodiment, a native copy of the gene encoding a transporter of a bile salt is located in the chromosome of the bacterial cell, and a copy of the gene encoding a transporter of a bile salt from a different species of bacteria is located in the chromosome of the bacterial cell. 
     In some embodiments, the at least one native gene encoding the transporter of a bile salt in the bacterial cell is not modified, and one or more additional copies of the native transporter of a bile salt are inserted into the genome. In one embodiment, the one or more additional copies of the native transporter that is inserted into the genome are under the control of the same inducible promoter that controls expression of the gene encoding the bile salt hydrolase enzyme, e.g., the FNR responsive promoter, or a different inducible promoter than the one that controls expression of the bile salt hydrolase enzyme, or a constitutive promoter. In alternate embodiments, the at least one native gene encoding the transporter is not modified, and one or more additional copies of the transporter from a different bacterial species is inserted into the genome of the bacterial cell. In one embodiment, the one or more additional copies of the transporter inserted into the genome of the bacterial cell are under the control of the same inducible promoter that controls expression of the gene encoding the bile salt hydrolase enzyme, e.g., the FNR responsive promoter, or a different inducible promoter than the one that controls expression of the gene encoding the bile salt hydrolase enzyme, or a constitutive promoter. 
     In one embodiment, when the transporter of a bile salt is expressed in the recombinant bacterial cells, the bacterial cells import 10% more bile salt 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 transporter of a bile salt is expressed in the recombinant bacterial cells, the bacterial cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more bile salt 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 transporter of a bile salt is expressed in the recombinant bacterial cells, the bacterial cells import two-fold more bile salt 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 transporter of a bile salt is expressed in the recombinant bacterial cells, the bacterial cells import three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, or ten-fold more bile salt into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions. 
     Generation of Bacterial Strains with Enhanced Ability to Transport Bile Salts 
     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 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 bile salts and/or over-produce bile salt hydrolase. 
     Exporters of Bile Salts 
     The export of bile salts is mediated by proteins well known to those of skill in the art. For example, the ATP-binding cassette, sub-family B member 11 (ABCB11, also called BSEP or “bile salt export pump”) is responsible for the export of taurochoate and other cholate conjugates from hepatocytes to the bile in mammals, and mutations in this gene have been associated with progressive familial intrahepatic cholestasis type 2 (PFIC2) and hepatocellular carcinoma (see Strautnieks et al.,  Nature Genetics,  20(3):233-238, 1998; Knisely et al.,  Hepatology,  44(2):478-486, 2006; and Ho et al.,  Pharmacogenet. Genomics,  20(1):45-57, 2010; SEQ ID NO: 113 and SEQ ID NO:114). In bacteria,  Streptococcus thermophilus  comprises a bile salt export pump (Msba subfamily ABC transporter ATP-binding protein; accession F8LYG6; SEQ ID NO: 116), and  Nostoc  spp. are known to comprise a bile salt export pump (As11293; accession Q8YXC2; SEQ ID NO: 117 and SEQ ID NO: 118). Multiple other bile salt exporters are known in the art. 
     Thus, in one embodiment of the invention, when the recombinant bacterial cell comprises an endogenous bile salt exporter gene, the recombinant bacterial cells may comprise a genetic modification that reduces export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cell comprises a genetic modification that reduces export of one or more bile salts from the bacterial cell and a heterologous gene encoding a bile salt catabolism enzyme. When the recombinant bacterial cells comprise a genetic modification that reduces export of a bile salt, the bacterial cells retain more bile salts in the bacterial cell than unmodified bacteria of the same bacterial subtype under the same conditions. Thus, the recombinant bacteria comprising a genetic modification that reduces export of a bile salt may be used to retain more bile salts in the bacterial cell so that any bile salt catabolism enzyme expressed in the organism can catabolize the bile salt(s) to treat diseases associated with bile salts, including cardiovascular disease. In one embodiment, the recombinant bacteria further comprise a heterologous gene encoding a transporter of one or more bile salts. 
     In one embodiment, the recombinant bacterial cell comprises a genetic modification in a gene encoding a bile salt exporter wherein said bile salt exporter comprises an amino acid sequence that has at least 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of a polypeptide encoded by a bile salt exporter gene disclosed herein. In one embodiment, the bile salt exporter has at least 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 115. In another embodiment, the bile salt exporter has at least 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 117. 
     In one embodiment, the genetic modification reduces export of a bile salt from the bacterial cell. In one embodiment, the bacterial cell is from a bacterial genus or species that includes but is not limited to,  Streptococcus thermophilus  or  Nostoc  spp. 
     In one embodiment, the genetic modification is a mutation in an endogenous gene encoding an exporter of one or more bile salts. In one embodiment, the genetic mutation results in an exporter having reduced activity as compared to a wild-type exporter protein. In one embodiment, the activity of the exporter is reduced at least 50%, at least 75%, or at least 100%. In another embodiment, the activity of the exporter is reduced at least two-fold, three-fold, four-fold, or five-fold. In another embodiment, the genetic mutation results in an exporter having no activity, i.e., results in an exporter which cannot export one or more bile salts from the bacterial cell. 
     It is routine for one of ordinary skill in the art to make mutations in a gene of interest. Mutations include substitutions, insertions, deletions, and/or truncations of one or more specific amino acid residues or of one or more specific nucleotides or codons in the polypeptide or polynucleotide of the exporter of an amino acid. Mutagenesis and directed evolution methods are well known in the art for creating variants. See, e.g., U.S. Pat. Nos. 7,783,428; 6,586,182; 6,117,679; and Ling, et al., 1999, “Approaches to DNA mutagenesis: an overview,”  Anal. Biochem.,  254(2):157-78; Smith, 1985, “In vitro mutagenesis,”  Ann. Rev. Genet.,  19:423-462; Carter, 1986, “Site-directed mutagenesis,”  Biochem. J.,  237:1-7; and Minshull, et al., 1999, “Protein evolution by molecular breeding,”  Current Opinion in Chemical Biology,  3:284-290. For example, the lambda red system can be used to knock-out genes in  E. coli  (see, for example, Datta et al.,  Gene,  379:109-115 (2006)). 
     The term “inactivated” as applied to a gene refers to any genetic modification that decreases or eliminates the expression of the gene and/or the functional activity of the corresponding gene product (mRNA and/or protein). The term “inactivated” encompasses complete or partial inactivation, suppression, deletion, interruption, blockage, promoter alterations, antisense RNA, dsRNA, or down-regulation of a gene. This can be accomplished, for example, by gene “knockout,” inactivation, mutation (e.g., insertion, deletion, point, or frameshift mutations that disrupt the expression or activity of the gene product), or by use of inhibitory RNAs (e.g., sense, antisense, or RNAi technology). A deletion may encompass all or part of a gene&#39;s coding sequence. The term “knockout” refers to the deletion of most (at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) or all (100%) of the coding sequence of a gene. In some embodiments, any number of nucleotides can be deleted, from a single base to an entire piece of a chromosome. 
     Assays for testing the activity of an exporter of one or more bile salts are well known to one of ordinary skill in the art. For example, export of one or more bile salts may be determined using the methods described by Telbisz and Homolya,  Expert Opinion Ther. Targets,  1-14, 2015, the entire contents of which are expressly incorporated herein by reference. 
     In another embodiment, the genetic modification is a mutation in a promoter of an endogenous gene encoding an exporter of one or more bile salts. In one embodiment, the genetic mutation results in decreased expression of the exporter gene. In one embodiment, exporter gene expression is reduced by about 50%, 75%, or 100%. In another embodiment, exporter gene expression is reduced about two-fold, three-fold, four-fold, or five-fold. In another embodiment, the genetic mutation completely inhibits expression of the exporter gene. 
     Assays for testing the level of expression of a gene, such as an exporter of one or more bile salts are well known to one of ordinary skill in the art. For example, reverse-transcriptase polymerase chain reaction may be used to detect the level of mRNA expression of a gene. Alternatively, Western blots using antibodies directed against a protein may be used to determine the level of expression of the protein. 
     In another embodiment, the genetic modification is an overexpression of a repressor of an exporter of one or more bile salts. In one embodiment, the overexpression of the repressor of the exporter is caused by a mutation which renders the promoter of the repressor constitutively active. In another embodiment, the overexpression of the repressor of the exporter is caused by the insertion of an inducible promoter in front of the repressor so that the expression of the repressor can be induced. Inducible promoters are described in more detail herein. 
     In one embodiment, the recombinant bacterial cells described herein comprise at least one genetic modification that reduces export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cells described herein comprise two genetic modifications that reduce export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cells described herein comprise three genetic modifications that reduce export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cells described herein comprise four genetic modifications that reduce export of one or more bile salts from the bacterial cell. In another embodiment, the recombinant bacterial cells described herein comprise five genetic modifications that reduce export of one or more bile salts from the bacterial cell. 
     Tryptophan, Tryptophan Metabolism, and Tryptophan Metabolites 
     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) in select populations of neurons by tryptophan hydroxylase. Serotonin can further be converted into the hormone melatonin. The majority of tryptophan, approximately 95%, 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). 
     AhR is a ligand-dependent cytosolic transcription factor that is able to translocate to the cell nucleus after ligand binding. The in additiona 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. 
     More recently, additional tryptophan metabolites, some of which are generated by the microbiota, some by the human host, which are also able to function as AhR agonists, see e.g., Table 13 and  FIG. 32  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. 
     In humans, 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). Along one side arm, KYN may be further metabolized to another bioactive metabolite, kynurenic acid, (KYNA) which can antagonize glutamate receptors and is generally considered neuroprotective or along a second arm to 3-hydroxykynurenine (3-HK). Along a third side arm 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. Consequently, the KP has two responsibilities in regard to TRP in the body; it depletes serum levels of TRP and converts TRP into other biologically active metabolites. These metabolites, along with the enzymes responsible for their production, have implications in a broad range of diseases, including, but not limited to, various neurological conditions, metabolic syndrome, and associated cardiovascular disorders, obesity and diabetes. 
     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. 
     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), thus allocating the bioactivity of serotonin into either the brain (Tph2) or the periphery (Tph1). Then, 5-HTP undergoes decarboxylation to serotonin. Intestinal serotonin (5-hydroxytryptamine, 5-HT) 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. In certain embodiments, the genetically engineered bacteria described herein may modulate serotonin levels in the intestine, e.g., decrease serotonin levels. 
     5-HT also functions a substrate for melatonin biosynthesis. The rate-limiting step of melatonin biosynthesis is 5-HT-N-acetylation resulting in the formation of N-acetyl-serotonin (NAS) with subsequent Omethylation into 5-methoxy-N-acetyltryptamine (melatonin). The deficient production of 5-HT, NAS, and melatonin contribute to depressed mood, disturbances of sleep and circadian rhythms. Melatonin acts as a neurohormone and is associated with the development of circadian rhythm and the sleep-wake cycle. 
     In certain embodiments, the genetically engineered bacteria influence 5-HT synthesis, release, and/or degradation. Gut microbiota are interconnected with serotonin signaling and care 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). In some embodiments, the genetically engineered bacteria may modulate the serotonin levels in the gut to ameliorate symptoms of a metabolic disease. In some embodiments, the genetically engineered bacteria take up serotonin from the environment, e.g., the gut. In a non limiting example, serotonin can be converted to melatonin by, e.g., TPH, tryptophan hydroxylase, HIOMT, hydroxyl-O-methyltransferase NAT, N-acetyltransferase, AAAD: aromatic-amino acid decarboxylase. In some embodiments, the genetically engineered influence serotonin levels produced by the host. 
     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 anon-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. 
     IDO, and 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 IDO1. 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 ( FIG. 27  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. 
     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 the amino acid 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 Reoports 5:12689). Table 13 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. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 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 
               
               
                   
                 Metabolism 
                 (I3S) 
               
               
                   
                 Host 
                 Kynurenine 
               
               
                   
                 Metabolism 
                 (Kyn) 
               
               
                   
                 Host 
                 Kynurenic acid 
               
               
                   
                 Metabolism 
                 (KA) 
               
               
                   
                 Host 
                 Xanthurenic acid 
               
               
                   
                 Metabolism 
                   
               
               
                   
                 Host 
                 Cinnabarinic acid 
               
               
                   
                 Metabolism 
                 (CA) 
               
               
                   
                 UV-Light 
                 6-formylindolo(3,2-b)carbazole 
               
               
                   
                 Oxidation 
                 (FICZ) 
               
               
                   
                   
               
            
           
         
       
     
     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 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, fourty-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 13. 
     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 comprises 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 comprises 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. 
     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 comprises one or more gene sequences for converting tryptophan to kynurenine. In some embodiments, the engineered bacteria comprises 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 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. 
     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 liver damage, metabolic disease, 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 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. The genetically engineered bacteria may comprise any suitable gene 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, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, 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 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 diseaseprogression 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 liver damage, metabolic disease, 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 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. In some embodiments, 
     The genetically engineered bacteria may comprise any suitable gene 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, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, 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 prevent the accumulation of post-kynurenine KP metabolites, e.g., neurotoxic metabolites, or diabetogenic metabolites. In some embodiments, the genetically engineered bacteria encode Kynureninase from  Pseudomonas fluorescens.    
     In some embodiments, the genetically engineered bacteria comprising one or more gene(s) or gene cassette(s) can alter the TRP:KYN ratio, e.g. in the circulation. In some embodiments the TRP:KYN ratio is increased. In some embodiments, TRP:KYN ratio is decreased. In some embodiments, the genetically engineered bacteria the genetically engineered bacteria comprising one or more gene(s) or gene cassette(s) can alter the KYNA:QUIN ratio, e.g., in the brain. 
     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, liver damage, or metabolic disease, or in the presence of some other metabolite that may or may not be present in the gut or the tumor micorenvironment, 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. 
     Increasing Exogenous Tryptophan 
     In some embodiments, the genetically engineered microorganisms of the present disclosure, are capable of producing tryptophan. 
     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 . 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. 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. 
     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. 
     As discussed above, studies have shown that the binding of kynurenine to the aryl hydrocarbon receptor results in the production of regulatory T cells (Tregs). Thus, in some embodiments, the genetically engineered bacteria comprise a mechanism for metabolizing or degrading kyurenine. In some embodiments, the genetically engineered bacteria comprise sequence encoding the enzyme kynureninase. Kynureninase is produced to metabolize Kynurenine to Anthranilic acid in the cell. Schwarcz et al.,  Nature Reviews Neuroscience,  13, 465-477; 2012 ; Chen  &amp;  Guillemin,  2009; 2; 1-19; Intl. J. Tryptophan Res. Exemplary kynureninase sequences are provided herein below in Table 3. 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 mtr gene. 
     In some embodiments, the genetically engineered bacteria comprise gene sequence(s) encoding enzymes of the tryptophan biosynthetic pathway and sequence encoding kynureninase. In some embodiments, the genetically engineered bacteria comprise a tryptophan operon, for example that of  E. coli . or  B. subtilis , and sequence encoding kynureninase. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes, for example, from  E. Coli  and sequence encoding kyureninase. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes, for example from  B. subtilis  and sequence encoding kyureninase. 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, for example, sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC. Thus, in some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypG-D, trypC-F, trypB, and trpA genes from  E. Coli , sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes, and sequence encoding kyureninase. In some embodiments, the genetically engineered bacteria comprise sequence(s) encoding trypE, trypD, trypC, trypF, trypB, and trpA genes from  B. subtilis , sequence(s) encoding aroG, aroF, aroH, aroB, aroD, aroE, aroK, and AroC genes, and sequence encoding kyureninase. In any of these embodiments, the genetically engineered bacterium may further comprise gene sequence for exporting or secreting tryptophan from the cell. Thus, in some embodiments, the engineered bacteria 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 any of these embodiments, the genetically engineered bacterium may further comprise gene sequence for importing or transporting kynurenine 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 mtr gene. 
     In some embodiments, the genetically engineered bacterium or genetically engineered microorganism comprises one or more genes for producing tryptophan and/or kynureninase, 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 and/or kynureninase, under the control of a cancer-specific promoter, a tissue-specific promoter, or a constitutive promoter, such as any of the promoters described herein. Table 14 lists exemplary tryptophan synthesis cassettes encoded by the genetically engineered bacteria of the disclosure. 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Tyrptophan Synthesis Cassette Sequences 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 Tet- 
                 taagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaaggccgaataagaagg 
               
               
                 regulated 
                 ctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcatactatcagtag 
               
               
                 Tryptophan 
                 taggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgc 
               
               
                 operon 
                 cccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcataaaaaggctaattg 
               
               
                 SEQ ID 
                 attttcgagagtttcatactgtttttctgtaggccgtgtacctaaatgtacttttgctccatcgcgatgact 
               
               
                 NO: 121 
                 tagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgccagctttccccttctaaaggg 
               
               
                   
                 caaaagtgagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagcccgcttattttt 
               
               
                   
                 tacatgccaatacaatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaaccttc 
               
               
                   
                 gattccgacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctagacatcatta 
               
               
                   
                 attcctaatttttgttgacactctatcattgatagagttattttaccactccctatcagtgatagagaaaag 
               
               
                   
                 tgaactctagaaataattttgtttaactttaagaaggagatatacatatgcaaacacaaaaaccgactc 
               
               
                   
                 tcgaactgctaacctgcgaaggcgcttatcgcgacaacccgactgcgctttttcaccagttgtgtgg 
               
               
                   
                 ggatcgtccggcaacgctgctgctggaatccgcagatatcgacagcaaagatgatttaaaaagcc 
               
               
                   
                 tgctgctggtagacagtgcgctgcgcattacagcattaagtgacactgtcacaatccaggcgctttc 
               
               
                   
                 cggcaatggagaagccctgttgacactactggataacgccttgcctgcgggtgtggaaaatgaac 
               
               
                   
                 aatcaccaaactgccgcgtactgcgcttcccgcctgtcagtccactgctggatgaagacgcccgct 
               
               
                   
                 tatgctccctttcggtttttgacgctttccgcttattacagaatctgttgaatgtaccgaaggaagaacg 
               
               
                   
                 agaagcaatgttcttcggcggcctgttctcttatgaccttgtggcgggatttgaaaatttaccgcaact 
               
               
                   
                 gtcagcggaaaatagctgccctgatttctgtttttatctcgctgaaacgctgatggtgattgaccatca 
               
               
                   
                 gaaaaaaagcactcgtattcaggccagcctgtttgctccgaatgaagaagaaaaacaacgtctcac 
               
               
                   
                 tgctcgcctgaacgaactacgtcagcaactgaccgaagccgcgccgccgctgccggtggtttcc 
               
               
                   
                 gtgccgcatatgcgttgtgaatgtaaccagagcgatgaagagttcggtggtgtagtgcgtttgttgc 
               
               
                   
                 aaaaagcgattcgcgccggagaaattttccaggtggtgccatctcgccgtttctctctgccctgccc 
               
               
                   
                 gtcaccgctggcagcctattacgtgctgaaaaagagtaatcccagcccgtacatgttttttatgcag 
               
               
                   
                 gataatgatttcaccctgtttggcgcgtcgccggaaagttcgctcaagtatgacgccaccagccgc 
               
               
                   
                 cagattgagatttacccgattgccggaacacgtccacgcggtcgtcgtgccgatggttcgctggac 
               
               
                   
                 agagacctcgacagccgcatcgaactggagatgcgtaccgatcataaagagctttctgaacatctg 
               
               
                   
                 atgctggtggatctcgcccgtaatgacctggcacgcatttgcacacccggcagccgctacgtcgc 
               
               
                   
                 cgatctcaccaaagttgaccgttactcttacgtgatgcacctagtctcccgcgttgttggtgagctgc 
               
               
                   
                 gccacgatctcgacgccctgcacgcttaccgcgcctgtatgaatatggggacgttaagcggtgca 
               
               
                   
                 ccgaaagtacgcgctatgcagttaattgccgaagcagaaggtcgtcgacgcggcagctacggcg 
               
               
                   
                 gcgcggtaggttattttaccgcgcatggcgatctcgacacctgcattgtgatccgctcggcgctggt 
               
               
                   
                 ggaaaacggtatcgccaccgtgcaagccggtgctggcgtagtccttgattctgttccgcagtcgga 
               
               
                   
                 agccgacgaaactcgtaataaagcccgcgctgtactgcgcgctattgccaccgcgcatcatgcac 
               
               
                   
                 aggagacgttctaatggctgacattctgctgctcgataatatcgactcttttacgtacaacctggcag 
               
               
                   
                 atcagttgcgcagcaatggtcataacgtggtgatttaccgcaaccatattccggcgcagaccttaatt 
               
               
                   
                 gaacgcctggcgacgatgagcaatccggtgctgatgctttctcctggccccggtgtgccgagcga 
               
               
                   
                 agccggttgtatgccggaactcctcacccgcttgcgtggcaagctgccaattattggcatttgcctc 
               
               
                   
                 ggacatcaggcgattgtcgaagcttacgggggctatgtcggtcaggcgggcgaaattcttcacgg 
               
               
                   
                 taaagcgtcgagcattgaacatgacggtcaggcgatgtttgccggattaacaaacccgctgccagt 
               
               
                   
                 ggcgcgttatcactcgctggttggcagtaacattccggccggtttaaccatcaacgcccattttaatg 
               
               
                   
                 gcatggtgatggcggtgcgtcacgatgcagatcgcgtttgtggattccagttccatccggaatccat 
               
               
                   
                 tcttactacccagggcgctcgcctgctggaacaaacgctggcctgggcgcagcagaaactagag 
               
               
                   
                 ccaaccaacacgctgcaaccgattctggaaaaactgtatcaggcacagacgcttagccaacaaga 
               
               
                   
                 aagccaccagctgttttcagcggtggtacgtggcgagctgaagccggaacaactggcggcggc 
               
               
                   
                 gctggtgagcatgaaaattcgcggtgaacacccgaacgagatcgccggggcagcaaccgcgct 
               
               
                   
                 actggaaaacgccgcgccattcccgcgcccggattatctgtttgccgatatcgtcggtactggcgg 
               
               
                   
                 tgacggcagcaacagcatcaatatttctaccgccagtgcgtttgtcgccgcggcctgcgggctgaa 
               
               
                   
                 agtggcgaaacacggcaaccgtagcgtctccagtaaatccggctcgtcggatctgctggcggcgt 
               
               
                   
                 tcggtattaatcttgatatgaacgccgataaatcgcgccaggcgctggatgagttaggcgtctgtttc 
               
               
                   
                 ctctttgcgccgaagtatcacaccggattccgccatgcgatgccggttcgccagcaactgaaaacc 
               
               
                   
                 cgcactctgttcaacgtgctgggaccattgattaacccggcgcatccgccgctggcgctaattggt 
               
               
                   
                 gtttatagtccggaactggtgctgccgattgccgaaaccttgcgcgtgctggggtatcaacgcgcg 
               
               
                   
                 gcagtggtgcacagcggcgggatggatgaagtttcattacacgcgccgacaatcgttgccgaact 
               
               
                   
                 acatgacggcgaaattaagagctatcaattgaccgctgaagattttggcctgacaccctaccacca 
               
               
                   
                 ggagcaattggcaggcggaacaccggaagaaaaccgtgacattttaacacgcttgttacaaggta 
               
               
                   
                 aaggcgacgccgcccatgaagcagccgtcgcggcgaatgtcgccatgttaatgcgcctgcatgg 
               
               
                   
                 ccatgaagatctgcaagccaatgcgcaaaccgttcttgaggtactgcgcagtggttccgcttacga 
               
               
                   
                 cagagtcaccgcactggcggcacgagggtaaatgatgcaaaccgttttagcgaaaatcgtcgca 
               
               
                   
                 gacaaggcgatttgggtagaaacccgcaaagagcagcaaccgctggccagttttcagaatgagg 
               
               
                   
                 ttcagccgagcacgcgacatttttatgatgcacttcagggcgcacgcacggcgtttattctggagtg 
               
               
                   
                 taaaaaagcgtcgccgtcaaaaggcgtgatccgtgatgatttcgatccggcacgcattgccgccat 
               
               
                   
                 ttataaacattacgcttcggcaatttcagtgctgactgatgagaaatattttcaggggagctttgatttc 
               
               
                   
                 ctccccatcgtcagccaaatcgccccgcagccgattttatgtaaagacttcattatcgatccttacca 
               
               
                   
                 gatctatctggcgcgctattaccaggccgatgcctgcttattaatgctttcagtactggatgacgaac 
               
               
                   
                 aatatcgccagcttgcagccgtcgcccacagtctggagatgggtgtgctgaccgaagtcagtaat 
               
               
                   
                 gaagaggaactggagcgcgccattgcattgggggcaaaggtcgttggcatcaacaaccgcgatc 
               
               
                   
                 tgcgcgatttgtcgattgatctcaaccgtacccgcgagcttgcgccgaaactggggcacaacgtga 
               
               
                   
                 cggtaatcagcgaatccggcatcaatacttacgctcaggtgcgcgagttaagccacttcgctaacg 
               
               
                   
                 gctttctgattggttcggcgttgatggcccatgacgatttgaacgccgccgtgcgtcgggtgttgctg 
               
               
                   
                 ggtgagaataaagtatgtggcctgacacgtgggcaagatgctaaagcagcttatgacgcgggcg 
               
               
                   
                 cgatttacggtgggttgatttttgttgcgacatcaccgcgttgcgtcaacgttgaacaggcgcagga 
               
               
                   
                 agtgatggctgcagcaccgttgcagtatgttggcgtgttccgcaatcacgatattgccgatgtggcg 
               
               
                   
                 gacaaagctaaggtgttatcgctggcggcagtgcaactgcatggtaatgaagatcagctgtatatc 
               
               
                   
                 gacaatctgcgtgaggctctgccagcacacgtcgccatctggaaggctttaagtgtcggtgaaact 
               
               
                   
                 cttcccgcgcgcgattttcagcacatcgataaatatgtattcgacaacggtcagggcgggagcgga 
               
               
                   
                 caacgtttcgactggtcactattaaatggtcaatcgcttggcaacgttctgctggcggggggcttag 
               
               
                   
                 gcgcagataactgcgtggaagcggcacaaaccggctgcgccgggcttgattttaattctgctgtag 
               
               
                   
                 agtcgcaaccgggtatcaaagacgcacgtcttttggcctcggttttccagacgctgcgcgcatatta 
               
               
                   
                 aggaaaggaacaatgacaacattacttaacccctattttggtgagtttggcggcatgtacgtgccac 
               
               
                   
                 aaatcctgatgcctgctctgcgccagctggaagaagcttttgtcagcgcgcaaaaagatcctgaatt 
               
               
                   
                 tcaggctcagttcaacgacctgctgaaaaactatgccgggcgtccaaccgcgctgaccaaatgcc 
               
               
                   
                 agaacattacagccgggacgaacaccacgctgtatctgaagcgcgaagatttgctgcacggcgg 
               
               
                   
                 cgcgcataaaactaaccaggtgctcggtcaggctttactggcgaagcggatgggtaaaactgaaa 
               
               
                   
                 ttattgccgaaaccggtgccggtcagcatggcgtggcgtcggcccttgccagcgccctgctcggc 
               
               
                   
                 ctgaaatgccgaatttatatgggtgccaaagacgttgaacgccagtcgcccaacgttttccggatgc 
               
               
                   
                 gcttaatgggtgcggaagtgatcccggtacatagcggttccgcgaccctgaaagatgcctgtaatg 
               
               
                   
                 aggcgctacgcgactggtccggcagttatgaaaccgcgcactatatgctgggtaccgcagctggc 
               
               
                   
                 ccgcatccttacccgaccattgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgca 
               
               
                   
                 gattctggaaagagaaggtcgcctgccggatgccgttatcgcctgtgttggcggtggttcgaatgc 
               
               
                   
                 catcggtatgtttgcagatttcatcaacgaaaccgacgtcggcctgattggtgtggagcctggcgg 
               
               
                   
                 ccacggtatcgaaactggcgagcacggcgcaccgttaaaacatggtcgcgtgggcatctatttcg 
               
               
                   
                 gtatgaaagcgccgatgatgcaaaccgaagacgggcaaattgaagagtcttactccatttctgccg 
               
               
                   
                 ggctggatttcccgtccgtcggcccgcaacatgcgtatctcaacagcactggacgcgctgattacg 
               
               
                   
                 tgtctattaccgacgatgaagccctggaagcctttaaaacgctttgcctgcatgaagggatcatccc 
               
               
                   
                 ggcgctggaatcctcccacgccctggcccatgcgctgaaaatgatgcgcgaaaatccggaaaaa 
               
               
                   
                 gagcagctactggtggttaacctttccggtcgcggcgataaagacatcttcaccgttcacgatatttt 
               
               
                   
                 gaaagcacgaggggaaatctgatggaacgctacgaatctctgtttgcccagttgaaggagcgcaa 
               
               
                   
                 agaaggcgcattcgttcctttcgtcaccctcggtgatccgggcattgagcagtcgttgaaaattatcg 
               
               
                   
                 atacgctaattgaagccggtgctgacgcgctggagttaggcatccccttctccgacccactggcgg 
               
               
                   
                 atggcccgacgattcaaaacgccacactgcgtgcttttgcggcgggagtaaccccggcgcagtg 
               
               
                   
                 ctttgagatgctggcactcattcgccagaagcacccgaccattcccatcggccttttgatgtatgcca 
               
               
                   
                 acctggtgtttaacaaaggcattgatgagttttatgccgagtgcgagaaagtcggcgtcgattcggt 
               
               
                   
                 gctggttgccgatgtgcccgtggaagagtccgcgcccttccgccaggccgcgttgcgtcataatgt 
               
               
                   
                 cgcacctatctttatttgcccgccgaatgccgacgatgatttgctgcgccagatagcctcttacggtc 
               
               
                   
                 gtggttacacctatttgctgtcgcgagcgggcgtgaccggcgcagaaaaccgcgccgcgttaccc 
               
               
                   
                 ctcaatcatctggttgcgaagctgaaagagtacaacgctgcgcctccattgcagggatttggtatttc 
               
               
                   
                 cgccccggatcaggtaaaagccgcgattgatgcaggagctgcgggcgcgatttctggttcggcc 
               
               
                   
                 atcgttaaaatcatcgagcaacatattaatgagccagagaaaatgctggcggcactgaaagcttttg 
               
               
                   
                 tacaaccgatgaaagcggcgacgcgcagttaatacgcatggcatggatgaCCGATGGTA 
               
               
                   
                 GTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGC 
               
               
                   
                 ATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTT 
               
               
                   
                 TCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAG 
               
               
                   
                 GACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAA 
               
               
                   
                 CGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTG 
               
               
                   
                 CCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGG 
               
               
                   
                 CCTTTTTGCGTGGCCAGTGCCAAGCTTGCATGCGTGC 
               
               
                   
               
               
                 Tet repressor 
                 taagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaaggccgaataagaagg 
               
               
                 SEQ ID NO: 22 
                 ctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaataatggcggcatactatcagtag 
               
               
                   
                 taggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgc 
               
               
                   
                 cccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcataaaaaggctaattg 
               
               
                   
                 attttcgagagtttcatactgtttttctgtaggccgtgtacctaaatgtacttttgctccatcgcgatgact 
               
               
                   
                 tagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgccagctttccccttctaaaggg 
               
               
                   
                 caaaagtgagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagcccgcttattttt 
               
               
                   
                 tacatgccaatacaatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaaccttc 
               
               
                   
                 gattccgacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctagacat 
               
               
                   
               
               
                 tetR/tetA 
                 cattaattcctaatttttgttgacactctatcattgatagagttattttaccactccctatcagtgatagag 
               
               
                 promoters 
                 aaaagtgaactctagaaataattttgtttaactttaagaaggagatatacat 
               
               
                 and RBS and 
                   
               
               
                 leader region 
                   
               
               
                 SEQ ID 
                   
               
               
                 NO: 123 
                   
               
               
                   
               
               
                 trpE 
                 atgcaaacacaaaaaccgactctcgaactgctaacctgcgaaggcgcttatcgcgacaacccgac 
               
               
                 SEQ ID 
                 tgcgctttttcaccagttgtgtggggatcgtccggcaacgctgctgctggaatccgcagatatcgac 
               
               
                 NO: 124 
                 agcaaagatgatttaaaaagcctgctgctggtagacagtgcgctgcgcattacagcattaagtgac 
               
               
                   
                 actgtcacaatccaggcgctttccggcaatggagaagccctgttgacactactggataacgccttg 
               
               
                   
                 cctgcgggtgtggaaaatgaacaatcaccaaactgccgcgtactgcgcttcccgcctgtcagtcca 
               
               
                   
                 ctgctggatgaagacgcccgcttatgctccctttcggtttttgacgctttccgcttattacagaatctgtt 
               
               
                   
                 gaatgtaccgaaggaagaacgagaagcaatgttcttcggcggcctgttctcttatgaccttgtggcg 
               
               
                   
                 ggatttgaaaatttaccgcaactgtcagcggaaaatagctgccctgatttctgtttttatctcgctgaaa 
               
               
                   
                 cgctgatggtgattgaccatcagaaaaaaagcactcgtattcaggccagcctgtttgctccgaatga 
               
               
                   
                 agaagaaaaacaacgtctcactgctcgcctgaacgaactacgtcagcaactgaccgaagccgcg 
               
               
                   
                 ccgccgctgccggtggtttccgtgccgcatatgcgttgtgaatgtaaccagagcgatgaagagttc 
               
               
                   
                 ggtggtgtagtgcgtttgttgcaaaaagcgattcgcgccggagaaattttccaggtggtgccatctc 
               
               
                   
                 gccgtttctctctgccctgcccgtcaccgctggcagcctattacgtgctgaaaaagagtaatcccag 
               
               
                   
                 cccgtacatgttttttatgcaggataatgatttcaccctgtttggcgcgtcgccggaaagttcgctcaa 
               
               
                   
                 gtatgacgccaccagccgccagattgagatttacccgattgccggaacacgtccacgcggtcgtc 
               
               
                   
                 gtgccgatggttcgctggacagagacctcgacagccgcatcgaactggagatgcgtaccgatcat 
               
               
                   
                 aaagagctttctgaacatctgatgctggtggatctcgcccgtaatgacctggcacgcatttgcacac 
               
               
                   
                 ccggcagccgctacgtcgccgatctcaccaaagttgaccgttactcttacgtgatgcacctagtctc 
               
               
                   
                 ccgcgttgttggtgagctgcgccacgatctcgacgccctgcacgcttaccgcgcctgtatgaatat 
               
               
                   
                 ggggacgttaagcggtgcaccgaaagtacgcgctatgcagttaattgccgaagcagaaggtcgt 
               
               
                   
                 cgacgcggcagctacggcggcgcggtaggttattttaccgcgcatggcgatctcgacacctgcat 
               
               
                   
                 tgtgatccgctcggcgctggtggaaaacggtatcgccaccgtgcaagccggtgctggcgtagtcc 
               
               
                   
                 ttgattctgttccgcagtcggaagccgacgaaactcgtaataaagcccgcgctgtactgcgcgctat 
               
               
                   
                 tgccaccgcgcatcatgcacaggagacgttcta 
               
               
                   
               
               
                 TrpE 
                 MQTQKPTLELLTCEGAYRDNPTALFHQLCGDRPATLLLESADI 
               
               
                 SEQ ID 
                 DSKDDLKSLLLVDSALRITALSDTVTIQALSGNGEALLTLLDN 
               
               
                 NO: 125 
                 ALPAGVENEQSPNCRVLRFPPVSPLLDEDARLCSLSVFDAFRL 
               
               
                   
                 LQNLLNVPKEEREAMFFGGLFSYDLVAGFENLPQLSAENSCP 
               
               
                   
                 DFCFYLAETLMVIDHQKKSTRIQASLFAPNEEEKQRLTARLNE 
               
               
                   
                 LRQQLTEAAPPLPVVSVPHMRCECNQSDEEFGGVVRLLQKAI 
               
               
                   
                 RAGEIFQVVPSRRFSLPCPSPLAAYYVLKKSNPSPYMFFMQDN 
               
               
                   
                 DFTLFGASPESSLKYDATSRQIEIYPIAGTRPRGRRADGSLDRD 
               
               
                   
                 LDSRIELEMRTDHKELSEHLMLVDLARNDLARICTPGSRYVA 
               
               
                   
                 DLTKVDRYSYVMHLVSRVVGELRHDLDALHAYRACMNMGT 
               
               
                   
                 LSGAPKVRAMQLIAEAEGRRRGSYGGAVGYFTAHGDLDTCIV 
               
               
                   
                 IRSALVENGIATVQAGAGVVLDSVPQSEADETRNKARAVLRA 
               
               
                   
                 IATAHHAQETF 
               
               
                   
               
               
                 trpD 
                 atggctgacattctgctgctcgataatatcgactcttttacgtacaacctggcagatcagttgcgcag 
               
               
                 SEQ ID 
                 caatggtcataacgtggtgatttaccgcaaccatattccggcgcagaccttaattgaacgcctggcg 
               
               
                 NO: 126 
                 acgatgagcaatccggtgctgatgctttctcctggccccggtgtgccgagcgaagccggttgtatg 
               
               
                   
                 ccggaactcctcacccgcttgcgtggcaagctgccaattattggcatttgcctcggacatcaggcg 
               
               
                   
                 attgtcgaagcttacgggggctatgtcggtcaggcgggcgaaattcttcacggtaaagcgtcgag 
               
               
                   
                 cattgaacatgacggtcaggcgatgtttgccggattaacaaacccgctgccagtggcgcgttatca 
               
               
                   
                 ctcgctggttggcagtaacattccggccggtttaaccatcaacgcccattttaatggcatggtgatgg 
               
               
                   
                 cggtgcgtcacgatgcagatcgcgtttgtggattccagttccatccggaatccattcttactacccag 
               
               
                   
                 ggcgctcgcctgctggaacaaacgctggcctgggcgcagcagaaactagagccaaccaacacg 
               
               
                   
                 ctgcaaccgattctggaaaaactgtatcaggcacagacgcttagccaacaagaaagccaccagct 
               
               
                   
                 gttttcagcggtggtacgtggcgagctgaagccggaacaactggcggcggcgctggtgagcatg 
               
               
                   
                 aaaattcgcggtgaacacccgaacgagatcgccggggcagcaaccgcgctactggaaaacgcc 
               
               
                   
                 gcgccattcccgcgcccggattatctgtttgccgatatcgtcggtactggcggtgacggcagcaac 
               
               
                   
                 agcatcaatatttctaccgccagtgcgtttgtcgccgcggcctgcgggctgaaagtggcgaaacac 
               
               
                   
                 ggcaaccgtagcgtctccagtaaatccggctcgtcggatctgctggcggcgttcggtattaatcttg 
               
               
                   
                 atatgaacgccgataaatcgcgccaggcgctggatgagttaggcgtctgtttcctctttgcgccgaa 
               
               
                   
                 gtatcacaccggattccgccatgcgatgccggttcgccagcaactgaaaacccgcactctgttcaa 
               
               
                   
                 cgtgctgggaccattgattaacccggcgcatccgccgctggcgctaattggtgtttatagtccggaa 
               
               
                   
                 ctggtgctgccgattgccgaaaccttgcgcgtgctggggtatcaacgcgcggcagtggtgcacag 
               
               
                   
                 cggcgggatggatgaagtttcattacacgcgccgacaatcgttgccgaactacatgacggcgaaa 
               
               
                   
                 ttaagagctatcaattgaccgctgaagattttggcctgacaccctaccaccaggagcaattggcag 
               
               
                   
                 gcggaacaccggaagaaaaccgtgacattttaacacgcttgttacaaggtaaaggcgacgccgc 
               
               
                   
                 ccatgaagcagccgtcgcggcgaatgtcgccatgttaatgcgcctgcatggccatgaagatctgc 
               
               
                   
                 aagccaatgcgcaaaccgttcttgaggtactgcgcagtggttccgcttacgacagagtcaccgca 
               
               
                   
                 ctggcggcacgagggtaa 
               
               
                   
               
               
                 TrpD 
                 MADILLLDNIDSFTYNLADQLRSNGHNVVIYRNHIPAQTLIERL 
               
               
                 SEQ ID 
                 ATMSNPVLMLSPGPGVPSEAGCMPELLTRLRGKLPIIGICLGH 
               
               
                 NO: 127 
                 QAIVEAYGGYVGQAGEILHGKASSIEHDGQAMFAGLTNPLPV 
               
               
                   
                 ARYHSLVGSNIPAGLTINAHFNGMVMAVRHDADRVCGFQFH 
               
               
                   
                 PESILTTQGARLLEQTLAWAQQKLEPTNTLQPILEKLYQAQTL 
               
               
                   
                 SQQESHQLFSAVVRGELKPEQLAAALVSMKIRGEHPNEIAGA 
               
               
                   
                 ATALLENAAPFPRPDYLFADIVGTGGDGSNSINISTASAFVAA 
               
               
                   
                 ACGLKVAKHGNRSVSSKSGSSDLLAAFGINLDMNADKSRQAL 
               
               
                   
                 DELGVCFLFAPKYHTGFRHAMPVRQQLKTRTLFNVLGPLINP 
               
               
                   
                 AHPPLALIGVYSPELVLPIAETLRVLGYQRAAVVHSGGMDEVS 
               
               
                   
                 LHAPTIVAELHDGEIKSYQLTAEDFGLTPYHQEQLAGGTPEEN 
               
               
                   
                 RDILTRLLQGKGDAAHEAAVAANVAMLMRLHGHEDLQANA 
               
               
                   
                 QTVLEVLRSGSAYDRVTALAARG 
               
               
                   
               
               
                 trpC 
                 atgcaaaccgttttagcgaaaatcgtcgcagacaaggcgatttgggtagaaacccgcaaagagca 
               
               
                 SEQ ID 
                 gcaaccgctggccagttttcagaatgaggttcagccgagcacgcgacatttttatgatgcacttcag 
               
               
                 NO: 128 
                 ggcgcacgcacggcgtttattctggagtgtaaaaaagcgtcgccgtcaaaaggcgtgatccgtga 
               
               
                   
                 tgatttcgatccggcacgcattgccgccatttataaacattacgcttcggcaatttcagtgctgactga 
               
               
                   
                 tgagaaatattttcaggggagctttgatttcctccccatcgtcagccaaatcgccccgcagccgattt 
               
               
                   
                 tatgtaaagacttcattatcgatccttaccagatctatctggcgcgctattaccaggccgatgcctgct 
               
               
                   
                 tattaatgctttcagtactggatgacgaacaatatcgccagcttgcagccgtcgcccacagtctgga 
               
               
                   
                 gatgggtgtgctgaccgaagtcagtaatgaagaggaactggagcgcgccattgcattgggggca 
               
               
                   
                 aaggtcgttggcatcaacaaccgcgatctgcgcgatttgtcgattgatctcaaccgtacccgcgag 
               
               
                   
                 cttgcgccgaaactggggcacaacgtgacggtaatcagcgaatccggcatcaatacttacgctca 
               
               
                   
                 ggtgcgcgagttaagccacttcgctaacggctttctgattggttcggcgttgatggcccatgacgatt 
               
               
                   
                 tgaacgccgccgtgcgtcgggtgttgctgggtgagaataaagtatgtggcctgacacgtgggcaa 
               
               
                   
                 gatgctaaagcagcttatgacgcgggcgcgatttacggtgggttgatttttgttgcgacatcaccgc 
               
               
                   
                 gttgcgtcaacgttgaacaggcgcaggaagtgatggctgcagcaccgttgcagtatgttggcgtgt 
               
               
                   
                 tccgcaatcacgatattgccgatgtggcggacaaagctaaggtgttatcgctggcggcagtgcaa 
               
               
                   
                 ctgcatggtaatgaagatcagctgtatatcgacaatctgcgtgaggctctgccagcacacgtcgcc 
               
               
                   
                 atctggaaggctttaagtgtcggtgaaactcttcccgcgcgcgattttcagcacatcgataaatatgt 
               
               
                   
                 attcgacaacggtcagggcgggagcggacaacgtttcgactggtcactattaaatggtcaatcgct 
               
               
                   
                 tggcaacgttctgctggcggggggcttaggcgcagataactgcgtggaagcggcacaaaccgg 
               
               
                   
                 ctgcgccgggcttgattttaattctgctgtagagtcgcaaccgggtatcaaagacgcacgtcttttgg 
               
               
                   
                 cctcggttttccagacgctgcgcgcatattaa 
               
               
                   
               
               
                 TrpC 
                 MQTVLAKIVADKAIWVETRKEQQPLASFQNEVQPSTRHFYDA 
               
               
                 SEQ ID 
                 LQGARTAFILECKKASPSKGVIRDDFDPARIAAIYKHYASAISV 
               
               
                 NO: 129 
                 LTDEKYFQGSFDFLPIVSQIAPQPILCKDFIIDPYQIYLARYYQA 
               
               
                   
                 DACLLMLSVLDDEQYRQLAAVAHSLEMGVLTEVSNEEELER 
               
               
                   
                 AIALGAKVVGINNRDLRDLSIDLNRTRELAPKLGHNVTVISES 
               
               
                   
                 GINTYAQVRELSHFANGFLIGSALMAHDDLNAAVRRVLLGEN 
               
               
                   
                 KVCGLTRGQDAKAAYDAGAIYGGLIFVATSPRCVNVEQAQE 
               
               
                   
                 VMAAAPLQYVGVFRNHDIADVADKAKVLSLAAVQLHGNED 
               
               
                   
                 QLYIDNLREALPAHVAIWKALSVGETLPARDFQHIDKYVFDN 
               
               
                   
                 GQGGSGQRFDWSLLNGQSLGNVLLAGGLGADNCVEAAQTG 
               
               
                   
                 CAGLDFNSAVESQPGIKDARLLASVFQTLRAY 
               
               
                   
               
               
                 trpB 
                 atgacaacattacttaacccctattttggtgagtttggcggcatgtacgtgccacaaatcctgatgcct 
               
               
                 SEQ ID 
                 gctctgcgccagctggaagaagcttttgtcagcgcgcaaaaagatcctgaatttcaggctcagttca 
               
               
                 NO: 130 
                 acgacctgctgaaaaactatgccgggcgtccaaccgcgctgaccaaatgccagaacattacagc 
               
               
                   
                 cgggacgaacaccacgctgtatctgaagcgcgaagatttgctgcacggcggcgcgcataaaact 
               
               
                   
                 aaccaggtgctcggtcaggctttactggcgaagcggatgggtaaaactgaaattattgccgaaacc 
               
               
                   
                 ggtgccggtcagcatggcgtggcgtcggcccttgccagcgccctgctcggcctgaaatgccgaa 
               
               
                   
                 tttatatgggtgccaaagacgttgaacgccagtcgcccaacgttttccggatgcgcttaatgggtgc 
               
               
                   
                 ggaagtgatcccggtacatagcggttccgcgaccctgaaagatgcctgtaatgaggcgctacgcg 
               
               
                   
                 actggtccggcagttatgaaaccgcgcactatatgctgggtaccgcagctggcccgcatccttacc 
               
               
                   
                 cgaccattgtgcgtgagtttcagcggatgattggcgaagaaacgaaagcgcagattctggaaaga 
               
               
                   
                 gaaggtcgcctgccggatgccgttatcgcctgtgttggcggtggttcgaatgccatcggtatgtttg 
               
               
                   
                 cagatttcatcaacgaaaccgacgtcggcctgattggtgtggagcctggcggccacggtatcgaa 
               
               
                   
                 actggcgagcacggcgcaccgttaaaacatggtcgcgtgggcatctatttcggtatgaaagcgcc 
               
               
                   
                 gatgatgcaaaccgaagacgggcaaattgaagagtcttactccatttctgccgggctggatttccc 
               
               
                   
                 gtccgtcggcccgcaacatgcgtatctcaacagcactggacgcgctgattacgtgtctattaccga 
               
               
                   
                 cgatgaagccctggaagcctttaaaacgctttgcctgcatgaagggatcatcccggcgctggaatc 
               
               
                   
                 ctcccacgccctggcccatgcgctgaaaatgatgcgcgaaaatccggaaaaagagcagctactg 
               
               
                   
                 gtggttaacctttccggtcgcggcgataaagacatcttcaccgttcacgatattttgaaagcacgag 
               
               
                   
                 gggaaatctga 
               
               
                   
               
               
                 TrpB 
                 MTTLLNPYFGEFGGMYVPQILMPALRQLEEAFVSAQKDPEFQ 
               
               
                 SEQ ID 
                 AQFNDLLKNYAGRPTALTKCQNITAGTNTTLYLKREDLLHGG 
               
               
                 NO: 131 
                 AHKTNQVLGQALLAKRMGKTEIIAETGAGQHGVASALASAL 
               
               
                   
                 LGLKCRIYMGAKDVERQSPNVFRMRLMGAEVIPVHSGSATLK 
               
               
                   
                 DACNEALRDWSGSYETAHYMLGTAAGPHPYPTIVREFQRMIG 
               
               
                   
                 EETKAQILEREGRLPDAVIACVGGGSNAIGMFADFINETDVGLI 
               
               
                   
                 GVEPGGHGIETGEHGAPLKHGRVGIYFGMKAPMMQTEDGQIE 
               
               
                   
                 ESYSISAGLDFPSVGPQHAYLNSTGRADYVSITDDEALEAFKT 
               
               
                   
                 LCLHEGIIPALESSHALAHALKMMRENPEKEQLLVVNLSGRG 
               
               
                   
                 DKDIFTVHDILKARGEI 
               
               
                   
               
               
                 trpA 
                 atggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgttcctttcg 
               
               
                 SEQ ID 
                 tcaccctcggtgatccgggcattgagcagtcgttgaaaattatcgatacgctaattgaagccggtgc 
               
               
                 NO: 132 
                 tgacgcgctggagttaggcatccccttctccgacccactggcggatggcccgacgattcaaaacg 
               
               
                   
                 ccacactgcgtgcttttgcggcgggagtaaccccggcgcagtgctttgagatgctggcactcattc 
               
               
                   
                 gccagaagcacccgaccattcccatcggccttttgatgtatgccaacctggtgtttaacaaaggcat 
               
               
                   
                 tgatgagttttatgccgagtgcgagaaagtcggcgtcgattcggtgctggttgccgatgtgcccgtg 
               
               
                   
                 gaagagtccgcgcccttccgccaggccgcgttgcgtcataatgtcgcacctatctttatttgcccgc 
               
               
                   
                 cgaatgccgacgatgatttgctgcgccagatagcctcttacggtcgtggttacacctatttgctgtcg 
               
               
                   
                 cgagcgggcgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcatctggttgcgaagc 
               
               
                   
                 tgaaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccggatcaggtaaaag ccgcgattgatgcaggagctgcgggcgcgatttctggttcggccatcgttaaaatcatcgagcaac 
               
               
                   
                 atattaatgagccagagaaaatgctggcggcactgaaagcttttgtacaaccgatgaaagcggcg 
               
               
                   
                 acgcgcagttaa 
               
               
                   
               
               
                 TrpA 
                 MERYESLFAQLKERKEGAFVPFVTLGDPGIEQSLKIIDTLIEAG 
               
               
                 SEQ ID 
                 ADALELGIPFSDPLADGPTIQNATLRAFAAGVTPAQCFEMLAL 
               
               
                 NO: 133 
                 IRQKHPTIPIGLLMYANLVFNKGIDEFYAECEKVGVDSVLVAD 
               
               
                   
                 VPVEESAPFRQAALRHNVAPIFICPPNADDDLLRQIASYGRGY 
               
               
                   
                 TYLLSRAGVTGAENRAALPLNHLVAKLKEYNAAPPLQGFGIS 
               
               
                   
                 APDQVKAAIDAGAAGAISGSAIVKIIEQHINEPEKMLAALKAF 
               
               
                   
                 VQPMKAATRS 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 14 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 14 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 14 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 14 or a functional fragment thereof. 
     In one embodiment, one or more polypeptides and/polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 121 through SEQ ID NO: 133. In one embodiment, one or more polypeptides and/polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 85% identity with with one or more of SEQ ID NO: 121 through SEQ ID NO: 133. In one embodiment, one or more polypeptides and/polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 90% identity with with one or more of SEQ ID NO: 121 through SEQ ID NO: 133. In one embodiment, one or more polypeptides and/polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 95% identity with with one or more of SEQ ID NO: 121 through SEQ ID NO: 133. In one embodiment, one or more polypeptides and/polynucleotides encoded and expressed by the genetically engineered bacteria have have at least about 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 121 through SEQ ID NO: 133. 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 with one or more of SEQ ID NO: 121 through SEQ ID NO: 133. 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: 121 through SEQ ID NO: 133. 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: 121 through SEQ ID NO: 133. 
     Table 15 lists exemplary genes encoding kynureninase which are encoded by the genetically engineered bacteria of the disclosure in certain embodiments. 
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Kynureninase protein sequences 
               
            
           
           
               
               
               
            
               
                 Description 
                 ID 
                 Sequence 
               
               
                   
               
               
                 
                   Pseudomonas 
                 
                 P83788 
                 MTTRNDCLALDAQDSLAPLRQQFALPEGVIYLD 
               
               
                 
                   kynureninase 
                 
                   
                 GNSLGARPVAALARAQAVIAEEWGNGLIRSWNS 
               
               
                 SEQ ID NO: 134 
                   
                 AGWRDLSERLGNRLATLIGARDGEVVVTDTTSIN 
               
               
                   
                   
                 LFKVLSAALRVQATRSPERRVIVTETSNFPTDLYI 
               
               
                   
                   
                 AEGLADMLQQGYTLRLVDSPEELPQAIDQDTAV 
               
               
                   
                   
                 VMLTHVNYKTGYMHDMQALTALSHECGALAIW 
               
               
                   
                   
                 DLAHSAGAVPVDLHQAGADYAIGCTYKYLNGG 
               
               
                   
                   
                 PGSQAFVWVSPQLCDLVPQPLSGWFGHSRQFAM 
               
               
                   
                   
                 EPRYEPSNGIARYLCGTQPITSLAMVECGLDVFA 
               
               
                   
                   
                 QTDMASLRRKSLALTDLFIELVEQRCAAHELTLV 
               
               
                   
                   
                 TPREHAKRGSHVSFEHPEGYAVIQALIDRGVIGD 
               
               
                   
                   
                 YREPRIMRFGFTPLYTTFTEVWDAVQILGEILDRK 
               
               
                   
                   
                 TWAQAQFQVRHSVT* 
               
               
                   
               
               
                 Human 
                 Q16719 
                 MEPSSLELPADTVQRIAAELKCHPTDERVALHLD 
               
               
                 SEQ ID NO: 135 
                   
                 EEDKLRHFRECFYIPKIQDLPPVDLSLVNKDENAI 
               
               
                   
                   
                 YFLGNSLGLQPKMVKTYLEEELDKWAKIAAYGH 
               
               
                   
                   
                 EVGKRPWITGDESIVGLMKDIVGANEKEIALMN 
               
               
                   
                   
                 ALTVNLHLLMLSFFKPTPKRYKILLEAKAFPSDH 
               
               
                   
                   
                 YAIESQLQLHGLNIEESMRMIKPREGEETLRIEDIL 
               
               
                   
                   
                 EVIEKEGDSIAVILFSGVHFYTGQHFNIPAITKAG 
               
               
                   
                   
                 QAKGCYVGFDLAHAVGNVELYLHDWGVDFAC 
               
               
                   
                   
                 WCSYKYLNAGAGGIAGAFIHEKHAHTIKPALVG 
               
               
                   
                   
                 WFGHELSTRFKMDNKLQLIPGVCGFRISNPPILLV 
               
               
                   
                   
                 CSLHASLEIFKQATMKALRKKSVLLTGYLEYLIK 
               
               
                   
                   
                 HNYGKDKAATKKPVVNIITPSHVEERGCQLTITF 
               
               
                   
                   
                 SVPNKDVFQELEKRGVVCDKRNPNGIRVAPVPL 
               
               
                   
                   
                 YNSFHDVYKFTNLLTSILDSAETKN* 
               
               
                   
               
               
                 
                   Shewanella 
                 
                 Q8E973 
                 MLLNVKQDFCLAGPGYLLNHSVGRPLKSTEQAL 
               
               
                 SEQ ID NO: 136 
                   
                 KQAFFAPWQESGREPWGQWLGVIDNFTAALASL 
               
               
                   
                   
                 FNGQPQDFCPQVNLSSALTKIVMSLDRLTRDLTR 
               
               
                   
                   
                 NGGAVVLMSEIDFPSMGFALKKALPASCELRFIP 
               
               
                   
                   
                 KSLDVTDPNVWDAHICDDVDLVFVSHAYSNTGQ 
               
               
                   
                   
                 QAPLAQIISLARERGCLSLVDVAQSAGILPLDLAK 
               
               
                   
                   
                 LQPDFMIGSSVKWLCSGPGAAYLWVNPAILPEC 
               
               
                   
                   
                 QPQDVGWFSHENPFEFDIHDFRYHPTALRFWGG 
               
               
                   
                   
                 TPSIAPYAIAAHSIEYFANIGSQVMREHNLQLMEP 
               
               
                   
                   
                 VVQALDNELVSPQEVDKRSGTIILQFGERQPQILA 
               
               
                   
                   
                 ALAAANISVDTRSLGIRVSPHIYNDEADIARLLGV 
               
               
                   
                   
                 IKANR* 
               
               
                   
               
               
                 *designates the position of the stop codon 
               
            
           
         
       
     
     Table 16 lists exemplary codon-optimized kynureninase cassette sequences. 
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Selected codon-optimized kynureninase cassette sequences 
               
            
           
           
               
               
            
               
                 Kynureninase 
                   
               
               
                 protein sequences 
                 Kynureninase protein sequences 
               
               
                   
               
               
                 Ptet- 
                 
                   atctaatctagacatcattaattcctaatttttgttgacactctatcattgatagagtta 
                 
               
               
                 kynU( Pseudomonas ) 
                 
                   ttttaccactccctatcagtgatagagaaaagtgaa 
                   ttatataaaagtgggaggtgcc 
                 
               
               
                 SEQ ID NO: 137 
                   cga atgacgacccgaaatgattgcctagcgttggatgcacaggacagtctggctccgct 
               
               
                   
                 gcgccaacaatttgcgctgccggagggtgtgatatacctggatggcaattcgctgggcg 
               
               
                   
                 cacgtccggtagctgcgctggctcgcgcgcaggctgtgatcgcagaagaatggggca 
               
               
                   
                 acgggttgatccgttcatggaactctgcgggctggcgtgatctgtctgaacgcctgggta 
               
               
                   
                 atcgcctggctaccctgattggtgcgcgcgatggggaagtagttgttactgataccacct 
               
               
                   
                 cgattaatctgtttaaagtgctgtcagcggcgctgcgcgtgcaagctacccgtagcccgg 
               
               
                   
                 agcgccgtgttatcgtgactgagacctcgaatttcccgaccgacctgtatattgcggaag 
               
               
                   
                 ggttggcggatatgctgcaacaaggttacactctgcgtttggtggattcaccggaagagc 
               
               
                   
                 tgccacaggctatagatcaggacaccgcggtggtgatgctgacgcacgtaaattataaa 
               
               
                   
                 accggttatatgcacgacatgcaggctctgaccgcgttgagccacgagtgtggggctct 
               
               
                   
                 ggcgatttgggatctggcgcactctgctggcgctgtgccggtggacctgcaccaagcg 
               
               
                   
                 ggcgcggactatgcgattggctgcacgtacaaatacctgaatggcggcccgggttcgc 
               
               
                   
                 aagcgtttgtttgggtttcgccgcaactgtgcgacctggtaccgcagccgctgtctggttg 
               
               
                   
                 gttcggccatagtcgccaattcgcgatggagccgcgctacgaaccttctaacggcattg 
               
               
                   
                 ctcgctatctgtgcggcactcagcctattactagcttggctatggtggagtgcggcctgga 
               
               
                   
                 tgtgtttgcgcagacggatatggcttcgctgcgccgtaaaagtctggcgctgactgatct 
               
               
                   
                 gttcatcgagctggttgaacaacgctgcgctgcacacgaactgaccctggttactccacg 
               
               
                   
                 tgaacacgcgaaacgcggctctcacgtgtcttttgaacaccccgagggttacgctgttatt 
               
               
                   
                 caagctctgattgatcgtggcgtgatcggcgattaccgtgagccacgtattatgcgtttcg 
               
               
                   
                 gtttcactcctctgtatactacttttacggaagtttgggatgcagtacaaatcctgggcgaa 
               
               
                   
                 atcctggatcgtaagacttgggcgcaggctcagtttcaggtgcgccactctgttacttaa a   
               
               
                   
                 
                   aataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttg 
                 
               
               
                   
               
               
                 Ptet-kynU(Human) 
                 
                   atctaatctagacatcattaattcctaatttttgttgacactctatcattgatagagtta 
                 
               
               
                 SEQ ID NO: 138 
                 
                   ttttaccactccctatcagtgatagagaaaagtgaa 
                   tatcaagacacgaggaggtaa 
                 
               
               
                   
                   gatt atggagccttcatctttagaactgccagcggacacggtgcagcgcatcgcggcgg 
               
               
                   
                 aactgaagtgccatccgactgatgagcgtgtggcgctgcatctggacgaagaagataa 
               
               
                   
                 actgcgccactttcgtgaatgtttttatattcctaaaattcaagacttgccgccggtagatttg 
               
               
                   
                 agtctcgttaacaaagatgaaaacgcgatctactttctgggcaactctctgggtctgcaac 
               
               
                   
                 caaaaatggttaaaacgtacctggaggaagaactggataaatgggcaaaaatcgcggc 
               
               
                   
                 ttatggtcacgaagtgggcaagcgtccttggattactggcgacgagtctattgtgggtttg 
               
               
                   
                 atgaaagatattgtgggcgcgaatgaaaaggaaattgcactgatgaatgctctgaccgtt 
               
               
                   
                 aatctgcacctgctgatgctgtctttttttaaaccgaccccgaaacgctacaaaatactgct 
               
               
                   
                 ggaagcgaaagcgtttccgtcggatcactatgctatagaaagtcaactgcagttgcatgg 
               
               
                   
                 tctgaatatcgaggaatctatgcgcatgattaaaccgcgtgagggtgaagaaacgctgc 
               
               
                   
                 gtattgaagacattctggaagttattgaaaaagaaggtgattctatcgcagttatactgtttt 
               
               
                   
                 ctggcgtgcacttttatacaggtcagcacttcaatatcccggcaatcactaaagcggggc 
               
               
                   
                 aggcaaaaggctgctatgttggttttgacctggcgcatgcagtggggaatgttgaactgt 
               
               
                   
                 atctgcacgattggggcgttgatttcgcgtgttggtgtagctacaaatatctgaacgctgg 
               
               
                   
                 cgcgggtggcattgctggcgcttttattcacgaaaaacacgcgcacaccattaaaccgg 
               
               
                   
                 ctctggttggctggttcggtcatgagctgagtactcgctttaaaatggataacaaactgca 
               
               
                   
                 attgattccgggtgtttgcggcttccgtatcagcaatccgccgattctgctggtttgcagcc 
               
               
                   
                 tgcacgctagtctggaaatctttaagcaggcgactatgaaagcgctgcgcaaaaaatctg 
               
               
                   
                 tgctgctgaccggctatctggagtatctgatcaaacacaattatggcaaagataaagctg 
               
               
                   
                 caactaaaaaaccggtagtgaacattatcaccccctcacacgtggaggagcgcggttgt 
               
               
                   
                 cagctgactattactttcagtgtacctaataaagatgtgttccaggaactggaaaaacgcg 
               
               
                   
                 gcgttgtttgtgataaacgtaacccgaatggtattcgcgtggctcctgtgccgctgtacaat 
               
               
                   
                 tcattccacgatgtttataaattcaccaacctgctgacttctattctcgacagtgctgagact 
               
               
                   
                 aaaaattaa aaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttg   
               
               
                   
               
               
                 ptet- 
                 
                   atctaatctagacatcattaattcctaatttttgttgacactctatcattgatagagtta 
                 
               
               
                 kynU( Shewanella ) 
                 
                   ttttaccactccctatcagtgatagagaaaagtgaa 
                   tggttcaccaccacaaggagg 
                 
               
               
                 SEQ ID NO: 139 
                   gatt atgctgctgaatgtaaaacaggacttttgcctggcaggcccgggctacctgctgaa 
               
               
                   
                 tcactcggttggccgtccgctgaaatcaactgagcaagcgctgaaacaagcattttttgct 
               
               
                   
                 ccgtggcaagagagcggtcgtgaaccgtggggccagtggctgggtgttattgataattt 
               
               
                   
                 cactgctgcgctggcatctctgtttaatggtcaaccgcaggatttttgtccgcaggttaacc 
               
               
                   
                 tgagcagcgcgctgactaaaattgtgatgtcactggatcgtctgactcgcgatctgaccc 
               
               
                   
                 gcaatggcggtgctgttgtgctgatgtctgaaatcgatttcccatctatgggcttcgcgttg 
               
               
                   
                 aaaaaagcgctgccagcgagctgcgaactgcgttttatcccgaaaagtctggacgtgac 
               
               
                   
                 tgatccgaacgtatgggatgcacacatctgtgatgatgtagacctggtttttgtgtctcacg 
               
               
                   
                 cctatagtaatacgggccaacaggctccgctggcgcaaatcatctctctggcgcgtgaa 
               
               
                   
                 cgtggctgcctgtcactggtggatgtagcgcaatcagcggggattttgccgctggatctg 
               
               
                   
                 gcgaaactgcaaccggacttcatgatcggcagttcggttaaatggctgtgctcgggccct 
               
               
                   
                 ggtgcggcatatctgtgggttaatccggcgattctgccggaatgtcagccgcaggatgt 
               
               
                   
                 gggctggttttcacatgagaatccctttgaattcgacatccacgatttccgctaccacccg 
               
               
                   
                 actgcactgcgcttttggggtggtacgccgtcgatcgcgccttatgcgatcgcggcgca 
               
               
                   
                 ctcgatcgaatattttgccaatatcggctcgcaagtgatgcgtgaacacaacctgcaactg 
               
               
                   
                 atggaaccggtggttcaggcgctggacaatgaactggtgagcccgcaggaagtggata 
               
               
                   
                 aacgctcaggcactattattctgcaattcggtgaacgtcaaccgcaaattctggcggctct 
               
               
                   
                 ggctgcggcgaacatttcggtggacactcgttctttggggattcgtgttagtccgcacattt 
               
               
                   
                 ataatgatgaggcggacattgcgcgcctgctgggtgtgatcaaagcaaatcgctaaaaa 
               
               
                   
                 taaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttg 
               
               
                   
               
               
                 The ptet-promoter is in bold, designed Ribosome binding site is underlined, codon-optimized protein coding sequence is in plain text, and the terminator is in italics. 
               
            
           
         
       
     
     In one embodiment, one or more polypeptides and/polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 121 through SEQ ID NO: 133. In one embodiment, one or more polypeptides and/polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 85% identity with with one or more of SEQ ID NO: 121 through SEQ ID NO: 133. In one embodiment, one or more polypeptides and/polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 90% identity with with one or more of SEQ ID NO: 121 through SEQ ID NO: 133. In one embodiment, one or more polypeptides and/polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 95% identity with with one or more of SEQ ID NO: 121 through SEQ ID NO: 133. In one embodiment, one or more polypeptides and/polynucleotides encoded and expressed by the genetically engineered bacteria have have at least about 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 121 through SEQ ID NO: 133. 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 with one or more of SEQ ID NO: 121 through SEQ ID NO: 133. 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: 121 through SEQ ID NO: 133. 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: 121 through SEQ ID NO: 133. 
     In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 16 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 16 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 16 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 16 or a functional fragment thereof. 
     In one embodiment, one or more polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 80% identity with one or more of SEQ ID NO: 137 through SEQ ID NO: 139. In one embodiment, one or more polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 85% identity with with one or more of SEQ ID NO: 137 through SEQ ID NO: 139. In one embodiment, one or more polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 90% identity with with one or more of SEQ ID NO: 137 through SEQ ID NO: 139. In one embodiment, one or more polynucleotides encoded and expressed by the genetically engineered bacteria have at least about 95% identity with with one or more of SEQ ID NO: 137 through SEQ ID NO: 139. In one embodiment, one or more polynucleotides encoded and expressed by the genetically engineered bacteria have have at least about 96%, 97%, 98%, or 99% identity with with one or more of SEQ ID NO: 137 through SEQ ID NO: 139. Accordingly, in one embodiment, one or more 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 with one or more of SEQ ID NO: 137 through SEQ ID NO: 139. In another embodiment, one or more polynucleotides encoded and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 137 through SEQ ID NO: 139. In another embodiment, one or more polynucleotides encoded and expressed by the genetically engineered bacteria comprise the sequence of one or more of SEQ ID NO: 137 through SEQ ID NO: 139. 
     The genetically engineered bacteria may comprise any suitable gene for producing kynureninase. In some embodiments, the gene for producing kynureninase is modified and/or mutated, e.g., to enhance stability, increase kynureninase production. 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 kynureninase under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynureninase in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, 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 genetically engineered bacteria may comprise any suitable gene for producing kynureninase. In some embodiments, the gene for producing kynureninase is modified and/or mutated, e.g., to enhance stability, increase kynureninase production. 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 kynureninase under inducing conditions, e.g., under a condition(s) associated with inflammation. In some embodiments, the genetically engineered bacteria are capable of producing kynureninase in low-oxygen conditions. In some embodiments, the genetically engineered bacteria are capable of producing kynureninase in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, 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. 
     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 liver damage, metabolic disease, 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 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 diseaseprogression 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 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. 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 liver damage, metabolic disease, 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 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, metabolic disease, 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. 
     Increasing Indole Tryptophan Metabolites 
     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 (JAM) pathway, see eg.,  FIG. 31  and  FIG. 32 . 
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Sequences for Tryptophan to tryptamine conversion 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 Tryptophan Decarboxylase 
                 MSQVIKKKRNTFMIGTEYILNSTQLEEAIKSFV 
               
               
                 (EC4.1.1.28) 
                 HDFCAEKHEIHDQPVVVEAKEHQEDKIKQIKIP 
               
               
                 Chain A,  Ruminococcus   
                 EKGRPVNEVVSEMMNEVYRYRGDANHPRFFS 
               
               
                   Gnavus  Tryptophan 
                 FVPGPASSVSWLGDIMTSAYNIHAGGSKLAPM 
               
               
                 Decarboxylase Rumgna_0152 
                 VNCIEQEVLKWLAKQVGFTENPGGVFVSGGS 
               
               
                 6 (alpha-fmt) 
                 MANITALTAARDNKLTDINLHLGTAYISDQTH 
               
               
                 SEQ ID NO: 140 
                 SSVAKGLRIIGITDSRIRRIPTNSHFQMDTTKLE 
               
               
                   
                 EAIETDKKSGYIPFVVIGTAGTTNTGSIDPLTEIS 
               
               
                   
                 ALCKKHDMWFHIDGAYGASVLLSPKYKSLLT 
               
               
                   
                 GTGLADSISWDAHKWLFQTYGCAMVLVKDIR 
               
               
                   
                 NLFHSFHVNPEYLKDLENDIDNVNTWDIGMEL 
               
               
                   
                 TRPARGLKLWLTLQVLGSDLIGSAIEHGFQLA 
               
               
                   
                 VWAEEALNPKKDWEIVSPAQMAMINFRYAPK 
               
               
                   
                 DLTKEEQDILNEKISHRILESGYAAIFTTVLNGK 
               
               
                   
                 TVLRICAIHPEATQEDMQHTIDLLDQYGREIYT 
               
               
                   
                 EMKKa 
               
               
                   
               
               
                 Tryptophan Decarboxylase 
                 ATGAGTCAAGTGATTAAGAAGAAACGTAAC 
               
               
                 (EC4.1.1.28) 
                 ACCTTTATGATCGGAACGGAGTACATTCTTA 
               
               
                 Chain A.  Ruminococcus   
                 ACAGTACACAATTGGAGGAAGCGATTAAAT 
               
               
                   Gnavus  Tryptophan 
                 CATTCGTACATGATTTCTGCGCAGAGAAGCA 
               
               
                 Decarboxylase Rumgna_0152 
                 TGAGATCCATGATCAACCTGTGGTAGTAGAA 
               
               
                 6 (alpha-fmt); codon 
                 GCTAAAGAACATCAGGAGGACAAAATCAAA 
               
               
                 optimized for the expression 
                 CAAATCAAAATCCCGGAAAAGGGACGTCCT 
               
               
                 in  E. coli   
                 GTAAATGAAGTCGTTTCTGAGATGATGAATG 
               
               
                 SEQ ID NO: 141 
                 AAGTGTATCGCTACCGCGGAGACGCCAAC 
               
               
                   
                 ATCCTCGCTTTTTTTCTTTTGTGCCCGGACCT 
               
               
                   
                 GCAAGCAGTGTGTCGTGGTTGGGGGATATTA 
               
               
                   
                 TGACGTCCGCCTACAATATTCATGCTGGAGG 
               
               
                   
                 CTCAAAGCTGGCACCGATGGTTAACTGCATT 
               
               
                   
                 GAGCAGGAAGTTCTGAAGTGGTTAGCAAAG 
               
               
                   
                 CAAGTGGGGTTCACAGAAATCCAGGTGGC 
               
               
                   
                 GTATTTGTGTCGGGCGGTTCAATGGCGAATA 
               
               
                   
                 TTACGGCACTTACTGCGGCTCGTGACAATAA 
               
               
                   
                 ACTGACCGACATTAACCTTCATTTGGGAACT 
               
               
                   
                 GCTTATATTAGTGACCAGACTCATAGTTCAG 
               
               
                   
                 TTGCGAAAGGATTACGCATTATTGGAATCAC 
               
               
                   
                 TGACAGTCGCATCCGTCGCATTCCCACTAAC 
               
               
                   
                 TCCCACTTCCAGATGGATACCACCAAGCTGG 
               
               
                   
                 AGGAAGCCATCGAGACCGACAAGAAGTCTG 
               
               
                   
                 GCTACATTCCGTTCGTCGTTATCGGAACAGC 
               
               
                   
                 AGGTACCACCAACACTGGTTCGATTGACCCC 
               
               
                   
                 CTGACAGAAATCTCTGCMIATGTAAGAAGC 
               
               
                   
                 ATGACATGTGGTTTCATATCGACGGAGCGTA 
               
               
                   
                 TGGAGCTAGTGTTCTGCTGTCACCTAAGTAC 
               
               
                   
                 AAGAGCCTTCTTACCGGAACCGGCTTGGCTG 
               
               
                   
                 ACAGTATTTCGTGGGATGCTCATAAATGGTT 
               
               
                   
                 GTTCCAAACGTACGGCTGTGCAATGGTACTT 
               
               
                   
                 GTCAAAGATATCCGTAATTTATTCCACTCTTT 
               
               
                   
                 TCATGTGAATCCCGAGTATCTTAAGGATCTG 
               
               
                   
                 GAAAACGACATCGATAACGTTAATACATGG 
               
               
                   
                 GACATCGGCATGGAGCTGACGCGCCCTGCA 
               
               
                   
                 CGCGGTCTTAAATTGTGGCTTACTTTACAGG 
               
               
                   
                 TCCTTGGATCTGACTTGATTGGGAGTGCCAT 
               
               
                   
                 TGAACACGGTTTCCAGCTGGCAGTTTGGGCT 
               
               
                   
                 GAGGAAGCATTGAATCCAAAGAAAGACTGG 
               
               
                   
                 GAGATCGTTTCTCCAGCTCAGATGGCTATGA 
               
               
                   
                 TTAATTTCCGTTATGCCCCTAAGGATTTAAC 
               
               
                   
                 CAAAGAGGAACAGGATATTCTGAATGAAAA 
               
               
                   
                 GATCTCCCACCGCATTTTAGAGAGCGGATAC 
               
               
                   
                 GCTGCAATTTTCACTACTGTATTAAACGGCA 
               
               
                   
                 AGACCGTTTTACGCATCTGTGCAATTCACCC 
               
               
                   
                 GGAGGCAACTCAAGAGGATATGCAACACAC 
               
               
                   
                 AATCGACTTATTAGACCAATACGGTCGTGAA 
               
               
                   
                 ATCTATACCGAGATGAAGAAAGCG 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 17 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 17 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 17 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 17 or a functional fragment thereof. 
     In one embodiment, the Tryptophan Decarboxylase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 140 or SEQ ID NO. 141: In another embodiment, the Tryptophan Decarboxylase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 140 or SEQ ID NO. 141. In one embodiment, the Tryptophan Decarboxylase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 140 or SEQ ID NO. 141. In one embodiment, the Tryptophan Decarboxylase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 140 or SEQ ID NO. 141. In another embodiment, the Tryptophan Decarboxylase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 140 or SEQ ID NO. 141. Accordingly, in one embodiment, the Tryptophan Decarboxylase 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 the entire sequence of SEQ ID NO: 140 or SEQ ID NO. 141. In another embodiment, the Tryptophan Decarboxylase gene comprises the sequence of SEQ ID NO: 140 or SEQ ID NO. 141. In yet another embodiment the Tryptophan Decarboxylase gene consists of the sequence of SEQ ID NO: 140 or SEQ ID NO. 141. 
     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, and further produce tryptamine from tryptophan. In some embodiments, the genetically engineered bacteria optionally comprise a tryptophan exporter. 
     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 Table 18. In some embodiments, the genetically engineered bacteria take up tryptophan through a 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 exporter. 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Exemplary tryptophan aminotransferase sequences 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 Trp 
                 MTATTISIETVPQAPAAGTKTNGTSGKYNPRTYLSDRA 
               
               
                 aminotransferase 
                 KVTEIDGSDAGRPNPDTFPFNSITLNLKPPLGLPESSNN 
               
               
                 (EC2.6.1.27); 
                 MPVSITIEDPDLATALQYAPSAGIPKLREWLADLQAHV 
               
               
                 tryptophan 
                 HERPRGDYAISVGSGSQDLMFKGFQAVLNPGDPVLLE 
               
               
                 aminotransferase 
                 TPMYSGVLPALRILKADYAEVDVDDQGLSAKNLEKV 
               
               
                 [ Cryptococcus   
                 LSEWPADKKRPRVLYTSPIGSNPSGCSASKERKLEVLK 
               
               
                   deuterogattii  R265] 
                 VCKKYDVLIFEDDPYYYLAQELIPSYFALEKQVYPEG 
               
               
                 SEQ ID NO: 142 
                 GHVVRFDSFSKLLSAGMRLGFATGPKEILHAIDVSTAG 
               
               
                   
                 ANLHTSAVSQGVALRLMQYWGIEGFLAHGRAVAKLY 
               
               
                   
                 TERRAQFEATAHKYLDGLATWVSPVAGMFLWIDLRP 
               
               
                   
                 AGIEDSYELIRHEALAKGVLGVPGMAFYPTGRKSSHV 
               
               
                   
                 RVSFSIVDLEDESDLGFQRLAEAIKDKRKALGLA 
               
               
                   
               
               
                 Trp 
                 ATGACGGCAACTACAATTTCTATTGAGACCGTACCT 
               
               
                 aminotransferase 
                 CAGGCCCCGGCGGCGGGGACCAAAACTAATGGGAC 
               
               
                 (EC2.6.1.27); 
                 TTCAGGAAAATACAACCCCCGCACTTACCTGTCCGA 
               
               
                 tryptophan 
                 CCGCGCCAAAGTCACTGAGATTGATGGATCTGACGC 
               
               
                 aminotransferase 
                 CGGTCGCCCCAATCCCGATACTTTCCCATTTAACTC 
               
               
                 [ Cryptococcus   
                 GATTACCTTAAATTTGAAACCACCTTTAGGCTTGCC 
               
               
                   deuterogattii  R265], 
                 CGAGAGTTCAAATAACATGCCGGTCTCTATCACGAT 
               
               
                 codon optimized for 
                 TGAAGACCCCGATTTAGCGACGGCCTTACAATATGC 
               
               
                 expression in  E. coli   
                 ACCTAGCGCCGGTATTCCTAAGCTGCGCGAATGGCT 
               
               
                 SEQ ID NO: 143 
                 GGCTGACTTACAAGCTCACGTTCATGAGCGCCCCCG 
               
               
                   
                 TGGCGATTATGCCATCTCGGTCGGGTCGGGGTCACA 
               
               
                   
                 GGATTTGATGTTTAAGGGCTTCCAAGCTGTCTTGAA 
               
               
                   
                 TCCAGGTGATCCAGTCCTTCTGGAAACCCCAATGTA 
               
               
                   
                 TTCAGGTGTTCTGCCAGCGCTGCGCATTCTGAAGGC 
               
               
                   
                 GGATTATGCAGAAGTTGATGTAGACGACCAGGGGT 
               
               
                   
                 TATCTGCTAAAAACCTTGAAAAAGTTTTATCAGAGT 
               
               
                   
                 GGCCCGCAGATAAGAAGCGTCCTCGTGTCCTGTATA 
               
               
                   
                 CGTCGCCAATCGGCTCCAATCCTTCCGGATGTTCAG 
               
               
                   
                 CATCCAAGGAACGCAAGTTAGAGGTACTGAAAGTC 
               
               
                   
                 TGTAAGAAGTACGATGTGCTGATCTTCGAAGACGAT 
               
               
                   
                 CCGTATTATTACCTTGCTCAAGAGCTTATTCCATCCT 
               
               
                   
                 ATTTTGCGTTGGAAAAACAAGTTTATCCGGAGGGTG 
               
               
                   
                 GGCACGTTGTACGCTTTGACTCATTTAGTAAATTGC 
               
               
                   
                 TTTCTGCTGGGATGCGCTTGGGATTTGCTACAGGGC 
               
               
                   
                 CGAAGGAAATTCTTCATGCGATTGACGTCAGTACAG 
               
               
                   
                 CAGGCGCAAATTTACATACTTCAGCGGTCTCTCAAG 
               
               
                   
                 GTGTCGCTCTTCGCCTGATGCAGTATTGGGGGATCG 
               
               
                   
                 AGGGATTCCTTGCACATGGCCGCGCGGTGGCCAAA 
               
               
                   
                 CTTTACACGGAGCGCCGCGCTCAGTTCGAGGCAACC 
               
               
                   
                 GCACATAAGTACCTGGACGGGCTGGCCACTTGGGT 
               
               
                   
                 ATCTCCCGTAGCGGGAATGTTTTTATGGATCGATCT 
               
               
                   
                 TCGTCCAGCAGGAATCGAAGATTCTTACGAATTAAT 
               
               
                   
                 TCGCCATGAAGCATTAGCCAAAGGCGTTTTAGGCGT 
               
               
                   
                 TCCAGGGATGGCGTTTTATCCGACAGGCCGTAAGTC 
               
               
                   
                 TTCCCATGTTCGTGTCAGTTTCAGTATCGTCGACCTG 
               
               
                   
                 GAAGACGAATCTGACCTTGGTTTTCAACGCCTGGCT 
               
               
                   
                 GAAGCTATTAAGGATAAACGCAAGGCTTTAGGGCT 
               
               
                   
                 GGCT 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 18 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 18 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 18 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 18 or a functional fragment thereof. 
     In one embodiment, the Trp aminotransferase gene has at least about 80% identity with the entire sequence of SEQ ID NO: 142 or SEQ ID NO: 143. In another embodiment, the Trp aminotransferase gene has at least about 85% identity with the entire sequence of SEQ ID NO: 142 or SEQ ID NO: 143. In one embodiment, the Trp aminotransferase gene has at least about 90% identity with the entire sequence of SEQ ID NO: 142 or SEQ ID NO: 143. In one embodiment, the Trp aminotransferase gene has at least about 95% identity with the entire sequence of SEQ ID NO: 142 or SEQ ID NO: 143. In another embodiment, the Trp aminotransferase gene has at least about 96%, 97%, 98%, or 99% identity with the entire sequence of SEQ ID NO: 142 or SEQ ID NO: 143. Accordingly, in one embodiment, the Trp aminotransferase 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 the entire sequence of SEQ ID NO: 142 or SEQ ID NO: 143. In another embodiment, the Trp aminotransferase gene comprises the sequence of SEQ ID NO: 142 or SEQ ID NO: 143. In yet another embodiment the Trp aminotransferase gene consists of the sequence of SEQ ID NO: 142 or SEQ ID NO: 143. 
     The genetically engineered bacteria may comprise any suitable gene for producing Indole-3-aldehyde and/or Indole Acetic Acidand/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 Acidand/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 genetically engineered bacteria are capable of producing Indole-3-aldehyde and/or Indole Acetic Acidand/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 liver damage, metabolic disease, 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. 
     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. 
     Tryptophan Catabolic Pathway Enzymes 
     In some embodiments the genetically engineered bacteria comprise one or more gene(s) or gene cassette(s) which encode one or more tryptophan pathway enzymes, e.g., from the indole pathway, the kynurenine pathway, or the serotonin arm. 
     Table 19 comprises polypeptide sequences of such enzymes which are encoded by the genetically engineered bacteria of the disclosure. 
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 Tryptophan Pathway Catabolic Enzymes 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 TDC: Tryptophan 
                 MGSIDSTNVAMSNSPVGEFKPLEAEEFRKQAHRMVDFIADY 
               
               
                 decarboxylase from 
                 YKNVETYPVLSEVEPGYLRKRIPETAPYLPEPLDDIMKDIQK 
               
               
                 
                   Catharanthus roseus 
                 
                 DIIPGMTNWMSPNFYAFFPATVSSAAFLGEMLSTALNSVGFT 
               
               
                 SEQ ID NO: 144 
                 WVSSPAATELEMIVMDWLAQILKLPKSFMFSGTGGGVIQNT 
               
               
                   
                 TSESILCTIIAARERALEKLGPDSIGKLVCYGSDQTHTMFPKT 
               
               
                   
                 CKLAGIYPNNIRLIPTTVETDFGISPQVLRKMVEDDVAAGYV 
               
               
                   
                 PLFLCATLGTTSTTATDPVDSLSEIANEFGIWIHVDAAYAGS 
               
               
                   
                 ACICPEFRHYLDGIERVDSLSLSPHKWLLAYLDCTCLWVKQ 
               
               
                   
                 PHLLLRALTTNPEYLKNKQSDLDKVVDFKNWQIATGRKFRS 
               
               
                   
                 LKLWLILRSYGVVNLQSHIRSDVAMGKMFEEWVRSDSRFEI 
               
               
                   
                 VVPRNFSLVCFRLKPDVSSLHVEEVNKKLLDMLNSTGRVY 
               
               
                   
                 MTHTIVGGIYMLRLAVGSSLTEEHHVRRVWDLIQKLTDDLL 
               
               
                   
                 KEA 
               
               
                   
               
               
                 TYNA: Monoamine 
                 MGSPSLYSARKTTLALAVALSFAWQAPVFAHGGEAHMVPM 
               
               
                 oxidase from  E. coli   
                 DKTLKEFGADVQWDDYAQLFTLIKDGAYVKVKPGAQTAIV 
               
               
                 SEQ ID NO: 145 
                 NGQPLALQVPVVMKDNKAWVSDTFINDVFQSGLDQTFQVE 
               
               
                   
                 KRPHPLNALTADEIKQAVEIVKASADFKPNTRFTEISLLPPDK 
               
               
                   
                 EAVWAFALENKPVDQPRKADVIMLDGKHIIEAVVDLQNNK 
               
               
                   
                 LLSWQPIKDAHGMVLLDDFASVQNIINNSEEFAAAVKKRGIT 
               
               
                   
                 DAKKVITTPLTVGYFDGKDGLKQDARLLKVISYLDVGDGN 
               
               
                   
                 YWAHPIENLVAVVDLEQKKIVKIEEGPVVPVPMTARPFDGR 
               
               
                   
                 DRVAPAVKPMQIIEPEGKNYTITGDMIHWRNWDFHLSMNSR 
               
               
                   
                 VGPMISTVTYNDNGTKRKVMYEGSLGGMIVPYGDPDIGWY 
               
               
                   
                 FKAYLDSGDYGMGTLTSPIARGKDAPSNAVLLNETIADYTG 
               
               
                   
                 VPMEIPRAIAVFERYAGPEYKHQEMGQPNVSTERRELVVRW 
               
               
                   
                 ISTVGNYDYIFDWIFHENGTIGIDAGATGIEAVKGVKAKTMH 
               
               
                   
                 DETAKDDTRYGTLIDHNIVGTTHQHIYNFRLDLDVDGENNS 
               
               
                   
                 LVAMDPVVKPNTAGGPRTSTMQVNQYNIGNEQDAAQKFDP 
               
               
                   
                 GTIRLLSNPNKENRMGNPVSYQIIPYAGGTHPVAKGAQFAPD 
               
               
                   
                 EWIYHRLSFMDKQLWVTRYHPGERFPEGKYPNRSTHDTGL 
               
               
                   
                 GQYSKDNESLDNTDAVVWMTTGTTHVARAEEWPIMPTEW 
               
               
                   
                 VHTLLKPWNFFDETPTLGALKKDK 
               
               
                   
               
               
                 AAO1: Indole-3- 
                 MGEKAIDEDKVEAMKSSKTSLVFAINGQRFELELSSIDPSTTL 
               
               
                 acetaldehyde oxidase 
                 VDFLRNKTPFKSVKLGCGEGGCGACVVLLSKYDPLLEKVDE 
               
               
                 from  Arabidopsis   
                 FTISSCLTLLCSIDGCSITTSDGLGNSRVGFHAVHERIAGFHAT 
               
               
                 
                   thaliana 
                 
                 QCGFCTPGMSVSMFSALLNADKSHPPPRSGFSNLTAVEAEK 
               
               
                 SEQ ID NO: 146 
                 AVSGNLCRCTGYRPLVDACKSFAADVDIEDLGFNAFCKKGE 
               
               
                   
                 NRDEVLRRLPCYDHTSSHVCTFPEFLKKEIKNDMSLHSRKY 
               
               
                   
                 RWSSPVSVSELQGLLEVENGLSVKLVAGNTSTGYYKEEKER 
               
               
                   
                 KYERFIDIRKIPEFTMVRSDEKGVELGACVTISKAIEVLREEK 
               
               
                   
                 NVSVLAKIATHMEKIANRFVRNTGTIGGNIMMAQRKQFPSD 
               
               
                   
                 LATILVAAQATVKIMTSSSSQEQFTLEEFLQQPPLDAKSLLLS 
               
               
                   
                 LEIPSWHSAKKNGSSEDSILLFETYRAAPRPLGNALAFLNAA 
               
               
                   
                 FSAEVTEALDGIVVNDCQLVFGAYGTKHAHRAKKVEEFLTG 
               
               
                   
                 KVISDEVLMEAISLLKDEIVPDKGTSNPGYRSSLAVTFLFEFF 
               
               
                   
                 GSLTKKNAKTTNGWLNGGCKEIGFDQNVESLKPEAMLSSA 
               
               
                   
                 QQIVENQEHSPVGKGITKAGACLQASGEAVYVDDIPAPENC 
               
               
                   
                 LYGAFIYSTMPLARIKGIRFKQNRVPEGVLGIITYKDIPKGGQ 
               
               
                   
                 NIGTNGFFTSDLLFAEEVTHCAGQIIAFLVADSQKHADIAAN 
               
               
                   
                 LVVIDYDTKDLKPPILSLEEAVENFSLFEVPPPLRGYPVGDIT 
               
               
                   
                 KGMDEAEHKILGSKISFGSQYFFYMETQTALAVPDEDNCMV 
               
               
                   
                 VYSSTQTPEFVHQTIAGCLGVPENNVRVITRRVGGGFGGKA 
               
               
                   
                 VKSMPVAAACALAASKMQRPVRTYVNRKTDMITTGGRHP 
               
               
                   
                 MKVTYSVGFKSNGKITALDVEVLLDAGLTEDISPLMPKGIQ 
               
               
                   
                 GALMKYDWGALSFNVKVCKTNTVSRTALRAPGDVQGSYIG 
               
               
                   
                 EAIIEKVASYLSVDVDEIRKVNLHTYESLRLFHSAKAGEFSE 
               
               
                   
                 YTLPLLWDRIDEFSGFNKRRKVVEEFNASNKWRKRGISRVP 
               
               
                   
                 AVYAVNMRSTPGRVSVLGDGSIVVEVQGIEIGQGLWTKVK 
               
               
                   
                 QMAAYSLGLIQCGTTSDELLKKIRVIQSDTLSMVQGSMTAG 
               
               
                   
                 STTSEASSEAVRICCDGLVERLLPVKTALVEQTGGPVTWDSL 
               
               
                   
                 ISQAYQQSINMSVSSKYMPDSTGEYLNYGIAASEVEVNVLT 
               
               
                   
                 GETTILRTDIIYDCGKSLNPAVDLGQIEGAFVQGLGFFMLEEF 
               
               
                   
                 LMNSDGLVVTDSTWTYKIPTVDTIPRQFNVEILNSGQHKNR 
               
               
                   
                 VLSSKASGEPPLLLAASVHCAVRAAVKEARKQILSWNSNKQ 
               
               
                   
                 GTDMYFELPVPATMPIVKEFCGLDVVEKYLEWKIQQRKNV 
               
               
                   
               
               
                 ARO9: L-tryptophan 
                 MTAGSAPPVDYTSLKKNFQPFLSRRVENRSLKSFWDASDISD 
               
               
                 aminotransferase 
                 DVIELAGGMPNERFFPIESMDLKISKVPFNDNPKWHNSFTTA 
               
               
                 from  S. cerevisae   
                 HLDLGSPSELPIARSFQYAETKGLPPLLHFVKDFVSRINRPAF 
               
               
                 SEQ ID NO: 147 
                 SDETESNWDVILSGGSNDSMFKVFETICDESTTVMIEEFTFTP 
               
               
                   
                 AMSNVEATGAKVIPIKMNLTFDRESQGIDVEYLTQLLDNWS 
               
               
                   
                 TGPYKDLNKPRVLYTIATGQNPTGMSVPQWKREKIYQLAQR 
               
               
                   
                 HDFLIVEDDPYGYLYFPSYNPQEPLENPYHSSDLTTERYLND 
               
               
                   
                 FLMKSFLTLDTDARVIRLETFSKIFAPGLRLSFIVANKFLLQKI 
               
               
                   
                 LDLADITTRAPSGTSQAIVYSTIKAMAESNLSSSLSMKEAMF 
               
               
                   
                 EGWIRWIMQIASKYNHRKNLTLKALYETESYQAGQFTVMEP 
               
               
                   
                 SAGMFIIIKINWGNFDRPDDLPQQMDILDKFLLKNGVKVVLG 
               
               
                   
                 YKMAVCPNYSKQNSDFLRLTIAYARDDDQLIEASKRIGSGIK 
               
               
                   
                 EFFDNYKS 
               
               
                   
               
               
                 aspC: aspartate 
                 MFENITAAPADPILGLADLFRADERPGKINLGIGVYKDETGK 
               
               
                 aminotransferase 
                 TPVLTSVKKAEQYLLENETTKNYLGIDGIPEFGRCTQELLFG 
               
               
                 from  E. coli   
                 KGSALINDKRARTAQTPGGTGALRVAADFLAKNTSVKRVW 
               
               
                 SEQ ID NO: 148 
                 VSNPSWPNHKSVFNSAGLEVREYAYYDAENHTLDFDALINS 
               
               
                   
                 LNEAQAGDVVLFHGCCHNPTGIDPTLEQWQTLAQLSVEKG 
               
               
                   
                 WLPLFDFAYQGFARGLEEDAEGLRAFAAMHKELIVASSYSK 
               
               
                   
                 NFGLYNERVGACTLVAADSETVDRAFSQMKAAIRANYSNPP 
               
               
                   
                 AHGASVVATILSNDALRAIWEQELTDMRQRIQRMRQLFVNT 
               
               
                   
                 LQEKGANRDFSFIIKQNGMFSFSGLTKEQVLRLREEFGVYAV 
               
               
                   
                 ASGRVNVAGMTPDNMAPLCEAIVAVL 
               
               
                   
               
               
                 TAA1: L-tryptophan- 
                 MVKLENSRKPEKISNKNIPMSDFVVNLDHGDPTAYEEYWRK 
               
               
                 pyruvate 
                 MGDRCTVTIRGCDLMSYFSDMTNLCWFLEPELEDAIKDLHG 
               
               
                 aminotransferase 
                 VVGNAATEDRYIVVGTGSTQLCQAAVHALSSLARSQPVSVV 
               
               
                 from  Arabidopsis   
                 AAAPFYSTYVEETTYVRSGMYKWEGDAWGFDKKGPYIELV 
               
               
                 
                   thaliana 
                 
                 TSPNNPDGTIRETVVNRPDDDEAKVIHDFAYYWPHYTPITRR 
               
               
                 SEQ ID NO: 149 
                 QDHDIMLFTFSKITGHAGSRIGWALVKDKEVAKKMVEYIIV 
               
               
                   
                 NSIGVSKESQVRTAKILNVLKETCKSESESENFFKYGREMMK 
               
               
                   
                 NRWEKLREVVKESDAFTLPKYPEAFCNYFGKSLESYPAFAW 
               
               
                   
                 LGTKEETDLVSELRRHKVMSRAGERCGSDKKHVRVSMLSR 
               
               
                   
                 EDVFNVFLERLANMKLIKSIDL 
               
               
                   
               
               
                 STAO: L-tryptophan 
                 MTAPLQDSDGPDDAIGGPKQVTVIGAGIAGLVTAYELERLG 
               
               
                 oxidase from 
                 HHVQIIEGSDDIGGRIHTHRFSGAGGPGPFAEMGAMRIPAGH 
               
               
                   streptomyces  sp. TP- 
                 RLTMHYIAELGLQNQVREFRTLFSDDAAYLPSSAGYLRVRE 
               
               
                 A0274 
                 AHDTLVDEFATGLPSAHYRQDTLLFGAWLDASIRAIAPRQF 
               
               
                 SEQ ID NO: 150 
                 YDGLHNDIGVELLNLVDDIDLTPYRCGTARNRIDLHALFAD 
               
               
                   
                 HPRVRASCPPRLERFLDDVLDETSSSIVRLKDGMDELPRRLA 
               
               
                   
                 SRIRGKISLGQEVTGIDVHDDTVTLTVRQGLRTVTRTCDYVV 
               
               
                   
                 CTIPFTVLRTLRLTGFDQDKLDIVHETKYWPATKIAFHCREPF 
               
               
                   
                 WEKDGISGGASFTGGHVRQTYYPPAEGDPALGAVLLASYTI 
               
               
                   
                 GPDAEALARMDEAERDALVAKELSVMHPELRRPGMVLAV 
               
               
                   
                 AGRDWGARRWSRGAATVRWGQEAALREAERRECARPQKG 
               
               
                   
                 LFFAGEHCSSKPAWIEGAIESAIDAAHEIEWYEPRASRVFAAS 
               
               
                   
                 RLSRSDRSA 
               
               
                   
               
               
                 ipdC: Indole-3- 
                 MRTPYCVADYLLDRLTDCGADHLFGVPGDYNLQFLDHVID 
               
               
                 pyruvate 
                 SPDICWVGCANELNASYAADGYARCKGFAALLTTFGVGELS 
               
               
                 decarboxylase from 
                 AMNGIAGSYAEHVPVLHIVGAPGTAAQQRGELLHHTLGDG 
               
               
                 
                   Enterobacter cloacae 
                 
                 EFRHFYHMSEPITVAQAVLTEQNACYEIDRVLTTMLRERRP 
               
               
                 SEQ ID NO: 151 
                 GYLMLPADVAKKAATPPVNALTHKQAHADSACLKAFRDA 
               
               
                   
                 AENKLAMSKRTALLADFLVLRHGLKHALQKWVKEVPMAH 
               
               
                   
                 ATMLMGKGIFDERQAGFYGTYSGSASTGAVKEAIEGADTVL 
               
               
                   
                 CVGTRFTDTLTAGFTHQLTPAQTIEVQPHAARVGDVWFTGI 
               
               
                   
                 PMNQAIETLVELCKQHVHAGLMSSSSGAIPFPQPDGSLTQEN 
               
               
                   
                 FWRTLQTFIRPGDIILADQGTSAFGAIDLRLPADVNFIVQPLW 
               
               
                   
                 GSIGYTLAAAFGAQTACPNRRVIVLTGDGAAQLTIQELGSM 
               
               
                   
                 LRDKQHPIILVLNNEGYTVERAIHGAEQRYNDIALWNWTHIP 
               
               
                   
                 QALSLDPQSECWRVSEAEQLADVLEKVAHHERLSLIEVMLP 
               
               
                   
                 KADIPPLLGALTKALEACNNA 
               
               
                   
               
               
                 IAD1 : Indo1e-3- 
                 MPTLNLDLPNGIKSTIQADLFINNKFVPALDGKTFATINPSTG 
               
               
                 acetaldehyde 
                 KEIGQVAEASAKDVDLAVKAAREAFETTWGENTPGDARGR 
               
               
                 dehydrogenase 
                 LLIKLAELVEANIDELAAIESLDNGKAFSIAKSFDVAAVAAN 
               
               
                 from  Ustilago maydis   
                 LRYYGGWADKNHGKVMEVDTKRLNYTRHEPIGVCGQIIPW 
               
               
                 SEQ ID NO: 152 
                 NFPLLMFAWKLGPALATGNTIVLKTAEQTPLSAIKMCELIVE 
               
               
                   
                 AGFPPGVVNVISGFGPVAGAAISQHMDIDKIAFTGSTLVGRN 
               
               
                   
                 IMKAAASTNLKKVTLELGGKSPNIIFKDADLDQAVRWSAFGI 
               
               
                   
                 MFNHGQCCCAGSRVYVEESIYDAFMEKMTAHCKALQVGDP 
               
               
                   
                 FSANTFQGPQVSQLQYDRIMEYIESGKKDANLALGGVRKGN 
               
               
                   
                 EGYFIEPTIFTDVPHDAKIAKEEIFGPVVVVSKFKDEKDLIRIA 
               
               
                   
                 NDSIYGLAAAVFSRDISRAIETAHKLKAGTVWVNCYNQLIPQ 
               
               
                   
                 VPFGGYKASGIGRELGEYALSNYTNIKAVHVNLSQPAPI 
               
               
                   
               
               
                 YUC2: indole-3- 
                 MEFVTETLGKRIHDPYVEETRCLMIPGPIIVGSGPSGLATAAC 
               
               
                 pyruvate 
                 LKSRDIPSLILERSTCIASLWQHKTYDRLRLHLPKDFCELPLM 
               
               
                 monoxygenase from 
                 PFPSSYPTYPTKQQFVQYLESYAEHFDLKPVFNQTVEEAKFD 
               
               
                 
                   Arabidopsis thaliana 
                 
                 RRCGLWRVRTTGGKKDETMEYVSRWLVVATGENAEEVMP 
               
               
                 SEQ ID NO: 153 
                 EIDGIPDFGGPILHTSSYKSGEIFSEKKILVVGCGNSGMEVCL 
               
               
                   
                 DLCNFNALPSLVVRDSVHVLPQEMLGISTFGISTSLLKWFPV 
               
               
                   
                 HVVDRFLLRMSRLVLGDTDRLGLVRPKLGPLERKIKCGKTP 
               
               
                   
                 VLDVGTLAKIRSGHIKVYPELKRVMHYSAEFVDGRVDNFDA 
               
               
                   
                 IILATGYKSNVPMWLKGVNMFSEKDGFPHKPFPNGWKGES 
               
               
                   
                 GLYAVGFTKLGLLGAAIDAKKIAEDIEVQRHFLPLARPQHC 
               
               
                   
               
               
                 IaaM: Tryptophan 2- 
                 MYDHFNSPSIDILYDYGPFLKKCEMTGGIGSYSAGTPTPRVA 
               
               
                 monooxygenase from 
                 IVGAGISGLVAATELLRAGVKDVVLYESRDRIGGRVWSQVF 
               
               
                 
                   Pseudomonas 
                 
                 DQTRPRYIAEMGAMRFPPSATGLFHYLKKFGISTSTTFPDPG 
               
               
                 
                   savastanoi 
                 
                 VVDTELHYRGKRYHWPAGKKPPELFRRVYEGWQSLLSEGY 
               
               
                 SEQ ID NO: 154 
                 LLEGGSLVAPLDITAMLKSGRLEEAAIAWQGWLNVFRDCSF 
               
               
                   
                 YNAIVCIFTGRHPPGGDRWARPEDFELFGSLGIGSGGFLPVF 
               
               
                   
                 QAGFTEILRMVINGYQSDQRLIPDGISSLAARLADQSFDGKA 
               
               
                   
                 LRDRVCFSRVGRISREAEKIIIQTEAGEQRVFDRVIVTSSNRA 
               
               
                   
                 MQMIHCLTDSESFLSRDVARAVRETHLTGSSKLFILTRTKFW 
               
               
                   
                 IKNKLPTTIQSDGLVRGVYCLDYQPDEPEGHGVVLLSYTWE 
               
               
                   
                 DDAQKMLAMPDKKTRCQVLVDDLAAIHPTFASYLLPVDGD 
               
               
                   
                 YERYVLHHDWLTDPHSAGAFKLNYPGEDVYSQRLFFQPMT 
               
               
                   
                 ANSPNKDTGLYLAGCSCSFAGGWIEGAVQTALNSACAVLRS 
               
               
                   
                 TGGQLSKGNPLDCINASYRY 
               
               
                   
               
               
                 iaaH: 
                 MHEIITLESLCQALADGEIAAAELRERALDTEARLARLNCFIR 
               
               
                 Indoleacetamide 
                 EGDAVSQFGEADHAMKGTPLWGMPVSFKDNICVRGLPLTA 
               
               
                 hydrolase from 
                 GTRGMSGFVSDQDAAIVSQLRALGAVVAGKNNMHELSFGV 
               
               
                 
                   Pseudomonas 
                 
                 TSINPHWGTVGNPVAPGYCAGGSSGGSAAAVASGIVPLSVG 
               
               
                 
                   savastanoi 
                 
                 TDTGGSIRIPAAFCGITGFRPTTGRWSTAGIIPVSHTKDCVGL 
               
               
                 SEQ ID NO: 155 
                 LTRTAGDAGFLYGLLSGKQQSFPLSRTAPCRIGLPVSMWSDL 
               
               
                   
                 DGEVERACVNALSLLRKTGFEFIEIDDADIVELNQTLTFTVPL 
               
               
                   
                 YEFFADLAQSLLSLGWKHGIHHIFAQVDDANVKGIINHHLG 
               
               
                   
                 EGAIKPAHYLSSLQNGELLKRKMDELFARHNIELLGYPTVPC 
               
               
                   
                 RVPHLDHADRPEFFSQAIRNTDLASNAMLPSITIPVGPEGRLP 
               
               
                   
                 VGLSFDALRGRDALLLSRVSAIEQVLGFVRKVLPHTT 
               
               
                   
               
               
                 TrpDH: Tryptophan 
                 MLLFETVREMGHEQVLFCHSKNPEIKAIIAIHDTTLGPAMGA 
               
               
                 dehydrogenase from 
                 TRILPYINEEAALKDALRLSRGMTYKAACANIPAGGGKAVII 
               
               
                 
                   Nostoc punctiforme 
                 
                 ANPENKTDDLLRAYGRFVDSLNGRFITGQDVNITPDDVRTIS 
               
               
                 NIES-2108 
                 QETKYVVGVSEKSGGPAPITSLGVFLGIKAAVESRWQSKRL 
               
               
                 SEQ ID NO: 156 
                 DGMKVAVQGLGNVGKNLCRHLHEHDVQLFVSDVDPIKAEE 
               
               
                   
                 VKRLFGATVVEPTEIYSLDVDIFAPCALGGILNSHTIPFLQASI 
               
               
                   
                 IAGAANNQLENEQLHSQMLAKKGILYSPDYVINAGGLINVY 
               
               
                   
                 NEMIGYDEEKAFKQVHNIYDTLLAIFEIAKEQGVTTNDAAR 
               
               
                   
                 RLAEDRINNSKRSKSKAIAA 
               
               
                   
               
               
                 CYP79B2: 
                 MNTFTSNSSDLTTTATETSSFSTLYLLSTLQAFVAITLVMLLK 
               
               
                 tryptophan N- 
                 KLMTDPNKKKPYLPPGPTGWPIIGMIPTMLKSRPVFRWLHSI 
               
               
                 monooxygenase from 
                 MKQLNTEIACVKLGNTHVITVTCPKIAREILKQQDALFASRP 
               
               
                 
                   Arabidopsis thaliana 
                 
                 LTYAQKILSNGYKTCVITPFGDQFKKMRKVVMTELVCPARH 
               
               
                 SEQ ID NO: 157 
                 RWLHQKRSEENDHLTAWVYNMVKNSGSVDFRFMTRHYCG 
               
               
                   
                 NAIKKLMFGTRTFSKNTAPDGGPTVEDVEHMEAMFEALGFT 
               
               
                   
                 FAFCISDYLPMLTGLDLNGHEKIMRESSAIMDKYHDPIIDERI 
               
               
                   
                 KMWREGKRTQIEDFLDIFISIKDEQGNPLLTADEIKPTIKELV 
               
               
                   
                 MAAPDNPSNAVEWAMAEMVNKPEILRKAMEEIDRVVGKE 
               
               
                   
                 RLVQESDIPKLNYVKAILREAFRLHPVAAFNLPHVALSDTTV 
               
               
                   
                 AGYHIPKGSQVLLSRYGLGRNPKVWADPLCFKPERHLNECS 
               
               
                   
                 EVTLTENDLRFISFSTGKRGCAAPALGTALTTMMLARLLQG 
               
               
                   
                 FTWKLPENETRVELMESSHDMFLAKPLVMVGDLRLPEHLYP 
               
               
                   
                 TVK 
               
               
                   
               
               
                 CYP79B3: 
                 MDTLASNSSDLTTKSSLGMSSFTNMYLLTTLQALAALCFLM 
               
               
                 tryptophan N- 
                 ILNKIKSSSRNKKLHPLPPGPTGFPIVGMIPAMLKNRPVFRWL 
               
               
                 monooxygenase from 
                 HSLMKELNTEIACVRLGNTHVIPVTCPKIAREIFKQQDALFAS 
               
               
                 
                   Arabidopsis thaliana 
                 
                 RPLTYAQKILSNGYKTCVITPFGEQFKKMRKVIMTEIVCPAR 
               
               
                 SEQ ID NO: 158 
                 HRWLHDNRAEETDHLTAWLYNMVKNSEPVDLRFVTRHYC 
               
               
                   
                 GNAIKRLMFGTRTFSEKTEADGGPTLEDIEHMDAMFEGLGF 
               
               
                   
                 TFAFCISDYLPMLTGLDLNGHEKIMRESSAIMDKYHDPIIDER 
               
               
                   
                 IKMWREGKRTQIEDFLDIFISIKDEAGQPLLTADEIKPTIKELV 
               
               
                   
                 MAAPDNPSNAVEWAIAEMINKPEILHKAMEEIDRVVGKERF 
               
               
                   
                 VQESDIPKLNYVKAIIREAFRLHPVAAFNLPHVALSDTTVAG 
               
               
                   
                 YHIPKGSQVLLSRYGLGRNPKVWSDPLSFKPERHLNECSEVT 
               
               
                   
                 LTENDLRFISFSTGKRGCAAPALGTAITTMMLARLLQGFKW 
               
               
                   
                 KLAGSETRVELMESSHDMFLSKPLVLVGELRLSEDLYPMVK 
               
               
                   
               
               
                 CYP71A13: 
                 MSNIQEMEMILSISLCLTTLITLLLLRRFLKRTATKVNLPPSP 
               
               
                 indoleacetaldoxime 
                 WRLPVIGNLHQLSLHPHRSLRSLSLRYGPLMLLHFGRVPILV 
               
               
                 dehydratase from 
                 VSSGEAAQEVLKTHDHKFANRPRSKAVHGLMNGGRDVVFA 
               
               
                 
                   Arabidopis thaliana 
                 
                 PYGEYWRQMKSVCILNLLTNKMVESFEKVREDEVNAMIEK 
               
               
                 SEQ ID NO: 159 
                 LEKASSSSSSENLSELFITLPSDVTSRVALGRKHSEDETARDL 
               
               
                   
                 KKRVRQIMELLGEFPIGEYVPILAWIDGIRGFNNKIKEVSRGF 
               
               
                   
                 SDLMDKVVQEHLEASNDKADFVDILLSIEKDKNSGFQVQRN 
               
               
                   
                 DIKFMILDMFIGGTSTTSTLLEWTMTELIRSPKSMKKLQDEIR 
               
               
                   
                 STIRPHGSYIKEKEVENMKYLKAVIKEVLRLHPSLPMILPRLL 
               
               
                   
                 SEDVKVKGYNIAAGTEVIINAWAIQRDTAIWGPDAEEFKPER 
               
               
                   
                 HLDSGLDYHGKNLNYIPFGSGRRICPGINLALGLAEVTVANL 
               
               
                   
                 VGRFDWRVEAGPNGDQPDLTEAIGIDVCRKFPLIAFPSSVV 
               
               
                   
               
               
                 PEN2: myrosinase 
                 MAHLQRTFPTEMSKGRASFPKGFLFGTASSSYQYEGAVNEG 
               
               
                 from  Arabidopsis   
                 ARGQSVWDHFSNRFPHRISDSSDGNVAVDFYHRYKEDIKRM 
               
               
                 
                   thaliana 
                 
                 KDINMDSFRLSIAWPRVLPYGKRDRGVSEEGIKFYNDVIDEL 
               
               
                 SEQ ID NO: 160 
                 LANEITPLVTIFHWDIPQDLEDEYGGFLSEQIIDDFRDYASLC 
               
               
                   
                 FERFGDRVSLWCTMNEPWVYSVAGYDTGRKAPGRCSKYV 
               
               
                   
                 NGASVAGMSGYEAYIVSHNMLLAHAEAVEVFRKCDHIKNG 
               
               
                   
                 QIGIAHNPLWYEPYDPSDPDDVEGCNRAMDFMLGWHQHPT 
               
               
                   
                 ACGDYPETMKKSVGDRLPSFTPEQSKKLIGSCDYVGINYYSS 
               
               
                   
                 LFVKSIKHVDPTQPTWRTDQGVDWMKTNIDGKQIAKQGGS 
               
               
                   
                 EWSFTYPTGLRNILKYVKKTYGNPPILITENGYGEVAEQSQS 
               
               
                   
                 LYMYNPSIDTERLEYIEGHIHAIHQAIHEDGVRVEGYYVWSL 
               
               
                   
                 LDNFEWNSGYGVRYGLYYIDYKDGLRRYPKMSALWLKEFL 
               
               
                   
                 RFDQEDDSSTSKKEEKKESYGKQLLHSVQDSQFVHSIKDSG 
               
               
                   
                 ALPAVLGSLFVVSATVGTSLFFKGANN 
               
               
                   
               
               
                 Nit1: Nitrilase from 
                 MSSTKDMSTVQNATPFNGVAPSTTVRVTIVQSSTVYNDTPA 
               
               
                 
                   Arabidopsis thaliana 
                 
                 TIDKAEKYIVEAASKGAELVLFPEGFIGGYPRGFRFGLAVGV 
               
               
                 SEQ ID NO: 161 
                 HNEEGRDEFRKYHASAIHVPGPEVARLADVARKNHVYLVM 
               
               
                   
                 GAIEKEGYTLYCTVLFFSPQGQFLGKHRKLMPTSLERCIWGQ 
               
               
                   
                 GDGSTIPVYDTPIGKLGAAICWENRMPLYRTALYAKGIELYC 
               
               
                   
                 APTADGSKEWQSSMLHIAIEGGCFVLSACQFCQRKHFPDHP 
               
               
                   
                 DYLFTDWYDDKEHDSIVSQGGSVIISPLGQVLAGPNFESEGL 
               
               
                   
                 VTADIDLGDIARAKLYFDSVGHYSRPDVLHLTVNEHPRKSV 
               
               
                   
                 TFVTKVEKAEDDSNK 
               
               
                   
               
               
                 IDO1: indoleamine 
                 MAHAMENSWTISKEYHIDEEVGFALPNPQENLPDFYNDWM 
               
               
                 2,3-dioxygenase 
                 FIAKHLPDLIESGQLRERVEKLNMLSIDHLTDHKSQRLARLV 
               
               
                 from  homo sapiens   
                 LGCITMAYVWGKGHGDVRKVLPRNIAVPYCQLSKKLELPPI 
               
               
                 SEQ ID NO: 162 
                 LVYADCVLANWKKKDPNKPLTYENMDVLFSFRDGDCSKGF 
               
               
                   
                 FLVSLLVEIAAASAIKVIPTVFKAMQMQERDTLLKALLEIAS 
               
               
                   
                 CLEKALQVFHQIHDHVNPKAFFSVLRIYLSGWKGNPQLSDG 
               
               
                   
                 LVYEGFWEDPKEFAGGSAGQSSVFQCFDVLLGIQQTAGGGH 
               
               
                   
                 AAQFLQDMRRYMPPAHRNFLCSLESNPSVREFVLSKGDAGL 
               
               
                   
                 REAYDACVKALVSLRSYHLQIVTKYILIPASQQPKENKTSED 
               
               
                   
                 PSKLEAKGTGGTDLMNFLKTVRSTTEKSLLKEG 
               
               
                   
               
               
                 TDO2: tryptophan 
                 MSGCPFLGNNFGYTFKKLPVEGSEEDKSQTGVNRASKGGLI 
               
               
                 2,3-dioxygenase 
                 YGNYLHLEKVLNAQELQSETKGNKIHDEHLFIITHQAYELW 
               
               
                 from  homo sapiens   
                 FKQILWELDSVREIFQNGHVRDERNMLKVVSRMHRVSVILK 
               
               
                 SEQ ID NO: 163 
                 LLVQQFSILETMTALDFNDFREYLSPASGFQSLQFRLLENKIG 
               
               
                   
                 VLQNMRVPYNRRHYRDNFKGEENELLLKSEQEKTLLELVE 
               
               
                   
                 AWLERTPGLEPHGFNFWGKLEKNITRGLEEEFIRIQAKEESE 
               
               
                   
                 EKEEQVAEFQKQKEVLLSLFDEKRHEHLLSKGERRLSYRAL 
               
               
                   
                 QGALMIYFYREEPRFQVPFQLLTSLMDIDSLMTKWRYNHVC 
               
               
                   
                 MVHRMLGSKAGTGGSSGYHYLRSTVSDRYKVFVDLFNLST 
               
               
                   
                 YLIPRHWIPKMNPTIHKFLYTAEYCDSSYFSSDESD 
               
               
                   
               
               
                 BNA2: indoleamine 
                 MNNTSITGPQVLHRTKMRPLPVLEKYCISPHHGFLDDRLPLT 
               
               
                 2,3-dioxygenase 
                 RLSSKKYMKWEEIVADLPSLLQEDNKVRSVIDGLDVLDLDE 
               
               
                 from  S. cerevisiae   
                 TILGDVRELRRAYSILGFMAHAYIWASGTPRDVLPECIARPL 
               
               
                 SEQ ID NO: 164 
                 LETAHILGVPPLATYSSLVLWNFKVTDECKKTETGCLDLENI 
               
               
                   
                 TTINTFTGTVDESWFYLVSVRFEKIGSACLNHGLQILRAIRSG 
               
               
                   
                 DKGDANVIDGLEGLAATIERLSKALMEMELKCEPNVFYFKI 
               
               
                   
                 RPFLAGWTNMSHMGLPQGVRYGAEGQYRIFSGGSNAQSSLI 
               
               
                   
                 QTLDILLGVKHTANAAHSSQGDSKINYLDEMKKYMPREHR 
               
               
                   
                 EFLYHLESVCNIREYVSRNASNRALQEAYGRCISMLKIFRDN 
               
               
                   
                 HIQIVTKYIILPSNSKQHGSNKPNVLSPIEPNTKASGCLGHKV 
               
               
                   
                 ASSKTIGTGGTRLMPFLKQCRDETVATADIKNEDKN 
               
               
                   
               
               
                 Afmid: Kynurenine 
                 MAFPSLSAGQNPWRNLSSEELEKQYSPSRWVIHTKPEEVVG 
               
               
                 formamidase from 
                 NFVQIGSQATQKARATRRNQLDVPYGDGEGEKLDIYFPDED 
               
               
                 mouse 
                 SKAFPLFLFLHGGYWQSGSKDDSAFMVNPLTAQGIVVVIVA 
               
               
                 SEQ ID NO: 165 
                 YDIAPKGTLDQMVDQVTRSVVFLQRRYPSNEGIYLCGHSAG 
               
               
                   
                 AHLAAMVLLARWTKHGVTPNLQGFLLVSGIYDLEPLIATSQ 
               
               
                   
                 NDPLRMTLEDAQRNSPQRHLDVVPAQPVAPACPVLVLVGQ 
               
               
                   
                 HDSPEFHRQSKEFYETLLRVGWKASFQQLRGVDHFDIIENLT 
               
               
                   
                 REDDVLTQIILKTVFQKL 
               
               
                   
               
               
                 BNA3: kynurenine-- 
                 MKQRFIRQFTNLMSTSRPKVVANKYFTSNTAKDVWSLTNE 
               
               
                 oxoglutarate 
                 AAAKAANNSKNQGRELINLGQGFFSYSPPQFAIKEAQKALDI 
               
               
                 transaminase from  S.   
                 PMVNQYSPTRGRPSLINSLIKLYSPIYNTELKAENVTVTTGA 
               
               
                 
                   cerevisae 
                 
                 NEGILSCLMGLLNAGDEVIVFEPFFDQYIPNIELCGGKVVYV 
               
               
                 SEQ ID NO: 166 
                 PINPPKELDQRNTRGEEWTIDFEQFEKAITSKTKAVIINTPHN 
               
               
                   
                 PIGKVFTREELTTLGNICVKHNVVIISDEVYEHLYFTDSFTRIA 
               
               
                   
                 TLSPEIGQLTLTVGSAGKSFAATGWRIGWVLSLNAELLSYAA 
               
               
                   
                 KAHTRICFASPSPLQEACANSINDALKIGYFEKMRQEYINKF 
               
               
                   
                 KIFTSIFDELGLPYTAPEGTYFVLVDFSKVKIPEDYPYPEEILN 
               
               
                   
                 KGKDFRISHWLINELGVVAIPPTEFYIKEHEKAAENLLRFAV 
               
               
                   
                 CKDDAYLENAVERLKLLKDYL 
               
               
                   
               
               
                 GOT2: Aspartate 
                 MALLHSGRVLPGIAAAFHPGLAAAASARASSWWTHVEMGP 
               
               
                 aminotransferase, 
                 PDPILGVTEAFKRDTNSKKMNLGVGAYRDDNGKPYVLPSV 
               
               
                 mitochondrial from 
                 RKAEAQIAAKNLDKEYLPIGGLAEFCKASAELALGENSEVL 
               
               
                 
                   homo sapiens 
                 
                 KSGRFVTVQTISGTGALRIGASFLQRFFKFSRDVFLPKPTWG 
               
               
                 SEQ ID NO: 167 
                 NHTPIFRDAGMQLQGYRYYDPKTCGFDFTGAVEDISKIPEQS 
               
               
                   
                 VLLLHACAHNPTGVDPRPEQWKEIATVVKKRNLFAFFDMA 
               
               
                   
                 YQGFASGDGDKDAWAVRHFIEQGINVCLCQSYAKNMGLYG 
               
               
                   
                 ERVGAFTMVCKDADEAKRVESQLKILIRPMYSNPPLNGARI 
               
               
                   
                 AAAILNTPDLRKQWLQEVKVMADRIIGMRTQLVSNLKKEGS 
               
               
                   
                 THNWQHITDQIGMFCFTGLKPEQVERLIKEFSIYMTKDGRIS 
               
               
                   
                 VAGVTSSNVGYLAHAIHQVTK 
               
               
                   
               
               
                 AADAT: 
                 MNYARFITAASAARNPSPIRTMTDILSRGPKSMISLAGGLPNP 
               
               
                 Kynurenine/alpha- 
                 NMFPFKTAVITVENGKTIQFGEEMMKRALQYSPSAGIPELLS 
               
               
                 aminoadipate 
                 WLKQLQIKLHNPPTIHYPPSQGQMDLCVTSGSQQGLCKVFE 
               
               
                 aminotransferase, 
                 MIINPGDNVLLDEPAYSGTLQSLHPLGCNIINVASDESGIVPD 
               
               
                 mitochondrial 
                 SLRDILSRWKPEDAKNPQKNTPKFLYTVPNGNNPTGNSLTSE 
               
               
                 SEQ ID NO: 168 
                 RKKEIYELARKYDFLIIEDDPYYFLQFNKFRVPTFLSMDVDG 
               
               
                   
                 RVIRADSFSKIISSGLRIGFLTGPKPLIERVILHIQVSTLHPSTFN 
               
               
                   
                 QLMISQLLHEWGEEGFMAHVDRVIDFYSNQKDAILAAADK 
               
               
                   
                 WLTGLAEWHVPAAGMFLWIKVKGINDVKELIEEKAVKMG 
               
               
                   
                 VLMLPGNAFYVDSSAPSPYLRASFSSASPEQMDVAFQVLAQ 
               
               
                   
                 LIKESL 
               
               
                   
               
               
                 CCLB1: Kynurenine- 
                 MAKQLQARRLDGIDYNPWVEFVKLASEHDVVNLGQGFPDF 
               
               
                 -oxoglutarate 
                 PPPDFAVEAFQHAVSGDFMLNQYTKTFGYPPLTKILASFFGE 
               
               
                 transaminase 1 from 
                 LLGQEIDPLRNVLVTVGGYGALFTAFQALVDEGDEVIIIEPFF 
               
               
                 
                   homo sapiens 
                 
                 DCYEPMTMMAGGRPVFVSLKPGPIQNGELGSSSNWQLDPM 
               
               
                 SEQ ID NO: 169 
                 ELAGKFTSRTKALVLNTPNNPLGKVFSREELELVASLCQQH 
               
               
                   
                 DVVCITDEVYQWMVYDGHQHISIASLPGMWERTLTIGSAGK 
               
               
                   
                 TFSATGWKVGWVLGPDHIMKHLRTVHQNSVFHCPTQSQAA 
               
               
                   
                 VAESFEREQLLFRQPSSYFVQFPQAMQRCRDHMIRSLQSVGL 
               
               
                   
                 KPIIPQGSYFLITDISDFKRKMPDLPGAVDEPYDRRFVKWMI 
               
               
                   
                 KNKGLVAIPVSIFYSVPHQKHFDHYIRFCFVKDEATLQAMDE 
               
               
                   
                 KLRKWKVEL 
               
               
                   
               
               
                 CCLB2: kynurenine- 
                 MFLAQRSLCSLSGRAKFLKTISSSKILGFSTSAKMSLKFTNAK 
               
               
                 -oxoglutarate 
                 RIEGLDSNVWIEFTKLAADPSVVNLGQGFPDISPPTYVKEELS 
               
               
                 transaminase 3 from 
                 KIAAIDSLNQYTRGFGHPSLVKALSYLYEKLYQKQIDSNKEI 
               
               
                 
                   homo sapiens 
                 
                 LVTVGAYGSLFNTIQALIDEGDEVILIVPFYDCYEPMVRMAG 
               
               
                 SEQ ID NO: 170 
                 ATPVFIPLRSKPVYGKRWSSSDWTLDPQELESKFNSKTKAIIL 
               
               
                   
                 NTPHNPLGKVYNREELQVIADLCIKYDTLCISDEVYEWLVYS 
               
               
                   
                 GNKHLKIATFPGMWERTITIGSAGKTFSVTGWKLGWSIGPN 
               
               
                   
                 HLIKHLQTVQQNTIYTCATPLQEALAQAFWIDIKRMDDPEC 
               
               
                   
                 YFNSLPKELEVKRDRMVRLLESVGLKPIVPDGGYFIIADVSL 
               
               
                   
                 LDPDLSDMKNNEPYDYKFVKWMTKHKKLSAIPVSAFCNSE 
               
               
                   
                 TKSQFEKFVRFCFIKKDSTLDAAEEIIKAWSVQKS 
               
               
                   
               
               
                 TnaA: tryptophanase 
                 MENFKHLPEPFRIRVIEPVKRTTRAYREEAIIKSGMNPFLLDS 
               
               
                 from  E. coli   
                 EDVFIDLLTDSGTGAVTQSMQAAMMRGDEAYSGSRSYYAL 
               
               
                 SEQ ID NO: 171 
                 AESVKNIFGYQYTIPTHQGRGAEQIYIPVLIKKREQEKGLDRS 
               
               
                   
                 KMVAFSNYFFDTTQGHSQINGCTVRNVYIKEAFDTGVRYDF 
               
               
                   
                 KGNFDLEGLERGIEEVGPNNVPYIVATITSNSAGGQPVSLAN 
               
               
                   
                 LKAMYSIAKKYDIPVVMDSARFAENAYFIKQREAEYKDWTI 
               
               
                   
                 EQITRETYKYADMLAMSAKKDAMVPMGGLLCMKDDSFFD 
               
               
                   
                 VYTECRTLCVVQEGFPTYGGLEGGAMERLAVGLYDGMNLD 
               
               
                   
                 WLAYRIAQVQYLVDGLEEIGVVCQQAGGHAAFVDAGKLLP 
               
               
                   
                 HIPADQFPAQALACELYKVAGIRAVEIGSFLLGRDPKTGKQL 
               
               
                   
                 PCPAELLRLTIPRATYTQTHMDFIIEAFKHVKENAANIKGLTF 
               
               
                   
                 TYEPKVLRHFTAKLKEV 
               
               
                   
               
            
           
         
       
     
     In one embodiment, the tryptophan pathway catabolic enzyme has at least about 80% identity with the entire sequence of one or more of SEQ ID NO: 144 through SEQ ID NO: 171. 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: 144 through SEQ ID NO: 171. 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: 144 through SEQ ID NO: 171. 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: 144 through SEQ ID NO: 171. 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: 144 through SEQ ID NO: 171. 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: 144 through SEQ ID NO: 171. In another embodiment, the tryptophan pathway catabolic enzyme comprises the sequence of one or more SEQ ID NO: 144 through SEQ ID NO: 171. In yet another embodiment the tryptophan pathway catabolic enzyme consists of the sequence of one or more SEQ ID NO: 144 through SEQ ID NO: 171. 
     ALE 
       E. coli  Nissle can be engineered to efficiently import KYN and convert it to TRP. While Nissle does not typically utilize KYN, by introducing the Kynureninase (KYNase) from  Pseudomonas fluorescens  (kynU) on a medium-copy plasmid under the control of the tetracycline promoter (Ptet) a new strain with this plasmid (Ptet-KYNase) is able to convert L-kynurenine into anthranilate.  E. coli  naturally utilizes anthranilate in its TRP biosynthetic pathway. Briefly, the TrpE (in complex with TrpD) enzyme converts chorismate into anthranilate. TrpD, TrpC, TrpA and TrpB then catalyzes a five-step reaction ending with the condensation of an indole with serine to form tryptophan. By replacing the TrpE enzyme via lambda-RED recombineering, the subsequent strain of Nissle (ΔtrpE::Cm) is an auxotroph unable to grow in minimal media without supplementation of TRP or anthranilate. By expressing kynureninase in ΔtrpE::Cm (KYNase-trpE), this auxotrophy can be alternatively rescued by providing KYN. 
     Leveraging the growth-limiting nature of KYN in KYNase-trpE, adaptive laboratory evolution was employed to further evolve a strain capable of increasingly efficient utilization of KYN. First a lower limit of KYN concentration was established and mutants were evolved by passaging in lowering concentrations of KYN. While this can select for mutants capable of increasing KYN import, the bacterial cells still prefer to utilize free, exogenous TRP. In the tumor environment, dual-therapeutic functions can be provided by depletion of KYN and increasing local concentrations of TRP. Therefore, to evolve a strain which prefers KYN over TRP, a toxic analogue of TRP—5-fluoro-L-tryptophan (ToxTRP)—can be incorporated into the ALE experiment. The resulting best performing strain is then whole genome sequenced in order to deconvolute the contributing mutations. Lambda-RED can be performed in order to reintroduce TrpE, to inactivate Trp regulation (trpR, tyrR, transcriptional attenuators) to up-regulate TrpABCDE expression and increase chorismate production. The resulting strain is now insensitive to external TRP, efficiently converts KYN into TRP, and also now overproduces TRP. 
     In some embodiments, the genetically engineered bacteria comprise one or more nucleic acid sequence of Table 3B 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 3B 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 3B 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 3B or a functional fragment thereof. 
     In some embodiments, the genetically engineered bacteria encode a gene or gene cassette, which promotes anti-inflammatory activity. In some embodiments, the genetically engineered bacteria are capable of producing kynurenine. 
     In some embodiments, 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). The genetically engineered bacteria may comprise any suitable gene for producing kynurenine. In some embodiments, the genetically engineered bacteria may comprise one or more gene(s) or gene cassette(s) 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 genetically engineered bacteria comprise a gene encoding kynurenine formamidase. 
     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 diseaseprogression 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 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. In some embodiments, 
     The genetically engineered bacteria may comprise any suitable gene 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. 
     In some embodiments, the genetically engineered bacteria comprising one or more gene(s) or gene cassette(s) can alter the TRP:KYN ratio, e.g. in the circulation. In some embodimetns the TRP:KYN ratio is increased. In some embodiments, TRP:KYN ratio is decreased. some embodiments, the genetically engineered bacteria the genetically engineered bacteria comprising one or more gene(s) or gene cassette(s) can alter the KYNA:QUIN ratio. 
     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, liver damage, metabolic disease, or in the presence of some other metabolite that may or may not be present in the gut or the tumor micorenvironment, 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. 
     Tryptophan 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. 
     Tryptophan and Tryptophan MetaboliteTransport 
     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 mtr 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 transporte 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 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 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 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. 
     Inhibitory and Targeting Molecules 
     In some embodiments, the genetically engineered bacteria of the invention are capable of producing a molecule that is capable of inhibiting a metabolic disease-promoting 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 metabolic disease-promoting molecules, e.g., dipeptidyl peptidase-4 (DPP-4) or ghrelin receptor. The genetically engineered bacteria may inhibit one or more metabolic disease-promoting molecules. 
     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 metabolic disease-promoting molecules in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, 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 produce siRNA targeting DPP-4 in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, 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. 
     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 a variety of molecules, 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 metabolic disease-promoting molecules in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, 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 produce scFv targeting DPP-4 in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, 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 produce both scFv and siRNA targeting one or more metabolic disease-promoting molecules in low-oxygen conditions, in the presence of certain molecules or metabolites, in the presence of molecules or metabolites associated with liver damage, metabolic disease, 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 (see, e.g., Xiao et al., 2014). 
     Generation of Bacterial Strains with Enhanced 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. O. 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 and/or kynurenine. 
     Inducible Promoters 
     In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the gene or genes for producing one or more payload molecules or the gene or genes for encoding one or more payload polypeptides. Herein the term “payload” is used to refer to butyrate, propionate, acetate, GLP-1, GLP-2, a manganese transporter, a GABA transporter, a tryptophan transporter, aromatic amino acid transporter, a kynureninase, a kynurenine-oxoglutarate transaminase (kynurenine aminotransferase, e.g., KAT I, II, III), polypeptides for metabolizing (catabolizing) GABA, and a gut-barrier enhancing molecule. In some embodiments, the gene, gene(s), or gene cassettes for producing the payload(s) is present on a plasmid and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene gene, gene(s), or gene cassettes for producing the payload(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, gene(s), or gene cassettes for producing the payload(s) is present on a chromosome and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene, gene(s), or gene cassettes for producing the payload(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, gene(s), or gene cassettes for producing the payload(s) is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline or arabinose. 
     In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying at least one gene, gene(s), or gene cassettes for producing the 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 copies of the at least one gene, gene(s), or gene cassettes for producing the payload(s). In some embodiments, the genetically engineered bacteria comprise multiple copies of the same at least one gene, gene(s), or gene cassettes for producing the payload(s). In some embodiments, the at least one gene, gene(s), or gene cassettes for producing the payload(s) is present on a plasmid and operably linked to a directly or indirectly inducible promoter. In some embodiments, the at least one gene, gene(s), or gene cassettes for producing the payload(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 at least one gene, gene(s), or gene cassettes for producing the payload(s) is present on a chromosome and operably linked to a directly or indirectly inducible promoter. In some embodiments, the at least one gene, gene(s), or gene cassettes for producing the payload(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 at least one gene, gene(s), or gene cassettes for producing the payload(s) is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline. 
     In some embodiments, the promoter that is operably linked to the gene, gene(s), or gene cassettes for producing the payload(s) is directly induced by exogenous environmental conditions. In some embodiments, the promoter that is operably linked to the gene, gene(s), or gene cassettes for producing the payload(s) is indirectly induced by exogenous environmental 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 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 gut of a mammal, e.g., propionate. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the bacterial cell. 
     In some embodiments, the bacterial cell comprises a stably maintained plasmid or chromosome carrying the at least one gene, gene(s), or gene cassettes for producing the 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 copies of the at least one gene, gene(s), or gene cassettes for producing the payload(s). In some embodiments, the genetically engineered bacteria comprise multiple copies of the same at least one gene, gene(s), or gene cassettes for producing the payload(s). In some embodiments, the at least one gene, gene(s), or gene cassettes for producing the payload(s) is present on a plasmid and operably linked to a directly or indirectly inducible promoter. In some embodiments, the at least one gene, gene(s), or gene cassettes for producing the payload(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 at least one gene, gene(s), or gene cassettes for producing the payload(s) is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline. 
     In some embodiments, the promoter that is operably linked to the gene, gene(s), or gene cassettes for producing the payload(s) is directly induced by exogenous environmental conditions. In some embodiments, the promoter that is operably linked to the gene, gene(s), or gene cassettes for producing the payload(s) is indirectly induced by exogenous environmental 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 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 gut of a mammal, e.g., propionate. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the bacterial cell. 
     FNR-Dependent Regulation 
     In certain embodiments, the bacterial cell comprises a gene, gene(s), or gene cassettes for producing the payload(s) is expressed under the control of the fumarate and nitrate reductase regulator (FNR) promoter. In certain embodiments, the bacterial cell comprises at least one gene, gene(s), or gene cassettes for producing the payload(s) is expressed under the control of the fumarate and nitrate reductase regulator (FNR) promoter. In certain embodiments, the bacterial cell comprises at least one gene, gene(s), or gene cassettes for producing the payload(s) is expressed under the control of the fumarate and nitrate reductase regulator (FNR) 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 the chart, below. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning. 
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 FNR Promoter Sequences 
               
            
           
           
               
               
            
               
                 FNR Responsive 
                   
               
               
                 Sequence 
                 Promoter 
               
               
                   
               
               
                 SEQ ID NO: 
                 GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGC 
               
               
                 172 
                 GGCACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGT 
               
               
                   
                 ACATCTATTTCTATAAATCCGTTCAATTTGTCTGTTTTTTGCAC 
               
               
                   
                 AAACATGAAATATCAGACAATTCCGTGACTTAAGAAAATTTATA 
               
               
                   
                 CAAATCAGCAATATACCCCTTAAGGAGTATATAAAGGTGAATTT 
               
               
                   
                 GATTTACATCAATAAGCGGGGTTGCTGAATCGTTAAGGTAGGCG 
               
               
                   
                 GTAATAG AAAAGAAATCGAGGCAAAA   
               
               
                   
               
               
                 SEQ ID NO: 
                 ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACT 
               
               
                 173 
                 TATGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAA 
               
               
                   
                 ACAAAAAATATTTCACTCGACAGGAGTATTTATATTGCGCCCGT 
               
               
                   
                 TACGTGGGCTTCGACTGTAAATC AGAAAGGAGAAAACACCT   
               
               
                   
               
               
                 SEQ ID NO: 
                 GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGC 
               
               
                 174 
                 GGCACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGT 
               
               
                   
                 ACATCTATTTCTATAAATCCGTTCAATTTGTCTGTTTTTTGCAC 
               
               
                   
                 AAACATGAAATATCAGACAATTCCGTGACTTAAGAAAATTTATA 
               
               
                   
                 CAAATCAGCAATATACCCCTTAAGGAGTATATAAAGGTGAATTT 
               
               
                   
                 GATTTACATCAATAAGCGGGGTTGCTGAATCGTTAA GGATCC   CT   
               
               
                   
                 
                   CTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT 
                 
               
               
                   
               
               
                 SEQ ID NO: 
                 CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGAC 
               
               
                 175 
                 TTATGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAA 
               
               
                   
                 AACAAAAAATATTTCACTCGACAGGAGTATTTATATTGCGCCC G   
               
               
                   
                 
                   GATCC 
                   CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATAT 
                 
               
               
                   
                 
                   ACAT 
                 
               
               
                   
               
               
                 SEQ ID NO: 
                 AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTA 
               
               
                 176 
                 AATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACAAA 
               
               
                   
                 AACGCCGTAAAGTTTGAGCGAAGTCAATAAACTCTCTACCCATT 
               
               
                   
                 CAGGGCAATATCTCTCTT GGATCC   CTCTAGAAATAATTTTGTTT   
               
               
                   
                 
                   AACTTTAAGAAGGAGATATACAT 
                 
               
               
                   
               
            
           
         
       
     
     In one embodiment, the FNR responsive promoter comprises SEQ ID NO: 172. In another embodiment, the FNR responsive promoter comprises SEQ ID NO: 173. In another embodiment, the FNR responsive promoter comprises SEQ ID NO: 174. In another embodiment, the FNR responsive promoter comprises SEQ ID NO: 175. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 176. Additional FNR responsive promoters are shown below. 
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 FNR Promoter sequences 
               
            
           
           
               
               
            
               
                 FNR- 
                   
               
               
                 responsive 
                   
               
               
                 regulatory 
                 1234567890123456789012345678901234567890123 
               
               
                 region 
                 4567890 
               
               
                   
               
               
                 SEQ ID NO: 
                 ATCCCCATCACTCTTGATGGAGATCAATTCCCCAAGCTGCTAG 
               
               
                 177 
                 AGCGTTACCTTGCCCTTAAACATTAGCAATGTCGATTTATCAG 
               
               
                   
                 AGGGCCGACAGGCTCCCACAGGAGAAAACCG 
               
               
                   
               
               
                 SEQ ID NO: 
                 CTCTTGATCGTTATCAATTCCCACGCTGTTTCAGAGCGTTACC 
               
               
                 178 
                 TTGCCCTTAAACATTAGCAATGTCGATTTATCAGAGGGCCGAC 
               
               
                   
                 AGGCTCCCACAGGAGAAAACCG 
               
               
                   
               
               
                 nirB1 
                 GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGG 
               
               
                 SEQ ID NO: 
                 CGGCACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTAC 
               
               
                 179 
                 GTACATCTATTTCTATAAATCCGTTCAATTTGTCTGTTTTTTG 
               
               
                   
                 CACAAACATGAAATATCAGACAATTCCGTGACTTAAGAAAATT 
               
               
                   
                 TATACAAATCAGCAATATACCCCTTAAGGAGTATATAAAGGTG 
               
               
                   
                 AATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTAAGG 
               
               
                   
                 TAGGCGGTAATAG AAAAGAAATCGAGGCAAAA   
               
               
                   
               
               
                 nirB2 
                 CGGCCCGATCGTTGAACATAGCGGTCCGCAGGCGGCACTGCTT 
               
               
                 SEQ ID NO: 
                 ACAGCAAACGGTCTGTACGCTGTCGTCTTTGTGATGTGCTTCC 
               
               
                 180 
                 TGTTAGGTTTCGTCAGCCGTCACCGTCAGCATAACACCCTGAC 
               
               
                   
                 CTCTCATTAATTGCTCATGCCGGACGGCACTATCGTCGTCCGG 
               
               
                   
                 CCTTTTCCTCTCTTCCCCCGCTACGTGCATCTATTTCTATAAA 
               
               
                   
                 CCCGCTCATTTTGTCTATTTTTTGCACAAACATGAAATATCAG 
               
               
                   
                 ACAATTCCGTGACTTAAGAAAATTTATACAAATCAGCAATATA 
               
               
                   
                 CCCATTAAGGAGTATATAAAGGTGAATTTGATTTACATCAATA 
               
               
                   
                 AGCGGGGTTGCTGAATCGTTAAGGTAGGCGGTAATAGAAAAGA 
               
               
                   
                 AATCGAGGCAAAAatgtttgtttaactttaagaaggagatata 
               
               
                   
                 cat 
               
               
                   
               
               
                 nirB3 
                 GTCAGCATAACACCCTGACCTCTCATTAATTGCTCATGCCGGA 
               
               
                 SEQ ID NO: 
                 CGGCACTATCGTCGTCCGGCCTTTTCCTCTCTTCCCCCGCTAC 
               
               
                 181 
                 GTGCATCTATTTCTATAAACCCGCTCATTTTGTCTATTTTTTG 
               
               
                   
                 CACAAACATGAAATATCAGACAATTCCGTGACTTAAGAAAATT 
               
               
                   
                 TATACAAATCAGCAATATACCCATTAAGGAGTATATAAAGGTG 
               
               
                   
                 AATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTAAGG 
               
               
                   
                 TAGGCGGTAATAGAAAAGAAATCGAGGCAAAA 
               
               
                   
               
               
                 ydfZ 
                 ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGAC 
               
               
                 SEQ ID NO: 
                 TTATGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAA 
               
               
                 182 
                 AAACAAAAAATATTTCACTCGACAGGAGTATTTATATTGCGCC 
               
               
                   
                 CGTTACGTGGGCTTCGACTGTAAATC AGAAAGGAGAAAACACC   
               
               
                   
                 
                   T 
                 
               
               
                   
               
               
                 nirB + RBS 
                 GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGG 
               
               
                 SEQ ID NO: 
                 CGGCACTATCGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTAC 
               
               
                 183 
                 GTACATCTATTTCTATAAATCCGTTCAATTTGTCTGTTTTTTG 
               
               
                   
                 CACAAACATGAAATATCAGACAATTCCGTGACTTAAGAAAATT 
               
               
                   
                 TATACAAATCAGCAATATACCCCTTAAGGAGTATATAAAGGTG 
               
               
                   
                 AATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTAA GG   
               
               
                   
                 
                   ATCC 
                   CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATAT 
                 
               
               
                   
                 
                   ACAT 
                 
               
               
                   
               
               
                 ydfZ + RBS 
                 CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGA 
               
               
                 SEQ ID NO: 
                 CTTATGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCA 
               
               
                 184 
                 AAAACAAAAAATATTTCACTCGACAGGAGTATTTATATTGCGC 
               
               
                   
                 CC GGATCC   CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAG   
               
               
                   
                 
                   ATATACAT 
                 
               
               
                   
               
               
                 fnrS1 
                 AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGT 
               
               
                 SEQ ID NO: 
                 AAATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACA 
               
               
                 185 
                 AAAACGCCGTAAAGTTTGAGCGAAGTCAATAAACTCTCTACCC 
               
               
                   
                 ATTCAGGGCAATATCTCTCTT GGATCC   CTCTAGAAATAATTTT   
               
               
                   
                 
                   GTTTAACTTTAAGAAGGAGATATACAT 
                 
               
               
                   
               
               
                 fnrS2 
                 AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGT 
               
               
                 SEQ ID NO: 
                 AAATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACA 
               
               
                 186 
                 AAAACGCCGCAAAGTTTGAGCGAAGTCAATAAACTCTCTACCC 
               
               
                   
                 ATTCAGGGCAATATCTCTCTT GGATCCAAAGTGAACTCTAGAA   
               
               
                   
                 
                   ATAATTTTGTTTAACTTTAAGAAGGAGATATACAT 
                 
               
               
                   
               
               
                 nirB + crp 
                 TCGTCTTTGTGATGTGCTTCCTGTTAGGTTTCGTCAGCCGTCA 
               
               
                 SEQ ID NO: 
                 CCGTCAGCATAACACCCTGACCTCTCATTAATTGCTCATGCCG 
               
               
                 187 
                 GACGGCACTATCGTCGTCCGGCCTTTTCCTCTCTTCCCCCGCT 
               
               
                   
                 ACGTGCATCTATTTCTATAAACCCGCTCATTTTGTCTATTTTT 
               
               
                   
                 TGCACAAACATGAAATATCAGACAATTCCGTGACTTAAGAAAA 
               
               
                   
                 TTTATACAAATCAGCAATATACCCATTAAGGAGTATATAAAGG 
               
               
                   
                 TGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTAA 
               
               
                   
                 GGTAGaaatgtgatctagttcacatttGCGGTAATAGAAAAGA 
               
               
                   
                 AATCGAGGCAAAA atgtttgtttaactttaagaaggagatata   
               
               
                   
                 
                   cat 
                 
               
               
                   
               
               
                 fnrS + crp 
                 AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGT 
               
               
                 SEQ ID NO: 
                 AAATGGTTGTAACAAAAGCAATTTTTCCGGCTGTCTGTATACA 
               
               
                 188 
                 AAAACGCCGCAAAGTTTGAGCGAAGTCAATAAACTCTCTACCC 
               
               
                   
                 ATTCAGGGCAATATCTCTCaaatgtgatctagttcacattt tt   
               
               
                   
                 
                   tgtttaactttaagaaggagatatacat 
                 
               
               
                   
               
            
           
         
       
     
     In some embodiments, multiple distinct FNR nucleic acid sequences are inserted in the genetically engineered bacteria. In alternate embodiments, the genetically engineered bacteria comprising a gene, gene(s), or gene cassettes for producing the payload(s) is 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 alternate embodiments, the genetically engineered bacteria comprising at least one gene, gene(s), or gene cassettes for producing the payload(s) is 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 alternate embodiments, the genetically engineered bacteria comprise at least one gene, gene(s), or gene cassettes for producing the payload(s) is 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 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 some embodiments, the bacterial cell comprises an oxygen-level dependent transcriptional regulator, e.g., FNR, ANR, or DNR, and corresponding promoter from a different bacterial species. The heterologous oxygen-level dependent transcriptional regulator and promoter increase the transcription of genes operably linked to said promoter, e.g., the gene, gene(s), or gene cassettes 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, 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 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, the bacterial cells disclosed herein 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, gene(s), or gene cassettes for producing the payload(s) are present on different plasmids. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene, gene(s), or gene cassettes for producing the payload(s) are present on different plasmids. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene, gene(s), or gene cassettes for producing the payload(s) 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, gene(s), or gene cassettes for producing the payload(s) are present on different chromosomes. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene, gene(s), or gene cassettes for producing the payload(s) 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, gene(s), or gene cassettes for producing the payload(s). In some embodiments, expression of the transcriptional regulator is controlled by the same promoter that controls expression of the gene, gene(s), or gene cassettes for producing the payload(s). In some embodiments, the transcriptional regulator and the payload(s) are divergently transcribed from a promoter region. 
     RNS-Dependent Regulation 
     In some embodiments, the genetically engineered bacteria comprise a gene, gene(s), or gene cassettes for producing the payload(s) that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium that expresses a gene, gene(s), or gene cassettes for producing the payload(s) is under the control of a promoter that is activated by inflammatory conditions. In one embodiment, the gene, gene(s), or gene cassettes for producing the payload(s) 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, gene(s), or gene cassettes for producing the payload(s). 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, gene(s), or gene cassettes for producing the payload(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 22. 
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 Examples of RNS-sensing transcription  
               
               
                 factors and RNS-responsive genes 
               
            
           
           
               
               
               
            
               
                 RNS-sensing 
                 Primarily 
                 Examples of responsive 
               
               
                 transcription 
                 capable 
                 genes, promoters, and/ 
               
               
                 factor: 
                 of sensing: 
                 or regulatory 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, gene(s), or gene cassettes for producing the payload(s), thus controlling expression of the payload(s) relative to RNS levels. For example, the tunable regulatory region is a RNS-inducible regulatory region, and the payload is any of the payloads described 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(s). Subsequently, when inflammation is ameliorated, RNS levels are reduced, and production of the payload(s) 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; Table 1). 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, gene(s), or gene cassettes for producing the payload(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, gene(s), or gene cassettes 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; Table 1). 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 amino acid catabolism enzymes. In some embodiments, the DNR is  Pseudomonas aeruginosa  DNR. 
     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; Table 1). 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. 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 gene, gene(s), or gene cassettes for producing the payload(s) and producing the 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 an amino acid catabolism enzyme. The two repressor activation regulatory circuit comprises a first RNS-sensing repressor and a second repressor, which is operatively linked to a gene, gene(s), or gene cassettes for producing the payload(s). 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, gene(s), or gene cassettes for producing the payload(s) 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(s) 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(s) 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(s) 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 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 a payload(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 amino acid catabolism enzyme(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. 
     ROS-Dependent Regulation 
     In some embodiments, the genetically engineered bacteria comprise gene, gene(s), or gene cassettes for producing the payload(s) that is expressed under the control of an inducible promoter. In some embodiments, the genetically engineered bacterium that expresses a payload(s) under the control of a promoter that is activated by conditions of cellular damage. In one embodiment, the gene, gene(s), or gene cassettes for producing the payload(s) is expressed under the control of a 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 (lO2), 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, gene(s), or gene cassettes for producing the 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, gene(s), or gene cassettes for producing the 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 23. 
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 Examples of ROS-sensing transcription  
               
               
                 factors and ROS-responsive genes 
               
            
           
           
               
               
               
            
               
                 ROS-sensing 
                 Primarily 
                 Examples of responsive 
               
               
                 transcription 
                 capable 
                 genes, promoters, and/ 
               
               
                 factor: 
                 of sensing: 
                 or regulatory 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 
                 ohrA 
               
               
                   
                 peroxides 
                   
               
               
                   
                 NaOCl 
                   
               
               
                 SoxR 
                 O 2 —NO 
                 soxS 
               
               
                   
                 (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 an amino acid catabolism enzyme, thus controlling expression of the payload(s) relative to ROS levels. For example, the tunable regulatory region is a ROS-inducible regulatory region, and the molecule is an 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(s) thereby producing the payload(s). Subsequently, when inflammation is ameliorated, ROS levels are reduced, and production of the payload(s) 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; Table 1). 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, gene(s), or gene cassettes for producing the payload(s). 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(s) and producing the payload(s). 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; Table 1). 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. In the presence of ROS, the SoxR transcription factor senses ROS and activates the soxS regulatory region, thereby driving expression of the operatively linked gene, gene(s), or gene cassettes for producing the payload(s) and producing the payload(s). 
     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 H2O2 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; Table 1). 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. In the presence of ROS, e.g., NaOCI, an OhrR transcription factor senses ROS and no longer binds to the ohrA regulatory region, thereby derepressing the operatively linked gene, gene(s), or gene cassettes for producing the payload(s) and producing the payload(s). 
     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; Table 1). 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. 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 gene, gene(s), or gene cassettes for producing the payload(s) and producing the payload(s). 
     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; Table 1). 
     In these embodiments, the genetically engineered bacteria may comprise a two repressor activation regulatory circuit, which is used to express an amino acid catabolism enzyme. 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., an amino acid catabolism enzyme. 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., an amino acid catabolism enzyme. 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., an amino acid catabolism enzyme. In the presence of PerR binding (which occurs in the presence of ROS), tetR expression is repressed, and the gene or gene cassette 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; Table 1). 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 24. 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: 189, 190, 191, or 192, or a functional fragment thereof. 
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 Nucleotide sequences of exemplary OxyR-regulated regulatory regions 
               
            
           
           
               
               
            
               
                 Regulatory 
                   
               
               
                 sequence 
                 01234567890123456789012345678901234567890123456789 
               
               
                   
               
               
                 katG 
                 TGTGGCTTTTATGAAAATCACACAGTGATCACAAATTTT 
               
               
                 (SEQ ID NO: 
                 AAACAGAGCACAAAATGCTGCCTCGAAATGAGGGCGG 
               
               
                 189) 
                 GAAAATAAGGTTATCAGCCTTGTTTTCTCCCTCATTACT 
               
               
                   
                 TGAAGGATATGAAGCTAAAACCCTTTTTTATAAAGCATT 
               
               
                   
                 TGTCCGAATTCGGACATAATCAAAAAAGCTTAATTAAG 
               
               
                   
                 ATCAATTTGATCTACATCTCTTTAACCAACAATAT   
               
               
                   
                    GCATC  AT 
               
               
                   
                 AACTTCTCTCTAACGCTGTGTATCGTAACGGTAACACTG 
               
               
                   
                 TAGAGGGGAGCACATTGATGCGAATTCATTAAAGAGGA 
               
               
                   
                 GAAAGGTACC 
               
               
                   
               
               
                 dps 
                 TTCCGAAAATTCCTGGCGAGCAGATAAATAAGAATTGT 
               
               
                 (SEQ ID NO: 
                 TCTTATCAATATATCTAACTCATTGAATCTTTATTAGTTT 
               
               
                 190) 
                 TGTTTTTCA  AGTGT   
               
               
                   
                    TAGCGGAACACATAGCCGGTGCTATAC 
               
               
                   
                 TTAATCTCGTTAATTACTGGGACATAACATCAAGAGGA 
               
               
                   
                 TATGAAATTCGAATTCATTAAAGAGGAGAAAGGTACC 
               
               
                   
               
               
                 ahpC 
                 GCTTAGATCAGGTGATTGCCCTTTGTTTATGAGGGTGTT 
               
               
                 (SEQ ID NO: 
                 GTAATCCATGTCGTTGTTGCATTTGTAAGGGCAACACCT 
               
               
                 191) 
                 CAGCCTGCAGGCAGGCACTGAAGATACCAAAGGGTAGT 
               
               
                   
                 TCAGATTACACGGTCACCTGGAAAGGGGGCCATTTTAC 
               
               
                   
                 TTTTTATCGCCGCTGGCGGTGCAAAGTTCACAAAGTTGT 
               
               
                   
                 CTTACGAAGGTT  GATTT   
               
               
                   
                    AGCCGAATCGGCAAAAATTGGTTA 
               
               
                   
                 CCTTACATCTCATCGAAAACACGGAGGAAGTATAGATG 
               
               
                   
                 CGAATTCATTAAAGAGGAGAAAGGTACC 
               
               
                   
               
               
                 oxyS 
                 CTCGAGTTCATTATCCATCCTCCATCGCCAC   
               
               
                 (SEQ ID NO: 
                    GGTAG  ATCCCT 
               
               
                 192) 
                 ATCAAGCATTCTGACTGATAATTGCTCACACGAATTCAT 
               
               
                   
                 TAAAGAGGAGAAAGGTACC 
               
               
                   
               
            
           
         
       
     
     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 gene, gene(s), or gene cassettes for producing the payload(s) 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 gene, gene(s), or gene cassettes for producing the payload(s) 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(s) in the presence of ROS. 
     In some embodiments, the gene, gene(s), or gene cassettes for producing the payload(s) is present on a plasmid and operably linked to a promoter that is induced by ROS. In some embodiments, the gene, gene(s), or gene cassettes for producing the payload(s) is present in the chromosome and operably linked to a promoter that is induced by ROS. In some embodiments, the gene, gene(s), or gene cassettes for producing the payload(s) is present on a chromosome and operably linked to a promoter that is induced by exposure to tetracycline. In some embodiments, the gene, gene(s), or gene cassettes for producing the payload(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 may comprise multiple copies of the gene, gene(s), or gene cassettes for producing the payload(s). In some embodiments, the gene, gene(s), or gene cassettes for producing the payload(s) is present on a plasmid and operatively linked to a ROS-responsive regulatory region. In some embodiments, the gene, gene(s), or gene cassettes for producing the payload(s) 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 amino acid catabolism enzymes 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, gene(s), or gene cassettes for producing the payload(s) such that the gene, gene(s), or gene cassettes for producing 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. In some embodiments, a bacterium may comprise multiple copies of the gene, gene(s), or gene cassettes for producing the payload(s). In some embodiments, the gene, gene(s), or gene cassettes for producing the payload(s) 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, gene(s), or gene cassettes for producing the payload(s) is expressed on a high-copy plasmid. In some embodiments, the high-copy plasmid may be useful for increasing expression of the gene, gene(s), or gene cassettes for producing the payload(s). In some embodiments, the gene, gene(s), or gene cassettes for producing the payload(s) is expressed on a chromosome. 
     Regulation of Expression 
     The genetically engineered bacteria of the invention comprise a gene or gene cassette for producing a metabolic and/or satiety effector molecule, 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 metabolic and/or satiety effector molecule 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 metabolic and/or satiety effector molecule. 
     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 genetically engineered bacteria comprise the gene or gene cassette for producing the metabolic and/or satiety effector molecule expressed under the control of the fumarate and nitrate reductase regulator (FNR). 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. In alternate embodiments, the genetically engineered bacteria comprise a gene or gene cassette for producing the metabolic and/or satiety effector molecule expressed under the control of an alternate oxygen level-dependent promoter, e.g., an anaerobic regulation of arginine deiminiase and nitrate reduction ANR promoter (Ray et al., 1997), a dissimilatory nitrate respiration regulator DNR promoter (Trunk et al., 2010). In these embodiments, expression of the payload is particularly activated in a low-oxygen or anaerobic environment, such as in the gut. 
     In another embodiment, the genetically engineered bacteria comprise the gene or gene cassette for producing the metabolic and/or satiety effector molecule 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. Pseudomonasfluorescens,  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 another embodiment, the genetically engineered bacteria comprise the gene or gene cassette for producing the metabolic and/or satiety effector molecule 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 genetically engineered bacteria comprise the gene or gene cassette for producing the metabolic and/or satiety effector molecule expressed under the control of an inducible promoter that is responsive to specific molecules or metabolites in the environment, e.g., 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 the metabolic and/or satiety effector molecule is under the control of a propionate-inducible promoter. In a more specific embodiment, the gene or gene cassette for producing the metabolic and/or satiety effector molecule 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 the metabolic and/or satiety effector molecule 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 metabolic and/or satiety effector molecule 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 metabolic and/or satiety effector molecule 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 metabolic and/or satiety effector molecule 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 metabolic and/or satiety effector molecule is present on a chromosome 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 metabolic and/or satiety effector molecule 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 metabolic and/or satiety effector molecule 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 variant or mutated oxygen level-dependent transcriptional regulator, e.g., FNR, ANR, or DNR, in addition to the corresponding oxygen level-dependent promoter. The variant or mutated oxygen level-dependent transcriptional regulator increases the transcription of operably linked genes in a low-oxygen or anaerobic environment. In some embodiments, the corresponding wild-type transcriptional regulator 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 certain embodiments, the mutant oxygen level-dependent transcriptional regulator is a FNR protein comprising amino acid substitutions that enhance dimerization and FNR activity (see, e.g., Moore et al., 2006). 
     In some embodiments, the genetically engineered bacteria comprise an oxygen level-dependent transcriptional regulator from a different bacterial species. In certain embodiments, the mutant oxygen level-dependent transcriptional regulator is a 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 express the gene or gene cassette for producing the metabolic and/or satiety molecule on a plasmid and/or a chromosome. In some embodiments, the gene or gene cassette is expressed under the control of a constitutive promoter. In some embodiments, the gene or gene cassette is expressed under the control of an inducible promoter. In one embodiment, the gene or gene cassette is expressed under the control of an oxygen level-dependent promoter that is activated under low-oxygen or anaerobic environments, e.g., a FNR-responsive promoter. 
     FNR-responsive promoter sequences are known in the art, and any suitable FNR-responsive promoter sequence(s) may be used in the genetically engineered bacteria of the invention. Any suitable FNR-responsive promoter(s) may be combined with any suitable gene(s) of interest. Non-limiting FNR-responsive promoter sequences are provided in Table 21. In some embodiments, the genetically engineered bacteria of the invention comprise one or more of: SEQ ID NO: 177, SEQ ID NO: 178, nirB1 promoter (SEQ ID NO: 179), nirB2 promoter (SEQ ID NO: 180), nirB3 promoter SEQ ID NO: 181, ydfZ promoter (SEQ ID NO: 182), nirB promoter fused to a strong ribosome binding site (SEQ ID NO: 183), ydfZ promoter fused to a strong ribosome binding site (SEQ ID NO: 184), fnrS, an anaerobically induced small RNA gene (fnrS1 promoter SEQ ID NO: 185 or fnrS2 promoter SEQ ID NO: 186), nirB promoter fused to a crp binding site (SEQ ID NO: 187), andfnrS fused to a crp binding site (SEQ ID NO: 188). 
     In other embodiments, the gene or gene cassette for producing the metabolic and/or satiety molecule is 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, expression of the gene or gene cassette is controlled by an oxygen level-dependent promoter fused to a CRP binding site. In some embodiments, expression of the gene or gene cassette is 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 gene transcription is repressed. In some embodiments, an oxygen level-dependent promoter (e.g., a FNR-responsive promoter) fused to a binding site for a transcriptional activator is used to ensure that the gene or gene cassette 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 a stably maintained plasmid or chromosome carrying the gene or gene cassette for producing the metabolic and/or satiety effector molecule, 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 metabolic and/or satiety effector molecule. 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 metabolic and/or satiety effector molecule 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 metabolic and/or satiety effector molecule is expressed on a chromosome. 
     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. 47 . For example, the genetically engineered bacteria may include four copies of GLP-1 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-1 inserted at three different insertion sites, e.g., malE/K, insB/I, and lacZ, and three copies of a propionate gene cassette inserted at three different insertion sites, e.g., dapA, cea, and araC/BAD. 
     In some embodiments, the genetically engineered bacteria of the invention produce at least one metabolic and/or satiety effector molecule under inducing conditions and are capable of reducing one or more symptoms of metabolic disease in a subject by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more as compared to unmodified bacteria of the same subtype under the same conditions. Symptoms and manifestations of metabolic diseases may be measured by methods known in the art, e.g., glucose tolerance testing, insulin tolerance testing. 
     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 metabolic and/or satiety effector molecule under inducing conditions than unmodified bacteria of the same subtype under the same conditions. Certain unmodified bacteria will not have detectable levels of the metabolic and/or satiety effector molecule. In embodiments using genetically modified forms of these bacteria, the metabolic and/or satiety effector molecule will be detectable under inducing conditions. 
     In certain embodiments, the metabolic and/or satiety effector 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 metabolic and/or satiety effector 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 μM, 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. 
     Table 25 lists a propionate promoter sequence. In some embodiments, the propionate promoter is induced in the mammalian gut. 
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 Propionate promoter sequence 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 Pip (Propionate) 
                 
                   TTACCCGTCTGGATTTTCAGTACGCGCTTTTAAACGACGCCA 
                 
               
               
                 promoter 
                 
                   CAGCGTGGTACGGCTGATCCCCAAATAACGTGCGGCGGCGCG 
                 
               
               
                 Bold: prpR 
                 
                   CTTATCGCCATTAAAGCGTGCGAGCACCTCCTGCAATGGAAG 
                 
               
               
                 Lower case: 
                 
                   CGCTTCTGCTGACGAGGGCGTGATTTCTGCTGTGGTCCCCAC 
                 
               
               
                 ribosome binding 
                 
                   CAGTTCAGGTAATAATTGCCGCATAAATTGTCTGTCCAGTGT 
                 
               
               
                 site 
                 
                   TGGTGCGGGATCGACGCTTAAAAAAAGCGCCAGGCGTTCCAT 
                 
               
               
                 ATG underlined: 
                 
                   CATATTCCGCAGTTCGCGAATATTACCGGGCCAATGATAGTT 
                 
               
               
                 start of gene of 
                 
                   CAGTAGAAGCGGCTGACACTGCGTCAGCCCATGACGCACCGA 
                 
               
               
                 interest 
                 
                   TTCGGTAAAAGGGATCTCCATCGCGGCCAGCGATTGTTTTAA 
                 
               
               
                 SEQ ID NO: 193 
                 
                   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   
               
               
                   
               
            
           
         
       
     
     Mutagenesis 
     In some embodiments, an inducible promoter is operably linked to a detectable product, e.g., GFP, and can be used to screen for mutants. In some embodiments, an oxygen level-dependent promoter is operably linked to a detectable product, e.g., GFP, and can be used to screen for mutants. In some embodiments, the oxygen level-dependent 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 metabolic and/or satiety effector molecule in low-oxygen conditions, as compared to unmutated bacteria of the same subtype under the same conditions. In some embodiments, the oxygen level-sensing transcription factor and/or the oxygen level-dependent promoter is a synthetic, non-naturally occurring sequence. In some embodiments, the transcription factor regulating the mutated promoter senses the presence of certain molecules or metabolites, the presence of molecules or metabolites associated with liver damage, inflammation or an inflammatory response, or the presence of some other metabolite that may or may not be present in the gut, such as arabinose. 
     In some embodiments, the gene encoding a metabolic and/or satiety effector molecule is mutated to increase expression and/or stability of said molecule in low oxygen 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 a metabolic and/or satiety effector molecule is mutated to increase expression of said molecule in low oxygen conditions, as compared to unmutated bacteria of the same subtype under the same conditions. 
     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. 47 . For example, the genetically engineered bacteria may include four copies of GLP-1 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-1 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/BAD. 
     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 one or more of GLP-1 and GLP-1 analogs, as described herein (4) one or more gene(s) or gene cassette(s) for the production of one or more bile salt hydrolases, as described herein (5) one or more gene(s) or gene cassette(s) for the production of one or more transporters, e.g. for the import of bile salts and/or metabolites, e.g. tryptophan and/or tryptophan metabolites, as described herein (6) one or more polypetides for secretion, including but not limited to.GLP-1 and its analogs, bile salt hydrolases, and tryptophan synthesis and/or catabolic enzymes of the tryptophan degradation pathways, in wild type or in mutated form (for increased stability or metabolic activity) (3) one or more components of secretion machinery, as described herein (4) one or more auxotrophies, e.g., deltaThyA (5) one more more antibiotic resistances, including but not limited to, kanamycin or chloramphenicol resistance (6) 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 (7) one or more mutations/deletions in one or more genes of the endogenous metabolic pathways, e.g., tryptophan synthesis pathway. 
     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, 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). 
     Secretion 
     In some embodiments, the genetically engineered bacteria further comprise a native secretion mechanism (e.g., gram positive bacteria) or non-native secretion mechanism (e.g., gram negative bacteria) that is capable of secreting a molecule from the bacterial cytoplasm. 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  FIGS. 3-6 . 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 (T5SS), the curli secretion system, and the chaperone-usher pathway for pili assembly (Saier, 2006; Costa et al., 2015). 
     In some embodiments, the genetically engineered bacteria of the invention further comprise a type III or a type III-like secretion system (T3SS) from  Shigella, Salmonella, E. coli, Bivrio, Burkholderia, Yersinia, Chlamydia , or  Pseudomonas . The T3SS is capable of transporting a protein from the bacterial cytoplasm to the host cytoplasm through a needle complex. 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 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 heterologouse protein or peptide comprises a type III secretion sequence that allows the molecule of interest o be secreted from the bacteria. 
     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. 
     In some embodiments, a Type V Autotransporter Secretion System is used to secrete the molecule of interest, e.g., therapeutic peptide. 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. 10 , 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 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 thread through the hollow pore of the beta-barrel structure ahead of the linker sequence. Once exposed to the extracellular environment, the therapeutic peptide can be freed from the linker system by an autocatalytic cleavage (left side of Bam complex) or by targeting of a membrane-associated peptidase (black scissors; right side of Bam complex) to a complimentary protease cut site in the linker. Thus, in some embodiments, the secreted molecule, such as a heterologouse 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. 
     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. 11  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. 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. 
     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 periplasm in a bacteria with a destabilized 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 deactived. 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, 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, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl, under the control of an inducible promoterFor 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., over expression of colicins or the third topological domain of TolA, which 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. 
     The Tables 26 and 27 below lists secretion systems for Gram positive bacteria and Gram negative bacteria. 
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 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 
                 Sec pathway 
               
               
                   
                 (Gram+) 
                 Twin-arginine (TAT) pathway 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 27 
               
             
            
               
                   
               
               
                 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- 
                 2.A.64 
                 + 
                 + 
                 + 
                 2-4 
                 PMF 
               
               
                 (IISP) 
                 arginine 
                   
                   
                   
                 (chloroplasts) 
                   
                   
               
               
                   
                 targeting 
                   
                   
                   
                   
                   
                   
               
               
                   
                 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· 
                 + 
                 − 
                 − 
                 1 
                 None 
               
               
                   
                 functional 
                 21 
                   
                   
                   
                   
                   
               
               
                   
                 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; PMF 
               
               
                 (IISP) 
                 terminal 
                   
                   
                   
                   
                   
                   
               
               
                   
                 branch of 
                   
                   
                   
                   
                   
                   
               
               
                   
                 the general 
                   
                   
                   
                   
                   
                   
               
               
                   
                 secretory 
                   
                   
                   
                   
                   
                   
               
               
                   
                 translocase 
                   
                   
                   
                   
                   
                   
               
               
                 FUP 
                 Fimbrial 
                 1.B.11 
                 + b   
                 − 
                 − 
                 1 
                 None 
               
               
                 AT-1 
                 usher protein 
                 1.B.12 
                 + b   
                   
                 − 
                 1 
                 None 
               
               
                   
                 Autotrans- 
                   
                   
                   
                   
                   
                   
               
               
                   
                 porter-1 
                   
                   
                   
                   
                   
                   
               
               
                 AT-2 
                 Autotrans- 
                 1.B.40 
                 + b   
                 − 
                 − 
                 1 
                 None 
               
               
                 OMF 
                 porter-2 
                 1.B.17 
                 + b   
                   
                 +(?) 
                 1 
                 None 
               
               
                 (ISP) 
                   
                   
                   
                   
                   
                   
                   
               
               
                 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. 
     Any of the secretion systems described herein may according to the disclosure be employed to secrete the proteins of interest. Non-limiting examples of proteins of interest include GLP-1 peptides, GLP-1 analogs, proglucagon peptides, catabolic enzymes, including but not limited to IDO, TDO, kynureninase, other tryptophan pathway catabolic enzymes, e.g. in the ndole pathway and/or the kynurenine pathway as described herein, and bile salt hydrolases as described herein. These polypeptides may be mutated to increase stability, resistance to protease digestion, and/or activity. 
     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, ginA, 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 28 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 28 
               
             
            
               
                   
               
               
                 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 28 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 28 
               
             
            
               
                   
               
               
                 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 at., 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, rnc, ftsB, eno, pyrG, chpR, lgt, fbaA, pgk, yqgD, metK, yqgF, plsC, ygiT, pare, ribB, cca, ygjD, tdcF, yraL, yihA, ftsN, murI, 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, rpsI, 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, ffh, 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, lnt, glnS, fldA, cydA, infA, cydC, ftsK, lolA, serS, rpsA, msbA, lpxK, kdsB, mukF, mukE, mukB, asnS, fabA, mviN, rne, yceQ, fabD, fabG, acpP, tmk, holB, lolC, lolD, lolE, purB, ymfK, minE, mind, pth, rsA, ispE, lolB, hemA, prfA, prmC, kdsA, topA, ribA, fabI, 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, 149G, 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 14L, L51 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. 56 . 
     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 metabolic or satiety effector 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, incorporated herein by reference in its entirety). The genetic regulatory circuits are useful to screen for mutant bacteria that produce a metabolic or satiety effector 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 metabolic or satiety effector molecule 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 fumarate and nitrate reductase regulator (FNR)-responsive promoter; a second gene or gene cassette for producing a metabolic or satiety effector molecule, 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 metabolic or satiety effector molecule 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 metabolic or satiety effector molecule 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 metabolic or satiety effector molecule 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 metabolic or satiety effector 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-lon 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 metabolic or satiety effector molecule is not expressed. In the absence of oxygen, FNR dimerizes and binds the FNR-responsive promoter, thereby inducing expression of mf-lon protease. The mf-lon protease recognizes the mf-lon degradation signal and degrades the tetR, and the metabolic or satiety effector molecule is expressed. 
     In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector 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 metabolic or satiety effector 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 metabolic or satiety effector 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 metabolic or satiety effector 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 metabolic or satiety effector 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 metabolic or satiety effector 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 metabolic or satiety effector 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 metabolic or satiety effector 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 metabolic or satiety effector molecule is expressed. 
     In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector 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 metabolic or satiety effector 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 metabolic or satiety effector 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 metabolic or satiety effector molecule is expressed. 
     In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector 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 metabolic or satiety effector 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 metabolic or satiety effector 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 functional metabolic or satiety effector molecule is produced. 
     In some embodiments, the invention provides genetically engineered bacteria comprising a gene or gene cassette for producing a metabolic or satiety effector 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 metabolic or satiety effector 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 metabolic or satiety effector 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 metabolic or satiety effector 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 metabolic or satiety effector molecule is expressed. 
     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 a metabolic or satiety effector molecule and further comprise a toxin-antitoxin system that simultaneously produces a toxin (hok) and a short-lived antitoxin (sok), wherein loss of the plasmid causes the cell to be killed by the long-lived toxin (Danino et al., 2015;  FIG. 29 ). 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 and 62/263,329, 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, a metabolic or satiety effector 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 oxygen level-dependent expression of the metabolic or satiety effector molecule, e.g., GLP-1. In some embodiments, the kill switch is activated in a delayed fashion following oxygen level-dependent expression of the metabolic or satiety effector 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 a low-oxygen environment. 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. 56-61 . 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 toxing 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 arbinoase 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. 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 antitoxin, 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 antitoxin 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 antitoxin 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 antitoxin 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/anto-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 is 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 is 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, MccEC TD , MccF, Cai, ImmEl, 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, ginA, 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 AthyA and AdapA 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 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 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 metabolic or satiety effector molecule is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions. In other embodiments, the gene or gene cassette for producing the metabolic or satiety effector molecule is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low-oxygen or anaerobic conditions. 
     Pharmaceutical Compositions and Formulations 
     Pharmaceutical compositions comprising the genetically engineered bacteria of the invention may be used to treat, manage, ameliorate, and/or prevent a metabolic disease, e.g., obesity, type 2 diabetes. Pharmaceutical compositions of the invention comprising one or more genetically engineered bacteria, alone or in combination with prophylactic agents, therapeutic agents, and/or and pharmaceutically acceptable carriers are provided. 
     In certain embodiments, the pharmaceutical composition comprises one species, strain, or subtype of bacteria described herein that are engineered to treat, manage, ameliorate, and/or prevent a metabolic disease. In alternate embodiments, the pharmaceutical composition comprises two or more species, strains, and/or subtypes of bacteria described herein that are each engineered to treat, manage, ameliorate, and/or prevent a metabolic disease. 
     The pharmaceutical compositions of the invention 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 bacteria of the invention 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, immediate-release, pulsatile-release, delayed-release, or sustained release). Suitable dosage amounts for the genetically engineered bacteria may range from about 105 to 10 12  bacteria, e.g., approximately 10 5  bacteria, approximately 10 6  bacteria, approximately 10 7  bacteria, approximately 10 8  bacteria, approximately 109 bacteria, approximately 10 10  bacteria, approximately 10 11  bacteria, or approximately 10 11  bacteria. The composition may be administered once or more daily, weekly, or monthly. The genetically engineered bacteria may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents. 
     The genetically engineered bacteria of the invention 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. 
     The genetically engineered bacteria of the invention 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 bacteria are enterically coated for release into the gut or a particular region of the gut, for example, the small or large intestines. 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. 
     In some embodiments, enteric coating materials may be used, in one or more coating layers (e.g., outer, inner and/o intermediate coating layers). Enteric coated polymers remain unionised at low pH, and therefore remain insoluble. But as the pH increases in the gastrointestinal tract, the acidic functional groups are capable of ionisation, and the polymer swells or becomes soluble in the intestinal fluid. 
     Materials used for enteric coatings include Cellulose acetate phthalate (CAP), Poly(methacrylic acid-co-methyl methacrylate), Cellulose acetate trimellitate (CAT), Poly(vinyl acetate phthalate) (PVAP) and Hydroxypropyl methylcellulose phthalate (HPMCP), fatty acids, waxes, Shellac (esters of aleurtic acid), plastics and plant fibers. Additionally, Zein, Aqua-Zein (an aqueous zein formulation containing no alcohol), amylose starch and starch derivatives, and dextrins (e.g., maltodextrin) are also used. Other known enteric coatings include ethylcellulose, methylcellulose, hydroxypropyl methylcellulose, amylose acetate phthalate, cellulose acetate phthalate, hydroxyl propyl methyl cellulose phthalate, an ethylacrylate, and a methylmethacrylate. 
     Coating polymers also may comprise one or more of, phthalate derivatives, CAT, HPMCAS, polyacrylic acid derivatives, copolymers comprising acrylic acid and at least one acrylic acid ester, Eudragit™ S (poly(methacrylic acid, methyl methacrylate) 1:2); Eudragit L100™ S (poly(methacrylic acid, methyl methacrylate) 1:1); Eudragit L30D™, (poly(methacrylic acid, ethyl acrylate) 1:1); and (Eudragit L100-55) (poly(methacrylic acid, ethyl acrylate) 1:1) (Eudragit™ L is an anionic polymer synthesized from methacrylic acid and methacrylic acid methyl ester), polymethyl methacrylate blended with acrylic acid and acrylic ester copolymers, alginic acid, ammonia alginate, sodium, potassium, magnesium or calcium alginate, vinyl acetate copolymers, polyvinyl acetate 30D (30% dispersion in water), a neutral methacrylic ester comprising poly(dimethylaminoethylacrylate) (“Eudragit E™), a copolymer of methylmethacrylate and ethylacrylate with trimethylammonioethyl methacrylate chloride, a copolymer of methylmethacrylate and ethylacrylate, Zein, shellac, gums, or polysaccharides, or a combination thereof. 
     Coating layers may also include polymers which contain Hydroxypropylmethylcellulose (HPMC), Hydroxypropylethylcellulose (HPEC), Hydroxypropylcellulose (HPC), hydroxypropylethylcellulose (HPEC), hydroxymethylpropylcellulose (HMPC), ethylhydroxyethylcellulose (EHEC) (Ethulose), hydroxyethylmethylcellulose (HEMC), hydroxymethylethylcellulose (HMEC), propylhydroxyethylcellulose (PHEC), methylhydroxyethylcellulose (M H EC), hydrophobically modified hydroxyethylcellulose (NEXTON), carboxymethyl hydroxyethylcellulose (CMHEC), Methylcellulose, Ethylcellulose, water soluble vinyl acetate copolymers, gums, polysaccharides such as alginic acid and alginates such as ammonia alginate, sodium alginate, potassium alginate, acid phthalate of carbohydrates, amylose acetate phthalate, cellulose acetate phthalate (CAP), cellulose ester phthalates, cellulose ether phthalates, hydroxypropylcellulose phthalate (HPCP), hydroxypropylethylcellulose phthalate (HPECP), hydroxyproplymethylcellulose phthalate (HPMCP), hydroxyproplymethylcellulose acetate succinate (HPMCAS). 
     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 bacteria of the invention. 
     In certain embodiments, the genetically engineered bacteria of the invention 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 of the invention 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 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 of the present invention 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 bacteria of the invention 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 bacteria of the invention may be administered and formulated as depot preparations. Such long acting formulations may be administered by implantation or by injection. 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, the invention provides 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 of the invention 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. 
     Dosage regimens may be adjusted to provide a therapeutic response. 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. 
     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. 
     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 of the invention 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 of the invention 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 of the invention 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. 
     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. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or animal models. For example, LD 50 , ED 50 , EC 50 , and IC 50  may be determined, and the dose ratio between toxic and therapeutic effects (LD 50 /ED 50 ) 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. 
     Methods of Treatment 
     Another aspect of the invention provides methods of treating metabolic disease, e.g., obesity, type 2 diabetes. In some embodiments, the metabolic disease is selected from the group consisting of type 1 diabetes; type 2 diabetes; metabolic syndrome; Bardet-Biedel syndrome; Prader-Willi syndrome; non-alcoholic fatty liver disease; tuberous sclerosis; Albright hereditary osteodystrophy; brain-derived neurotrophic factor (BDNF) deficiency; Single-minded 1 (SIM1) deficiency; leptin deficiency; leptin receptor deficiency; pro-opiomelanocortin (POMC) defects; proprotein convertase subtilisin/kexin type 1 (PCSK1) deficiency; Src homology 2B1 (SH2B1) deficiency; pro-hormone convertase 1/3 deficiency; melanocortin-4-receptor (MC4R) deficiency; Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome; pseudohypoparathyroidism type 1A; Fragile X syndrome; Borjeson-Forsmann-Lehmann syndrome; Alstrom syndrome; Cohen syndrome; and ulnar-mammary syndrome. 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 weight gain, obesity, fatigue, hyperlipidemia, hyperphagia, hyperdipsia, polyphagia, polydipsia, polyuria, pain of the extremities, numbness of the extremities, blurry vision, nystagmus, hearing loss, cardiomyopathy, insulin resistance, light sensitivity, pulmonary disease, liver disease, liver cirrhosis, liver failure, kidney disease, kidney failure, seizures, hypogonadism, and infertility. In some embodiments, the subject to be treated is a human patient. 
     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, e.g., 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 or gastric shunt. 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 certain embodiments, the pharmaceutical composition described herein is administered to treat, manage, ameliorate, or prevent metabolic disease in a subject. In some embodiments, the method of treating or ameliorating metabolic disease allows one or more symptoms of the disease to improve by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more as compared to levels in an untreated or control subject. In some embodiments, the symptom (e.g., obesity, insulin resistance) is measured by comparing measurements in a subject before and after administration of the pharmaceutical composition. 
     Before, during, and after the administration of the pharmaceutical composition in a subject, metabolic symptoms and manifestations may be measured in a biological sample, e.g., blood, serum, plasma, urine, fecal matter, peritoneal fluid, a sample collected from a tissue, such as liver, skeletal muscle, pancreas, epididymal fat, subcutaneous fat, and beige fat. The biological samples may be analyzed to measure symptoms and manifestations of metabolic disease. Useful measurements include measures of lean mass, fat mass, body weight, food intake, GLP-1 levels, endotoxin levels, insulin levels, lipid levels, HbAlc levels, short-chain fatty acid levels, triglyceride levels, and nonesterified fatty acid levels. Useful assays include, but are not limited to, insulin tolerance tests, glucose tolerance tests, pyruvate tolerance tests, assays for intestinal permeability, and assays for glycaemia upon multiple fasting and refeeding time points. In some embodiments, the methods may include administration of the compositions of the invention to reduce metabolic symptoms and manifestations 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 metabolic symptoms and manifestations 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 certain embodiments, the genetically engineered bacteria are  E. coli  Nissle. The genetically engineered 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 genetically engineered bacteria may be re-administered at a therapeutically effective dose and frequency. Length of Nissle residence in vivo in mice is shown in  FIG. 17 . In alternate embodiments, the genetically engineered 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., insulin. 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. 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 disorder. 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 metabolic disease may be used (see, e.g., Mizoguchi 2012). In some embodiments, the animal is a C57BL/6J mouse that is fed a high fat diet in order to induce obesity and T2DM-related symptoms such as hyperinsulinemia and hyperglycemia. In alternate embodiments, an animal harboring a genetic deficiency that causes a metabolic disease, e.g., a B6.BKS(D)-Lepr db/db  mouse, is used. 
     The genetically engineered bacteria of the invention are administered to the mice before, during, or after the onset of obesity and disease. Body weight, food intake, and blood plasma (e.g., triglyceride levels, insulin tolerance tests, glucose tolerance tests, pyruvate tolerance tests) may be assayed to determine the severity and amelioration of disease. Metabolism and physical activity may be measured in metabolic cages. Animals may be sacrificed to assay metabolic tissues such as liver, skeletal muscle, epididymal fat, subcutaneous fat, brown fat, pancreas, and brain, are collected for analysis of histology and gene expression. 
     
       
         
           
               
             
               
                 TABLE 29 
               
             
            
               
                   
               
               
                 Summary of rodent models of type 2 diabetes 
               
            
           
           
               
               
               
               
            
               
                 Induction 
                   
                   
                   
               
               
                 mechanism 
                 Model 
                 Main features 
                 Possible uses 
               
               
                   
               
               
                 Obese models 
                 Lep ob/ob  mice 
                 Obesity-induced 
                 Treatments to improve 
               
               
                 (monogenic) 
                   
                 hyperglycaemia 
                 insulin resistance 
               
               
                   
                 Lepr db/db  mice 
                   
                 Treatments to improve 
               
               
                   
                   
                   
                 beta cell function 
               
               
                   
                 ZDF Rats 
                   
                   
               
               
                 Obese models 
                 KK mice 
                 Obesity-induced 
                 Treatments to improve 
               
               
                 (polygenic) 
                   
                 hyperglycaemia 
                 insulin resistance 
               
               
                   
                 OLETF rat 
                   
                 Treatments to improve 
               
               
                   
                   
                   
                 beta cell function 
               
               
                   
                 NZO mice 
                   
                 Some models show 
               
               
                   
                 TallyHo/Jng mice 
                   
                 diabetic complications 
               
               
                   
                 NoncNZO10/ 
                   
                   
               
               
                   
                 LtJ mice 
                   
                   
               
               
                 Induced 
                 High fat feeding  
                 Obesity-induced 
                 Treatments to improve 
               
               
                 obesity 
                 (mice or rats) 
                 hyperglycaemia 
                 insulin resistance 
               
               
                   
                 Desert gerbil 
                   
                 Treatments to improve 
               
               
                   
                   
                   
                 beta cell function 
               
               
                   
                 Nile grass rat 
                   
                 Treatments to prevent 
               
               
                   
                   
                   
                 diet-induced obesity 
               
               
                 Non-obese 
                 GK rat 
                 Hyperglycaemia 
                 Treatments to improve 
               
               
                 models 
                   
                 induced by 
                 beta cell function 
               
               
                   
                   
                 insufficient beta 
                 Treatments to improve 
               
               
                   
                   
                 cell function/mass 
                 beta cell survival 
               
               
                 Genetically 
                 hIAPP mice 
                 Amyloid 
                 Treatments to prevent 
               
               
                 induced  
                   
                 deposition in  
                 amyloid deposition 
               
               
                 models 
                   
                 islets 
                 Treatments to improve 
               
               
                 of beta cell 
                   
                   
                 beta cell survival 
               
               
                 dysfunction 
                 AKITA mice 
                 Beta cell 
                 Treatments to prevent 
               
               
                   
                   
                 destruction due to 
                 ER stress 
               
               
                   
                   
                 ER stress. 
                 Treatments to improve 
               
               
                   
                   
                   
                 beta cell survival 
               
               
                   
               
            
           
         
       
     
     As described in Aileen J F King, The use of animal models in diabetes research, Br J Pharmacol. 2012 June; 166(3): 877-894. 
     The engineered bacteria may be evaluated in vivo, e.g., in an animal model for NASH. Any suitable animal model of a disease associated with Non-Alcoholic Fatty Liver Disease/Non-Alcoholic Steatohepatitis (NAFLD/NASH) may be used. For example, the effects of liver steatosis and hepatic inflammation in an in vivo mouse model have been described (Jun Jin, et al., Brit. J. Nutrition, 114:145-1755 (2015)). To briefly summarize, female C57BL/6J mice can be fasted and fed either a standard liquid diet of carbohydrates, fat, and protein; or a liquid Western style diet (WSD) fortified with fructose, fat, cholesterol, and a sodium butyrate supplement for six weeks. Butyrate is a short chain fatty acid naturally produced by intestinal bacteria effective in maintaining intestinal homoeostasis. Body weight and plasma samples can be taken throughout the duration of the study. Upon conclusion of the study, the mice can be killed, and the liver and intestine can be removed and assayed. 
     An in vivo rat model of choline deficient/L-amino acid defined (CDAA) diet has also been described (Endo, et al., PLoS One, 8(5):e63388 (2013)). In this model, rats are fed the CDAA diet for eight weeks and then treated with a strain of  Clostridium butyricum  (MIYAIRI 588) two weeks after. The diet induces NAFLD/NASH symptoms such as liver steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocarcinogenesis. The rats are killed at 8, 16, and 50 weeks after completion of the diet regiments, and liver tissues removed and assayed. 
     Other models are known in the art, including a Lepob/Lepob and C57BL6 (B6) mouse model used to study the effects of high fat diet and GLP-1 administration within the NASH setting. See, for example, Trevaskis et al., Am. J. Physiology-Gastrointestinal and Liver Physiology, 302(8):G762-G772, 2012, and Takahashi et al., World J. Gastroenterol., 18(19):2300-2308, 2012, the entire contents of each of which are expressly incorporated herein by reference. 
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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 Propionate 
     To facilitate inducible production of propionate in  Escherichia coli  Nissle, a propionate gene cassette comprising the genes encoding the enzymes of the acrylate pathway, i.e., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC, as well as transcriptional and translational elements, are synthesized (Gen9, Cambridge, Mass.) and cloned into vector pBR322. The genes are codon-optimized for  E. coli  codon usage using Integrated DNA Technologies online codon optimization tool (https://www.idtdna.com/CodonOpt). A second clone is generated as described above using a propionate gene cassette comprising the genes encoding the enzymes of the pyruvate pathway, i.e., thrA fbr , thrB, thrC, ilvA fbr , aceE, aceF, and lpd; NCBI; Tseng et al., 2012). A third clone is generated as described above that comprises thrAg r , thrB, thrC, ilvA fbr , aceE, aceF, lpd, and  E. coli  tesB. Each propionate gene cassette is expressed under the control of each of the following regulatory regions: a FNR-inducible regulatory region selected from the sequences listed in Table 21, a tetracycline-inducible promoter, and an arabinose-inducible promoter. In certain constructs, the FNR-responsive promoter is further fused to a strong ribosome binding site sequence. For efficient translation of propionate 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. Each gene cassette and regulatory region construct is expressed on a high-copy plasmid, a low-copy plasmid, or a chromosome. 
     The propionate construct 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. 47 ). 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. At the site of insertion, DNA primers that are homologous to the site of insertion and to the propionate construct are designed. A linear DNA fragment containing the construct with homology to the target site is generated by PCR, and lambda red recombination is performed as described below. The resulting  E. coli  Nissle bacteria are genetically engineered to express a propionate biosynthesis cassette and produce propionate. 
     Example 2. Lambda Red Recombination 
     Lambda red recombination is used to make chromosomal modifications, e.g., to express a propionate biosynthesis cassette in  E. coli  Nissle. Lambda red is a procedure using recombination enzymes from a bacteriophage lambda to insert a piece of custom DNA into the chromosome of  E. coli . A pKD46 plasmid is transformed into the  E. coli  Nissle host strain.  E. coli  Nissle cells are grown overnight in LB media. The overnight culture is diluted 1:100 in 5 mL of LB media and grown until it reaches an OD 600  of 0.4-0.6. All tubes, solutions, and cuvettes are pre-chilled to 4° C. The  E. coli  cells are 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 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 centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C. water. The electroporator is set to 2.5 kV. 1 ng of pKD46 plasmid DNA is added to the  E. coli  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. 1 mL of room-temperature SOC media is immediately added, and the mixture is transferred to a culture tube and incubated at 30° C. for 1 hr. The cells are spread out on a selective media plate and incubated overnight at 30° C. 
     DNA sequences comprising the desired propionate biosynthesis genes shown above were ordered from a gene synthesis company. The lambda enzymes are used to insert this construct into the genome of  E. coli  Nissle through homologous recombination. The construct is inserted into a specific site in the genome of  E. coli  Nissle based on its DNA sequence. In some embodiments, the construct is in the  E. coli  Nissle genome at the malP/T site ( FIG. 47 ). To insert the construct into a specific site, the homologous DNA sequence flanking the construct is identified, and includes approximately 50 bases on either side of the sequence. The homologous sequences are ordered as part of the synthesized gene. Alternatively, the homologous sequences may be added by PCR. The construct includes an antibiotic resistance marker that may be removed by recombination. The resulting construct comprises approximately 50 bases of homology upstream, a kanamycin resistance marker that can be removed by recombination, the propionate biosynthesis genes, and approximately 50 bases of homology downstream. 
     Example 3. Transforming  E. coli    
     Each of the constructs above is transformed into  E. coli  Nissle comprising pKD46. All tubes, solutions, and cuvettes are pre-chilled to 4° C. An overnight culture is diluted 1:100 in 5 mL of LB media containing ampicillin and grown until it reaches an OD 600  of 0.1. 0.05 mL of 100× L-arabinose stock solution is added to induce pKD46 lambda red expression. The culture is grown until it reaches an OD 600  of 0.4-0.6. The  E. coli  cells are 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 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 centrifuged at 2,000 rpm for 5 min. at 4° C., the supernatant is removed, and the cells are resuspended in 0.1 mL of 4° C. water. The electroporator is set to 2.5 kV. 0.5 μg of the construct 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. 1 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 kanamycin and incubated overnight. 
     In alternate embodiments, the propionate cassette may be inserted into the Nissle genome through homologous recombination (Genewiz, Cambridge, Mass.). Organization of the constructs and nucleotide sequences are shown in  FIGS. 1-5 . To create a vector capable of integrating the synthesized propionate 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 propionate 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. Verifying Mutants 
     The presence of the propionate gene cassette is verified by colony PCR. Colonies are picked with a pipette tip and resuspended in 20 μl of cold ddH 2 O by pipetting up and down. 3 μl of the suspension is pipetted onto an index plate with appropriate antibiotic for use later. The index plate is grown at 37° C. overnight. A PCR master mix is made using 5 μl of 10×PCR buffer, 0.6 μl of 10 mM dNTPs, 0.4 μl of 50 mM Mg 2 SO 4 , 6.0 μl of 10× enhancer, and 3.0 μl of ddH 2 O (15 μl of master mix per PCR reaction). A 10 NM primer mix is made by mixing 2 μL of primers unique to the propionate construct (100 NM stock) into 16 μL of ddH 2 O. For each 20 μl reaction, 15 μL of the PCR master mix, 2.0 μL of the colony suspension (template), 2.0 μL of the primer mix, and 1.0 μL of Pfx Platinum DNA Pol are mixed in a PCR tube. The PCR thermocycler is programmed as follows, with steps 2-4 repeating 34 times: 1) 94° C. at 5:00 min., 2) 94° C. at 0:15 min., 3) 55° C. at 0:30 min., 4) 68° C. at 2:00 min., 5) 68° C. at 7:00 min., and then cooled to 4° C. The PCR products are analyzed by gel electrophoresis using 10 μL of each amplicon and 2.5 μL 5× dye. The PCR product only forms if the heterologous sequence has been inserted. 
     Example 5. Generation of AThyA 
     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 30. 
     
       
         
           
               
             
               
                 TABLE 30 
               
             
            
               
                   
               
               
                 Primer Sequences 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 SEQ ID 
               
               
                 Name 
                 Sequence 
                 Description 
                 NO 
               
               
                   
               
               
                 SR36 
                 tagaactgatgcaaaaagtgctcgacgaaggcacacagaTGTGTAGG 
                 Round 1: binds 
                 SEQ ID 
               
               
                   
                 CTGGAGCTGCTTC 
                 on pKD3 
                 NO: 194 
               
               
                   
               
               
                 SR38 
                 gtttcgtaattagatagccaccggcgctttaatgcccggaCATATGAAT 
                 Round 1: binds 
                 SEQ ID 
               
               
                   
                 ATCCTCCTTAG 
                 on pKD3 
                 NO: 195 
               
               
                   
               
               
                 SR33 
                 caacacgtttcctgaggaaccatgaaacagtatttagaactgatgcaaaaag 
                 Round 2: binds to 
                 SEQ ID 
               
               
                   
                   
                 round 1 PCR 
                 NO: 196 
               
               
                   
                   
                 product 
                   
               
               
                   
               
               
                 SR34 
                 cgcacactggcgtcggctctggcaggatgtttcgtaattagatagc 
                 Round 2: binds to 
                 SEQ ID 
               
               
                   
                   
                 round 1 PCR 
                 NO: 197 
               
               
                   
                   
                 product 
                   
               
               
                   
               
               
                 SR43 
                 atatcgtcgcagcccacagcaacacgtttcctgagg 
                 Round 3: binds to 
                 SEQ ID 
               
               
                   
                   
                 round 2 PCR 
                 NO: 198 
               
               
                   
                   
                 product 
                   
               
               
                   
               
               
                 SR44 
                 aagaatttaacggagggcaaaaaaaaccgacgcacactggcgtcggc 
                 Round 3: binds to 
                 SEQ ID 
               
               
                   
                   
                 round 2 PCR 
                 NO: 199 
               
               
                   
                   
                 product 
               
               
                   
               
            
           
         
       
     
     For the first PCR round, 4×50 ul PCR reactions containing Ing 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 ing 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 6. Production of Propionate in Genetically Engineered  E. coli    
     Production of propionate is assessed in  E. coli  Nissle strains containing the propionate cassettes described above. All incubations are performed at 37° C. Cultures of  E. coli  strains DH5a and Nissle transformed with the propionate 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 the inducible constructs are induced as follows: (1) bacteria comprising a propionate gene cassette driven by a FNR-inducible promoter are induced in LB at 37 C for up to 4 hours in anaerobic conditions in a Coy anaerobic chamber (supplying 90% N2, 5% CO2, 5% H2, and 20 mM nitrate) at 37° C.; (2) bacteria comprising a propionate gene cassette driven by a tetracycline-inducible promoter are induced with anhydrotetracycline (100 ng/mL); (3) bacteria comprising a propionate gene cassette driven by a arabinose-inducible promoter are induced with 1% arabinose in media lacking glucose. One mL culture aliquots are prepared in 1.5 mL capped tubes and FNR-inducible constructs are 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 propionate concentration by LC-MS to confirm that propionate production in these recombinant strains can be achieved in a low-oxygen environment. 
     Example 7. Efficacy of Propionate-Expressing Bacteria in a Mouse Model of Obesity and Type 2 Diabetes Mellitus (T2DM) 
     For in vivo studies to assess the efficacy of the genetically engineered bacteria in an animal model of obesity and type 2 diabetes, C57BL/6J mice are fed a high fat diet (60 kcal % fat, Research Diets Inc.) starting from 4-5 weeks of age for 8 weeks or until body weight is at least 45 g in order to induce obesity and T2DM-related symptoms such as hyperinsulinemia and hyperglycemia, e.g., glycaemia above 160 mg/dL and plasma insulin above 4000 pg/mL. Alternatively, B6.BKS(D)-Lep db/db  mice (Lepr db/db ) are obtained from The Jackson Laboratory; these mice typically become obese and display T2DM-related symptoms beginning at 10 weeks of age. 
     Bacteria harboring the propionate gene cassette 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. To analyze the efficacy of the bacteria in vivo, bacteria are resuspended in phosphate buffered saline (PBS) and 100 microliters is administered by oral gavage to mice daily for 8 weeks. Alternatively, the bacteria can be supplemented in the drinking water (5×10 9  CFU bacteria/mL). 
     Body weight and food intake are measured weekly before, during, and after the administration of the bacteria. In addition, mice are subjected to insulin tolerance tests (ITT), glucose tolerance tests (GTT) and pyruvate tolerance tests (PTT) to determine the severity of T2DM during treatment, e.g., amelioration of insulin resistance. For ITT, mice are fasted overnight and injected with insulin (1 U/kg, diluted in PBS). Blood glucose levels are measured prior to the injection and at 20, 40, 60, and 90 min. post injection via tail bleeding. For GTT, mice are fasted overnight and injected with glucose solution (1 g/kg, dissolved in PBS); blood glucose levels are measured as described above in order to determine changes. For PTT, mice are fasted overnight and injected with sodium pyruvate solution (1 g/kg, dissolved in PBS); blood glucose levels are measured as described above. Whole-body metabolic functions are analyzed by placing the mice in a Comprehensive Lab Animal Monitoring System (CLAMS), which monitors physical activity, food intake, metabolic rate (as a function of O 2  consumption and CO 2  production). Mice are sacrificed and metabolic tissues such as liver, skeletal muscle, epididymal fat, subcutaneous fat, brown fat, pancreas, and brain, are collected for analysis of histology, e.g., Oil Red O staining of the liver, and gene expression. 
     Example 8. 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 31. 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 31 
               
             
            
               
                   
               
               
                 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 14). 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 32. 
       FIG. 65  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 32 
               
               
                   
               
               
                 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 9. Intestinal Residence and Survival of Bacterial Strains In Vivo 
     Localization and intestinal residence time of streptomycin resistant Nissle,  FIG. 66 ) 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 109 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. 66 . 
     Example 33. Construction of Vectors for Overproducing Butyrate 
     In addition to the ammonia conversion circuit, GABA transport circuit, GABA metabolic circuit, and/or manganese transport circuit described above, the  E. coli  Nissle bacteria further comprise one or more circuits for producing a gut barrier enhancer molecule. 
     To facilitate inducible production of butyrate in  E. 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. The butyrate gene cassette is placed under the control of a FNR regulatory region selected from (SEQ ID NOs: 177-188) (Table 21) In certain constructs, the FNR-responsive promoter is further fused to a strong ribosome binding site sequence. For efficient translation ofbutyrate 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 constructs, 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., Tables 33 and 34). In certain constructs, the butyrate gene cassette 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., Tables 14-17). In certain constructs, the butyrate gene cassette is placed under the control of a tetracycline-inducible or constitutive promoter. 
     
       
         
           
               
             
               
                 TABLE 33 
               
             
            
               
                   
               
               
                 pLogic031-nsrR-norB-butyrate construct (SEQ ID NO: 200) 
               
            
           
           
               
               
            
               
                   
                 Nucleotide sequences of pLogic031-nsrR-norB- 
               
               
                 Description 
                 butyrate construct (SEQ ID NO: 200) 
               
               
                   
               
               
                 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- 
                 atttctagagcaacatacgagccggaagcataaagtgtaaagc 
               
               
                   
               
               
                 norB-butyrate construct; 
                 ctggggtgcctaatgagttgagttgaggaattataacaggaag 
               
               
                   
               
               
                 SEQ ID NO: 79). 
                 aaatattcctcatacgcttgtaattcctctatggttgttgaca 
               
               
                   
               
               
                 The 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 sequence 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 encoding NsrR is 
                 gatatacatatggatttaaattctaaaaaatatcagatgctta 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 aagagctatatgtaagcttcgctgaaaatgaagttaaaccttt 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 agcaacagaacttgatgaagaagaaagatttccttatgaaaca 
               
               
                   
               
               
                 NsrR binding site 
                 gtggaaaaaatggcaaaagcaggaatgatgggtataccatatc 
               
               
                   
               
               
                 i.e., a regulatory 
                 caaaagaatatggtggagaaggtggagacactgtaggatatat 
               
               
                   
               
               
                 region of norB is 
                 aatggcagttgaagaattgtctagagtttgtggtactacagga 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 gttatattatcagctcatacatctcttggctcatggcctatat 
               
               
                   
               
               
                   
                 atcaatatggtaatgaagaacaaaaacaaaaattcttaagacc 
               
               
                   
               
               
                   
                 actagcaagtggagaaaaattaggagcatttggtcttactgag 
               
               
                   
               
               
                   
                 cctaatgctggtacagatgcgtctggccaacaaacaactgctg 
               
               
                   
               
               
                   
                 ttttagacggggatgaatacatacttaatggctcaaaaatatt 
               
               
                   
               
               
                   
                 tataacaaacgcaatagctggtgacatatatgtagtaatggca 
               
               
                   
               
               
                   
                 atgactgataaatctaaggggaacaaaggaatatcagcattta 
               
               
                   
               
               
                   
                 tagttgaaaaaggaactcctgggtttagctttggagttaaaga 
               
               
                   
               
               
                   
                 aaagaaaatgggtataagaggttcagctacgagtgaattaata 
               
               
                   
               
               
                   
                 tttgaggattgcagaatacctaaagaaaatttacttggaaaag 
               
               
                   
               
               
                   
                 aaggtcaaggatttaagatagcaatgtctactcttgatggtgg 
               
               
                   
               
               
                   
                 tagaattggtatagctgcacaagctttaggtttagcacaaggt 
               
               
                   
               
               
                   
                 gctcttgatgaaactgttaaatatgtaaaagaaagagtacaat 
               
               
                   
               
               
                   
                 ttggtagaccattatcaaaattccaaaatacacaattccaatt 
               
               
                   
               
               
                   
                 agctgatatggaagttaaggtacaagcggctagacaccttgta 
               
               
                   
               
               
                   
                 tatcaagcagctataaataaagacttaggaaaaccttatggag 
               
               
                   
               
               
                   
                 tagaagcagcaatggcaaaattatttgcagctgaaacagctat 
               
               
                   
               
               
                   
                 ggaagttactacaaaagctgtacaacttcatggaggatatgga 
               
               
                   
               
               
                   
                 tacactcgtgactatccagtagaaagaatgatgagagatgcta 
               
               
                   
               
               
                   
                 agataactgaaatatatgaaggaactagtgaagttcaaagaat 
               
               
                   
               
               
                   
                 ggttatttcaggaaaactattaaaatagtaagaaggagatata 
               
               
                   
               
               
                   
                 catatggaggaaggatttatgaatatagtcgtttgtataaaac 
               
               
                   
               
               
                   
                 aagttccagatacaacagaagttaaactagatcctaatacagg 
               
               
                   
               
               
                   
                 tactttaattagagatggagtaccaagtataataaaccctgat 
               
               
                   
               
               
                   
                 gataaagcaggtttagaagaagctataaaattaaaagaagaaa 
               
               
                   
               
               
                   
                 tgggtgctcatgtaactgttataacaatgggacctcctcaagc 
               
               
                   
               
               
                   
                 agatatggctttaaaagaagctttagcaatgggtgcagataga 
               
               
                   
               
               
                   
                 ggtatattattaacagatagagcatttgcgggtgctgatactt 
               
               
                   
               
               
                   
                 gggcaacttcatcagcattagcaggagcattaaaaaatataga 
               
               
                   
               
               
                   
                 ttttgatattataatagctggaagacaggcgatagatggagat 
               
               
                   
               
               
                   
                 actgcacaagttggacctcaaatagctgaacatttaaatcttc 
               
               
                   
               
               
                   
                 catcaataacatatgctgaagaaataaaaactgaaggtgaata 
               
               
                   
               
               
                   
                 tgtattagtaaaaagacaatttgaagattgttgccatgactta 
               
               
                   
               
               
                   
                 aaagttaaaatgccatgccttataacaactcttaaagatatga 
               
               
                   
               
               
                   
                 acacaccaagatacatgaaagttggaagaatatatgatgcttt 
               
               
                   
               
               
                   
                 cgaaaatgatgtagtagaaacatggactgtaaaagatatagaa 
               
               
                   
               
               
                   
                 gttgacccttctaatttaggtcttaaaggttctccaactagtg 
               
               
                   
               
               
                   
                 tatttaaatcatttacaaaatcagttaaaccagctggtacaat 
               
               
                   
               
               
                   
                 atacaatgaagatgcgaaaacatcagctggaattatcatagat 
               
               
                   
               
               
                   
                 aaattaaaagagaagtatatcatataataagaaggagatatac 
               
               
                   
               
               
                   
                 atatgggtaacgttttagtagtaatagaacaaagagaaaatgt 
               
               
                   
               
               
                   
                 aattcaaactgtttctttagaattactaggaaaggctacagaa 
               
               
                   
               
               
                   
                 atagcaaaagattatgatacaaaagtttctgcattacttttag 
               
               
                   
               
               
                   
                 gtagtaaggtagaaggtttaatagatacattagcacactatgg 
               
               
                   
               
               
                   
                 tgcagatgaggtaatagtagtagatgatgaagctttagcagtg 
               
               
                   
               
               
                   
                 tatacaactgaaccatatacaaaagcagcttatgaagcaataa 
               
               
                   
               
               
                   
                 aagcagctgaccctatagttgtattatttggtgcaacttcaat 
               
               
                   
               
               
                   
                 aggtagagatttagcgcctagagtttctgctagaatacataca 
               
               
                   
               
               
                   
                 ggtcttactgctgactgtacaggtcttgcagtagctgaagata 
               
               
                   
               
               
                   
                 caaaattattattaatgacaagacctgcctttggtggaaatat 
               
               
                   
               
               
                   
                 aatggcaacaatagtttgtaaagatttcagacctcaaatgtct 
               
               
                   
               
               
                   
                 acagttagaccaggggttatgaagaaaaatgaacctgatgaaa 
               
               
                   
               
               
                   
                 ctaaagaagctgtaattaaccgtttcaaggtagaatttaatga 
               
               
                   
               
               
                   
                 tgctgataaattagttcaagttgtacaagtaataaaagaagct 
               
               
                   
               
               
                   
                 aaaaaacaagttaaaatagaagatgctaagatattagtttctg 
               
               
                   
               
               
                   
                 ctggacgtggaatgggtggaaaagaaaacttagacatacttta 
               
               
                   
               
               
                   
                 tgaattagctgaaattataggtggagaagtttctggttctcgt 
               
               
                   
               
               
                   
                 gccactatagatgcaggttggttagataaagcaagacaagttg 
               
               
                   
               
               
                   
                 gtcaaactggtaaaactgtaagaccagacctttatatagcatg 
               
               
                   
               
               
                   
                 tggtatatctggagcaatacaacatatagctggtatggaagat 
               
               
                   
               
               
                   
                 gctgagtttatagttgctataaataaaaatccagaagctccaa 
               
               
                   
               
               
                   
                 tatttaaatatgctgatgttggtatagttggagatgttcataa 
               
               
                   
               
               
                   
                 agtgcttccagaacttatcagtcagttaagtgttgcaaaagaa 
               
               
                   
               
               
                   
                 aaaggtgaagttttagctaactaataagaaggagatatacata 
               
               
                   
               
               
                   
                 tgagagaagtagtaattgccagtgcagctagaacagcagtagg 
               
               
                   
               
               
                   
                 aagttttggaggagcatttaaatcagtttcagcggtagagtta 
               
               
                   
               
               
                   
                 ggggtaacagcagctaaagaagctataaaaagagctaacataa 
               
               
                   
               
               
                   
                 ctccagatatgatagatgaatctcttttagggggagtacttac 
               
               
                   
               
               
                   
                 agcaggtcttggacaaaatatagcaagacaaatagcattagga 
               
               
                   
               
               
                   
                 gcaggaataccagtagaaaaaccagctatgactataaatatag 
               
               
                   
               
               
                   
                 tttgtggttctggattaagatctgtttcaatggcatctcaact 
               
               
                   
               
               
                   
                 tatagcattaggtgatgctgatataatgttagttggtggagct 
               
               
                   
               
               
                   
                 gaaaacatgagtatgtctccttatttagtaccaagtgcgagat 
               
               
                   
               
               
                   
                 atggtgcaagaatgggtgatgctgcttttgttgattcaatgat 
               
               
                   
               
               
                   
                 aaaagatggattatcagacatatttaataactatcacatgggt 
               
               
                   
               
               
                   
                 attactgctgaaaacatagcagagcaatggaatataactagag 
               
               
                   
               
               
                   
                 aagaacaagatgaattagctcttgcaagtcaaaataaagctga 
               
               
                   
               
               
                   
                 aaaagctcaagctgaaggaaaatttgatgaagaaatagttcct 
               
               
                   
               
               
                   
                 gttgttataaaaggaagaaaaggtgacactgtagtagataaag 
               
               
                   
               
               
                   
                 atgaatatattaagcctggcactacaatggagaaacttgctaa 
               
               
                   
               
               
                   
                 gttaagacctgcatttaaaaaagatggaacagttactgctggt 
               
               
                   
               
               
                   
                 aatgcatcaggaataaatgatggtgctgctatgttagtagtaa 
               
               
                   
               
               
                   
                 tggctaaagaaaaagctgaagaactaggaatagagcctcttgc 
               
               
                   
               
               
                   
                 aactatagtttcttatggaacagctggtgttgaccctaaaata 
               
               
                   
               
               
                   
                 atgggatatggaccagttccagcaactaaaaaagctttagaag 
               
               
                   
               
               
                   
                 ctgctaatatgactattgaagatatagatttagttgaagctaa 
               
               
                   
               
               
                   
                 tgaggcatttgctgcccaatctgtagctgtaataagagactta 
               
               
                   
               
               
                   
                 aatatagatatgaataaagttaatgttaatggtggagcaatag 
               
               
                   
               
               
                   
                 ctataggacatccaataggatgctcaggagcaagaatacttac 
               
               
                   
               
               
                   
                 tacacttttatatgaaatgaagagaagagatgctaaaactggt 
               
               
                   
               
               
                   
                 cttgctacactttgtataggcggtggaatgggaactactttaa 
               
               
                   
               
               
                   
                 tagttaagagatagtaagaaggagatatacatatgaaattagc 
               
               
                   
               
               
                   
                 tgtaataggtagtggaactatgggaagtggtattgtacaaact 
               
               
                   
               
               
                   
                 tttgcaagttgtggacatgatgtatgtttaaagagtagaactc 
               
               
                   
               
               
                   
                 aaggtgctatagataaatgtttagctttattagataaaaattt 
               
               
                   
               
               
                   
                 aactaagttagttactaagggaaaaatggatgaagctacaaaa 
               
               
                   
               
               
                   
                 gcagaaatattaagtcatgttagttcaactactaattatgaag 
               
               
                   
               
               
                   
                 atttaaaagatatggatttaataatagaagcatctgtagaaga 
               
               
                   
               
               
                   
                 catgaatataaagaaagatgttttcaagttactagatgaatta 
               
               
                   
               
               
                   
                 tgtaaagaagatactatcttggcaacaaatacttcatcattat 
               
               
                   
               
               
                   
                 ctataacagaaatagcttcttctactaagcgcccagataaagt 
               
               
                   
               
               
                   
                 tataggaatgcatttctttaatccagttcctatgatgaaatta 
               
               
                   
               
               
                   
                 gttgaagttataagtggtcagttaacatcaaaagttacttttg 
               
               
                   
               
               
                   
                 atacagtatttgaattatctaagagtatcaataaagtaccagt 
               
               
                   
               
               
                   
                 agatgtatctgaatctcctggatttgtagtaaatagaatactt 
               
               
                   
               
               
                   
                 atacctatgataaatgaagctgttggtatatatgcagatggtg 
               
               
                   
               
               
                   
                 ttgcaagtaaagaagaaatagatgaagctatgaaattaggagc 
               
               
                   
               
               
                   
                 aaaccatccaatgggaccactagcattaggtgatttaatcgga 
               
               
                   
               
               
                   
                 ttagatgttgttttagctataatgaacgttttatatactgaat 
               
               
                   
               
               
                   
                 ttggagatactaaatatagacctcatccacttttagctaaaat 
               
               
                   
               
               
                   
                 ggttagagctaatcaattaggaagaaaaactaagataggattc 
               
               
                   
               
               
                   
                 tatgattataataaataataagaaggagatatacatatgagta 
               
               
                   
               
               
                   
                 caagtgatgttaaagtttatgagaatgtagctgttgaagtaga 
               
               
                   
               
               
                   
                 tggaaatatatgtacagtgaaaatgaatagacctaaagccctt 
               
               
                   
               
               
                   
                 aatgcaataaattcaaagactttagaagaactttatgaagtat 
               
               
                   
               
               
                   
                 ttgtagatattaataatgatgaaactattgatgttgtaatatt 
               
               
                   
               
               
                   
                 gacaggggaaggaaaggcatttgtagctggagcagatattgca 
               
               
                   
               
               
                   
                 tacatgaaagatttagatgctgtagctgctaaagattttagta 
               
               
                   
               
               
                   
                 tcttaggagcaaaagcttttggagaaatagaaaatagtaaaaa 
               
               
                   
               
               
                   
                 agtagtgatagctgctgtaaacggatttgctttaggtggagga 
               
               
                   
               
               
                   
                 tgtgaacttgcaatggcatgtgatataagaattgcatctgcta 
               
               
                   
               
               
                   
                 aagctaaatttggtcagccagaagtaactcttggaataactcc 
               
               
                   
               
               
                   
                 aggatatggaggaactcaaaggcttacaagattggttggaatg 
               
               
                   
               
               
                   
                 gcaaaagcaaaagaattaatctttacaggtcaagttataaaag 
               
               
                   
               
               
                   
                 ctgatgaagctgaaaaaatagggctagtaaatagagtcgttga 
               
               
                   
               
               
                   
                 gccagacattttaatagaagaagttgagaaattagctaagata 
               
               
                   
               
               
                   
                 atagctaaaaatgctcagcttgcagttagatactctaaagaag 
               
               
                   
               
               
                   
                 caatacaacttggtgctcaaactgatataaatactggaataga 
               
               
                   
               
               
                   
                 tatagaatctaatttatttggtctttgtttttcaactaaagac 
               
               
                   
               
               
                   
                 caaaaagaaggaatgtcagctttcgttgaaaagagagaagcta 
               
               
                   
               
               
                   
                 actttataaaagggtaataagaaggagatatacatatgagaag 
               
               
                   
               
               
                   
                 ttttgaagaagtaattaagtttgcaaaagaaagaggacctaaa 
               
               
                   
               
               
                   
                 actatatcagtagcatgttgccaagataaagaagttttaatgg 
               
               
                   
               
               
                   
                 cagttgaaatggctagaaaagaaaaaatagcaaatgccatttt 
               
               
                   
               
               
                   
                 agtaggagatatagaaaagactaaagaaattgcaaaaagcata 
               
               
                   
               
               
                   
                 gacatggatatcgaaaattatgaactgatagatataaaagatt 
               
               
                   
               
               
                   
                 tagcagaagcatctctaaaatctgttgaattagtttcacaagg 
               
               
                   
               
               
                   
                 aaaagccgacatggtaatgaaaggcttagtagacacatcaata 
               
               
                   
               
               
                   
                 atactaaaagcagttttaaataaagaagtaggtcttagaactg 
               
               
                   
               
               
                   
                 gaaatgtattaagtcacgtagcagtatttgatgtagagggata 
               
               
                   
               
               
                   
                 tgatagattatttttcgtaactgacgcagctatgaacttagct 
               
               
                   
               
               
                   
                 cctgatacaaatactaaaaagcaaatcatagaaaatgcttgca 
               
               
                   
               
               
                   
                 cagtagcacattcattagatataagtgaaccaaaagttgctgc 
               
               
                   
               
               
                   
                 aatatgcgcaaaagaaaaagtaaatccaaaaatgaaagataca 
               
               
                   
               
               
                   
                 gttgaagctaaagaactagaagaaatgtatgaaagaggagaaa 
               
               
                   
               
               
                   
                 tcaaaggttgtatggttggtgggccttttgcaattgataatgc 
               
               
                   
               
               
                   
                 agtatctttagaagcagctaaacataaaggtataaatcatcct 
               
               
                   
               
               
                   
                 gtagcaggacgagctgatatattattagccccagatattgaag 
               
               
                   
               
               
                   
                 gtggtaacatattatataaagctttggtattcttctcaaaatc 
               
               
                   
               
               
                   
                 aaaaaatgcaggagttatagttggggctaaagcaccaataata 
               
               
                   
               
               
                   
                 ttaacttctagagcagacagtgaagaaactaaactaaactcaa 
               
               
                   
               
               
                   
                 tagctttaggtgttttaatggcagcaaaggcataataagaagg 
               
               
                   
               
               
                   
                 agatatacatatgagcaaaatatttaaaatcttaacaataaat 
               
               
                   
               
               
                   
                 cctggttcgacatcaactaaaatagctgtatttgataatgagg 
               
               
                   
               
               
                   
                 atttagtatttgaaaaaactttaagacattcttcagaagaaat 
               
               
                   
               
               
                   
                 aggaaaatatgagaaggtgtctgaccaatttgaatttcgtaaa 
               
               
                   
               
               
                   
                 caagtaatagaagaagctctaaaagaaggtggagtaaaaacat 
               
               
                   
               
               
                   
                 ctgaattagatgctgtagtaggtagaggaggacttcttaaacc 
               
               
                   
               
               
                   
                 tataaaaggtggtacttattcagtaagtgctgctatgattgaa 
               
               
                   
               
               
                   
                 gatttaaaagtgggagttttaggagaacacgcttcaaacctag 
               
               
                   
               
               
                   
                 gtggaataatagcaaaacaaataggtgaagaagtaaatgttcc 
               
               
                   
               
               
                   
                 ttcatacatagtagaccctgttgttgtagatgaattagaagat 
               
               
                   
               
               
                   
                 gttgctagaatttctggtatgcctgaaataagtagagcaagtg 
               
               
                   
               
               
                   
                 tagtacatgctttaaatcaaaaggcaatagcaagaagatatgc 
               
               
                   
               
               
                   
                 tagagaaataaacaagaaatatgaagatataaatcttatagtt 
               
               
                   
               
               
                   
                 gcacacatgggtggaggagtttctgttggagctcataaaaatg 
               
               
                   
               
               
                   
                 gtaaaatagtagatgttgcaaacgcattagatggagaaggacc 
               
               
                   
               
               
                   
                 tttctctccagaaagaagtggtggactaccagtaggtgcatta 
               
               
                   
               
               
                   
                 gtaaaaatgtgctttagtggaaaatatactcaagatgaaatta 
               
               
                   
               
               
                   
                 aaaagaaaataaaaggtaatggcggactagttgcatacttaaa 
               
               
                   
               
               
                   
                 cactaatgatgctagagaagttgaagaaagaattgaagctggt 
               
               
                   
               
               
                   
                 gatgaaaaagctaaattagtatatgaagctatggcatatcaaa 
               
               
                   
               
               
                   
                 tctctaaagaaataggagctagtgctgcagttcttaagggaga 
               
               
                   
               
               
                   
                 tgtaaaagcaatattattaactggtggaatcgcatattcaaaa 
               
               
                   
               
               
                   
                 atgtttacagaaatgattgcagatagagttaaatttatagcag 
               
               
                   
               
               
                   
                 atgtaaaagtttatccaggtgaagatgaaatgattgcattagc 
               
               
                   
               
               
                   
                 tcaaggtggacttagagttttaactggtgaagaagaggctcaa 
               
               
                   
               
               
                   
                 gtttatgataactaataa 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 34 
               
             
            
               
                   
               
               
                 Nucleotide sequences of pLogic046-nsrR-norB-butyrate construct 
               
            
           
           
               
               
            
               
                   
                 Nucleotide sequences of pLogic046-nsrR-norB-butyrate  
               
               
                 Description 
                 construct(SEQ ID NO: 201) 
               
               
                   
               
               
                 Nucleic acid 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 sequence of an 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 exemplary RNS- 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 regulated 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 construct 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 comprising a 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 gene encoding 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 nsrR, a 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 regulatory 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 region of norB 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 and a 
                 acctccttagtacatgcaaaattatttctagagcaacatacgagc 
               
               
                   
               
               
                 butyrogenic 
                 cggaagcataaagtgtaaagcctggggtgcctaatgagttgagtt 
               
               
                   
               
               
                 gene cassette 
                 gaggaattataacaggaagaaatattcctcatacgcttgtaattc 
               
               
                   
               
               
                 (pLogic046- 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 nsrR-norB- 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 butyrate 
                 actttaagaaggagatatacatatgatcgtaaaacctatggtacg 
               
               
                   
               
               
                 construct;  
                 caacaatatctgcctgaacgcccatcctcagggctgcaagaaggg 
               
               
                   
               
               
                 SEQ ID NO: 80). 
                 agtggaagatcagattgaatataccaagaaacgcattaccgcaga 
               
               
                   
               
               
                   
                 agtcaaagctggcgcaaaagctccaaaaaacgttctggtgcttgg 
               
               
                   
               
               
                   
                 ctgctcaaatggttacggcctggcgagccgcattactgctgcgtt 
               
               
                   
               
               
                   
                 cggatacggggctgcgaccatcggcgtgtcctttgaaaaagcggg 
               
               
                   
               
               
                   
                 ttcagaaaccaaatatggtacaccgggatggtacaataatttggc 
               
               
                   
               
               
                   
                 atttgatgaagcggcaaaacgcgagggtctttatagcgtgacgat 
               
               
                   
               
               
                   
                 cgacggcgatgcgttttcagacgagatcaaggcccaggtaattga 
               
               
                   
               
               
                   
                 ggaagccaaaaaaaaaggtatcaaatttgatctgatcgtatacag 
               
               
                   
               
               
                   
                 cttggccagcccagtacgtactgatcctgatacaggtatcatgca 
               
               
                   
               
               
                   
                 caaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaac 
               
               
                   
               
               
                   
                 agtagatccgtttactggcgagctgaaggaaatctccgcggaacc 
               
               
                   
               
               
                   
                 agcaaatgacgaggaagcagccgccactgttaaagttatgggggg 
               
               
                   
               
               
                   
                 tgaagattgggaacgttggattaagcagctgtcgaaggaaggcct 
               
               
                   
               
               
                   
                 cttagaagaaggctgtattaccttggcctatagttatattggccc 
               
               
                   
               
               
                   
                 tgaagctacccaagctttgtaccgtaaaggcacaatcggcaaggc 
               
               
                   
               
               
                   
                 caaagaacacctggaggccacagcacaccgtctcaacaaagagaa 
               
               
                   
               
               
                   
                 cccgtcaatccgtgccttcgtgagcgtgaataaaggcctggtaac 
               
               
                   
               
               
                   
                 ccgcgcaagcgccgtaatcccggtaatccctctgtatctcgccag 
               
               
                   
               
               
                   
                 cttgttcaaagtaatgaaagagaagggcaatcatgaaggttgtat 
               
               
                   
               
               
                   
                 tgaacagatcacgcgtctgtacgccgagcgcctgtaccgtaaaga 
               
               
                   
               
               
                   
                 tggtacaattccagttgatgaggaaaatcgcattcgcattgatga 
               
               
                   
               
               
                   
                 ttgggagttagaagaagacgtccagaaagcggtatccgcgttgat 
               
               
                   
               
               
                   
                 ggagaaagtcacgggtgaaaacgcagaatctctcactgacttagc 
               
               
                   
               
               
                   
                 ggggtaccgccatgatttcttagctagtaacggctttgatgtaga 
               
               
                   
               
               
                   
                 aggtattaattatgaagcggaagttgaacgcttcgaccgtatctg 
               
               
                   
               
               
                   
                 ataagaaggagatatacatatgagagaagtagtaattgccagtgc 
               
               
                   
               
               
                   
                 agctagaacagcagtaggaagttttggaggagcatttaaatcagt 
               
               
                   
               
               
                   
                 ttcagcggtagagttaggggtaacagcagctaaagaagctataaa 
               
               
                   
               
               
                   
                 aagagctaacataactccagatatgatagatgaatctcttttagg 
               
               
                   
               
               
                   
                 gggagtacttacagcaggtcttggacaaaatatagcaagacaaat 
               
               
                   
               
               
                   
                 agcattaggagcaggaataccagtagaaaaaccagctatgactat 
               
               
                   
               
               
                   
                 aaatatagtttgtggttctggattaagatctgtttcaatggcatc 
               
               
                   
               
               
                   
                 tcaacttatagcattaggtgatgctgatataatgttagttggtgg 
               
               
                   
               
               
                   
                 agctgaaaacatgagtatgtctccttatttagtaccaagtgcgag 
               
               
                   
               
               
                   
                 atatggtgcaagaatgggtgatgctgcttttgttgattcaatgat 
               
               
                   
               
               
                   
                 aaaagatggattatcagacatatttaataactatcacatgggtat 
               
               
                   
               
               
                   
                 tactgctgaaaacatagcagagcaatggaatataactagagaaga 
               
               
                   
               
               
                   
                 acaagatgaattagctcttgcaagtcaaaataaagctgaaaaagc 
               
               
                   
               
               
                   
                 tcaagctgaaggaaaatttgatgaagaaatagttcctgttgttat 
               
               
                   
               
               
                   
                 aaaaggaagaaaaggtgacactgtagtagataaagatgaatatat 
               
               
                   
               
               
                   
                 taagcctggcactacaatggagaaacttgctaagttaagacctgc 
               
               
                   
               
               
                   
                 atttaaaaaagatggaacagttactgctggtaatgcatcaggaat 
               
               
                   
               
               
                   
                 aaatgatggtgctgctatgttagtagtaatggctaaagaaaaagc 
               
               
                   
               
               
                   
                 tgaagaactaggaatagagcctcttgcaactatagtttcttatgg 
               
               
                   
               
               
                   
                 aacagctggtgttgaccctaaaataatgggatatggaccagttcc 
               
               
                   
               
               
                   
                 agcaactaaaaaagctttagaagctgctaatatgactattgaaga 
               
               
                   
               
               
                   
                 tatagatttagttgaagctaatgaggcatttgctgcccaatctgt 
               
               
                   
               
               
                   
                 agctgtaataagagacttaaatatagatatgaataaagttaatgt 
               
               
                   
               
               
                   
                 taatggtggagcaatagctataggacatccaataggatgctcagg 
               
               
                   
               
               
                   
                 agcaagaatacttactacacttttatatgaaatgaagagaagaga 
               
               
                   
               
               
                   
                 tgctaaaactggtcttgctacactttgtataggcggtggaatggg 
               
               
                   
               
               
                   
                 aactactttaatagttaagagatagtaagaaggagatatacatat 
               
               
                   
               
               
                   
                 gaaattagctgtaataggtagtggaactatgggaagtggtattgt 
               
               
                   
               
               
                   
                 acaaacttttgcaagttgtggacatgatgtatgtttaaagagtag 
               
               
                   
               
               
                   
                 aactcaaggtgctatagataaatgtttagctttattagataaaaa 
               
               
                   
               
               
                   
                 tttaactaagttagttactaagggaaaaatggatgaagctacaaa 
               
               
                   
               
               
                   
                 agcagaaatattaagtcatgttagttcaactactaattatgaaga 
               
               
                   
               
               
                   
                 tttaaaagatatggatttaataatagaagcatctgtagaagacat 
               
               
                   
               
               
                   
                 gaatataaagaaagatgttttcaagttactagatgaattatgtaa 
               
               
                   
               
               
                   
                 agaagatactatcttggcaacaaatacttcatcattatctataac 
               
               
                   
               
               
                   
                 agaaatagcttcttctactaagcgcccagataaagttataggaat 
               
               
                   
               
               
                   
                 gcatttctttaatccagttcctatgatgaaattagttgaagttat 
               
               
                   
               
               
                   
                 aagtggtcagttaacatcaaaagttacttttgatacagtatttga 
               
               
                   
               
               
                   
                 attatctaagagtatcaataaagtaccagtagatgtatctgaatc 
               
               
                   
               
               
                   
                 tcctggatttgtagtaaatagaatacttatacctatgataaatga 
               
               
                   
               
               
                   
                 agctgttggtatatatgcagatggtgttgcaagtaaagaagaaat 
               
               
                   
               
               
                   
                 agatgaagctatgaaattaggagcaaaccatccaatgggaccact 
               
               
                   
               
               
                   
                 agcattaggtgatttaatcggattagatgttgttttagctataat 
               
               
                   
               
               
                   
                 gaacgttttatatactgaatttggagatactaaatatagacctca 
               
               
                   
               
               
                   
                 tccacttttagctaaaatggttagagctaatcaattaggaagaaa 
               
               
                   
               
               
                   
                 aactaagataggattctatgattataataaataataagaaggaga 
               
               
                   
               
               
                   
                 tatacatatgagtacaagtgatgttaaagtttatgagaatgtagc 
               
               
                   
               
               
                   
                 tgttgaagtagatggaaatatatgtacagtgaaaatgaatagacc 
               
               
                   
               
               
                   
                 taaagcccttaatgcaataaattcaaagactttagaagaacttta 
               
               
                   
               
               
                   
                 tgaagtatttgtagatattaataatgatgaaactattgatgttgt 
               
               
                   
               
               
                   
                 aatattgacaggggaaggaaaggcatttgtagctggagcagatat 
               
               
                   
               
               
                   
                 tgcatacatgaaagatttagatgctgtagctgctaaagattttag 
               
               
                   
               
               
                   
                 tatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaa 
               
               
                   
               
               
                   
                 agtagtgatagctgctgtaaacggatttgctttaggtggaggatg 
               
               
                   
               
               
                   
                 tgaacttgcaatggcatgtgatataagaattgcatctgctaaagc 
               
               
                   
               
               
                   
                 taaatttggtcagccagaagtaactcttggaataactccaggata 
               
               
                   
               
               
                   
                 tggaggaactcaaaggcttacaagattggttggaatggcaaaagc 
               
               
                   
               
               
                   
                 aaaagaattaatctttacaggtcaagttataaaagctgatgaagc 
               
               
                   
               
               
                   
                 tgaaaaaatagggctagtaaatagagtcgttgagccagacatttt 
               
               
                   
               
               
                   
                 aatagaagaagttgagaaattagctaagataatagctaaaaatgc 
               
               
                   
               
               
                   
                 tcagcttgcagttagatactctaaagaagcaatacaacttggtgc 
               
               
                   
               
               
                   
                 tcaaactgatataaatactggaatagatatagaatctaatttatt 
               
               
                   
               
               
                   
                 tggtctttgtttttcaactaaagaccaaaaagaaggaatgtcagc 
               
               
                   
               
               
                   
                 tttcgttgaaaagagagaagctaactttataaaagggtaataaga 
               
               
                   
               
               
                   
                 aggagatatacatatgagaagttttgaagaagtaattaagtttgc 
               
               
                   
               
               
                   
                 aaaagaaagaggacctaaaactatatcagtagcatgttgccaaga 
               
               
                   
               
               
                   
                 taaagaagttttaatggcagttgaaatggctagaaaagaaaaaat 
               
               
                   
               
               
                   
                 agcaaatgccattttagtaggagatatagaaaagactaaagaaat 
               
               
                   
               
               
                   
                 tgcaaaaagcatagacatggatatcgaaaattatgaactgataga 
               
               
                   
               
               
                   
                 tataaaagatttagcagaagcatctctaaaatctgttgaattagt 
               
               
                   
               
               
                   
                 ttcacaaggaaaagccgacatggtaatgaaaggcttagtagacac 
               
               
                   
               
               
                   
                 atcaataatactaaaagcagttttaaataaagaagtaggtcttag 
               
               
                   
               
               
                   
                 aactggaaatgtattaagtcacgtagcagtatttgatgtagaggg 
               
               
                   
               
               
                   
                 atatgatagattatttttcgtaactgacgcagctatgaacttagc 
               
               
                   
               
               
                   
                 tcctgatacaaatactaaaaagcaaatcatagaaaatgcttgcac 
               
               
                   
               
               
                   
                 agtagcacattcattagatataagtgaaccaaaagttgctgcaat 
               
               
                   
               
               
                   
                 atgcgcaaaagaaaaagtaaatccaaaaatgaaagatacagttga 
               
               
                   
               
               
                   
                 agctaaagaactagaagaaatgtatgaaagaggagaaatcaaagg 
               
               
                   
               
               
                   
                 ttgtatggttggtgggccttttgcaattgataatgcagtatcttt 
               
               
                   
               
               
                   
                 agaagcagctaaacataaaggtataaatcatcctgtagcaggacg 
               
               
                   
               
               
                   
                 agctgatatattattagccccagatattgaaggtggtaacatatt 
               
               
                   
               
               
                   
                 atataaagctttggtattcttctcaaaatcaaaaaatgcaggagt 
               
               
                   
               
               
                   
                 tatagttggggctaaagcaccaataatattaacttctagagcaga 
               
               
                   
               
               
                   
                 cagtgaagaaactaaactaaactcaatagctttaggtgttttaat 
               
               
                   
               
               
                   
                 ggcagcaaaggcataataagaaggagatatacatatgagcaaaat 
               
               
                   
               
               
                   
                 atttaaaatcttaacaataaatcctggttcgacatcaactaaaat 
               
               
                   
               
               
                   
                 agctgtatttgataatgaggatttagtatttgaaaaaactttaag 
               
               
                   
               
               
                   
                 acattcttcagaagaaataggaaaatatgagaaggtgtctgacca 
               
               
                   
               
               
                   
                 atttgaatttcgtaaacaagtaatagaagaagctctaaaagaagg 
               
               
                   
               
               
                   
                 tggagtaaaaacatctgaattagatgctgtagtaggtagaggagg 
               
               
                   
               
               
                   
                 acttcttaaacctataaaaggtggtacttattcagtaagtgctgc 
               
               
                   
               
               
                   
                 tatgattgaagatttaaaagtgggagttttaggagaacacgcttc 
               
               
                   
               
               
                   
                 aaacctaggtggaataatagcaaaacaaataggtgaagaagtaaa 
               
               
                   
               
               
                   
                 tgttccttcatacatagtagaccctgttgttgtagatgaattaga 
               
               
                   
               
               
                   
                 agatgttgctagaatttctggtatgcctgaaataagtagagcaag 
               
               
                   
               
               
                   
                 tgtagtacatgctttaaatcaaaaggcaatagcaagaagatatgc 
               
               
                   
               
               
                   
                 tagagaaataaacaagaaatatgaagatataaatcttatagttgc 
               
               
                   
               
               
                   
                 acacatgggtggaggagtttctgttggagctcataaaaatggtaa 
               
               
                   
               
               
                   
                 aatagtagatgttgcaaacgcattagatggagaaggacctttctc 
               
               
                   
               
               
                   
                 tccagaaagaagtggtggactaccagtaggtgcattagtaaaaat 
               
               
                   
               
               
                   
                 gtgctttagtggaaaatatactcaagatgaaattaaaaagaaaat 
               
               
                   
               
               
                   
                 aaaaggtaatggcggactagttgcatacttaaacactaatgatgc 
               
               
                   
               
               
                   
                 tagagaagttgaagaaagaattgaagctggtgatgaaaaagctaa 
               
               
                   
               
               
                   
                 attagtatatgaagctatggcatatcaaatctctaaagaaatagg 
               
               
                   
               
               
                   
                 agctagtgctgcagttcttaagggagatgtaaaagcaatattatt 
               
               
                   
               
               
                   
                 aactggtggaatcgcatattcaaaaatgtttacagaaatgattgc 
               
               
                   
               
               
                   
                 agatagagttaaatttatagcagatgtaaaagtttatccaggtga 
               
               
                   
               
               
                   
                 agatgaaatgattgcattagctcaaggtggacttagagttttaac 
               
               
                   
               
               
                   
                 tggtgaagaagaggctcaagtttatgataactaataa 
               
               
                   
               
            
           
         
       
     
     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), 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. The second butyrate gene cassette is placed under control of a FNR regulatory region as described above. In certain constructs, 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 22). In certain constructs, the butyrate gene cassette 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 23 and Table 35). 
     
       
         
           
               
             
               
                 TABLE 35 
               
             
            
               
                   
               
               
                 ROS regulated constructs, OxyR construct, Tet-regulated constructs 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 Nucleotide 
                 ctcgagttcattatccatcctccatcgccacgatagttcatggcgataggtaga 
               
               
                   
               
               
                 sequences of 
                 atagcaatgaacgattatccctatcaagcattctgactgataattgctcacacg 
               
               
                   
               
               
                 pLogic031- 
                 aattcattaaagaggagaaaggtaccatggatttaaattctaaaaaatatcaga 
               
               
                   
               
               
                 oxyS-butyrate 
                 tgcttaaagagctatatgtaagcttcgctgaaaatgaagttaaacctttagcaa 
               
               
                   
               
               
                 construct 
                 cagaacttgatgaagaagaaagatttccttatgaaacagtggaaaaaatggcaa 
               
               
                   
               
               
                 (SEQ ID NO: 202) 
                 aagcaggaatgatgggtataccatatccaaaagaatatggtggagaaggtggag 
               
               
                   
               
               
                   
                 acactgtaggatatataatggcagttgaagaattgtctagagtttgtggtacta 
               
               
                   
               
               
                   
                 caggagttatattatcagctcatacatctcttggctcatggcctatatatcaat 
               
               
                   
               
               
                   
                 atggtaatgaagaacaaaaacaaaaattcttaagaccactagcaagtggagaaa 
               
               
                   
               
               
                   
                 aattaggagcatttggtcttactgagcctaatgctggtacagatgcgtctggcc 
               
               
                   
               
               
                   
                 aacaaacaactgctgttttagacggggatgaatacatacttaatggctcaaaaa 
               
               
                   
               
               
                   
                 tatttataacaaacgcaatagctggtgacatatatgtagtaatggcaatgactg 
               
               
                   
               
               
                   
                 ataaatctaaggggaacaaaggaatatcagcatttatagttgaaaaaggaactc 
               
               
                   
               
               
                   
                 ctgggtttagctttggagttaaagaaaagaaaatgggtataagaggttcagcta 
               
               
                   
               
               
                   
                 cgagtgaattaatatttgaggattgcagaatacctaaagaaaatttacttggaa 
               
               
                   
               
               
                   
                 aagaaggtcaaggatttaagatagcaatgtctactcttgatggtggtagaattg 
               
               
                   
               
               
                   
                 gtatagctgcacaagctttaggtttagcacaaggtgctcttgatgaaactgtta 
               
               
                   
               
               
                   
                 aatatgtaaaagaaagagtacaatttggtagaccattatcaaaattccaaaata 
               
               
                   
               
               
                   
                 cacaattccaattagctgatatggaagttaaggtacaagcggctagacaccttg 
               
               
                   
               
               
                   
                 tatatcaagcagctataaataaagacttaggaaaaccttatggagtagaagcag 
               
               
                   
               
               
                   
                 caatggcaaaattatttgcagctgaaacagctatggaagttactacaaaagctg 
               
               
                   
               
               
                   
                 tacaacttcatggaggatatggatacactcgtgactatccagtagaaagaatga 
               
               
                   
               
               
                   
                 tgagagatgctaagataactgaaatatatgaaggaactagtgaagttcaaagaa 
               
               
                   
               
               
                   
                 tggttatttcaggaaaactattaaaatagtaagaaggagatatacatatggagg 
               
               
                   
               
               
                   
                 aaggatttatgaatatagtcgtttgtataaaacaagttccagatacaacagaag 
               
               
                   
               
               
                   
                 ttaaactagatcctaatacaggtactttaattagagatggagtaccaagtataa 
               
               
                   
               
               
                   
                 taaaccctgatgataaagcaggtttagaagaagctataaaattaaaagaagaaa 
               
               
                   
               
               
                   
                 tgggtgctcatgtaactgttataacaatgggacctcctcaagcagatatggctt 
               
               
                   
               
               
                   
                 taaaagaagctttagcaatgggtgcagatagaggtatattattaacagatagag 
               
               
                   
               
               
                   
                 catttgcgggtgctgatacttgggcaacttcatcagcattagcaggagcattaa 
               
               
                   
               
               
                   
                 aaaatatagattttgatattataatagctggaagacaggcgatagatggagata 
               
               
                   
               
               
                   
                 ctgcacaagttggacctcaaatagctgaacatttaaatcttccatcaataacat 
               
               
                   
               
               
                   
                 atgctgaagaaataaaaactgaaggtgaatatgtattagtaaaaagacaatttg 
               
               
                   
               
               
                   
                 aagattgttgccatgacttaaaagttaaaatgccatgccttataacaactctta 
               
               
                   
               
               
                   
                 aagatatgaacacaccaagatacatgaaagttggaagaatatatgatgctttcg 
               
               
                   
               
               
                   
                 aaaatgatgtagtagaaacatggactgtaaaagatatagaagttgacccttcta 
               
               
                   
               
               
                   
                 atttaggtcttaaaggttctccaactagtgtatttaaatcatttacaaaatcag 
               
               
                   
               
               
                   
                 ttaaaccagctggtacaatatacaatgaagatgcgaaaacatcagctggaatta 
               
               
                   
               
               
                   
                 tcatagataaattaaaagagaagtatatcatataataagaaggagatatacata 
               
               
                   
               
               
                   
                 tgggtaacgttttagtagtaatagaacaaagagaaaatgtaattcaaactgttt 
               
               
                   
               
               
                   
                 ctttagaattactaggaaaggctacagaaatagcaaaagattatgatacaaaag 
               
               
                   
               
               
                   
                 tttctgcattacttttaggtagtaaggtagaaggtttaatagatacattagcac 
               
               
                   
               
               
                   
                 actatggtgcagatgaggtaatagtagtagatgatgaagctttagcagtgtata 
               
               
                   
               
               
                   
                 caactgaaccatatacaaaagcagcttatgaagcaataaaagcagctgacccta 
               
               
                   
               
               
                   
                 tagttgtattatttggtgcaacttcaataggtagagatttagcgcctagagttt 
               
               
                   
               
               
                   
                 ctgctagaatacatacaggtcttactgctgactgtacaggtcttgcagtagctg 
               
               
                   
               
               
                   
                 aagatacaaaattattattaatgacaagacctgcctttggtggaaatataatgg 
               
               
                   
               
               
                   
                 caacaatagtttgtaaagatttcagacctcaaatgtctacagttagaccagggg 
               
               
                   
               
               
                   
                 ttatgaagaaaaatgaacctgatgaaactaaagaagctgtaattaaccgtttca 
               
               
                   
               
               
                   
                 aggtagaatttaatgatgctgataaattagttcaagttgtacaagtaataaaag 
               
               
                   
               
               
                   
                 aagctaaaaaacaagttaaaatagaagatgctaagatattagtttctgctggac 
               
               
                   
               
               
                   
                 gtggaatgggtggaaaagaaaacttagacatactttatgaattagctgaaatta 
               
               
                   
               
               
                   
                 taggtggagaagtttctggttctcgtgccactatagatgcaggttggttagata 
               
               
                   
               
               
                   
                 aagcaagacaagttggtcaaactggtaaaactgtaagaccagacctttatatag 
               
               
                   
               
               
                   
                 catgtggtatatctggagcaatacaacatatagctggtatggaagatgctgagt 
               
               
                   
               
               
                   
                 ttatagttgctataaataaaaatccagaagctccaatatttaaatatgctgatg 
               
               
                   
               
               
                   
                 ttggtatagttggagatgttcataaagtgcttccagaacttatcagtcagttaa 
               
               
                   
               
               
                   
                 gtgttgcaaaagaaaaaggtgaagttttagctaactaataagaaggagatatac 
               
               
                   
               
               
                   
                 atatgagagaagtagtaattgccagtgcagctagaacagcagtaggaagttttg 
               
               
                   
               
               
                   
                 gaggagcatttaaatcagtttcagcggtagagttaggggtaacagcagctaaag 
               
               
                   
               
               
                   
                 aagctataaaaagagctaacataactccagatatgatagatgaatctcttttag 
               
               
                   
               
               
                   
                 ggggagtacttacagcaggtcttggacaaaatatagcaagacaaatagcattag 
               
               
                   
               
               
                   
                 gagcaggaataccagtagaaaaaccagctatgactataaatatagtttgtggtt 
               
               
                   
               
               
                   
                 ctggattaagatctgtttcaatggcatctcaacttatagcattaggtgatgctg 
               
               
                   
               
               
                   
                 atataatgttagttggtggagctgaaaacatgagtatgtctccttatttagtac 
               
               
                   
               
               
                   
                 caagtgcgagatatggtgcaagaatgggtgatgctgcttttgttgattcaatga 
               
               
                   
               
               
                   
                 taaaagatggattatcagacatatttaataactatcacatgggtattactgctg 
               
               
                   
               
               
                   
                 aaaacatagcagagcaatggaatataactagagaagaacaagatgaattagctc 
               
               
                   
               
               
                   
                 ttgcaagtcaaaataaagctgaaaaagctcaagctgaaggaaaatttgatgaag 
               
               
                   
               
               
                   
                 aaatagttcctgttgttataaaaggaagaaaaggtgacactgtagtagataaag 
               
               
                   
               
               
                   
                 atgaatatattaagcctggcactacaatggagaaacttgctaagttaagacctg 
               
               
                   
               
               
                   
                 catttaaaaaagatggaacagttactgctggtaatgcatcaggaataaatgatg 
               
               
                   
               
               
                   
                 gtgctgctatgttagtagtaatggctaaagaaaaagctgaagaactaggaatag 
               
               
                   
               
               
                   
                 agcctcttgcaactatagtttcttatggaacagctggtgttgaccctaaaataa 
               
               
                   
               
               
                   
                 tgggatatggaccagttccagcaactaaaaaagctttagaagctgctaatatga 
               
               
                   
               
               
                   
                 ctattgaagatatagatttagttgaagctaatgaggcatttgctgcccaatctg 
               
               
                   
               
               
                   
                 tagctgtaataagagacttaaatatagatatgaataaagttaatgttaatggtg 
               
               
                   
               
               
                   
                 gagcaatagctataggacatccaataggatgctcaggagcaagaatacttacta 
               
               
                   
               
               
                   
                 cacttttatatgaaatgaagagaagagatgctaaaactggtcttgctacacttt 
               
               
                   
               
               
                   
                 gtataggcggtggaatgggaactactttaatagttaagagatagtaagaaggag 
               
               
                   
               
               
                   
                 atatacatatgaaattagctgtaataggtagtggaactatgggaagtggtattg 
               
               
                   
               
               
                   
                 tacaaacttttgcaagttgtggacatgatgtatgtttaaagagtagaactcaag 
               
               
                   
               
               
                   
                 gtgctatagataaatgtttagctttattagataaaaatttaactaagttagtta 
               
               
                   
               
               
                   
                 ctaagggaaaaatggatgaagctacaaaagcagaaatattaagtcatgttagtt 
               
               
                   
               
               
                   
                 caactactaattatgaagatttaaaagatatggatttaataatagaagcatctg 
               
               
                   
               
               
                   
                 tagaagacatgaatataaagaaagatgttttcaagttactagatgaattatgta 
               
               
                   
               
               
                   
                 aagaagatactatcttggcaacaaatacttcatcattatctataacagaaatag 
               
               
                   
               
               
                   
                 cttcttctactaagcgcccagataaagttataggaatgcatttctttaatccag 
               
               
                   
               
               
                   
                 ttcctatgatgaaattagttgaagttataagtggtcagttaacatcaaaagtta 
               
               
                   
               
               
                   
                 cttttgatacagtatttgaattatctaagagtatcaataaagtaccagtagatg 
               
               
                   
               
               
                   
                 tatctgaatctcctggatttgtagtaaatagaatacttatacctatgataaatg 
               
               
                   
               
               
                   
                 aagctgttggtatatatgcagatggtgttgcaagtaaagaagaaatagatgaag 
               
               
                   
               
               
                   
                 ctatgaaattaggagcaaaccatccaatgggaccactagcattaggtgatttaa 
               
               
                   
               
               
                   
                 tcggattagatgttgttttagctataatgaacgttttatatactgaatttggag 
               
               
                   
               
               
                   
                 atactaaatatagacctcatccacttttagctaaaatggttagagctaatcaat 
               
               
                   
               
               
                   
                 taggaagaaaaactaagataggattctatgattataataaataataagaaggag 
               
               
                   
               
               
                   
                 atatacatatgagtacaagtgatgttaaagtttatgagaatgtagctgttgaag 
               
               
                   
               
               
                   
                 tagatggaaatatatgtacagtgaaaatgaatagacctaaagcccttaatgcaa 
               
               
                   
               
               
                   
                 taaattcaaagactttagaagaactttatgaagtatttgtagatattaataatg 
               
               
                   
               
               
                   
                 atgaaactattgatgttgtaatattgacaggggaaggaaaggcatttgtagctg 
               
               
                   
               
               
                   
                 gagcagatattgcatacatgaaagatttagatgctgtagctgctaaagatttta 
               
               
                   
               
               
                   
                 gtatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtga 
               
               
                   
               
               
                   
                 tagctgctgtaaacggatttgctttaggtggaggatgtgaacttgcaatggcat 
               
               
                   
               
               
                   
                 gtgatataagaattgcatctgctaaagctaaatttggtcagccagaagtaactc 
               
               
                   
               
               
                   
                 ttggaataactccaggatatggaggaactcaaaggcttacaagattggttggaa 
               
               
                   
               
               
                   
                 tggcaaaagcaaaagaattaatctttacaggtcaagttataaaagctgatgaag 
               
               
                   
               
               
                   
                 ctgaaaaaatagggctagtaaatagagtcgttgagccagacattttaatagaag 
               
               
                   
               
               
                   
                 aagttgagaaattagctaagataatagctaaaaatgctcagcttgcagttagat 
               
               
                   
               
               
                   
                 actctaaagaagcaatacaacttggtgctcaaactgatataaatactggaatag 
               
               
                   
               
               
                   
                 atatagaatctaatttatttggtctttgtttttcaactaaagaccaaaaagaag 
               
               
                   
               
               
                   
                 gaatgtcagctttcgttgaaaagagagaagctaactttataaaagggtaataag 
               
               
                   
               
               
                   
                 aaggagatatacatatgagaagttttgaagaagtaattaagtttgcaaaagaaa 
               
               
                   
               
               
                   
                 gaggacctaaaactatatcagtagcatgttgccaagataaagaagttttaatgg 
               
               
                   
               
               
                   
                 cagttgaaatggctagaaaagaaaaaatagcaaatgccattttagtaggagata 
               
               
                   
               
               
                   
                 tagaaaagactaaagaaattgcaaaaagcatagacatggatatcgaaaattatg 
               
               
                   
               
               
                   
                 aactgatagatataaaagatttagcagaagcatctctaaaatctgttgaattag 
               
               
                   
               
               
                   
                 tttcacaaggaaaagccgacatggtaatgaaaggcttagtagacacatcaataa 
               
               
                   
               
               
                   
                 tactaaaagcagttttaaataaagaagtaggtcttagaactggaaatgtattaa 
               
               
                   
               
               
                   
                 gtcacgtagcagtatttgatgtagagggatatgatagattatttttcgtaactg 
               
               
                   
               
               
                   
                 acgcagctatgaacttagctcctgatacaaatactaaaaagcaaatcatagaaa 
               
               
                   
               
               
                   
                 atgcttgcacagtagcacattcattagatataagtgaaccaaaagttgctgcaa 
               
               
                   
               
               
                   
                 tatgcgcaaaagaaaaagtaaatccaaaaatgaaagatacagttgaagctaaag 
               
               
                   
               
               
                   
                 aactagaagaaatgtatgaaagaggagaaatcaaaggttgtatggttggtgggc 
               
               
                   
               
               
                   
                 cttttgcaattgataatgcagtatctttagaagcagctaaacataaaggtataa 
               
               
                   
               
               
                   
                 atcatcctgtagcaggacgagctgatatattattagccccagatattgaaggtg 
               
               
                   
               
               
                   
                 gtaacatattatataaagctttggtattcttctcaaaatcaaaaaatgcaggag 
               
               
                   
               
               
                   
                 ttatagttggggctaaagcaccaataatattaacttctagagcagacagtgaag 
               
               
                   
               
               
                   
                 aaactaaactaaactcaatagctttaggtgttttaatggcagcaaaggcataat 
               
               
                   
               
               
                   
                 aagaaggagatatacatatgagcaaaatatttaaaatcttaacaataaatcctg 
               
               
                   
               
               
                   
                 gttcgacatcaactaaaatagctgtatttgataatgaggatttagtatttgaaa 
               
               
                   
               
               
                   
                 aaactttaagacattcttcagaagaaataggaaaatatgagaaggtgtctgacc 
               
               
                   
               
               
                   
                 aatttgaatttcgtaaacaagtaatagaagaagctctaaaagaaggtggagtaa 
               
               
                   
               
               
                   
                 aaacatctgaattagatgctgtagtaggtagaggaggacttcttaaacctataa 
               
               
                   
               
               
                   
                 aaggtggtacttattcagtaagtgctgctatgattgaagatttaaaagtgggag 
               
               
                   
               
               
                   
                 ttttaggagaacacgcttcaaacctaggtggaataatagcaaaacaaataggtg 
               
               
                   
               
               
                   
                 aagaagtaaatgttccttcatacatagtagaccctgttgttgtagatgaattag 
               
               
                   
               
               
                   
                 aagatgttgctagaatttctggtatgcctgaaataagtagagcaagtgtagtac 
               
               
                   
               
               
                   
                 atgctttaaatcaaaaggcaatagcaagaagatatgctagagaaataaacaaga 
               
               
                   
               
               
                   
                 aatatgaagatataaatcttatagttgcacacatgggtggaggagtttctgttg 
               
               
                   
               
               
                   
                 gagctcataaaaatggtaaaatagtagatgttgcaaacgcattagatggagaag 
               
               
                   
               
               
                   
                 gacctttctctccagaaagaagtggtggactaccagtaggtgcattagtaaaaa 
               
               
                   
               
               
                   
                 tgtgctttagtggaaaatatactcaagatgaaattaaaaagaaaataaaaggta 
               
               
                   
               
               
                   
                 atggcggactagttgcatacttaaacactaatgatgctagagaagttgaagaaa 
               
               
                   
               
               
                   
                 gaattgaagctggtgatgaaaaagctaaattagtatatgaagctatggcatatc 
               
               
                   
               
               
                   
                 aaatctctaaagaaataggagctagtgctgcagttcttaagggagatgtaaaag 
               
               
                   
               
               
                   
                 caatattattaactggtggaatcgcatattcaaaaatgtttacagaaatgattg 
               
               
                   
               
               
                   
                 cagatagagttaaatttatagcagatgtaaaagtttatccaggtgaagatgaaa 
               
               
                   
               
               
                   
                 tgattgcattagctcaaggtggacttagagttttaactggtgaagaagaggctc 
               
               
                   
               
               
                   
                 aagtttatgataactaataa 
               
               
                   
               
               
                 Nucleotide 
                 ctcgagttcattatccatcctccatcgccacgatagttcatggcgataggtaga 
               
               
                   
               
               
                 sequences of 
                 atagcaatgaacgattatccctatcaagcattctgactgataattgctcacacg 
               
               
                   
               
               
                 pLogic046- 
                 aattcattaaagaggagaaaggtaccatgatcgtaaaacctatggtacgcaaca 
               
               
                   
               
               
                 oxyS-butyrate 
                 atatctgcctgaacgcccatcctcagggctgcaagaagggagtggaagatcaga 
               
               
                   
               
               
                 construct 
                 ttgaatataccaagaaacgcattaccgcagaagtcaaagctggcgcaaaagctc 
               
               
                   
               
               
                 (SEQ ID NO: 203) 
                 caaaaaacgttctggtgcttggctgctcaaatggttacggcctggcgagccgca 
               
               
                   
               
               
                   
                 ttactgctgcgttcggatacggggctgcgaccatcggcgtgtcctttgaaaaag 
               
               
                   
               
               
                   
                 cgggttcagaaaccaaatatggtacaccgggatggtacaataatttggcatttg 
               
               
                   
               
               
                   
                 atgaagcggcaaaacgcgagggtctttatagcgtgacgatcgacggcgatgcgt 
               
               
                   
               
               
                   
                 tttcagacgagatcaaggcccaggtaattgaggaagccaaaaaaaaaggtatca 
               
               
                   
               
               
                   
                 aatttgatctgatcgtatacagcttggccagcccagtacgtactgatcctgata 
               
               
                   
               
               
                   
                 caggtatcatgcacaaaagcgttttgaaaccctttggaaaaacgttcacaggca 
               
               
                   
               
               
                   
                 aaacagtagatccgtttactggcgagctgaaggaaatctccgcggaaccagcaa 
               
               
                   
               
               
                   
                 atgacgaggaagcagccgccactgttaaagttatggggggtgaagattgggaac 
               
               
                   
               
               
                   
                 gttggattaagcagctgtcgaaggaaggcctcttagaagaaggctgtattacct 
               
               
                   
               
               
                   
                 tggcctatagttatattggccctgaagctacccaagctttgtaccgtaaaggca 
               
               
                   
               
               
                   
                 caatcggcaaggccaaagaacacctggaggccacagcacaccgtctcaacaaag 
               
               
                   
               
               
                   
                 agaacccgtcaatccgtgccttcgtgagcgtgaataaaggcctggtaacccgcg 
               
               
                   
               
               
                   
                 caagcgccgtaatcccggtaatccctctgtatctcgccagcttgttcaaagtaa 
               
               
                   
               
               
                   
                 tgaaagagaagggcaatcatgaaggttgtattgaacagatcacgcgtctgtacg 
               
               
                   
               
               
                   
                 ccgagcgcctgtaccgtaaagatggtacaattccagttgatgaggaaaatcgca 
               
               
                   
               
               
                   
                 ttcgcattgatgattgggagttagaagaagacgtccagaaagcggtatccgcgt 
               
               
                   
               
               
                   
                 tgatggagaaagtcacgggtgaaaacgcagaatctctcactgacttagcggggt 
               
               
                   
               
               
                   
                 accgccatgatttcttagctagtaacggctttgatgtagaaggtattaattatg 
               
               
                   
               
               
                   
                 aagcggaagttgaacgcttcgaccgtatctgataagaaggagatatacatatga 
               
               
                   
               
               
                   
                 gagaagtagtaattgccagtgcagctagaacagcagtaggaagttttggaggag 
               
               
                   
               
               
                   
                 catttaaatcagtttcagcggtagagttaggggtaacagcagctaaagaagcta 
               
               
                   
               
               
                   
                 taaaaagagctaacataactccagatatgatagatgaatctcttttagggggag 
               
               
                   
               
               
                   
                 tacttacagcaggtcttggacaaaatatagcaagacaaatagcattaggagcag 
               
               
                   
               
               
                   
                 gaataccagtagaaaaaccagctatgactataaatatagtttgtggttctggat 
               
               
                   
               
               
                   
                 taagatctgtttcaatggcatctcaacttatagcattaggtgatgctgatataa 
               
               
                   
               
               
                   
                 tgttagttggtggagctgaaaacatgagtatgtctccttatttagtaccaagtg 
               
               
                   
               
               
                   
                 cgagatatggtgcaagaatgggtgatgctgcttttgttgattcaatgataaaag 
               
               
                   
               
               
                   
                 atggattatcagacatatttaataactatcacatgggtattactgctgaaaaca 
               
               
                   
               
               
                   
                 tagcagagcaatggaatataactagagaagaacaagatgaattagctcttgcaa 
               
               
                   
               
               
                   
                 gtcaaaataaagctgaaaaagctcaagctgaaggaaaatttgatgaagaaatag 
               
               
                   
               
               
                   
                 ttcctgttgttataaaaggaagaaaaggtgacactgtagtagataaagatgaat 
               
               
                   
               
               
                   
                 atattaagcctggcactacaatggagaaacttgctaagttaagacctgcattta 
               
               
                   
               
               
                   
                 aaaaagatggaacagttactgctggtaatgcatcaggaataaatgatggtgctg 
               
               
                   
               
               
                   
                 ctatgttagtagtaatggctaaagaaaaagctgaagaactaggaatagagcctc 
               
               
                   
               
               
                   
                 ttgcaactatagtttcttatggaacagctggtgttgaccctaaaataatgggat 
               
               
                   
               
               
                   
                 atggaccagttccagcaactaaaaaagctttagaagctgctaatatgactattg 
               
               
                   
               
               
                   
                 aagatatagatttagttgaagctaatgaggcatttgctgcccaatctgtagctg 
               
               
                   
               
               
                   
                 taataagagacttaaatatagatatgaataaagttaatgttaatggtggagcaa 
               
               
                   
               
               
                   
                 tagctataggacatccaataggatgctcaggagcaagaatacttactacacttt 
               
               
                   
               
               
                   
                 tatatgaaatgaagagaagagatgctaaaactggtcttgctacactttgtatag 
               
               
                   
               
               
                   
                 gcggtggaatgggaactactttaatagttaagagatagtaagaaggagatatac 
               
               
                   
               
               
                   
                 atatgaaattagctgtaataggtagtggaactatgggaagtggtattgtacaaa 
               
               
                   
               
               
                   
                 cttttgcaagttgtggacatgatgtatgtttaaagagtagaactcaaggtgcta 
               
               
                   
               
               
                   
                 tagataaatgtttagctttattagataaaaatttaactaagttagttactaagg 
               
               
                   
               
               
                   
                 gaaaaatggatgaagctacaaaagcagaaatattaagtcatgttagttcaacta 
               
               
                   
               
               
                   
                 ctaattatgaagatttaaaagatatggatttaataatagaagcatctgtagaag 
               
               
                   
               
               
                   
                 acatgaatataaagaaagatgttttcaagttactagatgaattatgtaaagaag 
               
               
                   
               
               
                   
                 atactatcttggcaacaaatacttcatcattatctataacagaaatagcttctt 
               
               
                   
               
               
                   
                 ctactaagcgcccagataaagttataggaatgcatttctttaatccagttccta 
               
               
                   
               
               
                   
                 tgatgaaattagttgaagttataagtggtcagttaacatcaaaagttacttttg 
               
               
                   
               
               
                   
                 atacagtatttgaattatctaagagtatcaataaagtaccagtagatgtatctg 
               
               
                   
               
               
                   
                 aatctcctggatttgtagtaaatagaatacttatacctatgataaatgaagctg 
               
               
                   
               
               
                   
                 ttggtatatatgcagatggtgttgcaagtaaagaagaaatagatgaagctatga 
               
               
                   
               
               
                   
                 aattaggagcaaaccatccaatgggaccactagcattaggtgatttaatcggat 
               
               
                   
               
               
                   
                 tagatgttgttttagctataatgaacgttttatatactgaatttggagatacta 
               
               
                   
               
               
                   
                 aatatagacctcatccacttttagctaaaatggttagagctaatcaattaggaa 
               
               
                   
               
               
                   
                 gaaaaactaagataggattctatgattataataaataataagaaggagatatac 
               
               
                   
               
               
                   
                 atatgagtacaagtgatgttaaagtttatgagaatgtagctgttgaagtagatg 
               
               
                   
               
               
                   
                 gaaatatatgtacagtgaaaatgaatagacctaaagcccttaatgcaataaatt 
               
               
                   
               
               
                   
                 caaagactttagaagaactttatgaagtatttgtagatattaataatgatgaaa 
               
               
                   
               
               
                   
                 ctattgatgttgtaatattgacaggggaaggaaaggcatttgtagctggagcag 
               
               
                   
               
               
                   
                 atattgcatacatgaaagatttagatgctgtagctgctaaagattttagtatct 
               
               
                   
               
               
                   
                 taggagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatagctg 
               
               
                   
               
               
                   
                 ctgtaaacggatttgctttaggtggaggatgtgaacttgcaatggcatgtgata 
               
               
                   
               
               
                   
                 taagaattgcatctgctaaagctaaatttggtcagccagaagtaactcttggaa 
               
               
                   
               
               
                   
                 taactccaggatatggaggaactcaaaggcttacaagattggttggaatggcaa 
               
               
                   
               
               
                   
                 aagcaaaagaattaatctttacaggtcaagttataaaagctgatgaagctgaaa 
               
               
                   
               
               
                   
                 aaatagggctagtaaatagagtcgttgagccagacattttaatagaagaagttg 
               
               
                   
               
               
                   
                 agaaattagctaagataatagctaaaaatgctcagcttgcagttagatactcta 
               
               
                   
               
               
                   
                 aagaagcaatacaacttggtgctcaaactgatataaatactggaatagatatag 
               
               
                   
               
               
                   
                 aatctaatttatttggtctttgtttttcaactaaagaccaaaaagaaggaatgt 
               
               
                   
               
               
                   
                 cagctttcgttgaaaagagagaagctaactttataaaagggtaataagaaggag 
               
               
                   
               
               
                   
                 atatacatatgagaagttttgaagaagtaattaagtttgcaaaagaaagaggac 
               
               
                   
               
               
                   
                 ctaaaactatatcagtagcatgttgccaagataaagaagttttaatggcagttg 
               
               
                   
               
               
                   
                 aaatggctagaaaagaaaaaatagcaaatgccattttagtaggagatatagaaa 
               
               
                   
               
               
                   
                 agactaaagaaattgcaaaaagcatagacatggatatcgaaaattatgaactga 
               
               
                   
               
               
                   
                 tagatataaaagatttagcagaagcatctctaaaatctgttgaattagtttcac 
               
               
                   
               
               
                   
                 aaggaaaagccgacatggtaatgaaaggcttagtagacacatcaataatactaa 
               
               
                   
               
               
                   
                 aagcagttttaaataaagaagtaggtcttagaactggaaatgtattaagtcacg 
               
               
                   
               
               
                   
                 tagcagtatttgatgtagagggatatgatagattatttttcgtaactgacgcag 
               
               
                   
               
               
                   
                 ctatgaacttagctcctgatacaaatactaaaaagcaaatcatagaaaatgctt 
               
               
                   
               
               
                   
                 gcacagtagcacattcattagatataagtgaaccaaaagttgctgcaatatgcg 
               
               
                   
               
               
                   
                 caaaagaaaaagtaaatccaaaaatgaaagatacagttgaagctaaagaactag 
               
               
                   
               
               
                   
                 aagaaatgtatgaaagaggagaaatcaaaggttgtatggttggtgggccttttg 
               
               
                   
               
               
                   
                 caattgataatgcagtatctttagaagcagctaaacataaaggtataaatcatc 
               
               
                   
               
               
                   
                 ctgtagcaggacgagctgatatattattagccccagatattgaaggtggtaaca 
               
               
                   
               
               
                   
                 tattatataaagctttggtattcttctcaaaatcaaaaaatgcaggagttatag 
               
               
                   
               
               
                   
                 ttggggctaaagcaccaataatattaacttctagagcagacagtgaagaaacta 
               
               
                   
               
               
                   
                 aactaaactcaatagctttaggtgttttaatggcagcaaaggcataataagaag 
               
               
                   
               
               
                   
                 gagatatacatatgagcaaaatatttaaaatcttaacaataaatcctggttcga 
               
               
                   
               
               
                   
                 catcaactaaaatagctgtatttgataatgaggatttagtatttgaaaaaactt 
               
               
                   
               
               
                   
                 taagacattcttcagaagaaataggaaaatatgagaaggtgtctgaccaatttg 
               
               
                   
               
               
                   
                 aatttcgtaaacaagtaatagaagaagctctaaaagaaggtggagtaaaaacat 
               
               
                   
               
               
                   
                 ctgaattagatgctgtagtaggtagaggaggacttcttaaacctataaaaggtg 
               
               
                   
               
               
                   
                 gtacttattcagtaagtgctgctatgattgaagatttaaaagtgggagttttag 
               
               
                   
               
               
                   
                 gagaacacgcttcaaacctaggtggaataatagcaaaacaaataggtgaagaag 
               
               
                   
               
               
                   
                 taaatgttccttcatacatagtagaccctgttgttgtagatgaattagaagatg 
               
               
                   
               
               
                   
                 ttgctagaatttctggtatgcctgaaataagtagagcaagtgtagtacatgctt 
               
               
                   
               
               
                   
                 taaatcaaaaggcaatagcaagaagatatgctagagaaataaacaagaaatatg 
               
               
                   
               
               
                   
                 aagatataaatcttatagttgcacacatgggtggaggagtttctgttggagctc 
               
               
                   
               
               
                   
                 ataaaaatggtaaaatagtagatgttgcaaacgcattagatggagaaggacctt 
               
               
                   
               
               
                   
                 tctctccagaaagaagtggtggactaccagtaggtgcattagtaaaaatgtgct 
               
               
                   
               
               
                   
                 ttagtggaaaatatactcaagatgaaattaaaaagaaaataaaaggtaatggcg 
               
               
                   
               
               
                   
                 gactagttgcatacttaaacactaatgatgctagagaagttgaagaaagaattg 
               
               
                   
               
               
                   
                 aagctggtgatgaaaaagctaaattagtatatgaagctatggcatatcaaatct 
               
               
                   
               
               
                   
                 ctaaagaaataggagctagtgctgcagttcttaagggagatgtaaaagcaatat 
               
               
                   
               
               
                   
                 tattaactggtggaatcgcatattcaaaaatgtttacagaaatgattgcagata 
               
               
                   
               
               
                   
                 gagttaaatttatagcagatgtaaaagtttatccaggtgaagatgaaatgattg 
               
               
                   
               
               
                   
                 cattagctcaaggtggacttagagttttaactggtgaagaagaggctcaagttt 
               
               
                   
               
               
                   
                 atgataactaataa 
               
               
                   
               
               
                 Nucleotide 
                 ctcgagatgctagcaattgtgagcggataacaattgacattgtgagcggataac 
               
               
                   
               
               
                 sequences of 
                 aagatactgagcacatcagcaggacgcactgaccttaattaaaagaattcatta 
               
               
                   
               
               
                 pZA22-oxyR 
                 aagaggagaaaggtaccatgaatattcgtgatcttgagtacctggtggcattgg 
               
               
                   
               
               
                 construct  
                 ctgaacaccgccattttcggcgtgcggcagattcctgccacgttagccagccga 
               
               
                   
               
               
                 (SEQ ID NO: 204) 
                 cgcttagcgggcaaattcgtaagctggaagatgagctgggcgtgatgttgctgg 
               
               
                   
               
               
                   
                 agcggaccagccgtaaagtgttgttcacccaggcgggaatgctgctggtggatc 
               
               
                   
               
               
                   
                 aggcgcgtaccgtgctgcgtgaggtgaaagtccttaaagagatggcaagccagc 
               
               
                   
               
               
                   
                 agggcgagacgatgtccggaccgctgcacattggtttgattcccacagttggac 
               
               
                   
               
               
                   
                 cgtacctgctaccgcatattatccctatgctgcaccagacctttccaaagctgg 
               
               
                   
               
               
                   
                 aaatgtatctgcatgaagcacagacccaccagttactggcgcaactggacagcg 
               
               
                   
               
               
                   
                 gcaaactcgattgcgtgatcctcgcgctggtgaaagagagcgaagcattcattg 
               
               
                   
               
               
                   
                 aagtgccgttgtttgatgagccaatgttgctggctatctatgaagatcacccgt 
               
               
                   
               
               
                   
                 gggcgaaccgcgaatgcgtaccgatggccgatctggcaggggaaaaactgctga 
               
               
                   
               
               
                   
                 tgctggaagatggtcactgtttgcgcgatcaggcaatgggtttctgttttgaag 
               
               
                   
               
               
                   
                 ccggggcggatgaagatacacacttccgcgcgaccagcctggaaactctgcgca 
               
               
                   
               
               
                   
                 acatggtggcggcaggtagcgggatcactttactgccagcgctggctgtgccgc 
               
               
                   
               
               
                   
                 cggagcgcaaacgcgatggggttgtttatctgccgtgcattaagccggaaccac 
               
               
                   
               
               
                   
                 gccgcactattggcctggtttatcgtcctggctcaccgctgcgcagccgctatg 
               
               
                   
               
               
                   
                 agcagctggcagaggccatccgcgcaagaatggatggccatttcgataaagttt 
               
               
                   
               
               
                   
                 taaaacaggcggtttaaggatcccatggtacgcgtgctagaggcatcaaataaa 
               
               
                   
               
               
                   
                 acgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggt 
               
               
                   
               
               
                   
                 gaacgctctcctgagtaggacaaatccgccgccctagacctaggggatatattc 
               
               
                   
               
               
                   
                 cgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagcgga 
               
               
                   
               
               
                   
                 aatggcttacgaacggggcggagatttcctggaagatgccaggaagatacttaa 
               
               
                   
               
               
                   
                 cagggaagtgagagggccgcggcaaagccgtttttccataggctccgcccccct 
               
               
                   
               
               
                   
                 gacaagcatcacgaaatctgacgctcaaatcagtggtggcgaaacccgacagga 
               
               
                   
               
               
                   
                 ctataaagataccaggcgtttccccctggcggctccctcgtgcgctctcctgtt 
               
               
                   
               
               
                   
                 cctgcctttcggtttaccggtgtcattccgctgttatggccgcgtttgtctcat 
               
               
                   
               
               
                   
                 tccacgcctgacactcagttccgggtaggcagttcgctccaagctggactgtat 
               
               
                   
               
               
                   
                 gcacgaaccccccgttcagtccgaccgctgcgccttatccggtaactatcgtct 
               
               
                   
               
               
                   
                 tgagtccaacccggaaagacatgcaaaagcaccactggcagcagccactggtaa 
               
               
                   
               
               
                   
                 ttgatttagaggagttagtcttgaagtcatgcgccggttaaggctaaactgaaa 
               
               
                   
               
               
                   
                 ggacaagttttggtgactgcgctcctccaagccagttacctcggttcaaagagt 
               
               
                   
               
               
                   
                 tggtagctcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgtttt 
               
               
                   
               
               
                   
                 cagagcaagagattacgcgcagaccaaaacgatctcaagaagatcatcttatta 
               
               
                   
               
               
                   
                 atcagataaaatatttctagatttcagtgcaatttatctcttcaaatgtagcac 
               
               
                   
               
               
                   
                 ctgaagtcagccccatacgatataagttgttactagtgcttggattctcaccaa 
               
               
                   
               
               
                   
                 taaaaaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctg 
               
               
                   
               
               
                   
                 aggtcattactggatctatcaacaggagtccaagcgagctctcgaaccccagag 
               
               
                   
               
               
                   
                 tcccgctcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatc 
               
               
                   
               
               
                   
                 gggagcggcgataccgtaaagcacgaggaagcggtcagcccattcgccgccaag 
               
               
                   
               
               
                   
                 ctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccgccac 
               
               
                   
               
               
                   
                 acccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgat 
               
               
                   
               
               
                   
                 attcggcaagcaggcatcgccatgggtcacgacgagatcctcgccgtcgggcat 
               
               
                   
               
               
                   
                 gcgcgccttgagcctggcgaacagttcggctggcgcgagcccctgatgctcttc 
               
               
                   
               
               
                   
                 gtccagatcatcctgatcgacaagaccggcttccatccgagtacgtgctcgctc 
               
               
                   
               
               
                   
                 gatgcgatgtttcgcttggtggtcgaatgggcaggtagccggatcaagcgtatg 
               
               
                   
               
               
                   
                 cagccgccgcattgcatcagccatgatggatactttctcggcaggagcaaggtg 
               
               
                   
               
               
                   
                 agatgacaggagatcctgccccggcacttcgcccaatagcagccagtcccttcc 
               
               
                   
               
               
                   
                 cgcttcagtgacaacgtcgagcacagctgcgcaaggaacgcccgtcgtggccag 
               
               
                   
               
               
                   
                 ccacgatagccgcgctgcctcgtcctgcagttcattcagggcaccggacaggtc 
               
               
                   
               
               
                   
                 ggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacacggcggc 
               
               
                   
               
               
                   
                 atcagagcagccgattgtctgttgtgcccagtcatagccgaatagcctctccac 
               
               
                   
               
               
                   
                 ccaagcggccggagaacctgcgtgcaatccatcttgttcaatcatgcgaaacga 
               
               
                   
               
               
                   
                 tcctcatcctgtctcttgatcagatcttgatcccctgcgccatcagatccttgg 
               
               
                   
               
               
                   
                 cggcaagaaagccatccagtttactttgcagggcttcccaaccttaccagaggg 
               
               
                   
               
               
                   
                 cgccccagctggcaattccgacgtctaagaaaccattattatcatgacattaac 
               
               
                   
               
               
                   
                 ctataaaaataggcgtatcacgaggccctttcgtcttcac 
               
               
                   
               
               
                 Nucleotide 
                 gtaaaacgacggccagtgaattcg ttaagacccactttcacatttaagttgttt   
               
               
                   
               
               
                 sequences of 
                 
                   ttctaatccgcatatgatcaattcaaggccgaataagaaggctggctctgcacc 
                 
               
               
                   
               
               
                 pLogic031-tet- 
                 
                   ttggtgatcaaataattcgatagcttgtcgtaataatggcggcatactatcagt 
                 
               
               
                   
               
               
                 butyrate 
                 
                   agtaggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatac 
                 
               
               
                   
               
               
                 construct  
                 
                   gcaacctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctag 
                 
               
               
                   
               
               
                 (SEQ ID NO: 205) 
                 
                   tgaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcatactg 
                 
               
               
                   
               
               
                 The sequence 
                 
                   tttttctgtaggccgtgtacctaaatgtacttttgctccatcgcgatgacttag 
                 
               
               
                   
               
               
                 encoding TetR 
                 
                   taaagcacatctaaaacttttagcgttattacgtaaaaaatcttgccagctttc 
                 
               
               
                   
               
               
                 is  underlined , 
                 
                   cccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaa 
                 
               
               
                   
               
               
                 and the 
                 
                   ggcgtcgagcaaagcccgcttattttttacatgccaatacaatgtaggctgctc 
                 
               
               
                   
               
               
                 overlapping 
                 
                   tacacctagcttctgggcgagtttacgggttgttaaaccttcgattccgacctc 
                 
               
               
                   
               
               
                 tetR/tetA 
                 
                   attaagcagctctaatgcgctgttaatcactttacttttatctaatctagacat 
                 
               
               
                   
               
               
                 promoters are 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                   
                 agaaggagatatacatatggatttaaattctaaaaaatatcagatgcttaaaga 
               
               
                   
               
               
                   
                 gctatatgtaagcttcgctgaaaatgaagttaaacctttagcaacagaacttga 
               
               
                   
               
               
                   
                 tgaagaagaaagatttccttatgaaacagtggaaaaaatggcaaaagcaggaat 
               
               
                   
               
               
                   
                 gatgggtataccatatccaaaagaatatggtggagaaggtggagacactgtagg 
               
               
                   
               
               
                   
                 atatataatggcagttgaagaattgtctagagtttgtggtactacaggagttat 
               
               
                   
               
               
                   
                 attatcagctcatacatctcttggctcatggcctatatatcaatatggtaatga 
               
               
                   
               
               
                   
                 agaacaaaaacaaaaattcttaagaccactagcaagtggagaaaaattaggagc 
               
               
                   
               
               
                   
                 atttggtcttactgagcctaatgctggtacagatgcgtctggccaacaaacaac 
               
               
                   
               
               
                   
                 tgctgttttagacggggatgaatacatacttaatggctcaaaaatatttataac 
               
               
                   
               
               
                   
                 aaacgcaatagctggtgacatatatgtagtaatggcaatgactgataaatctaa 
               
               
                   
               
               
                   
                 ggggaacaaaggaatatcagcatttatagttgaaaaaggaactcctgggtttag 
               
               
                   
               
               
                   
                 ctttggagttaaagaaaagaaaatgggtataagaggttcagctacgagtgaatt 
               
               
                   
               
               
                   
                 aatatttgaggattgcagaatacctaaagaaaatttacttggaaaagaaggtca 
               
               
                   
               
               
                   
                 aggatttaagatagcaatgtctactcttgatggtggtagaattggtatagctgc 
               
               
                   
               
               
                   
                 acaagctttaggtttagcacaaggtgctcttgatgaaactgttaaatatgtaaa 
               
               
                   
               
               
                   
                 agaaagagtacaatttggtagaccattatcaaaattccaaaatacacaattcca 
               
               
                   
               
               
                   
                 attagctgatatggaagttaaggtacaagcggctagacaccttgtatatcaagc 
               
               
                   
               
               
                   
                 agctataaataaagacttaggaaaaccttatggagtagaagcagcaatggcaaa 
               
               
                   
               
               
                   
                 attatttgcagctgaaacagctatggaagttactacaaaagctgtacaacttca 
               
               
                   
               
               
                   
                 tggaggatatggatacactcgtgactatccagtagaaagaatgatgagagatgc 
               
               
                   
               
               
                   
                 taagataactgaaatatatgaaggaactagtgaagttcaaagaatggttatttc 
               
               
                   
               
               
                   
                 aggaaaactattaaaatagtaagaaggagatatacatatggaggaaggatttat 
               
               
                   
               
               
                   
                 gaatatagtcgtttgtataaaacaagttccagatacaacagaagttaaactaga 
               
               
                   
               
               
                   
                 tcctaatacaggtactttaattagagatggagtaccaagtataataaaccctga 
               
               
                   
               
               
                   
                 tgataaagcaggtttagaagaagctataaaattaaaagaagaaatgggtgctca 
               
               
                   
               
               
                   
                 tgtaactgttataacaatgggacctcctcaagcagatatggctttaaaagaagc 
               
               
                   
               
               
                   
                 tttagcaatgggtgcagatagaggtatattattaacagatagagcatttgcggg 
               
               
                   
               
               
                   
                 tgctgatacttgggcaacttcatcagcattagcaggagcattaaaaaatataga 
               
               
                   
               
               
                   
                 ttttgatattataatagctggaagacaggcgatagatggagatactgcacaagt 
               
               
                   
               
               
                   
                 tggacctcaaatagctgaacatttaaatcttccatcaataacatatgctgaaga 
               
               
                   
               
               
                   
                 aataaaaactgaaggtgaatatgtattagtaaaaagacaatttgaagattgttg 
               
               
                   
               
               
                   
                 ccatgacttaaaagttaaaatgccatgccttataacaactcttaaagatatgaa 
               
               
                   
               
               
                   
                 cacaccaagatacatgaaagttggaagaatatatgatgctttcgaaaatgatgt 
               
               
                   
               
               
                   
                 agtagaaacatggactgtaaaagatatagaagttgacccttctaatttaggtct 
               
               
                   
               
               
                   
                 taaaggttctccaactagtgtatttaaatcatttacaaaatcagttaaaccagc 
               
               
                   
               
               
                   
                 tggtacaatatacaatgaagatgcgaaaacatcagctggaattatcatagataa 
               
               
                   
               
               
                   
                 attaaaagagaagtatatcatataataagaaggagatatacatatgggtaacgt 
               
               
                   
               
               
                   
                 tttagtagtaatagaacaaagagaaaatgtaattcaaactgtttctttagaatt 
               
               
                   
               
               
                   
                 actaggaaaggctacagaaatagcaaaagattatgatacaaaagtttctgcatt 
               
               
                   
               
               
                   
                 acttttaggtagtaaggtagaaggtttaatagatacattagcacactatggtgc 
               
               
                   
               
               
                   
                 agatgaggtaatagtagtagatgatgaagctttagcagtgtatacaactgaacc 
               
               
                   
               
               
                   
                 atatacaaaagcagcttatgaagcaataaaagcagctgaccctatagttgtatt 
               
               
                   
               
               
                   
                 atttggtgcaacttcaataggtagagatttagcgcctagagtttctgctagaat 
               
               
                   
               
               
                   
                 acatacaggtcttactgctgactgtacaggtcttgcagtagctgaagatacaaa 
               
               
                   
               
               
                   
                 attattattaatgacaagacctgcctttggtggaaatataatggcaacaatagt 
               
               
                   
               
               
                   
                 ttgtaaagatttcagacctcaaatgtctacagttagaccaggggttatgaagaa 
               
               
                   
               
               
                   
                 aaatgaacctgatgaaactaaagaagctgtaattaaccgtttcaaggtagaatt 
               
               
                   
               
               
                   
                 taatgatgctgataaattagttcaagttgtacaagtaataaaagaagctaaaaa 
               
               
                   
               
               
                   
                 acaagttaaaatagaagatgctaagatattagtttctgctggacgtggaatggg 
               
               
                   
               
               
                   
                 tggaaaagaaaacttagacatactttatgaattagctgaaattataggtggaga 
               
               
                   
               
               
                   
                 agtttctggttctcgtgccactatagatgcaggttggttagataaagcaagaca 
               
               
                   
               
               
                   
                 agttggtcaaactggtaaaactgtaagaccagacctttatatagcatgtggtat 
               
               
                   
               
               
                   
                 atctggagcaatacaacatatagctggtatggaagatgctgagtttatagttgc 
               
               
                   
               
               
                   
                 tataaataaaaatccagaagctccaatatttaaatatgctgatgttggtatagt 
               
               
                   
               
               
                   
                 tggagatgttcataaagtgcttccagaacttatcagtcagttaagtgttgcaaa 
               
               
                   
               
               
                   
                 agaaaaaggtgaagttttagctaactaataagaaggagatatacatatgagaga 
               
               
                   
               
               
                   
                 agtagtaattgccagtgcagctagaacagcagtaggaagttttggaggagcatt 
               
               
                   
               
               
                   
                 taaatcagtttcagcggtagagttaggggtaacagcagctaaagaagctataaa 
               
               
                   
               
               
                   
                 aagagctaacataactccagatatgatagatgaatctcttttagggggagtact 
               
               
                   
               
               
                   
                 tacagcaggtcttggacaaaatatagcaagacaaatagcattaggagcaggaat 
               
               
                   
               
               
                   
                 accagtagaaaaaccagctatgactataaatatagtttgtggttctggattaag 
               
               
                   
               
               
                   
                 atctgtttcaatggcatctcaacttatagcattaggtgatgctgatataatgtt 
               
               
                   
               
               
                   
                 agttggtggagctgaaaacatgagtatgtctccttatttagtaccaagtgcgag 
               
               
                   
               
               
                   
                 atatggtgcaagaatgggtgatgctgcttttgttgattcaatgataaaagatgg 
               
               
                   
               
               
                   
                 attatcagacatatttaataactatcacatgggtattactgctgaaaacatagc 
               
               
                   
               
               
                   
                 agagcaatggaatataactagagaagaacaagatgaattagctcttgcaagtca 
               
               
                   
               
               
                   
                 aaataaagctgaaaaagctcaagctgaaggaaaatttgatgaagaaatagttcc 
               
               
                   
               
               
                   
                 tgttgttataaaaggaagaaaaggtgacactgtagtagataaagatgaatatat 
               
               
                   
               
               
                   
                 taagcctggcactacaatggagaaacttgctaagttaagacctgcatttaaaaa 
               
               
                   
               
               
                   
                 agatggaacagttactgctggtaatgcatcaggaataaatgatggtgctgctat 
               
               
                   
               
               
                   
                 gttagtagtaatggctaaagaaaaagctgaagaactaggaatagagcctcttgc 
               
               
                   
               
               
                   
                 aactatagtttcttatggaacagctggtgttgaccctaaaataatgggatatgg 
               
               
                   
               
               
                   
                 accagttccagcaactaaaaaagctttagaagctgctaatatgactattgaaga 
               
               
                   
               
               
                   
                 tatagatttagttgaagctaatgaggcatttgctgcccaatctgtagctgtaat 
               
               
                   
               
               
                   
                 aagagacttaaatatagatatgaataaagttaatgttaatggtggagcaatagc 
               
               
                   
               
               
                   
                 tataggacatccaataggatgctcaggagcaagaatacttactacacttttata 
               
               
                   
               
               
                   
                 tgaaatgaagagaagagatgctaaaactggtcttgctacactttgtataggcgg 
               
               
                   
               
               
                   
                 tggaatgggaactactttaatagttaagagatagtaagaaggagatatacatat 
               
               
                   
               
               
                   
                 gaaattagctgtaataggtagtggaactatgggaagtggtattgtacaaacttt 
               
               
                   
               
               
                   
                 tgcaagttgtggacatgatgtatgtttaaagagtagaactcaaggtgctataga 
               
               
                   
               
               
                   
                 taaatgtttagctttattagataaaaatttaactaagttagttactaagggaaa 
               
               
                   
               
               
                   
                 aatggatgaagctacaaaagcagaaatattaagtcatgttagttcaactactaa 
               
               
                   
               
               
                   
                 ttatgaagatttaaaagatatggatttaataatagaagcatctgtagaagacat 
               
               
                   
               
               
                   
                 gaatataaagaaagatgttttcaagttactagatgaattatgtaaagaagatac 
               
               
                   
               
               
                   
                 tatcttggcaacaaatacttcatcattatctataacagaaatagcttcttctac 
               
               
                   
               
               
                   
                 taagcgcccagataaagttataggaatgcatttctttaatccagttcctatgat 
               
               
                   
               
               
                   
                 gaaattagttgaagttataagtggtcagttaacatcaaaagttacttttgatac 
               
               
                   
               
               
                   
                 agtatttgaattatctaagagtatcaataaagtaccagtagatgtatctgaatc 
               
               
                   
               
               
                   
                 tcctggatttgtagtaaatagaatacttatacctatgataaatgaagctgttgg 
               
               
                   
               
               
                   
                 tatatatgcagatggtgttgcaagtaaagaagaaatagatgaagctatgaaatt 
               
               
                   
               
               
                   
                 aggagcaaaccatccaatgggaccactagcattaggtgatttaatcggattaga 
               
               
                   
               
               
                   
                 tgttgttttagctataatgaacgttttatatactgaatttggagatactaaata 
               
               
                   
               
               
                   
                 tagacctcatccacttttagctaaaatggttagagctaatcaattaggaagaaa 
               
               
                   
               
               
                   
                 aactaagataggattctatgattataataaataataagaaggagatatacatat 
               
               
                   
               
               
                   
                 gagtacaagtgatgttaaagtttatgagaatgtagctgttgaagtagatggaaa 
               
               
                   
               
               
                   
                 tatatgtacagtgaaaatgaatagacctaaagcccttaatgcaataaattcaaa 
               
               
                   
               
               
                   
                 gactttagaagaactttatgaagtatttgtagatattaataatgatgaaactat 
               
               
                   
               
               
                   
                 tgatgttgtaatattgacaggggaaggaaaggcatttgtagctggagcagatat 
               
               
                   
               
               
                   
                 tgcatacatgaaagatttagatgctgtagctgctaaagattttagtatcttagg 
               
               
                   
               
               
                   
                 agcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatagctgctgt 
               
               
                   
               
               
                   
                 aaacggatttgctttaggtggaggatgtgaacttgcaatggcatgtgatataag 
               
               
                   
               
               
                   
                 aattgcatctgctaaagctaaatttggtcagccagaagtaactcttggaataac 
               
               
                   
               
               
                   
                 tccaggatatggaggaactcaaaggcttacaagattggttggaatggcaaaagc 
               
               
                   
               
               
                   
                 aaaagaattaatctttacaggtcaagttataaaagctgatgaagctgaaaaaat 
               
               
                   
               
               
                   
                 agggctagtaaatagagtcgttgagccagacattttaatagaagaagttgagaa 
               
               
                   
               
               
                   
                 attagctaagataatagctaaaaatgctcagcttgcagttagatactctaaaga 
               
               
                   
               
               
                   
                 agcaatacaacttggtgctcaaactgatataaatactggaatagatatagaatc 
               
               
                   
               
               
                   
                 taatttatttggtctttgtttttcaactaaagaccaaaaagaaggaatgtcagc 
               
               
                   
               
               
                   
                 tttcgttgaaaagagagaagctaactttataaaagggtaataagaaggagatat 
               
               
                   
               
               
                   
                 acatatgagaagttttgaagaagtaattaagtttgcaaaagaaagaggacctaa 
               
               
                   
               
               
                   
                 aactatatcagtagcatgttgccaagataaagaagttttaatggcagttgaaat 
               
               
                   
               
               
                   
                 ggctagaaaagaaaaaatagcaaatgccattttagtaggagatatagaaaagac 
               
               
                   
               
               
                   
                 taaagaaattgcaaaaagcatagacatggatatcgaaaattatgaactgataga 
               
               
                   
               
               
                   
                 tataaaagatttagcagaagcatctctaaaatctgttgaattagtttcacaagg 
               
               
                   
               
               
                   
                 aaaagccgacatggtaatgaaaggcttagtagacacatcaataatactaaaagc 
               
               
                   
               
               
                   
                 agttttaaataaagaagtaggtcttagaactggaaatgtattaagtcacgtagc 
               
               
                   
               
               
                   
                 agtatttgatgtagagggatatgatagattatttttcgtaactgacgcagctat 
               
               
                   
               
               
                   
                 gaacttagctcctgatacaaatactaaaaagcaaatcatagaaaatgcttgcac 
               
               
                   
               
               
                   
                 agtagcacattcattagatataagtgaaccaaaagttgctgcaatatgcgcaaa 
               
               
                   
               
               
                   
                 agaaaaagtaaatccaaaaatgaaagatacagttgaagctaaagaactagaaga 
               
               
                   
               
               
                   
                 aatgtatgaaagaggagaaatcaaaggttgtatggttggtgggccttttgcaat 
               
               
                   
               
               
                   
                 tgataatgcagtatctttagaagcagctaaacataaaggtataaatcatcctgt 
               
               
                   
               
               
                   
                 agcaggacgagctgatatattattagccccagatattgaaggtggtaacatatt 
               
               
                   
               
               
                   
                 atataaagctttggtattcttctcaaaatcaaaaaatgcaggagttatagttgg 
               
               
                   
               
               
                   
                 ggctaaagcaccaataatattaacttctagagcagacagtgaagaaactaaact 
               
               
                   
               
               
                   
                 aaactcaatagctttaggtgttttaatggcagcaaaggcataataagaaggaga 
               
               
                   
               
               
                   
                 tatacatatgagcaaaatatttaaaatcttaacaataaatcctggttcgacatc 
               
               
                   
               
               
                   
                 aactaaaatagctgtatttgataatgaggatttagtatttgaaaaaactttaag 
               
               
                   
               
               
                   
                 acattcttcagaagaaataggaaaatatgagaaggtgtctgaccaatttgaatt 
               
               
                   
               
               
                   
                 tcgtaaacaagtaatagaagaagctctaaaagaaggtggagtaaaaacatctga 
               
               
                   
               
               
                   
                 attagatgctgtagtaggtagaggaggacttcttaaacctataaaaggtggtac 
               
               
                   
               
               
                   
                 ttattcagtaagtgctgctatgattgaagatttaaaagtgggagttttaggaga 
               
               
                   
               
               
                   
                 acacgcttcaaacctaggtggaataatagcaaaacaaataggtgaagaagtaaa 
               
               
                   
               
               
                   
                 tgttccttcatacatagtagaccctgttgttgtagatgaattagaagatgttgc 
               
               
                   
               
               
                   
                 tagaatttctggtatgcctgaaataagtagagcaagtgtagtacatgctttaaa 
               
               
                   
               
               
                   
                 tcaaaaggcaatagcaagaagatatgctagagaaataaacaagaaatatgaaga 
               
               
                   
               
               
                   
                 tataaatcttatagttgcacacatgggtggaggagtttctgttggagctcataa 
               
               
                   
               
               
                   
                 aaatggtaaaatagtagatgttgcaaacgcattagatggagaaggacctttctc 
               
               
                   
               
               
                   
                 tccagaaagaagtggtggactaccagtaggtgcattagtaaaaatgtgctttag 
               
               
                   
               
               
                   
                 tggaaaatatactcaagatgaaattaaaaagaaaataaaaggtaatggcggact 
               
               
                   
               
               
                   
                 agttgcatacttaaacactaatgatgctagagaagttgaagaaagaattgaagc 
               
               
                   
               
               
                   
                 tggtgatgaaaaagctaaattagtatatgaagctatggcatatcaaatctctaa 
               
               
                   
               
               
                   
                 agaaataggagctagtgctgcagttcttaagggagatgtaaaagcaatattatt 
               
               
                   
               
               
                   
                 aactggtggaatcgcatattcaaaaatgtttacagaaatgattgcagatagagt 
               
               
                   
               
               
                   
                 taaatttatagcagatgtaaaagtttatccaggtgaagatgaaatgattgcatt 
               
               
                   
               
               
                   
                 agctcaaggtggacttagagttttaactggtgaagaagaggctcaagtttatga 
               
               
                   
               
               
                   
                 taactaataa 
               
               
                   
               
               
                 Nucleotide 
                 gtaaaacgacggccagtgaattcg ttaagacccactttcacatttaagttgttt   
               
               
                   
               
               
                 sequences of 
                 
                   ttctaatccgcatatgatcaattcaaggccgaataagaaggctggctctgcacc 
                 
               
               
                   
               
               
                 pLogic046-tet-  
                 
                   ttggtgatcaaataattcgatagcttgtcgtaataatggcggcatactatcagt 
                 
               
               
                   
               
               
                 butyrate 
                 
                   agtaggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatac 
                 
               
               
                   
               
               
                 construct 
                 
                   gcaacctaaagtaaaatgccccacagcgctgagtgcatataatgcattctctag 
                 
               
               
                   
               
               
                 (SEQ ID NO: 206) 
                 
                   tgaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcatactg 
                 
               
               
                   
               
               
                 The sequence 
                 
                   tttttctgtaggccgtgtacctaaatgtacttttgctccatcgcgatgacttag 
                 
               
               
                   
               
               
                 encoding TetR 
                 
                   taaagcacatctaaaacttttagcgttattacgtaaaaaatcttgccagctttc 
                 
               
               
                   
               
               
                 is  underlined   
                 
                   cccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaa 
                 
               
               
                   
               
               
                 and the 
                 
                   ggcgtcgagcaaagcccgcttattttttacatgccaatacaatgtaggctgctc 
                 
               
               
                   
               
               
                 overlapping 
                 
                   tacacctagcttctgggcgagtttacgggttgttaaaccttcgattccgacctc 
                 
               
               
                   
               
               
                 tetR/tetA 
                 
                   attaagcagctctaatgcgctgttaatcactttacttttatctaatctagacat 
                 
               
               
                   
               
               
                 promoters are 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                   
                 agaaggagatatacatatgatcgtaaaacctatggtacgcaacaatatctgcct 
               
               
                   
               
               
                   
                 gaacgcccatcctcagggctgcaagaagggagtggaagatcagattgaatatac 
               
               
                   
               
               
                   
                 caagaaacgcattaccgcagaagtcaaagctggcgcaaaagctccaaaaaacgt 
               
               
                   
               
               
                   
                 tctggtgcttggctgctcaaatggttacggcctggcgagccgcattactgctgc 
               
               
                   
               
               
                   
                 gttcggatacggggctgcgaccatcggcgtgtcctttgaaaaagcgggttcaga 
               
               
                   
               
               
                   
                 aaccaaatatggtacaccgggatggtacaataatttggcatttgatgaagcggc 
               
               
                   
               
               
                   
                 aaaacgcgagggtctttatagcgtgacgatcgacggcgatgcgttttcagacga 
               
               
                   
               
               
                   
                 gatcaaggcccaggtaattgaggaagccaaaaaaaaaggtatcaaatttgatct 
               
               
                   
               
               
                   
                 gatcgtatacagcttggccagcccagtacgtactgatcctgatacaggtatcat 
               
               
                   
               
               
                   
                 gcacaaaagcgttttgaaaccctttggaaaaacgttcacaggcaaaacagtaga 
               
               
                   
               
               
                   
                 tccgtttactggcgagctgaaggaaatctccgcggaaccagcaaatgacgagga 
               
               
                   
               
               
                   
                 agcagccgccactgttaaagttatggggggtgaagattgggaacgttggattaa 
               
               
                   
               
               
                   
                 gcagctgtcgaaggaaggcctcttagaagaaggctgtattaccttggcctatag 
               
               
                   
               
               
                   
                 ttatattggccctgaagctacccaagctttgtaccgtaaaggcacaatcggcaa 
               
               
                   
               
               
                   
                 ggccaaagaacacctggaggccacagcacaccgtctcaacaaagagaacccgtc 
               
               
                   
               
               
                   
                 aatccgtgccttcgtgagcgtgaataaaggcctggtaacccgcgcaagcgccgt 
               
               
                   
               
               
                   
                 aatcccggtaatccctctgtatctcgccagcttgttcaaagtaatgaaagagaa 
               
               
                   
               
               
                   
                 gggcaatcatgaaggttgtattgaacagatcacgcgtctgtacgccgagcgcct 
               
               
                   
               
               
                   
                 gtaccgtaaagatggtacaattccagttgatgaggaaaatcgcattcgcattga 
               
               
                   
               
               
                   
                 tgattgggagttagaagaagacgtccagaaagcggtatccgcgttgatggagaa 
               
               
                   
               
               
                   
                 agtcacgggtgaaaacgcagaatctctcactgacttagcggggtaccgccatga 
               
               
                   
               
               
                   
                 tttcttagctagtaacggctttgatgtagaaggtattaattatgaagcggaagt 
               
               
                   
               
               
                   
                 tgaacgcttcgaccgtatctgataagaaggagatatacatatgagagaagtagt 
               
               
                   
               
               
                   
                 aattgccagtgcagctagaacagcagtaggaagttttggaggagcatttaaatc 
               
               
                   
               
               
                   
                 agtttcagcggtagagttaggggtaacagcagctaaagaagctataaaaagagc 
               
               
                   
               
               
                   
                 taacataactccagatatgatagatgaatctcttttagggggagtacttacagc 
               
               
                   
               
               
                   
                 aggtcttggacaaaatatagcaagacaaatagcattaggagcaggaataccagt 
               
               
                   
               
               
                   
                 agaaaaaccagctatgactataaatatagtttgtggttctggattaagatctgt 
               
               
                   
               
               
                   
                 ttcaatggcatctcaacttatagcattaggtgatgctgatataatgttagttgg 
               
               
                   
               
               
                   
                 tggagctgaaaacatgagtatgtctccttatttagtaccaagtgcgagatatgg 
               
               
                   
               
               
                   
                 tgcaagaatgggtgatgctgcttttgttgattcaatgataaaagatggattatc 
               
               
                   
               
               
                   
                 agacatatttaataactatcacatgggtattactgctgaaaacatagcagagca 
               
               
                   
               
               
                   
                 atggaatataactagagaagaacaagatgaattagctcttgcaagtcaaaataa 
               
               
                   
               
               
                   
                 agctgaaaaagctcaagctgaaggaaaatttgatgaagaaatagttcctgttgt 
               
               
                   
               
               
                   
                 tataaaaggaagaaaaggtgacactgtagtagataaagatgaatatattaagcc 
               
               
                   
               
               
                   
                 tggcactacaatggagaaacttgctaagttaagacctgcatttaaaaaagatgg 
               
               
                   
               
               
                   
                 aacagttactgctggtaatgcatcaggaataaatgatggtgctgctatgttagt 
               
               
                   
               
               
                   
                 agtaatggctaaagaaaaagctgaagaactaggaatagagcctcttgcaactat 
               
               
                   
               
               
                   
                 agtttcttatggaacagctggtgttgaccctaaaataatgggatatggaccagt 
               
               
                   
               
               
                   
                 tccagcaactaaaaaagctttagaagctgctaatatgactattgaagatataga 
               
               
                   
               
               
                   
                 tttagttgaagctaatgaggcatttgctgcccaatctgtagctgtaataagaga 
               
               
                   
               
               
                   
                 cttaaatatagatatgaataaagttaatgttaatggtggagcaatagctatagg 
               
               
                   
               
               
                   
                 acatccaataggatgctcaggagcaagaatacttactacacttttatatgaaat 
               
               
                   
               
               
                   
                 gaagagaagagatgctaaaactggtcttgctacactttgtataggcggtggaat 
               
               
                   
               
               
                   
                 gggaactactttaatagttaagagatagtaagaaggagatatacatatgaaatt 
               
               
                   
               
               
                   
                 agctgtaataggtagtggaactatgggaagtggtattgtacaaacttttgcaag 
               
               
                   
               
               
                   
                 ttgtggacatgatgtatgtttaaagagtagaactcaaggtgctatagataaatg 
               
               
                   
               
               
                   
                 tttagctttattagataaaaatttaactaagttagttactaagggaaaaatgga 
               
               
                   
               
               
                   
                 tgaagctacaaaagcagaaatattaagtcatgttagttcaactactaattatga 
               
               
                   
               
               
                   
                 agatttaaaagatatggatttaataatagaagcatctgtagaagacatgaatat 
               
               
                   
               
               
                   
                 aaagaaagatgttttcaagttactagatgaattatgtaaagaagatactatctt 
               
               
                   
               
               
                   
                 ggcaacaaatacttcatcattatctataacagaaatagcttcttctactaagcg 
               
               
                   
               
               
                   
                 cccagataaagttataggaatgcatttctttaatccagttcctatgatgaaatt 
               
               
                   
               
               
                   
                 agttgaagttataagtggtcagttaacatcaaaagttacttttgatacagtatt 
               
               
                   
               
               
                   
                 tgaattatctaagagtatcaataaagtaccagtagatgtatctgaatctcctgg 
               
               
                   
               
               
                   
                 atttgtagtaaatagaatacttatacctatgataaatgaagctgttggtatata 
               
               
                   
               
               
                   
                 tgcagatggtgttgcaagtaaagaagaaatagatgaagctatgaaattaggagc 
               
               
                   
               
               
                   
                 aaaccatccaatgggaccactagcattaggtgatttaatcggattagatgttgt 
               
               
                   
               
               
                   
                 tttagctataatgaacgttttatatactgaatttggagatactaaatatagacc 
               
               
                   
               
               
                   
                 tcatccacttttagctaaaatggttagagctaatcaattaggaagaaaaactaa 
               
               
                   
               
               
                   
                 gataggattctatgattataataaataataagaaggagatatacatatgagtac 
               
               
                   
               
               
                   
                 aagtgatgttaaagtttatgagaatgtagctgttgaagtagatggaaatatatg 
               
               
                   
               
               
                   
                 tacagtgaaaatgaatagacctaaagcccttaatgcaataaattcaaagacttt 
               
               
                   
               
               
                   
                 agaagaactttatgaagtatttgtagatattaataatgatgaaactattgatgt 
               
               
                   
               
               
                   
                 tgtaatattgacaggggaaggaaaggcatttgtagctggagcagatattgcata 
               
               
                   
               
               
                   
                 catgaaagatttagatgctgtagctgctaaagattttagtatcttaggagcaaa 
               
               
                   
               
               
                   
                 agcttttggagaaatagaaaatagtaaaaaagtagtgatagctgctgtaaacgg 
               
               
                   
               
               
                   
                 atttgctttaggtggaggatgtgaacttgcaatggcatgtgatataagaattgc 
               
               
                   
               
               
                   
                 atctgctaaagctaaatttggtcagccagaagtaactcttggaataactccagg 
               
               
                   
               
               
                   
                 atatggaggaactcaaaggcttacaagattggttggaatggcaaaagcaaaaga 
               
               
                   
               
               
                   
                 attaatctttacaggtcaagttataaaagctgatgaagctgaaaaaatagggct 
               
               
                   
               
               
                   
                 agtaaatagagtcgttgagccagacattttaatagaagaagttgagaaattagc 
               
               
                   
               
               
                   
                 taagataatagctaaaaatgctcagcttgcagttagatactctaaagaagcaat 
               
               
                   
               
               
                   
                 acaacttggtgctcaaactgatataaatactggaatagatatagaatctaattt 
               
               
                   
               
               
                   
                 atttggtctttgtttttcaactaaagaccaaaaagaaggaatgtcagctttcgt 
               
               
                   
               
               
                   
                 tgaaaagagagaagctaactttataaaagggtaataagaaggagatatacatat 
               
               
                   
               
               
                   
                 gagaagttttgaagaagtaattaagtttgcaaaagaaagaggacctaaaactat 
               
               
                   
               
               
                   
                 atcagtagcatgttgccaagataaagaagttttaatggcagttgaaatggctag 
               
               
                   
               
               
                   
                 aaaagaaaaaatagcaaatgccattttagtaggagatatagaaaagactaaaga 
               
               
                   
               
               
                   
                 aattgcaaaaagcatagacatggatatcgaaaattatgaactgatagatataaa 
               
               
                   
               
               
                   
                 agatttagcagaagcatctctaaaatctgttgaattagtttcacaaggaaaagc 
               
               
                   
               
               
                   
                 cgacatggtaatgaaaggcttagtagacacatcaataatactaaaagcagtttt 
               
               
                   
               
               
                   
                 aaataaagaagtaggtcttagaactggaaatgtattaagtcacgtagcagtatt 
               
               
                   
               
               
                   
                 tgatgtagagggatatgatagattatttttcgtaactgacgcagctatgaactt 
               
               
                   
               
               
                   
                 agctcctgatacaaatactaaaaagcaaatcatagaaaatgcttgcacagtagc 
               
               
                   
               
               
                   
                 acattcattagatataagtgaaccaaaagttgctgcaatatgcgcaaaagaaaa 
               
               
                   
               
               
                   
                 agtaaatccaaaaatgaaagatacagttgaagctaaagaactagaagaaatgta 
               
               
                   
               
               
                   
                 tgaaagaggagaaatcaaaggttgtatggttggtgggccttttgcaattgataa 
               
               
                   
               
               
                   
                 tgcagtatctttagaagcagctaaacataaaggtataaatcatcctgtagcagg 
               
               
                   
               
               
                   
                 acgagctgatatattattagccccagatattgaaggtggtaacatattatataa 
               
               
                   
               
               
                   
                 agctttggtattcttctcaaaatcaaaaaatgcaggagttatagttggggctaa 
               
               
                   
               
               
                   
                 agcaccaataatattaacttctagagcagacagtgaagaaactaaactaaactc 
               
               
                   
               
               
                   
                 aatagctttaggtgttttaatggcagcaaaggcataataagaaggagatataca 
               
               
                   
               
               
                   
                 tatgagcaaaatatttaaaatcttaacaataaatcctggttcgacatcaactaa 
               
               
                   
               
               
                   
                 aatagctgtatttgataatgaggatttagtatttgaaaaaactttaagacattc 
               
               
                   
               
               
                   
                 ttcagaagaaataggaaaatatgagaaggtgtctgaccaatttgaatttcgtaa 
               
               
                   
               
               
                   
                 acaagtaatagaagaagctctaaaagaaggtggagtaaaaacatctgaattaga 
               
               
                   
               
               
                   
                 tgctgtagtaggtagaggaggacttcttaaacctataaaaggtggtacttattc 
               
               
                   
               
               
                   
                 agtaagtgctgctatgattgaagatttaaaagtgggagttttaggagaacacgc 
               
               
                   
               
               
                   
                 ttcaaacctaggtggaataatagcaaaacaaataggtgaagaagtaaatgttcc 
               
               
                   
               
               
                   
                 ttcatacatagtagaccctgttgttgtagatgaattagaagatgttgctagaat 
               
               
                   
               
               
                   
                 ttctggtatgcctgaaataagtagagcaagtgtagtacatgctttaaatcaaaa 
               
               
                   
               
               
                   
                 ggcaatagcaagaagatatgctagagaaataaacaagaaatatgaagatataaa 
               
               
                   
               
               
                   
                 tcttatagttgcacacatgggtggaggagtttctgttggagctcataaaaatgg 
               
               
                   
               
               
                   
                 taaaatagtagatgttgcaaacgcattagatggagaaggacctttctctccaga 
               
               
                   
               
               
                   
                 aagaagtggtggactaccagtaggtgcattagtaaaaatgtgctttagtggaaa 
               
               
                   
               
               
                   
                 atatactcaagatgaaattaaaaagaaaataaaaggtaatggcggactagttgc 
               
               
                   
               
               
                   
                 atacttaaacactaatgatgctagagaagttgaagaaagaattgaagctggtga 
               
               
                   
               
               
                   
                 tgaaaaagctaaattagtatatgaagctatggcatatcaaatctctaaagaaat 
               
               
                   
               
               
                   
                 aggagctagtgctgcagttcttaagggagatgtaaaagcaatattattaactgg 
               
               
                   
               
               
                   
                 tggaatcgcatattcaaaaatgtttacagaaatgattgcagatagagttaaatt 
               
               
                   
               
               
                   
                 tatagcagatgtaaaagtttatccaggtgaagatgaaatgattgcattagctca 
               
               
                   
               
               
                   
                 aggtggacttagagttttaactggtgaagaagaggctcaagtttatgataacta 
               
               
                   
               
               
                   
                 ataa 
               
               
                   
               
            
           
         
       
     
     In certain constructs, the butyrate gene cassette is placed under the control of a tetracycline-inducible or constitutive promoter. 
     In a third butyrate gene cassette, the pbt and buk genes are replaced with tesB. TesB is a thioesterase found in  E. Coli  that cleaves off the butyrate from butyryl-coA, thus obviating the need for pbt-buk. 
     In one embodiment, the tesB cassette is placed under the control of a FNR regulatory region selected from any of the sequences in Table 6. In an alternate embodiment, the tesB 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. In yet another embodiment, the tesB cassette 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. In certain constructs, the different described butyrate gene cassettes are each placed under the control of a tetracycline-inducible or constitutive promoter. For example, genetically engineered Nissle are generated comprising a butyrate gene cassette in which the pbt and buk genes are replaced with tesB expressed under the control of a nitric oxide-responsive regulatory element. SEQ ID NO: 207 comprises a reverse complement of the nsrR repressor gene from  Neisseria gonorrhoeae  (underlined), intergenic region containing divergent promoters controlling nsrR and the butyrogenic gene cassette and their respective RBS (bold), and the butyrate genes (ter-thiA-hbd-crt-tesB) separated by RBS. 
     
       
         
           
               
             
               
                 TABLE 36 
               
               
                   
               
               
                 SEQ ID NO: 207 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 SEQ ID NO: 207 
               
               
                 ttatta tcgcaccgcaatcgggattttcgattcataaagcaggtcgtaggtcggctt   
               
               
                   
               
               
                 
                   gttgagcaggtcttgcagcgtgaaaccgtccagatacgtgaaaaacgacttcattgc 
                 
               
               
                   
               
               
                 
                   accgccgagtatgcccgtcagccggcaggacggcgtaatcaggcattcgttgttcgg 
                 
               
               
                   
               
               
                 
                   gcccatacactcgaccagctgcatcggttcgaggtggcggacgaccgcgccgatatt 
                 
               
               
                   
               
               
                 
                   gatgcgttcgggcggcgcggccagcctcagcccgccgcctttcccgcgtacgctgtg 
                 
               
               
                   
               
               
                 
                   caagaacccgcctttgaccagcgcggtaaccactttcatcaaatggcttttggaaat 
                 
               
               
                   
               
               
                 
                   gccgtaggtcgaggcgatggtggcgatattgaccagcgcgtcgtcgttgacggcggt 
                 
               
               
                   
               
               
                 
                   gtagatgaggacgcgcagcccgtagtcggtatgttgggtcagatacat 
                   acaacctcc 
                 
               
               
                   
               
               
                 
                   ttagtacatgcaaaattatttctagagcaacatacgagccggaagcataaagtgtaa 
                 
               
               
                   
               
               
                 
                   agcctggggtgcctaatgagttgagttgaggaattataacaggaagaaatattcctc 
                 
               
               
                   
               
               
                 
                   atacgcttgtaattcctctatggttgttgacaattaatcatcggctcgtataatgta 
                 
               
               
                   
               
               
                 
                   taacattcatattttgtgaattttaaactctagaaataattttgtttaactttaaga 
                 
               
               
                   
               
               
                   aggagatatacat atgatcgtaaaacctatggtacgcaacaatatctgcctgaacgc 
               
               
                   
               
               
                 ccatcctcagggctgcaagaagggagtggaagatcagattgaatataccaagaaacg 
               
               
                   
               
               
                 cattaccgcagaagtcaaagctggcgcaaaagctccaaaaaacgttctggtgcttgg 
               
               
                   
               
               
                 ctgctcaaatggttacggcctggcgagccgcattactgctgcgttcggatacggggc 
               
               
                   
               
               
                 tgcgaccatcggcgtgtcctttgaaaaagcgggttcagaaaccaaatatggtacacc 
               
               
                   
               
               
                 gggatggtacaataatttggcatttgatgaagcggcaaaacgcgagggtctttatag 
               
               
                   
               
               
                 cgtgacgatcgacggcgatgcgttttcagacgagatcaaggcccaggtaattgagga 
               
               
                   
               
               
                 agccaaaaaaaaaggtatcaaatttgatctgatcgtatacagcttggccagcccagt 
               
               
                   
               
               
                 acgtactgatcctgatacaggtatcatgcacaaaagcgttttgaaaccctttggaaa 
               
               
                   
               
               
                 aacgttcacaggcaaaacagtagatccgtttactggcgagctgaaggaaatctccgc 
               
               
                   
               
               
                 ggaaccagcaaatgacgaggaagcagccgccactgttaaagttatggggggtgaaga 
               
               
                   
               
               
                 ttgggaacgttggattaagcagctgtcgaaggaaggcctcttagaagaaggctgtat 
               
               
                   
               
               
                 taccttggcctatagttatattggccctgaagctacccaagctttgtaccgtaaagg 
               
               
                   
               
               
                 cacaatcggcaaggccaaagaacacctggaggccacagcacaccgtctcaacaaaga 
               
               
                   
               
               
                 gaacccgtcaatccgtgccttcgtgagcgtgaataaaggcctggtaacccgcgcaag 
               
               
                   
               
               
                 cgccgtaatcccggtaatccctctgtatctcgccagcttgttcaaagtaatgaaaga 
               
               
                   
               
               
                 gaagggcaatcatgaaggttgtattgaacagatcacgcgtctgtacgccgagcgcct 
               
               
                   
               
               
                 gtaccgtaaagatggtacaattccagttgatgaggaaaatcgcattcgcattgatga 
               
               
                   
               
               
                 ttgggagttagaagaagacgtccagaaagcggtatccgcgttgatggagaaagtcac 
               
               
                   
               
               
                 gggtgaaaacgcagaatctctcactgacttagcggggtaccgccatgatttcttagc 
               
               
                   
               
               
                 tagtaacggctttgatgtagaaggtattaattatgaagcggaagttgaacgcttcga 
               
               
                   
               
               
                 ccgtatctgataagaaggagatatacatatgagagaagtagtaattgccagtgcagc 
               
               
                   
               
               
                 tagaacagcagtaggaagttttggaggagcatttaaatcagtttcagcggtagagtt 
               
               
                   
               
               
                 aggggtaacagcagctaaagaagctataaaaagagctaacataactccagatatgat 
               
               
                   
               
               
                 agatgaatctcttttagggggagtacttacagcaggtcttggacaaaatatagcaag 
               
               
                   
               
               
                 acaaatagcattaggagcaggaataccagtagaaaaaccagctatgactataaatat 
               
               
                   
               
               
                 agtttgtggttctggattaagatctgtttcaatggcatctcaacttatagcattagg 
               
               
                   
               
               
                 tgatgctgatataatgttagttggtggagctgaaaacatgagtatgtctccttattt 
               
               
                   
               
               
                 agtaccaagtgcgagatatggtgcaagaatgggtgatgctgcttttgttgattcaat 
               
               
                   
               
               
                 gataaaagatggattatcagacatatttaataactatcacatgggtattactgctga 
               
               
                   
               
               
                 aaacatagcagagcaatggaatataactagagaagaacaagatgaattagctcttgc 
               
               
                   
               
               
                 aagtcaaaataaagctgaaaaagctcaagctgaaggaaaatttgatgaagaaatagt 
               
               
                   
               
               
                 tcctgttgttataaaaggaagaaaaggtgacactgtagtagataaagatgaatatat 
               
               
                   
               
               
                 taagcctggcactacaatggagaaacttgctaagttaagacctgcatttaaaaaaga 
               
               
                   
               
               
                 tggaacagttactgctggtaatgcatcaggaataaatgatggtgctgctatgttagt 
               
               
                   
               
               
                 agtaatggctaaagaaaaagctgaagaactaggaatagagcctcttgcaactatagt 
               
               
                   
               
               
                 ttcttatggaacagctggtgttgaccctaaaataatgggatatggaccagttccagc 
               
               
                   
               
               
                 aactaaaaaagctttagaagctgctaatatgactattgaagatatagatttagttga 
               
               
                   
               
               
                 agctaatgaggcatttgctgcccaatctgtagctgtaataagagacttaaatataga 
               
               
                   
               
               
                 tatgaataaagttaatgttaatggtggagcaatagctataggacatccaataggatg 
               
               
                   
               
               
                 ctcaggagcaagaatacttactacacttttatatgaaatgaagagaagagatgctaa 
               
               
                   
               
               
                 aactggtcttgctacactttgtataggcggtggaatgggaactactttaatagttaa 
               
               
                   
               
               
                 gagatagtaagaaggagatatacatatgaaattagctgtaataggtagtggaactat 
               
               
                   
               
               
                 gggaagtggtattgtacaaacttttgcaagttgtggacatgatgtatgtttaaagag 
               
               
                   
               
               
                 tagaactcaaggtgctatagataaatgtttagctttattagataaaaatttaactaa 
               
               
                   
               
               
                 gttagttactaagggaaaaatggatgaagctacaaaagcagaaatattaagtcatgt 
               
               
                   
               
               
                 tagttcaactactaattatgaagatttaaaagatatggatttaataatagaagcatc 
               
               
                   
               
               
                 tgtagaagacatgaatataaagaaagatgttttcaagttactagatgaattatgtaa 
               
               
                   
               
               
                 agaagatactatcttggcaacaaatacttcatcattatctataacagaaatagcttc 
               
               
                   
               
               
                 ttctactaagcgcccagataaagttataggaatgcatttctttaatccagttcctat 
               
               
                   
               
               
                 gatgaaattagttgaagttataagtggtcagttaacatcaaaagttacttttgatac 
               
               
                   
               
               
                 agtatttgaattatctaagagtatcaataaagtaccagtagatgtatctgaatctcc 
               
               
                   
               
               
                 tggatttgtagtaaatagaatacttatacctatgataaatgaagctgttggtatata 
               
               
                   
               
               
                 tgcagatggtgttgcaagtaaagaagaaatagatgaagctatgaaattaggagcaaa 
               
               
                   
               
               
                 ccatccaatgggaccactagcattaggtgatttaatcggattagatgttgttttagc 
               
               
                   
               
               
                 tataatgaacgttttatatactgaatttggagatactaaatatagacctcatccact 
               
               
                   
               
               
                 tttagctaaaatggttagagctaatcaattaggaagaaaaactaagataggattcta 
               
               
                   
               
               
                 tgattataataaataataagaaggagatatacatatgagtacaagtgatgttaaagt 
               
               
                   
               
               
                 ttatgagaatgtagctgttgaagtagatggaaatatatgtacagtgaaaatgaatag 
               
               
                   
               
               
                 acctaaagcccttaatgcaataaattcaaagactttagaagaactttatgaagtatt 
               
               
                   
               
               
                 tgtagatattaataatgatgaaactattgatgttgtaatattgacaggggaaggaaa 
               
               
                   
               
               
                 ggcatttgtagctggagcagatattgcatacatgaaagatttagatgctgtagctgc 
               
               
                   
               
               
                 taaagattttagtatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaa 
               
               
                   
               
               
                 agtagtgatagctgctgtaaacggatttgctttaggtggaggatgtgaacttgcaat 
               
               
                   
               
               
                 ggcatgtgatataagaattgcatctgctaaagctaaatttggtcagccagaagtaac 
               
               
                   
               
               
                 tcttggaataactccaggatatggaggaactcaaaggcttacaagattggttggaat 
               
               
                   
               
               
                 ggcaaaagcaaaagaattaatctttacaggtcaagttataaaagctgatgaagctga 
               
               
                   
               
               
                 aaaaatagggctagtaaatagagtcgttgagccagacattttaatagaagaagttga 
               
               
                   
               
               
                 gaaattagctaagataatagctaaaaatgctcagcttgcagttagatactctaaaga 
               
               
                   
               
               
                 agcaatacaacttggtgctcaaactgatataaatactggaatagatatagaatctaa 
               
               
                   
               
               
                 tttatttggtctttgtttttcaactaaagaccaaaaagaaggaatgtcagctttcgt 
               
               
                   
               
               
                 tgaaaagagagaagctaactttataaaagggtaataagaaggagatatacatatgAG 
               
               
                   
               
               
                 TCAGGCGCTAAAAAATTTACTGACATTGTTAAATCTGGAAAAAATTGAGGAAGGACT 
               
               
                   
               
               
                 CTTTCGCGGCCAGAGTGAAGATTTAGGTTTACGCCAGGTGTTTGGCGGCCAGGTCGT 
               
               
                   
               
               
                 GGGTCAGGCCTTGTATGCTGCAAAAGAGACCGTCCCTGAAGAGCGGCTGGTACATTC 
               
               
                   
               
               
                 GTTTCACAGCTACTTTCTTCGCCCTGGCGATAGTAAGAAGCCGATTATTTATGATGT 
               
               
                   
               
               
                 CGAAACGCTGCGTGACGGTAACAGCTTCAGCGCCCGCCGGGTTGCTGCTATTCAAAA 
               
               
                   
               
               
                 CGGCAAACCGATTTTTTATATGACTGCCTCTTTCCAGGCACCAGAAGCGGGTTTCGA 
               
               
                   
               
               
                 ACATCAAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCCTCCCTTCGGAAACGCA 
               
               
                   
               
               
                 AATCGCCCAATCGCTGGCGCACCTGCTGCCGCCAGTGCTGAAAGATAAATTCATCTG 
               
               
                   
               
               
                 CGATCGTCCGCTGGAAGTCCGTCCGGTGGAGTTTCATAACCCACTGAAAGGTCACGT 
               
               
                   
               
               
                 CGCAGAACCACATCGTCAGGTGTGGATCCGCGCAAATGGTAGCGTGCCGGATGACCT 
               
               
                   
               
               
                 GCGCGTTCATCAGTATCTGCTCGGTTACGCTTCTGATCTTAACTTCCTGCCGGTAGC 
               
               
                   
               
               
                 TCTACAGCCGCACGGCATCGGTTTTCTCGAACCGGGGATTCAGATTGCCACCATTGA 
               
               
                   
               
               
                 CCATTCCATGTGGTTCCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATAGCGT 
               
               
                   
               
               
                 GGAGAGCACCTCGGCGTCCAGCGCACGTGGCTTTGTGCGCGGTGAGTTTTATACCCA 
               
               
                   
               
               
                 AGACGGCGTACTGGTTGCCTCGACCGTTCAGGAAGGGGTGATGCGTAATCACAATta 
               
               
                   
               
               
                 a 
               
               
                   
               
            
           
         
       
     
     Example 11. Construction of Vectors for Overproducing Butyrate Using a Tet-Inducible Promoter 
     To facilitate inducible production of butyrate in  Escherichia coli  Nissle, the eight genes of the butyrate production pathway from  Peptoclostridium difficile  (bed, etfB, etfA, thiA, hbd, crt, bpt, and buk; NCBI), as well as transcriptional and translational elements, were synthesized (Gen, Cambridge, Mass.) and cloned into vector pBR to create pLogic. As synthesized, the genes were placed under control of a tetracycline-inducible promoter, with the tet repressor (tetR) expressed constitutively, divergent from the tet-inducible synthetic butyrate operon. For efficient translation of butyrate genes, each synthetic gene in the operon was separated by a base pair ribosome binding site derived from the T promoter. 
     The gene products of bcd-etfA-etfB form a complex that convert crotonyl-CoA to butyryl-CoA, and may show some dependence on oxygen as a co-oxidant. Because an effective probiotic should be able to function in an oxygen-limited environment (e.g. the mammalian gut), and because it has been shown that a single gene from  Treponema denticola  can functionally replace this three gene complex in an oxygen-independent manner (trans—enoynl-CoA reductase; ter), we created a second plasmid capable of butyrate production in  E. coli . Inverse PCR was used to amplify the entire sequence of pLogic outside of the bcd-etfA-etfB region. The ter gene was codon optimized for  E. coli  codon usage using Integrated DNA technologies online codon optimization tool (https://www.idtdna.com/CodonOpt), synthesized (Genewiz, Cambridge, Mass.), and cloned into this inverse PCR fragment using Gibson assembly to create pLogic. 
     Example 12. 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. 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 13. Production of Butyrate in Recombinant  E. coli    
     Production of butyrate is assessed in  E. coli  Nissle strains containing the butyrate cassettes described above 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% N2, 5% CO2, 5% H2). 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 hrs) 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. 
     Example 14. Production of Butyrate in Recombinant  E. coli    
     Production of butyrate is assessed in  E. coli  Nissle strains containing the butyrate cassettes described above 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% N2, 5% CO2, 5% H2). 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 hrs) 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. 
     Example 15. Production of Butyrate in Recombinant  E. coli  Using Tet-Inducible Promoter 
       FIG. 2  shows butyrate cassettes described above under the control of a tet-inducible promoter. Production of butyrate is assessed using the methods described below in Example 22. 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-butyarte cassette in which tesB is substituted in place of pbt and buk genes. 
       FIG. 6A  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. 6B  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. 7  shows the BW25113 strain of  E. Coli , which is a common cloning strain and the background of the KEIO collection of  E. Coli  mutants. NuoB mutants having NuoB deletion were obtained. 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.  FIG. 7  shows that compared with wild-type Nissle, deletion of NuoB results in grater production of butyrate. 
     
       
         
           
               
             
               
                 TABLE 37 
               
               
                   
               
               
                 pLOGIC046-tesB-butyrate 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 pLOGIC046-tesB-butyrate: SEQ ID NO: 208 
               
               
                 gtaaaacgacggccagtgaattcgttaagacccactttcacatttaagttgtttttctaatccgcatatgatcaa 
               
               
                   
               
               
                 ttcaaggccgaataagaaggctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaataatggcgg 
               
               
                   
               
               
                 catactatcagtagtaggtgtttccctttcttctttagcgacttgatgctcttgatcttccaatacgcaacctaa 
               
               
                   
               
               
                 agtaaaatgccccacagcgctgagtgcatataatgcattctctagtgaaaaaccttgttggcataaaaaggctaa 
               
               
                   
               
               
                 ttgattttcgagagtttcatactgtttttctgtaggccgtgtacctaaatgtacttttgctccatcgcgatgact 
               
               
                   
               
               
                 tagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgccagctttccccttctaaagggcaaaa 
               
               
                   
               
               
                 gtgagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagcccgcttattttttacatgccaata 
               
               
                   
               
               
                 caatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaaccttcgattccgacctcattaag 
               
               
                   
               
               
                 cagctctaatgcgctgttaatcactttacttttatctaatctagacatcattaattcctaatttttgttgacact 
               
               
                   
               
               
                 ctatcattgatagagttattttaccactccctatcagtgatagagaaaagtgaactctagaaataattttgttta 
               
               
                   
               
               
                 actttaagaaggagatatacatatgatcgtaaaacctatggtacgcaacaatatctgcctgaacgcccatcctca 
               
               
                   
               
               
                 gggctgcaagaagggagtggaagatcagattgaatataccaagaaacgcattaccgcagaagtcaaagctggcgc 
               
               
                   
               
               
                 aaaagctccaaaaaacgttctggtgcttggctgctcaaatggttacggcctggcgagccgcattactgctgcgtt 
               
               
                   
               
               
                 cggatacggggctgcgaccatcggcgtgtcctttgaaaaagcgggttcagaaaccaaatatggtacaccgggatg 
               
               
                   
               
               
                 gtacaataatttggcatttgatgaagcggcaaaacgcgagggtctttatagcgtgacgatcgacggcgatgcgtt 
               
               
                   
               
               
                 ttcagacgagatcaaggcccaggtaattgaggaagccaaaaaaaaaggtatcaaatttgatctgatcgtatacag 
               
               
                   
               
               
                 cttggccagcccagtacgtactgatcctgatacaggtatcatgcacaaaagcgttttgaaaccctttggaaaaac 
               
               
                   
               
               
                 gttcacaggcaaaacagtagatccgtttactggcgagctgaaggaaatctccgcggaaccagcaaatgacgagga 
               
               
                   
               
               
                 agcagccgccactgttaaagttatggggggtgaagattgggaacgttggattaagcagctgtcgaaggaaggcct 
               
               
                   
               
               
                 cttagaagaaggctgtattaccttggcctatagttatattggccctgaagctacccaagctttgtaccgtaaagg 
               
               
                   
               
               
                 cacaatcggcaaggccaaagaacacctggaggccacagcacaccgtctcaacaaagagaacccgtcaatccgtgc 
               
               
                   
               
               
                 cttcgtgagcgtgaataaaggcctggtaacccgcgcaagcgccgtaatcccggtaatccctctgtatctcgccag 
               
               
                   
               
               
                 cttgttcaaagtaatgaaagagaagggcaatcatgaaggttgtattgaacagatcacgcgtctgtacgccgagcg 
               
               
                   
               
               
                 cctgtaccgtaaagatggtacaattccagttgatgaggaaaatcgcattcgcattgatgattgggagttagaaga 
               
               
                   
               
               
                 agacgtccagaaagcggtatccgcgttgatggagaaagtcacgggtgaaaacgcagaatctctcactgacttagc 
               
               
                   
               
               
                 ggggtaccgccatgatttcttagctagtaacggctttgatgtagaaggtattaattatgaagcggaagttgaacg 
               
               
                   
               
               
                 cttcgaccgtatctgataagaaggagatatacatatgagagaagtagtaattgccagtgcagctagaacagcagt 
               
               
                   
               
               
                 aggaagttttggaggagcatttaaatcagtttcagcggtagagttaggggtaacagcagctaaagaagctataaa 
               
               
                   
               
               
                 aagagctaacataactccagatatgatagatgaatctcttttagggggagtacttacagcaggtcttggacaaaa 
               
               
                   
               
               
                 tatagcaagacaaatagcattaggagcaggaataccagtagaaaaaccagctatgactataaatatagtttgtgg 
               
               
                   
               
               
                 ttctggattaagatctgtttcaatggcatctcaacttatagcattaggtgatgctgatataatgttagttggtgg 
               
               
                   
               
               
                 agctgaaaacatgagtatgtctccttatttagtaccaagtgcgagatatggtgcaagaatgggtgatgctgcttt 
               
               
                   
               
               
                 tgttgattcaatgataaaagatggattatcagacatatttaataactatcacatgggtattactgctgaaaacat 
               
               
                   
               
               
                 agcagagcaatggaatataactagagaagaacaagatgaattagctcttgcaagtcaaaataaagctgaaaaagc 
               
               
                   
               
               
                 tcaagctgaaggaaaatttgatgaagaaatagttcctgttgttataaaaggaagaaaaggtgacactgtagtaga 
               
               
                   
               
               
                 taaagatgaatatattaagcctggcactacaatggagaaacttgctaagttaagacctgcatttaaaaaagatgg 
               
               
                   
               
               
                 aacagttactgctggtaatgcatcaggaataaatgatggtgctgctatgttagtagtaatggctaaagaaaaagc 
               
               
                   
               
               
                 tgaagaactaggaatagagcctcttgcaactatagtttcttatggaacagctggtgttgaccctaaaataatggg 
               
               
                   
               
               
                 atatggaccagttccagcaactaaaaaagctttagaagctgctaatatgactattgaagatatagatttagttga 
               
               
                   
               
               
                 agctaatgaggcatttgctgcccaatctgtagctgtaataagagacttaaatatagatatgaataaagttaatgt 
               
               
                   
               
               
                 taatggtggagcaatagctataggacatccaataggatgctcaggagcaagaatacttactacacttttatatga 
               
               
                   
               
               
                 aatgaagagaagagatgctaaaactggtcttgctacactttgtataggcggtggaatgggaactactttaatagt 
               
               
                   
               
               
                 taagagatagtaagaaggagatatacatatgaaattagctgtaataggtagtggaactatgggaagtggtattgt 
               
               
                   
               
               
                 acaaacttttgcaagttgtggacatgatgtatgtttaaagagtagaactcaaggtgctatagataaatgtttagc 
               
               
                   
               
               
                 tttattagataaaaatttaactaagttagttactaagggaaaaatggatgaagctacaaaagcagaaatattaag 
               
               
                   
               
               
                 tcatgttagttcaactactaattatgaagatttaaaagatatggatttaataatagaagcatctgtagaagacat 
               
               
                   
               
               
                 gaatataaagaaagatgttttcaagttactagatgaattatgtaaagaagatactatcttggcaacaaatacttc 
               
               
                   
               
               
                 atcattatctataacagaaatagcttcttctactaagcgcccagataaagttataggaatgcatttctttaatcc 
               
               
                   
               
               
                 agttcctatgatgaaattagttgaagttataagtggtcagttaacatcaaaagttacttttgatacagtatttga 
               
               
                   
               
               
                 attatctaagagtatcaataaagtaccagtagatgtatctgaatctcctggatttgtagtaaatagaatacttat 
               
               
                   
               
               
                 acctatgataaatgaagctgttggtatatatgcagatggtgttgcaagtaaagaagaaatagatgaagctatgaa 
               
               
                   
               
               
                 attaggagcaaaccatccaatgggaccactagcattaggtgatttaatcggattagatgttgttttagctataat 
               
               
                   
               
               
                 gaacgttttatatactgaatttggagatactaaatatagacctcatccacttttagctaaaatggttagagctaa 
               
               
                   
               
               
                 tcaattaggaagaaaaactaagataggattctatgattataataaataataagaaggagatatacatatgagtac 
               
               
                   
               
               
                 aagtgatgttaaagtttatgagaatgtagctgttgaagtagatggaaatatatgtacagtgaaaatgaatagacc 
               
               
                   
               
               
                 taaagcccttaatgcaataaattcaaagactttagaagaactttatgaagtatttgtagatattaataatgatga 
               
               
                   
               
               
                 aactattgatgttgtaatattgacaggggaaggaaaggcatttgtagctggagcagatattgcatacatgaaaga 
               
               
                   
               
               
                 tttagatgctgtagctgctaaagattttagtatcttaggagcaaaagcttttggagaaatagaaaatagtaaaaa 
               
               
                   
               
               
                 agtagtgatagctgctgtaaacggatttgctttaggtggaggatgtgaacttgcaatggcatgtgatataagaat 
               
               
                   
               
               
                 tgcatctgctaaagctaaatttggtcagccagaagtaactcttggaataactccaggatatggaggaactcaaag 
               
               
                   
               
               
                 gcttacaagattggttggaatggcaaaagcaaaagaattaatctttacaggtcaagttataaaagctgatgaagc 
               
               
                   
               
               
                 tgaaaaaatagggctagtaaatagagtcgttgagccagacattttaatagaagaagttgagaaattagctaagat 
               
               
                   
               
               
                 aatagctaaaaatgctcagcttgcagttagatactctaaagaagcaatacaacttggtgctcaaactgatataaa 
               
               
                   
               
               
                 tactggaatagatatagaatctaatttatttggtctttgtttttcaactaaagaccaaaaagaaggaatgtcagc 
               
               
                   
               
               
                 tttcgttgaaaagagagaagctaactttataaaagggtaataagaaggagatatacatatgAGTCAGGCGCTAAA 
               
               
                   
               
               
                 AAATTTACTGACATTGTTAAATCTGGAAAAAATTGAGGAAGGACTCTTTCGCGGCCAGAGTGAAGATTTAGGTTT 
               
               
                   
               
               
                 ACGCCAGGTGTTTGGCGGCCAGGTCGTGGGTCAGGCCTTGTATGCTGCAAAAGAGACCGTCCCTGAAGAGCGGCT 
               
               
                   
               
               
                 GGTACATTCGTTTCACAGCTACTTTCTTCGCCCTGGCGATAGTAAGAAGCCGATTATTTATGATGTCGAAACGCT 
               
               
                   
               
               
                 GCGTGACGGTAACAGCTTCAGCGCCCGCCGGGTTGCTGCTATTCAAAACGGCAAACCGATTTTTTATATGACTGC 
               
               
                   
               
               
                 CTCTTTCCAGGCACCAGAAGCGGGTTTCGAACATCAAAAAACAATGCCGTCCGCGCCAGCGCCTGATGGCCTCCC 
               
               
                   
               
               
                 TTCGGAAACGCAAATCGCCCAATCGCTGGCGCACCTGCTGCCGCCAGTGCTGAAAGATAAATTCATCTGCGATCG 
               
               
                   
               
               
                 TCCGCTGGAAGTCCGTCCGGTGGAGTTTCATAACCCACTGAAAGGTCACGTCGCAGAACCACATCGTCAGGTGTG 
               
               
                   
               
               
                 GATCCGCGCAAATGGTAGCGTGCCGGATGACCTGCGCGTTCATCAGTATCTGCTCGGTTACGCTTCTGATCTTAA 
               
               
                   
               
               
                 CTTCCTGCCGGTAGCTCTACAGCCGCACGGCATCGGTTTTCTCGAACCGGGGATTCAGATTGCCACCATTGACCA 
               
               
                   
               
               
                 TTCCATGTGGTTCCATCGCCCGTTTAATTTGAATGAATGGCTGCTGTATAGCGTGGAGAGCACCTCGGCGTCCAG 
               
               
                   
               
               
                 CGCACGTGGCTTTGTGCGCGGTGAGTTTTATACCCAAGACGGCGTACTGGTTGCCTCGACCGTTCAGGAAGGGGT 
               
               
                   
               
               
                 GATGCGTAATCACAATtaa 
               
               
                   
               
            
           
         
       
     
     Example 16. Production of Butyrate in Recombinant  E. coli    
     Production of butyrate is assessed in  E. coli  Nissle strains containing the butyrate cassettes described above 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, 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 to assess levels of butyrate production. Genetically engineered Nissle comprising pLogic031-nsrR-norB-butyrate operon construct; SYN507) or (pLogic046-nsrR-norB-butyrate operon construct; SYN—508) produce significantly more butyrate as compared to wild-type Nissle. 
     Genetically engineered Nissle were generated comprising a butyrate gene cassette in which the pbt and buk genes are replaced with tesB (SEQ ID NO: 15) expressed under the control of a tetracycline promoter (pLOGIC046-tesB-butyrate; SEQ ID NO: 208). SEQ ID NO: 208 comprises 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 (ter-thiA1-hbd-crt2-tesB) separated by RBS. 
     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. Replacement of pbt and buk with tesB leads to greater levels of butyrate production. 
       FIG. 8C  shows butyrate production in strains comprising an FNR-butyrate cassette SYN501 (having the ter substitution) in the presence/absence of glucose and oxygen.  FIG. 8C  shows that bacteria need both glucose and anaerobic conditions for butyrate production from the FNR promoter. Cells were grown aerobically or anaerobically in media containg no glucose (LB) or in media containing glucose at 0.5% (RMC). Culture samples were taken at indicaed 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. 
     
       
         
           
               
             
               
                 TABLE 38 
               
             
            
               
                   
               
               
                 Butyrate cassette sequences 
               
            
           
           
               
               
               
            
               
                 Description 
                 Sequence 
                 SEQ ID NO 
               
               
                   
               
               
                 ydfZ + RBS 
                 CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGAC 
                 SEQ ID NO: 209 
               
               
                 (RBS is bolded) 
                 TTATGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAA 
                   
               
               
                   
                 AACAAAAAATATTTCACTCGACAGGAGTATTTATATTGCGCCCG 
                   
               
               
                   
                 GATCCCTCTAGAAATAA TTTTGTTTAACTTTAAGAAGGAGATAT   
                   
               
               
                   
                 
                   ACAT 
                 
                   
               
               
                   
               
               
                 First RBS (in 
                 TTTGTTTAACTTTAAGAAGGAGA 
                 SEQ ID NO: 210 
               
               
                 ydfZ = RBS) 
                   
                   
               
               
                   
               
               
                 Internal RBS 
                 taagaaggagatatacat 
                 SEQ ID NO: 211 
               
               
                 between genes 
                   
                   
               
               
                   
               
               
                 Butylate cassette 
                 CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGAC 
                 SEQ ID NO: 212 
               
               
                 under the control of 
                 TTATGGCTCATGCATGCATCAAAAAAGATGTGAGCTTGATCAAA 
                   
               
               
                 thd ydfZ promoter 
                 AACAAAAAATATTTCACTCGACAGGAGTATTTATATTGCGCCCG 
                   
               
               
                 (uppercase: ydfZ 
                 GATCCCTCTAGAAATAA TTTTGTTTAACTTTAAGAAGGAGATAT   
                   
               
               
                 promoter, with RBS in 
                   ACAT atgatcgtaaaacctatggtacgcaacaatatctgcctga 
                   
               
               
                 bold; lower case: 
                 acgcccatcctcagggctgcaagaagggagtggaagatcagatt 
                   
               
               
                 coding regions in the 
                 gaatataccaagaaacgcattaccgcagaagtcaaagctggcgc 
                   
               
               
                 following order: ter, 
                 aaaagctccaaaaaacgttctggtgcttggctgctcaaatggtt 
                   
               
               
                 thisA, hbd, crt3, 
                 acggcctggcgagccgcattactgctgcgttcggatacggggct 
                   
               
               
                 pbt, buk, separated 
                 gcgaccatcggcgtgtcctttgaaaaagcgggttcagaaaccaa 
                   
               
               
                 by internal RBS 
                 atatggtacaccgggatggtacaataatttggcatttgatgaag 
                   
               
               
                 (uppercase and 
                 cggcaaaacgcgagggtctttatagcgtgacgatcgacggcgat 
                   
               
               
                 underlined) 
                 gcgttttcagacgagatcaaggcccaggtaattgaggaagccaa 
                   
               
               
                   
                 aaaaaaaggtatcaaatttgatctgatcgtatacagcttggcca 
                   
               
               
                   
                 gcccagtacgtactgatcctgatacaggtatcatgcacaaaagc 
                   
               
               
                   
                 gttttgaaaccchtggaaaaacgttcacaggcaaaacagtagat 
                   
               
               
                   
                 ccgtttactggcgagctgaaggaaatctccgaaatgacgaggaa 
                   
               
               
                   
                 gcagccgccactgttaaagttatggggggtgaagattgggaacg 
                   
               
               
                   
                 ttggattaagcagctgtcgaaggaaggcctcttagaagaaggct 
                   
               
               
                   
                 gtattaccttggcctatagttatattggccctgaagctacccaa 
                   
               
               
                   
                 gctttgtaccgtaaaggcacaatcggcaaggccaaagaacacct 
                   
               
               
                   
                 ggaggccacagcacaccgtctcaacaaagagaacccgtcaatcc 
                   
               
               
                   
                 gtgccttcgtgagcgtgaataaaggcctggtaacccgcgcaagc 
                   
               
               
                   
                 gccgtaatcccggtaatccctctgtatctcgccagcttgttcaa 
                   
               
               
                   
                 agtaatgaaagagaagggcaatcatgaaggttgtattgaacaga 
                   
               
               
                   
                 tcacgcgtctgtacgccgagcgcctgtaccgtaaagatggtaca 
                   
               
               
                   
                 attccagttgatgaggaaaatcgcattcgcattgatgattggga 
                   
               
               
                   
                 gttagaagaagacgtccagaaagcggtatccgcgttgatggaga 
                   
               
               
                   
                 aagtcacgggtgaaaacgcagaatctctcactgacttagcgggg 
                   
               
               
                   
                 taccgccatgatttcttagctagtaacggctttgatgtagaagg 
                   
               
               
                   
                 tattaattatgaagcggaagttgaacgcttcgaccgtatctga T   
                   
               
               
                   
                   AAGAAGGAGATATACAT atgagagaagtagtaattgccagtgca 
                   
               
               
                   
                 gctagaacagcagtaggaagttttggaggagcatttaaatcagt 
                   
               
               
                   
                 ttcagcggtagagttaggggtaacagcagctaaagaagctataa 
                   
               
               
                   
                 aaagagctaacataactccagatatgatagatgaatctchttag 
                   
               
               
                   
                 ggggagtacttacagcaggtcttggacaaaatatagcaagacaa 
                   
               
               
                   
                 atagcattaggagcaggaataccagtagaaaaaccagctatgac 
                   
               
               
                   
                 tataaatatagtttgtggttctggattaagatctgtttcaatgg 
                   
               
               
                   
                 catctcaacttatagcattaggtgatgctgatataatgttagtt 
                   
               
               
                   
                 ggtggagctgaaaacatgagtatgtctccttatttagtaccaag 
                   
               
               
                   
                 tgcgagatatggtgcaagaatgggtgatgctgcttttgttgatt 
                   
               
               
                   
                 caatgataaaagatggattatcagacatatttaataactatcac 
                   
               
               
                   
                 atgggtattactgctgaaaacatagcagagcaatggaatataac 
                   
               
               
                   
                 tagagaagaacaagatgaattagctcttgcaagtcaaaataaag 
                   
               
               
                   
                 ctgaaaaagctcaagctgaaggaaaatttgatgaagaaatagtt 
                   
               
               
                   
                 cctgttgttataaaaggaagaaaaggtgacactgtagtagataa 
                   
               
               
                   
                 agatgaatatattaagcctggcactacaatggagaaacttgcta 
                   
               
               
                   
                 agttaagacctgcatttaaaaaagatggaacagttactgctggt 
                   
               
               
                   
                 aatgcatcaggaataaatgatggtgctgctatgttagtagtaat 
                   
               
               
                   
                 ggctaaagaaaaagctgaagaactaggaatagagcctatgcaac 
                   
               
               
                   
                 tatagtacttatggaacagctggtgagaccctaaaataatggga 
                   
               
               
                   
                 tatggaccagttccagcaactaaaaaagctttagaagctgctaa 
                   
               
               
                   
                 tatgactattgaagatatagatttagttgaagctaatgaggcat 
                   
               
               
                   
                 ttgctgcccaatctgtagctgtaataagagacttaaatatagat 
                   
               
               
                   
                 atgaataaagttaatgttaatggtggagcaatagctataggaca 
                   
               
               
                   
                 tccaataggatgctcaggagcaagaatacttactacacttttat 
                   
               
               
                   
                 atgaaatgaagagaagagatgctaaaactggtcttgctacacta 
                   
               
               
                   
                 gtataggcggtggaatgggaactacataatagttaagagatag T   
                   
               
               
                   
                   AAGAAGGAGATATACAT atgaaattagctgtaataggtagtgga 
                   
               
               
                   
                 actatgggaagtggtattgtacaaactatgcaagttgtggacat 
                   
               
               
                   
                 gatgtatgtttaaagagtagaactcaaggtgctatagataaatg 
                   
               
               
                   
                 tttagctttattagataaaaatttaactaagttagttactaagg 
                   
               
               
                   
                 gaaaaatggatgaagctacaaaagcagaaatattaagtcatgtt 
                   
               
               
                   
                 agttcaactactaattatgaagatttaaaagatatggatttaat 
                   
               
               
                   
                 aatagaagcatctgtagaagacatgaatataaagaaagatgttt 
                   
               
               
                   
                 tcaagttactagatgaattatgtaaagaagatactatcaggcaa 
                   
               
               
                   
                 caaatacttcatcattatctataacagaaatagcacactactaa 
                   
               
               
                   
                 gcgcccagataaagttataggaatgcatttattaatccagacct 
                   
               
               
                   
                 atgatgaaattagttgaagttataagtggtcagttaacatcaaa 
                   
               
               
                   
                 agttacttttgatacagtatttgaattatctaagagtatcaata 
                   
               
               
                   
                 aagtaccagtagatgtatctgaatctcctggatttgtagtaaat 
                   
               
               
                   
                 agaatacttatacctatgataaatgaagctgttggtatatatgc 
                   
               
               
                   
                 agatggtgttgcaagtaaagaagaaatagatgaagctatgaaat 
                   
               
               
                   
                 taggagcaaaccatccaatgggaccactagcattaggtgattta 
                   
               
               
                   
                 atcggattagatgttgttttagctataatgaacgttttatatac 
                   
               
               
                   
                 tgaataggagatactaaatatagacctcatccactatagctaaa 
                   
               
               
                   
                 atggttagagctaatcaattaggaagaaaaactaagataggatt 
                   
               
               
                   
                 ctatgattataataaataa TAAGAAGGAGATATACAT atgagta 
                   
               
               
                   
                 caagtgatgttaaagatatgagaatgtagctgagaagtagatgg 
                   
               
               
                   
                 aaatatatgtacagtgaaaatgaatagacctaaagcccttaatg 
                   
               
               
                   
                 caataaattcaaagactttagaagaacatatgaagtatagtaga 
                   
               
               
                   
                 tattaataatgatgaaactattgatgagtaatattgacagggga 
                   
               
               
                   
                 aggaaaggcatttgtagctggagcagatattgcatacatgaaag 
                   
               
               
                   
                 atttagatgctgtagctgctaaagattttagtatcttaggagca 
                   
               
               
                   
                 aaagcattggagaaatagaaaatagtaaaaaagtagtgatagct 
                   
               
               
                   
                 gctgtaaacggatttgattaggtggaggatgtgaacttgcaatg 
                   
               
               
                   
                 gcatgtgatataagaattgcatctgctaaagctaaatttggtca 
                   
               
               
                   
                 gccagaagtaactcttggaataactccaggatatggaggaactc 
                   
               
               
                   
                 aaaggcttacaagattggttggaatggcaaaagcaaaagaatta 
                   
               
               
                   
                 atctttacaggtcaagttataaaagctgatgaagctgaaaaaat 
                   
               
               
                   
                 agggctagtaaatagagtcgttgagccagacattttaatagaag 
                   
               
               
                   
                 aagttgagaaattagctaagataatagctaaaaatgctcagctt 
                   
               
               
                   
                 gcagttagatactctaaagaagcaatacaacttggtgctcaaac 
                   
               
               
                   
                 tgatataaatactggaatagatatagaatctaatttataggtat 
                   
               
               
                   
                 tgatttcaactaaagaccaaaaagaaggaatgtcagctttcgtt 
                   
               
               
                   
                 gaaaagagagaagctaactttataaaagggtaa TAAGAAGGAGA   
                   
               
               
                   
                   TATACAT atgagaagttttgaagaagtaattaagtttgcaaaag 
                   
               
               
                   
                 aaagaggacctaaaactatatcagtagcatgagccaagataaag 
                   
               
               
                   
                 aagattaatggcagttgaaatggctagaaaagaaaaaatagcaa 
                   
               
               
                   
                 atgccatatagtaggagatatagaaaagactaaagaaattgcaa 
                   
               
               
                   
                 aaagcatagacatggatatcgaaaattatgaactgatagatata 
                   
               
               
                   
                 aaagatttagcagaagcatctctaaaatctgagaattagatcac 
                   
               
               
                   
                 aaggaaaagccgacatggtaatgaaaggcttagtagacacatca 
                   
               
               
                   
                 ataatactaaaagcagttttaaataaagaagtaggtcttagaac 
                   
               
               
                   
                 tggaaatgtattaagtcacgtagcagtatttgatgtagagggat 
                   
               
               
                   
                 atgatagattattatcgtaactgacgcagctatgaacttagctc 
                   
               
               
                   
                 ctgatacaaatactaaaaagcaaatcatagaaaatgcttgcaca 
                   
               
               
                   
                 gtagcacattcattagatataagtgaaccaaaagagctgcaata 
                   
               
               
                   
                 tgcgcaaaagaaaaagtaaatccaaaaatgaaagatacagttga 
                   
               
               
                   
                 agctaaagaactagaagaaatgtatgaaagaggagaaatcaaag 
                   
               
               
                   
                 gagtatggaggtgggccattgcaattgataatgcagtatattag 
                   
               
               
                   
                 aagcagctaaacataaaggtataaatcatcctgtagcaggacga 
                   
               
               
                   
                 gctgatatattattagccccagatattgaaggtggtaacatatt 
                   
               
               
                   
                 atataaagctttggtattcttctcaaaatcaaaaaatgcaggag 
                   
               
               
                   
                 ttatagaggggctaaagcaccaataatattaacactagagcaga 
                   
               
               
                   
                 cagtgaagaaactaaactaaactcaatagctttaggtgttttaa 
                   
               
               
                   
                 tggcagcaaaggcataa TAAGAAGGAGATATACAT atgagcaaa 
                   
               
               
                   
                 atatttaaaatcttaacaataaatcctggacgacatcaactaaa 
                   
               
               
                   
                 atagctgtatttgataatgaggatttagtatttgaaaaaacttt 
                   
               
               
                   
                 aagacattatcagaagaaataggaaaatatgagaaggtgtctga 
                   
               
               
                   
                 ccaatttgaatttcgtaaacaagtaatagaagaagctctaaaag 
                   
               
               
                   
                 aaggtggagtaaaaacatctgaattagatgctgtagtaggtaga 
                   
               
               
                   
                 ggaggacttcttaaacctataaaaggtggtacttattcagtaag 
                   
               
               
                   
                 tgctgctatgattgaagatttaaaagtgggagattaggagaaca 
                   
               
               
                   
                 cgcttcaaacctaggtggaataatagcaaaacaaataggtgaag 
                   
               
               
                   
                 aagtaaatgaccacatacatagtagaccctgagagtagatgaat 
                   
               
               
                   
                 tagaagatgagctagaatactggtatgcctgaaataagtagagc 
                   
               
               
                   
                 aagtgtagtacatgattaaatcaaaaggcaatagcaagaagata 
                   
               
               
                   
                 tgctagagaaataaacaagaaatatgaagatataaatcttatag 
                   
               
               
                   
                 agcacacatgggtggaggagatctgaggagctcataaaaatggt 
                   
               
               
                   
                 aaaatagtagatgagcaaacgcattagatggagaaggacctttc 
                   
               
               
                   
                 tctccagaaagaagtggtggactaccagtaggtgcattagtaaa 
                   
               
               
                   
                 aatgtgattagtggaaaatatactcaagatgaaattaaaaagaa 
                   
               
               
                   
                 aataaaaggtaatggcggactagagcatacttaaacactaatga 
                   
               
               
                   
                 tgctagagaagttgaagaaagaattgaagctggtgatgaaaaag 
                   
               
               
                   
                 ctaaattagtatatgaagctatggcatatcaaatctctaaagaa 
                   
               
               
                   
                 ataggagctagtgctgcagacttaagggagatgtaaaagcaata 
                   
               
               
                   
                 ttattaactggtggaatcgcatattcaaaaatgtttacagaaat 
                   
               
               
                   
                 gattgcagatagagttaaatttatagcagatgtaaaagatatcc 
                   
               
               
                   
                 aggtgaagatgaaatgattgcattagctcaaggtggacttagag 
                   
               
               
                   
                 ttttaactggtgaagaagaggctcaagtttatgataactaataa 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the genetically engineered acteria comprise the, nucleic acid sequence of SEQ ID NO: 212 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 SEQ ID NO: 212 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 SEQ ID NO: 212 or a functional fragment thereof, or a nucleic acid sequence that, but for the redundancy of the genetic code, encodes the same polypeptide as SEQ ID NO: 212 or a functional fragment thereof. 
     In alternate embodiments, pbt and buk are replaced with TesB (SEQ ID NO: 15) 
     In some embodiments, the butyrate cassette is driven by an inducible promoter. For example, other FNR promotors can be used in lieu of ydfZ, e.g., in SEQ ID NO: 177-188. 
     Non-limiting FNR promoter sequences are provided herein. In some embodiments, the genetically engineered bacteria of the invention comprise a butyrate cassette under the control of one or more of promoter sequences found in Table 6, e.g., nirB promoter, ydfZ promoter, nirB promoter fused to a strong ribosome binding site, ydfZ promoter fused to a strong ribosome binding site, fnrS, an anaerobically induced small RNA gene (fnrS promoter), nirB promoter fused to a crp binding site, andfnrS fused to a crp binding site. 
     In some embodiments, the butyrate cassette is under the control of a promoter which is inducible by metabolites present in the gut. In some embodiments the butyrate cassette is induced by HE-specific molecules or metabolites indicative of liver damage, e.g., bilirubin. In some embodiments, the butyrate cassette is placed under the control of promoter, which is inducible by inflammation or an inflammatory response (e.g., RNS or ROS promoter). 
     In some embodiments, the genetically engineered bacteria comprise a butyrate cassette driven by a promoter induced by a molecule or metabolite. Promoters that respond to one of these molecules or their metabolites may be used in the genetically engineered bacteria provided herein. 
     In some embodiments, the butyrate cassette is inducible by arabinose and is driven by the AraBAD promoter. 
     Example 17. 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 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 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 concentrations (as described in Example 22). Results are depicted in  FIG. 11 , and show that SYN1001 and SYN1002 give comparable butyrate production to the plasmid strain SYN501. 
     
       
         
           
               
             
               
                 TABLE 39 
               
             
            
               
                   
               
               
                 FRNRs Butyrate Cassette Sequences 
               
            
           
           
               
               
            
               
                 Description 
                 Sequence 
               
               
                   
               
               
                 Pfnrs-ter-thiA1-hbd-ctr2-tesB 
                 GGTACCAGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGGTTGTA 
               
               
                 SEQ ID NO: 213, e.g. 
                 ACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGCAAAGTTTGAGCGAAGTCA 
               
               
                 integrated into the 
                 ATAAACTCTCTACCCATTCAGGGCAATATCTCTCTTGGATCCAAAGTGAACTCTAGAAAT 
               
               
                 chromosome in SYN1001 
                 AATTTTGTTTAACTTTAAGAAGGAGATATACATatgatcgtaaaacctatggtacgcaac 
               
               
                 Pfnrs: uppercase; butyrate 
                 aatatctgcctgaacgcccatcctcagggctgcaagaagggagtggaagatcagattgaa 
               
               
                 cassette: lower case 
                 tataccaagaaacgcattaccgcagaagtcaaagctggcgcaaaagctccaaaaaacgtt 
               
               
                   
                 ctggtgcttggctgctcaaatggttacggcctggcgagccgcattactgctgcgttcgga 
               
               
                   
                 tacggggctgcgaccatcggcgtgtcctttgaaaaagcgggttcagaaaccaaatatggt 
               
               
                   
                 acaccgggatggtacaataatttggcatttgatgaagcggcaaaacgcgagggtctttat 
               
               
                   
                 agcgtgacgatcgacggcgatgcgttttcagacgagatcaaggcccaggtaattgaggaa 
               
               
                   
                 gccaaaaaaaaaggtatcaaatttgatctgatcgtatacagcttggccagcccagtacgt 
               
               
                   
                 actgatcctgatacaggtatcatgcacaaaagcgttttgaaaccctttggaaaaacgttc 
               
               
                   
                 acaggcaaaacagtagatccgtttactggcgagctgaaggaaatctccgcggaaccagca 
               
               
                   
                 aatgacgaggaagcagccgccactgttaaagttatggggggtgaagattgggaacgttgg 
               
               
                   
                 attaagcagctgtcgaaggaaggcctcttagaagaaggctgtattaccttggcctatagt 
               
               
                   
                 tatattggccctgaagctacccaagctttgtaccgtaaaggcacaatcggcaaggccaaa 
               
               
                   
                 gaacacctggaggccacagcacaccgtctcaacaaagagaacccgtcaatccgtgccttc 
               
               
                   
                 gtgagcgtgaataaaggcctggtaacccgcgcaagcgccgtaatcccggtaatccctctg 
               
               
                   
                 tatctcgccagcttgttcaaagtaatgaaagagaagggcaatcatgaaggttgtattgaa 
               
               
                   
                 cagatcacgcgtctgtacgccgagcgcctgtaccgtaaagatggtacaattccagttgat 
               
               
                   
                 gaggaaaatcgcattcgcattgatgattgggagttagaagaagacgtccagaaagcggta 
               
               
                   
                 tccgcgttgatggagaaagtcacgggtgaaaacgcagaatctctcactgacttagcgggg 
               
               
                   
                 taccgccatgatttcttagctagtaacggctttgatgtagaaggtattaattatgaagcg 
               
               
                   
                 gaagttgaacgcttcgaccgtatctgataagaaggagatatacatatgagagaagtagta 
               
               
                   
                 attgccagtgcagctagaacagcagtaggaagttttggaggagcatttaaatcagtttca 
               
               
                   
                 gcggtagagttaggggtaacagcagctaaagaagctataaaaagagctaacataactcca 
               
               
                   
                 gatatgatagatgaatctcttttagggggagtacttacagcaggtcttggacaaaatata 
               
               
                   
                 gcaagacaaatagcattaggagcaggaataccagtagaaaaaccagctatgactataaat 
               
               
                   
                 atagtttgtggttctggattaagatctgtttcaatggcatctcaacttatagcattaggt 
               
               
                   
                 gatgctgatataatgttagttggtggagctgaaaacatgagtatgtctccttatttagta 
               
               
                   
                 ccaagtgcgagatatggtgcaagaatgggtgatgctgcttttgttgattcaatgataaaa 
               
               
                   
                 gatggattatcagacatatttaataactatcacatgggtattactgctgaaaacatagca 
               
               
                   
                 gagcaatggaatataactagagaagaacaagatgaattagctcttgcaagtcaaaataaa 
               
               
                   
                 gctgaaaaagctcaagctgaaggaaaatttgatgaagaaatagttcctgttgttataaaa 
               
               
                   
                 ggaagaaaaggtgacactgtagtagataaagatgaatatattaagcctggcactacaatg 
               
               
                   
                 gagaaacttgctaagttaagacctgcatttaaaaaagatggaacagttactgctggtaat 
               
               
                   
                 gcatcaggaataaatgatggtgctgctatgttagtagtaatggctaaagaaaaagctgaa 
               
               
                   
                 gaactaggaatagagcctcttgcaactatagtttcttatggaacagctggtgttgaccct 
               
               
                   
                 aaaataatgggatatggaccagttccagcaactaaaaaagctttagaagctgctaatatg 
               
               
                   
                 actattgaagatatagatttagttgaagctaatgaggcatttgctgcccaatctgtagct 
               
               
                   
                 gtaataagagacttaaatatagatatgaataaagttaatgttaatggtggagcaatagct 
               
               
                   
                 ataggacatccaataggatgctcaggagcaagaatacttactacacttttatatgaaatg 
               
               
                   
                 aagagaagagatgctaaaactggtcttgctacactttgtataggcggtggaatgggaact 
               
               
                   
                 actttaatagttaagagatagtaagaaggagatatacatatgaaattagctgtaataggt 
               
               
                   
                 agtggaactatgggaagtggtattgtacaaacttttgcaagttgtggacatgatgtatgt 
               
               
                   
                 ttaaagagtagaactcaaggtgctatagataaatgtttagctttattagataaaaattta 
               
               
                   
                 actaagttagttactaagggaaaaatggatgaagctacaaaagcagaaatattaagtcat 
               
               
                   
                 gttagttcaactactaattatgaagatttaaaagatatggatttaataatagaagcatct 
               
               
                   
                 gtagaagacatgaatataaagaaagatgttttcaagttactagatgaattatgtaaagaa 
               
               
                   
                 gatactatcttggcaacaaatacttcatcattatctataacagaaatagcttcttctact 
               
               
                   
                 aagcgcccagataaagttataggaatgcatttctttaatccagttcctatgatgaaatta 
               
               
                   
                 gttgaagttataagtggtcagttaacatcaaaagttacttttgatacagtatttgaatta 
               
               
                   
                 tctaagagtatcaataaagtaccagtagatgtatctgaatctcctggatttgtagtaaat 
               
               
                   
                 agaatacttatacctatgataaatgaagctgttggtatatatgcagatggtgttgcaagt 
               
               
                   
                 aaagaagaaatagatgaagctatgaaattaggagcaaaccatccaatgggaccactagca 
               
               
                   
                 ttaggtgatttaatcggattagatgttgttttagctataatgaacgttttatatactgaa 
               
               
                   
                 tttggagatactaaatatagacctcatccacttttagctaaaatggttagagctaatcaa 
               
               
                   
                 ttaggaagaaaaactaagataggattctatgattataataaataataagaaggagatata 
               
               
                   
                 catatgagtacaagtgatgttaaagtttatgagaatgtagctgttgaagtagatggaaat 
               
               
                   
                 atatgtacagtgaaaatgaatagacctaaagcccttaatgcaataaattcaaagacttta 
               
               
                   
                 gaagaactttatgaagtatttgtagatattaataatgatgaaactattgatgttgtaata 
               
               
                   
                 ttgacaggggaaggaaaggcatttgtagctggagcagatattgcatacatgaaagattta 
               
               
                   
                 gatgctgtagctgctaaagattttagtatcttaggagcaaaagcttttggagaaatagaa 
               
               
                   
                 aatagtaaaaaagtagtgatagctgctgtaaacggatttgctttaggtggaggatgtgaa 
               
               
                   
                 cttgcaatggcatgtgatataagaattgcatctgctaaagctaaatttggtcagccagaa 
               
               
                   
                 gtaactcttggaataactccaggatatggaggaactcaaaggcttacaagattggttgga 
               
               
                   
                 atggcaaaagcaaaagaattaatctttacaggtcaagttataaaagctgatgaagctgaa 
               
               
                   
                 aaaatagggctagtaaatagagtcgttgagccagacattttaatagaagaagttgagaaa 
               
               
                   
                 ttagctaagataatagctaaaaatgctcagcttgcagttagatactctaaagaagcaata 
               
               
                   
                 caacttggtgctcaaactgatataaatactggaatagatatagaatctaatttatttggt 
               
               
                   
                 ctttgtttttcaactaaagaccaaaaagaaggaatgtcagctttcgttgaaaagagagaa 
               
               
                   
                 gctaactttataaaagggtaataagaaggagatatacatatgagtcaggcgctaaaaaat 
               
               
                   
                 ttactgacattgttaaatctggaaaaaattgaggaaggactctttcgcggccagagtgaa 
               
               
                   
                 gatttaggtttacgccaggtgtttggcggccaggtcgtgggtcaggccttgtatgctgca 
               
               
                   
                 aaagagaccgtccctgaagagcggctggtacattcgtttcacagctactttcttcgccct 
               
               
                   
                 ggcgatagtaagaagccgattatttatgatgtcgaaacgctgcgtgacggtaacagcuca 
               
               
                   
                 gcgcccgccgggugctgctattcaaaacggcaaaccgattttttatatgactgcctcttt 
               
               
                   
                 ccaggcaccagaagcgggtttcgaacatcaaaaaacaatgccgtccgcgccagcgcctga 
               
               
                   
                 tggcctcccttcggaaacgcaaatcgcccaatcgctggcgcacctgctgccgccagtgct 
               
               
                   
                 gaaagataaattcatctgcgatcgtccgctggaagtccgtccggtggagtttcataaccc 
               
               
                   
                 actgaaaggtcacgtcgcagaaccacatcgtcaggtgtggatccgcgcaaatggtagcgt 
               
               
                   
                 gccggatgacctgcgcgttcatcagtatctgctcggttacgcttctgatcttaacttcct 
               
               
                   
                 gccggtagctctacagccgcacggcatcggttttctcgaaccggggattcagattgccac 
               
               
                   
                 cattgaccattccatgtggttccatcgcccgtttaatttgaatgaatggctgctgtatag 
               
               
                   
                 cgtggagagcacctcggcgtccagcgcacgtggctttgtgcgcggtgaguttatacccaa 
               
               
                   
                 gacggcgtactggugcctcgaccgucaggaaggggtgatgcgtaatcacaattaa 
               
               
                   
               
               
                 Pfnrs-ter-thiA1-hbd-ctr2- 
                 GGTACCAGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGGTTGTA 
               
               
                 pbt-buk 
                 ACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGCAAAGTTTGAGCGAAGTCA 
               
               
                 (SEQ ID NO: 214), e.g. 
                 ATAAACTCTCTACCCATTCAGGGCAATATCTCTCTTGGATCCAAAGTGAACTCTAGAAAT 
               
               
                 integrated into the 
                 AATTTTGTTTAACTTTAAGAAGGAGATATACATatgatcgtaaaacctatggtacgcaac 
               
               
                 chromosome in SYN1002 
                 aatatctgcctgaacgcccatcctcagggctgcaagaagggagtggaagatcagattgaa 
               
               
                 Pfnrs: uppercase; butyrate 
                 tataccaagaaacgcattaccgcagaagtcaaagctggcgcaaaagctccaaaaaacgtt 
               
               
                 cassette: lower case 
                 ctggtgcttggctgctcaaatggttacggcctggcgagccgcattactgctgcgttcgga 
               
               
                   
                 tacggggctgcgaccatcggcgtgtcctttgaaaaagcgggttcagaaaccaaatatggt 
               
               
                   
                 acaccgggatggtacaataatttggcatttgatgaagcggcaaaacgcgagggtctttat 
               
               
                   
                 agcgtgacgatcgacggcgatgcgttttcagacgagatcaaggcccaggtaattgaggaa 
               
               
                   
                 gccaaaaaaaaaggtatcaaatttgatctgatcgtatacagcttggccagcccagtacgt 
               
               
                   
                 actgatcctgatacaggtatcatgcacaaaagcgttttgaaaccctttggaaaaacgttc 
               
               
                   
                 acaggcaaaacagtagatccgtttactggcgagctgaaggaaatctccgcggaaccagca 
               
               
                   
                 aatgacgaggaagcagccgccactgttaaagttatggggggtgaagattgggaacgttgg 
               
               
                   
                 attaagcagctgtcgaaggaaggcctcttagaagaaggctgtattaccttggcctatagt 
               
               
                   
                 tatattggccctgaagctacccaagctagtaccgtaaaggcacaatcggcaaggccaaag 
               
               
                   
                 aacacctggaggccacagcacaccgtctcaacaaagagaacccgtcaatccgtgccttcg 
               
               
                   
                 tgagcgtgaataaaggcctggtaacccgcgcaagcgccgtaatcccggtaatccctctgt 
               
               
                   
                 atctcgccagcttgacaaagtaatgaaagagaagggcaatcatgaaggttgtattgaaca 
               
               
                   
                 gatcacgcgtctgtacgccgagcgcctgtaccgtaaagatggtacaattccagttgatga 
               
               
                   
                 ggaaaatcgcattcgcattgatgattgggagttagaagaagacgtccagaaagcggtatc 
               
               
                   
                 cgcgttgatggagaaagtcacgggtgaaaacgcagaatctctcactgacttagcggggta 
               
               
                   
                 ccgccatgatttcttagctagtaacggctttgatgtagaaggtattaattatgaagcgga 
               
               
                   
                 agttgaacgcttcgaccgtatctgataagaaggagatatacatatgagagaagtagtaat 
               
               
                   
                 tgccagtgcagctagaacagcagtaggaagattggaggagcatttaaatcagatcagcgg 
               
               
                   
                 tagagttaggggtaacagcagctaaagaagctataaaaagagctaacataactccagata 
               
               
                   
                 tgatagatgaatctcattagggggagtacttacagcaggtatggacaaaatatagcaaga 
               
               
                   
                 caaatagcattaggagcaggaataccagtagaaaaaccagctatgactataaatatagta 
               
               
                   
                 gtggactggattaagatctgatcaatggcatctcaacttatagcattaggtgatgctgat 
               
               
                   
                 ataatgttagttggtggagctgaaaacatgagtatgtctccttatttagtaccaagtgcg 
               
               
                   
                 agatatggtgcaagaatgggtgatgctgatttgagattcaatgataaaagatggattatc 
               
               
                   
                 agacatatttaataactatcacatgggtattactgctgaaaacatagcagagcaatggaa 
               
               
                   
                 tataactagagaagaacaagatgaattagctatgcaagtcaaaataaagctgaaaaagct 
               
               
                   
                 caagctgaaggaaaatttgatgaagaaatagttcctgttgttataaaaggaagaaaaggt 
               
               
                   
                 gacactgtagtagataaagatgaatatattaagcctggcactacaatggagaaacttgct 
               
               
                   
                 aagttaagacctgcatttaaaaaagatggaacagttactgctggtaatgcatcaggaata 
               
               
                   
                 aatgatggtgctgctatgttagtagtaatggctaaagaaaaagctgaagaactaggaata 
               
               
                   
                 gagcctatgcaactatagtacttatggaacagctggtgagaccctaaaataatgggatat 
               
               
                   
                 ggaccagttccagcaactaaaaaagctttagaagctgctaatatgactattgaagatata 
               
               
                   
                 gatttagagaagctaatgaggcatttgctgcccaatctgtagctgtaataagagacttaa 
               
               
                   
                 atatagatatgaataaagttaatgttaatggtggagcaatagctataggacatccaatag 
               
               
                   
                 gatgctcaggagcaagaatacttactacacttttatatgaaatgaagagaagagatgcta 
               
               
                   
                 aaactggtcttgctacactttgtataggcggtggaatgggaactactttaatagttaaga 
               
               
                   
                 gatagtaagaaggagatatacatatgaaattagctgtaataggtagtggaactatgggaa 
               
               
                   
                 gtggtattgtacaaacattgcaagagtggacatgatgtatgataaagagtagaactcaag 
               
               
                   
                 gtgctatagataaatgtttagctttattagataaaaatttaactaagttagttactaagg 
               
               
                   
                 gaaaaatggatgaagctacaaaagcagaaatattaagtcatgttagttcaactactaatt 
               
               
                   
                 atgaagatttaaaagatatggatttaataatagaagcatctgtagaagacatgaatataa 
               
               
                   
                 agaaagatgattcaagttactagatgaattatgtaaagaagatactatcaggcaacaaat 
               
               
                   
                 acttcatcattatctataacagaaatagcttcactactaagcgcccagataaagttatag 
               
               
                   
                 gaatgcatttctttaatccagttcctatgatgaaattagttgaagttataagtggtcagt 
               
               
                   
                 taacatcaaaagttacattgatacagtatttgaattatctaagagtatcaataaagtacc 
               
               
                   
                 agtagatgtatctgaatctcctggatttgtagtaaatagaatacttatacctatgataaa 
               
               
                   
                 tgaagctgaggtatatatgcagatggtgagcaagtaaagaagaaatagatgaagctatga 
               
               
                   
                 aattaggagcaaaccatccaatgggaccactagcattaggtgatttaatcggattagatg 
               
               
                   
                 agattagctataatgaacgattatatactgaataggagatactaaatatagacctcatcc 
               
               
                   
                 actatagctaaaatggttagagctaatcaattaggaagaaaaactaagataggattctat 
               
               
                   
                 gattataataaataataagaaggagatatacatatgagtacaagtgatgttaaagatatg 
               
               
                   
                 agaatgtagctgagaagtagatggaaatatatgtacagtgaaaatgaatagacctaaagc 
               
               
                   
                 ccttaatgcaataaattcaaagactttagaagaactttatgaagtatttgtagatattaa 
               
               
                   
                 taatgatgaaactattgatgagtaatattgacaggggaaggaaaggcatagtagctggag 
               
               
                   
                 cagatattgcatacatgaaagatttagatgctgtagctgctaaagattttagtatcttag 
               
               
                   
                 gagcaaaagcttttggagaaatagaaaatagtaaaaaagtagtgatagctgctgtaaacg 
               
               
                   
                 gatttgattaggtggaggatgtgaacttgcaatggcatgtgatataagaattgcatctgc 
               
               
                   
                 taaagctaaataggtcagccagaagtaactcaggaataactccaggatatggaggaactc 
               
               
                   
                 aaaggcttacaagattggttggaatggcaaaagcaaaagaattaatctttacaggtcaag 
               
               
                   
                 ttataaaagctgatgaagctgaaaaaatagggctagtaaatagagtcgttgagccagaca 
               
               
                   
                 tataatagaagaagttgagaaattagctaagataatagctaaaaatgctcagcttgcagt 
               
               
                   
                 tagatactctaaagaagcaatacaacttggtgctcaaactgatataaatactggaataga 
               
               
                   
                 tatagaatctaatttatttggtcatgatttcaactaaagaccaaaaagaaggaatgtcag 
               
               
                   
                 attcgttgaaaagagagaagctaactttataaaagggtaataagaaggagatatacatat 
               
               
                   
                 gagaagattgaagaagtaattaagtttgcaaaagaaagaggacctaaaactatatcagta 
               
               
                   
                 gcatgttgccaagataaagaagttttaatggcagttgaaatggctagaaaagaaaaaata 
               
               
                   
                 gcaaatgccatatagtaggagatatagaaaagactaaagaaattgcaaaaagcatagaca 
               
               
                   
                 tggatatcgaaaattatgaactgatagatataaaagatttagcagaagcatctctaaaat 
               
               
                   
                 ctgagaattagatcacaaggaaaagccgacatggtaatgaaaggcttagtagacacatca 
               
               
                   
                 ataatactaaaagcagttttaaataaagaagtaggtcttagaactggaaatgtattaagt 
               
               
                   
                 cacgtagcagtatttgatgtagagggatatgatagattattatcgtaactgacgcagcta 
               
               
                   
                 tgaacttagctcctgatacaaatactaaaaagcaaatcatagaaaatgcttgcacagtag 
               
               
                   
                 cacattcattagatataagtgaaccaaaagttgctgcaatatgcgcaaaagaaaaagtaa 
               
               
                   
                 atccaaaaatgaaagatacagttgaagctaaagaactagaagaaatgtatgaaagaggag 
               
               
                   
                 aaatcaaaggagtatggttggtgggccttttgcaattgataatgcagtatctttagaagc 
               
               
                   
                 agctaaacataaaggtataaatcatcctgtagcaggacgagctgatatattattagcccc 
               
               
                   
                 agatattgaaggtggtaacatattatataaagctaggtattcactcaaaatcaaaaaatg 
               
               
                   
                 caggagttatagttggggctaaagcaccaataatattaacttctagagcagacagtgaag 
               
               
                   
                 aaactaaactaaactcaatagctttaggtgttttaatggcagcaaaggcataataagaag 
               
               
                   
                 gagatatacatatgagcaaaatatttaaaatcttaacaataaatcctggacgacatcaac 
               
               
                   
                 taaaatagctgtatttgataatgaggatttagtatttgaaaaaacataagacattcacag 
               
               
                   
                 aagaaataggaaaatatgagaaggtgtctgaccaatttgaatttcgtaaacaagtaatag 
               
               
                   
                 aagaagctctaaaagaaggtggagtaaaaacatctgaattagatgctgtagtaggtagag 
               
               
                   
                 gaggacttcttaaacctataaaaggtggtacttattcagtaagtgctgctatgattgaag 
               
               
                   
                 atttaaaagtgggagattaggagaacacgcttcaaacctaggtggaataatagcaaaaca 
               
               
                   
                 aataggtgaagaagtaaatgaccacatacatagtagaccctgagagtagatgaattagaa 
               
               
                   
                 gatgagctagaatactggtatgcctgaaataagtagagcaagtgtagtacatgattaaat 
               
               
                   
                 caaaaggcaatagcaagaagatatgctagagaaataaacaagaaatatgaagatataaat 
               
               
                   
                 cttatagttgcacacatgggtggaggagtttctgttggagctcataaaaatggtaaaata 
               
               
                   
                 gtagatgttgcaaacgcattagatggagaaggacctttctctccagaaagaagtggtgga 
               
               
                   
                 ctaccagtaggtgcattagtaaaaatgtgattagtggaaaatatactcaagatgaaatta 
               
               
                   
                 aaaagaaaataaaaggtaatggcggactagagcatacttaaacactaatgatgctagaga 
               
               
                   
                 agttgaagaaagaattgaagctggtgatgaaaaagctaaattagtatatgaagctatggc 
               
               
                   
                 atatcaaatctctaaagaaataggagctagtgctgcagttcttaagggagatgtaaaagc 
               
               
                   
                 aatattattaactggtggaatcgcatattcaaaaatgatacagaaatgattgcagataga 
               
               
                   
                 gttaaatttatagcagatgtaaaagatatccaggtgaagatgaaatgattgcattagctc 
               
               
                   
                 aaggtggacttagagattaactggtgaagaagaggctcaagtttatgataactaa 
               
               
                   
               
               
                 PfNRS (ribosome binding site 
                 GGTACCAGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGGTTGTA 
               
               
                 is underlined) 
                 ACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGCAAAGTTTGAGCGAAGTCA 
               
               
                 (SEQ ID NO: 215) 
                 ATAAACTCTCTACCCATTCAGGGCAATATCTCTCTTGGATCCAAAGTGAA CTCTAGAAAT   
               
               
                   
                 
                   AATTTTGTTTAACTTTAAGAAGGAGATATACAT 
                 
               
               
                   
               
               
                 Ribosome binding site and 
                 CTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT 
               
               
                 leadter region 
                   
               
               
                 (SEQ ID NO: 216) 
               
               
                   
               
            
           
         
       
     
     Example 18. 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. 10 . 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: H20 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=5 d). 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 19. Measurement of Satiety Markers Upon Administration of SYN501 In Vivo 
     To determine whether administration of a butyrate producing strain might result in increased levels of satiety markers, SYN501 is administered to 10-week old C57BL6 (10 weeks) and blood levels of GLP1 and insulin are measured. Butyrate in H20 at 100 mM is used as a control (e.g., as described in Lin et al., Butyrate and Propionate Protect against Diet-Induced Obesity and Regulate Gut Hormones via Free Fatty Acid Receptor 3-Independent Mechanisms, PLOS One, April 2012|Volume 7|Issue 4|e35240). 
     On day 1, animals are randomized and distributed into 5 groups as follows: Group 1: Time 0 control (n=6); Group 2-H20 (+) 100 mM butyrate, 10 min (n=6); Group 3-SYN501, 30 min (n=6); Group 4-SYN501, 4 h (n=6); Group 5-H20 (+) 100 mM butyrate, 4 h (n=6). Mice are fasted overnight. On day 2, mice are gavaged with either H20(+) 100 mM butyrate or SYN501. Then, blood is harvested via cardiac bleed at the following time points post dose: Group 1 is Time 0; Group 2 (H20 (+) 100 mM butyrate) at 10 min; Group 3 (SYN501) at 30 min; Group 4 (SYN501) at 4 h; Group 5 (H20 (+) 100 mM butyrate) at 4 h. Serum is analyzed by ELISA for GLP-1 and insulin. Fecal samples are analyzed for butyrate by MS as described herein. 
     Example 20. 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 dilution and was grown in RCM (Reinforced Clostridial Media, which is similar to LB but contains 05% 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 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 concentrations (as described in Example 22). Results are depicted in  FIG. 12 , and show that SYN501 produces butyrate levels comparable to  Clostridium  spp. in RCM media 
     Example 22. 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 μg/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 40 and Table 41. Tandem Mass Spectrometry details are found in Table 42. 
     
       
         
           
               
             
               
                 TABLE 40 
               
             
            
               
                   
               
               
                 HPLC Details 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Thermo Aquasil C18 
               
               
                   
                   
                   
                 column, 5 μm (50 × 2.1 
               
               
                   
                   
                 Column 
                 mm) 
               
               
                   
                   
               
               
                   
                   
                 Mobile 
                 100% H2O, 0.1% Formic 
               
               
                   
                   
                 Phase A 
                 Acid 
               
               
                   
                   
                 Mobile 
                 100% ACN, 0.1% Formic 
               
               
                   
                   
                 Phase B  
                 Acid 
               
               
                   
                   
                 Injection 
                 10 uL 
               
               
                   
                   
                 volume 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 41 
               
             
            
               
                   
               
               
                 HPLC Method 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Total 
                 Flow 
                   
                   
               
               
                   
                 Time 
                 Rate 
                   
                   
               
               
                   
                 (min) 
                 (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 42 
               
               
                   
               
               
                 Tandem Mass Spectrometry Details 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Ion Source 
                 HESI-II 
               
               
                   
                   
                 Polarity 
                 Positive 
               
               
                   
                   
                 SRM 
                 Butyrate 170.0/71.1, 
               
               
                   
                   
                 transitions 
                 Butyrate d7 
               
               
                   
                   
                   
                 177.1/78.3 
               
               
                   
                   
               
            
           
         
       
     
     Example 23. 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 90 μL of the derivatizing solution containing 50 mM of 2-Hydrazinoquinoline (2-HQ), dipyridyl disulfide, and triphenylphospine in acetonitrile with 5 ug/mL of butyrate-d 7  were added to each sample. The plate was heat-sealed and incubated at 60° C. for lhr. 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 43 and Table 43. Tandem Mass Spectrometry details are found in Table 43. 
     
       
         
           
               
             
               
                 TABLE 43 
               
             
            
               
                   
               
               
                 HPLC Details 
               
            
           
           
               
               
               
            
               
                   
                   
                 Luna phenomenex C18 
               
               
                   
                   
                 column, 5 μm (100 × 2.1 
               
               
                   
                 Column 
                 mm) 
               
               
                   
                   
               
               
                   
                 Mobile 
                 100% H2O, 0.1% Formic 
               
               
                   
                 Phase A 
                 Acid 
               
               
                   
                 Mobile 
                 100% ACN, 0.1% Formic 
               
               
                   
                 Phase B 
                 Acid 
               
               
                   
                 Injection 
                 10 uL 
               
               
                   
                 volume 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 43 
               
             
            
               
                   
               
               
                 HPLC Method 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Total 
                 Flow 
                   
                   
               
               
                   
                 Time 
                 Rate 
                   
                   
               
               
                   
                 (min) 
                 (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 44 
               
               
                   
               
               
                 Tandem Mass Spectrometry Details 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Ion Source 
                 HESI-II 
               
               
                   
                   
                 Polarity 
                 Positive 
               
               
                   
                   
                 SRM 
                 Butyrate 230.1/143.1, 
               
               
                   
                   
                 transitions 
                 Butyrate d7 
               
               
                   
                   
                   
                 237.1/143.1 
               
               
                   
                   
               
            
           
         
       
     
     Example 24. 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-ygfG-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 45. Mutation of the lactate dehydrogenase gene (ldhA) reportedly increases propionate production, and this mutation is therefore also added in certain embodiments. 
                     TABLE 45                  SBM Construct Sequences                     Description   Sequence               BW25113 fnrS SBM construct             (BW25113 frt-cam-frt-Pfnrs-             sbm, ygfD, ygfG, ygfH),             comprising rrnB terminator 2     AGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGAC         (both italic, uppercase), cat     GCCCGCC           promoter and cam resistance       CCAGTGC         gene (encoded on the lagging     CAAGCTTGCATGCAGATTGCAGCATTACACGTCTTGAGCGATTGTGTAGGCTGGAGC         strand underlined uppercase),     TGCTTC       ATTT         frt sites (italic underlined),     AAATGGCGCGCCTTACGCCCCGCCCTGCCA           FNRS promoter bold lowercase,               with RBS and leader region bold               and underlined and FNR binding               site in bold and italics);               sleeping beauty operon (sbm,               ygfD, ygfG, ygfH) bold and               uppercase               (SEQ ID NO: 217)                                                                                       ACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGAC               ACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTAGGCGCGCC                   GGAATAGGAACTAAGGAGGATATTC               ATATGGACCATGGCTAATTCCCAGGTACCagttgttcttattggtggtgttgctttatg               gttgcatcgtagtaaatggttgtaacaaaagcaatttttccggctgtctgtatacaaaa               acgccgcaaagt     aactctctacccattcagggcaatatctctc               ttggatccaaagtgaa                   ATGTCTAACGTGCAGGAGTGGCAACAGCTTGCCAACAAGGAATTGAGCCGTCGGGAGAAA               ACTGTCGACTCGCTGGTTCATCAAACCGCGGAAGGGATCGCCATCAAGCCGCTGTATACC               GAAGCCGATCTCGATAATCTGGAGGTGACAGGTACCCTTCCTGGTTTGCCGCCCTACGTT               CGTGGCCCGCGTGCCACTATGTATACCGCCCAACCGTGGACCATCCGTCAGTATGCTGGT               TTTTCAACAGCAAAAGAGTCCAACGCTTTTTATCGCCGTAACCTGGCCGCCGGGCAAAAA               GGTCTTTCCGTTGCGTTTGACCTTGCCACCCACCGTGGCTACGACTCCGATAACCCGCGC               GTGGCGGGCGACGTCGGCAAAGCGGGCGTCGCTATCGACACCGTGGAAGATATGAAAGTC               CTGTTCGACCAGATCCCGCTGGATAAAATGTCGGTTTCGATGACCATGAATGGCGCAGTG               CTACCAGTACTGGCGTTTTATATCGTCGCCGCAGAAGAGCAAGGTGTTACACCTGATAAA               CTGACCGGCACCATTCAAAACGATATTCTCAAAGAGTACCTCTGCCGCAACACCTATATT               TACCCACCAAAACCGTCAATGCGCATTATCGCCGACATCATCGCCTGGTGTTCCGGCAAC               ATGCCGCGATTTAATACCATCAGTATCAGCGGTTACCACATGGGTGAAGCGGGTGCCAAC               TGCGTGCAGCAGGTAGCATTTACGCTCGCTGATGGGATTGAGTACATCAAAGCAGCAATC               TCTGCCGGACTGAAAATTGATGACTTCGCTCCTCGCCTGTCGTTCTTCTTCGGCATCGGC               ATGGATCTGTTTATGAACGTCGCCATGTTGCGTGCGGCACGTTATTTATGGAGCGAAGCG               GTCAGTGGATTTGGCGCACAGGACCCGAAATCACTGGCGCTGCGTACCCACTGCCAGACC               TCAGGCTGGAGCCTGACTGAACAGGATCCGTATAACAACGTTATCCGCACCACCATTGAA               GCGCTGGCTGCGACGCTGGGCGGTACTCAGTCACTGCATACCAACGCCTTTGACGAAGCG               CTTGGTTTGCCTACCGATTTCTCAGCACGCATTGCCCGCAACACCCAGATCATCATCCAG               GAAGAATCAGAACTCTGCCGCACCGTCGATCCACTGGCCGGATCCTATTACATTGAGTCG               CTGACCGATCAAATCGTCAAACAAGCCAGAGCTATTATCCAACAGATCGACGAAGCCGGT               GGCATGGCGAAAGCGATCGAAGCAGGTCTGCCAAAACGAATGATCGAAGAGGCCTCAGCG               CGCGAACAGTCGCTGATCGACCAGGGCAAGCGTGTCATCGTTGGTGTCAACAAGTACAAA               CTGGATCACGAAGACGAAACCGATGTACTTGAGATCGACAACGTGATGGTGCGTAACGAG               CAAATTGCTTCGCTGGAACGCATTCGCGCCACCCGTGATGATGCCGCCGTAACCGCCGCG               TTGAACGCCCTGACTCACGCCGCACAGCATAACGAAAACCTGCTGGCTGCCGCTGTTAAT               GCCGCTCGCGTTCGCGCCACCCTGGGTGAAATTTCCGATGCGCTGGAAGTCGCTTTCGAC               CGTTATCTGGTGCCAAGCCAGTGTGTTACCGGCGTGATTGCGCAAAGCTATCATCAGTCT               GAGAAATCGGCCTCCGAGTTCGATGCCATTGTTGCGCAAACGGAGCAGTTCCTTGCCGAC               AATGGTCGTCGCCCGCGCATTCTGATCGCTAAGATGGGCCAGGATGGACACGATCGCGGC               GCGAAAGTGATCGCCAGCGCCTATTCCGATCTCGGTTTCGACGTAGATTTAAGCCCGATG               TTCTCTACACCTGAAGAGATCGCCCGCCTGGCCGTAGAAAACGACGTTCACGTAGTGGGC               GCATCCTCACTGGCTGCCGGTCATAAAACGCTGATCCCGGAACTGGTCGAAGCGCTGAAA               AAATGGGGACGCGAAGATATCTGCGTGGTCGCGGGTGGCGTCATTCCGCCGCAGGATTAC               GCCTTCCTGCAAGAGCGCGGCGTGGCGGCGATTTATGGTCCAGGTACACCTATGCTCGAC               AGTGTGCGCGACGTACTGAATCTGATAAGCCAGCATCATGATTAATGAAGCCACGCTGGC               AGAAAGTATTCGCCGCTTACGTCAGGGTGAGCGTGCCACACTCGCCCAGGCCATGACGCT               GGTGGAAAGCCGTCACCCGCGTCATCAGGCACTAAGTACGCAGCTGCTTGATGCCATTAT               GCCGTACTGCGGTAACACCCTGCGACTGGGCGTTACCGGCACCCCCGGCGCGGGGAAAAG               TACCTTTCTTGAGGCCTTTGGCATGTTGTTGATTCGAGAGGGATTAAAGGTCGCGGTTAT               TGCGGTCGATCCCAGCAGCCCGGTCACTGGCGGTAGCATTCTCGGGGATAAAACCCGCAT               GAATGACCTGGCGCGTGCCGAAGCGGCGTTTATTCGCCCGGTACCATCCTCCGGTCATCT               GGGCGGTGCCAGTCAGCGAGCGCGGGAATTAATGCTGTTATGCGAAGCAGCGGGTTATGA               CGTAGTGATTGTCGAAACGGTTGGCGTCGGGCAGTCGGAAACAGAAGTCGCCCGCATGGT               GGACTGTTTTATCTCGTTGCAAATTGCCGGTGGCGGCGATGATCTGCAGGGCATTAAAAA               AGGGCTGATGGAAGTGGCTGATCTGATCGTTATCAACAAAGACGATGGCGATAACCATAC               CAATGTCGCCATTGCCCGGCATATGTACGAGAGTGCCCTGCATATTCTGCGACGTAAATA               CGACGAATGGCAGCCACGGGTTCTGACTTGTAGCGCACTGGAAAAACGTGGAATCGATGA               GATCTGGCACGCCATCATCGACTTCAAAACCGCGCTAACTGCCAGTGGTCGTTTACAACA               AGTGCGGCAACAACAATCGGTGGAATGGCTGCGTAAGCAGACCGAAGAAGAAGTACTGAA               TCACCTGTTCGCGAATGAAGATTTCGATCGCTATTACCGCCAGACGCTTTTAGCGGTCAA               AAACAATACGCTCTCACCGCGCACCGGCCTGCGGCAGCTCAGTGAATTTATCCAGACGCA               ATATTTTGATTAAAGGAATTTTTATGTCTTATCAGTATGTTAACGTTGTCACTATCAACA               AAGTGGCGGTCATTGAGTTTAACTATGGCCGAAAACTTAATGCCTTAAGTAAAGTCTTTA               TTGATGATCTTATGCAGGCGTTAAGCGATCTCAACCGGCCGGAAATTCGCTGTATCATTT               TGCGCGCACCGAGTGGATCCAAAGTCTTCTCCGCAGGTCACGATATTCACGAACTGCCGT               CTGGCGGTCGCGATCCGCTCTCCTATGATGATCCATTGCGTCAAATCACCCGCATGATCC               AAAAATTCCCGAAACCGATCATTTCGATGGTGGAAGGTAGTGTTTGGGGTGGCGCATTTG               AAATGATCATGAGTTCCGATCTGATCATCGCCGCCAGTACCTCAACCTTCTCAATGACGC               CTGTAAACCTCGGCGTCCCGTATAACCTGGTCGGCATTCACAACCTGACCCGCGACGCGG               GCTTCCACATTGTCAAAGAGCTGATTTTTACCGCTTCGCCAATCACCGCCCAGCGCGCGC               TGGCTGTCGGCATCCTCAACCATGTTGTGGAAGTGGAAGAACTGGAAGATTTCACCTTAC               AAATGGCGCACCACATCTCTGAGAAAGCGCCGTTAGCCATTGCCGTTATCAAAGAAGAGC               TGCGTGTACTGGGCGAAGCACACACCATGAACTCCGATGAATTTGAACGTATTCAGGGGA               TGCGCCGCGCGGTGTATGACAGCGAAGATTACCAGGAAGGGATGAACGCTTTCCTCGAAA               AACGTAAACCTAATTTCGTTGGTCATTAATCCCTGCGAACGAAGGAGTAAAAATGGAAAC               TCAGTGGACAAGGATGACCGCCAATGAAGCGGCAGAAATTATCCAGCATAACGACATGGT               GGCATTTAGCGGCTTTACCCCGGCGGGTTCGCCGAAAGCCCTACCCACCGCGATTGCCCG               CAGAGCTAACGAACAGCATGAGGCCAAAAAGCCGTATCAAATTCGCCTTCTGACGGGTGC               GTCAATCAGCGCCGCCGCTGACGATGTACTTTCTGACGCCGATGCTGTTTCCTGGCGTGC               GCCATATCAAACATCGTCCGGTTTACGTAAAAAGATCAATCAGGGCGCGGTGAGTTTCGT               TGACCTGCATTTGAGCGAAGTGGCGCAAATGGTCAATTACGGTTTCTTCGGCGACATTGA               TGTTGCCGTCATTGAAGCATCGGCACTGGCACCGGATGGTCGAGTCTGGTTAACCAGCGG               GATCGGTAATGCGCCGACCTGGCTGCTGCGGGCGAAGAAAGTGATCATTGAACTCAATCA               CTATCACGATCCGCGCGTTGCAGAACTGGCGGATATTGTGATTCCTGGCGCGCCACCGCG               GCGCAATAGCGTGTCGATCTTCCATGCAATGGATCGCGTCGGTACCCGCTATGTGCAAAT               CGATCCGAAAAAGATTGTCGCCGTCGTGGAAACCAACTTGCCCGACGCCGGTAATATGCT               GGATAAGCAAAATCCCATGTGCCAGCAGATTGCCGATAACGTGGTCACGTTCTTATTGCA               GGAAATGGCGCATGGGCGTATTCCGCCGGAATTTCTGCCGCTGCAAAGTGGCGTGGGCAA               TATCAATAATGCGGTAATGGCGCGTCTGGGGGAAAACCCGGTAATTCCTCCGTTTATGAT               GTATTCGGAAGTGCTACAGGAATCGGTGGTGCATTTACTGGAAACCGGCAAAATCAGCGG               GGCCAGCGCCTCCAGCCTGACAATCTCGGCCGATTCCCTGCGCAAGATTTACGACAATAT               GGATTACTTTGCCAGCCGCATTGTGTTGCGTCCGCAGGAGATTTCCAATAACCCGGAAAT               CATCCGTCGTCTGGGCGTCATCGCTCTGAACGTCGGCCTGGAGTTTGATATTTACGGGCA               TGCCAACTCAACACACGTAGCCGGGGTCGATCTGATGAACGGCATCGGCGGCAGCGGTGA               TTTTGAACGCAACGCGTATCTGTCGATCTTTATGGCCCCGTCGATTGCTAAAGAAGGCAA               GATCTCAACCGTCGTGCCAATGTGCAGCCATGTTGATCACAGCGAACACAGCGTCAAAGT               GATCATCACCGAACAAGGGATCGCCGATCTGCGCGGTCTTTCCCCGCTTCAACGCGCCCG               CACTATCATTGATAATTGTGCACATCCTATGTATCGGGATTATCTGCATCGCTATCTGGA               AAATGCGCCTGGCGGACATATTCACCACGATCTTAGCCACGTCTTCGACTTACACCGTAA               TTTAATTGCAACCGGCTCGATGCTGGGTTAA                 FNRS promoter bold lowercase,     agttgttcttattggtggtgttgctttatggttgcatcgtagtaaatggttgtaacaaa         with RBS and leader region     agcaatttttccggctgtctgtatacaaaaacgccgcaaagt         bold and underlined, and FNR       tggatccaaag         binding site bold and italics);         ATGTCTAACGTGCAGGA         sleeping beauty operon (sbm,     GTGGCAACAGCTTGCCAACAAGGAATTGAGCCGTCGGGAGAAAACTGTCGACTCGCTGGT         ygfD, ygfG, ygfH) bold and     TCATCAAACCGCGGAAGGGATCGCCATCAAGCCGCTGTATACCGAAGCCGATCTCGATAA         uppercase     TCTGGAGGTGACAGGTACCCTTCCTGGTTTGCCGCCCTACGTTCGTGGCCCGCGTGCCAC         (SEQ ID NO: 218)     TATGTATACCGCCCAACCGTGGACCATCCGTCAGTATGCTGGTTTTTCAACAGCAAAAGA               GTCCAACGCTTTTTATCGCCGTAACCTGGCCGCCGGGCAAAAAGGTCTTTCCGTTGCGTT               TGACCTTGCCACCCACCGTGGCTACGACTCCGATAACCCGCGCGTGGCGGGCGACGTCGG               CAAAGCGGGCGTCGCTATCGACACCGTGGAAGATATGAAAGTCCTGTTCGACCAGATCCC               GCTGGATAAAATGTCGGTTTCGATGACCATGAATGGCGCAGTGCTACCAGTACTGGCGTT               TTATATCGTCGCCGCAGAAGAGCAAGGTGTTACACCTGATAAACTGACCGGCACCATTCA               AAACGATATTCTCAAAGAGTACCTCTGCCGCAACACCTATATTTACCCACCAAAACCGTC               AATGCGCATTATCGCCGACATCATCGCCTGGTGTTCCGGCAACATGCCGCGATTTAATAC               CATCAGTATCAGCGGTTACCACATGGGTGAAGCGGGTGCCAACTGCGTGCAGCAGGTAGC               ATTTACGCTCGCTGATGGGATTGAGTACATCAAAGCAGCAATCTCTGCCGGACTGAAAAT               TGATGACTTCGCTCCTCGCCTGTCGTTCTTCTTCGGCATCGGCATGGATCTGTTTATGAA               CGTCGCCATGTTGCGTGCGGCACGTTATTTATGGAGCGAAGCGGTCAGTGGATTTGGCGC               ACAGGACCCGAAATCACTGGCGCTGCGTACCCACTGCCAGACCTCAGGCTGGAGCCTGAC               TGAACAGGATCCGTATAACAACGTTATCCGCACCACCATTGAAGCGCTGGCTGCGACGCT               GGGCGGTACTCAGTCACTGCATACCAACGCCTTTGACGAAGCGCTTGGTTTGCCTACCGA               TTTCTCAGCACGCATTGCCCGCAACACCCAGATCATCATCCAGGAAGAATCAGAACTCTG               CCGCACCGTCGATCCACTGGCCGGATCCTATTACATTGAGTCGCTGACCGATCAAATCGT               CAAACAAGCCAGAGCTATTATCCAACAGATCGACGAAGCCGGTGGCATGGCGAAAGCGAT               CGAAGCAGGTCTGCCAAAACGAATGATCGAAGAGGCCTCAGCGCGCGAACAGTCGCTGAT               CGACCAGGGCAAGCGTGTCATCGTTGGTGTCAACAAGTACAAACTGGATCACGAAGACGA               AACCGATGTACTTGAGATCGACAACGTGATGGTGCGTAACGAGCAAATTGCTTCGCTGGA               ACGCATTCGCGCCACCCGTGATGATGCCGCCGTAACCGCCGCGTTGAACGCCCTGACTCA               CGCCGCACAGCATAACGAAAACCTGCTGGCTGCCGCTGTTAATGCCGCTCGCGTTCGCGC               CACCCTGGGTGAAATTTCCGATGCGCTGGAAGTCGCTTTCGACCGTTATCTGGTGCCAAG               CCAGTGTGTTACCGGCGTGATTGCGCAAAGCTATCATCAGTCTGAGAAATCGGCCTCCGA               GTTCGATGCCATTGTTGCGCAAACGGAGCAGTTCCTTGCCGACAATGGTCGTCGCCCGCG               CATTCTGATCGCTAAGATGGGCCAGGATGGACACGATCGCGGCGCGAAAGTGATCGCCAG               CGCCTATTCCGATCTCGGTTTCGACGTAGATTTAAGCCCGATGTTCTCTACACCTGAAGA               GATCGCCCGCCTGGCCGTAGAAAACGACGTTCACGTAGTGGGCGCATCCTCACTGGCTGC               CGGTCATAAAACGCTGATCCCGGAACTGGTCGAAGCGCTGAAAAAATGGGGACGCGAAGA               TATCTGCGTGGTCGCGGGTGGCGTCATTCCGCCGCAGGATTACGCCTTCCTGCAAGAGCG               CGGCGTGGCGGCGATTTATGGTCCAGGTACACCTATGCTCGACAGTGTGCGCGACGTACT               GAATCTGATAAGCCAGCATCATGATTAATGAAGCCACGCTGGCAGAAAGTATTCGCCGCT               TACGTCAGGGTGAGCGTGCCACACTCGCCCAGGCCATGACGCTGGTGGAAAGCCGTCACC               CGCGTCATCAGGCACTAAGTACGCAGCTGCTTGATGCCATTATGCCGTACTGCGGTAACA               CCCTGCGACTGGGCGTTACCGGCACCCCCGGCGCGGGGAAAAGTACCTTTCTTGAGGCCT               TTGGCATGTTGTTGATTCGAGAGGGATTAAAGGTCGCGGTTATTGCGGTCGATCCCAGCA               GCCCGGTCACTGGCGGTAGCATTCTCGGGGATAAAACCCGCATGAATGACCTGGCGCGTG               CCGAAGCGGCGTTTATTCGCCCGGTACCATCCTCCGGTCATCTGGGCGGTGCCAGTCAGC               GAGCGCGGGAATTAATGCTGTTATGCGAAGCAGCGGGTTATGACGTAGTGATTGTCGAAA               CGGTTGGCGTCGGGCAGTCGGAAACAGAAGTCGCCCGCATGGTGGACTGTTTTATCTCGT               TGCAAATTGCCGGTGGCGGCGATGATCTGCAGGGCATTAAAAAAGGGCTGATGGAAGTGG               CTGATCTGATCGTTATCAACAAAGACGATGGCGATAACCATACCAATGTCGCCATTGCCC               GGCATATGTACGAGAGTGCCCTGCATATTCTGCGACGTAAATACGACGAATGGCAGCCAC               GGGTTCTGACTTGTAGCGCACTGGAAAAACGTGGAATCGATGAGATCTGGCACGCCATCA               TCGACTTCAAAACCGCGCTAACTGCCAGTGGTCGTTTACAACAAGTGCGGCAACAACAAT               CGGTGGAATGGCTGCGTAAGCAGACCGAAGAAGAAGTACTGAATCACCTGTTCGCGAATG               AAGATTTCGATCGCTATTACCGCCAGACGCTTTTAGCGGTCAAAAACAATACGCTCTCAC               CGCGCACCGGCCTGCGGCAGCTCAGTGAATTTATCCAGACGCAATATTTTGATTAAAGGA               ATTTTTATGTCTTATCAGTATGTTAACGTTGTCACTATCAACAAAGTGGCGGTCATTGAG               TTTAACTATGGCCGAAAACTTAATGCCTTAAGTAAAGTCTTTATTGATGATCTTATGCAG               GCGTTAAGCGATCTCAACCGGCCGGAAATTCGCTGTATCATTTTGCGCGCACCGAGTGGA               TCCAAAGTCTTCTCCGCAGGTCACGATATTCACGAACTGCCGTCTGGCGGTCGCGATCCG               CTCTCCTATGATGATCCATTGCGTCAAATCACCCGCATGATCCAAAAATTCCCGAAACCG               ATCATTTCGATGGTGGAAGGTAGTGTTTGGGGTGGCGCATTTGAAATGATCATGAGTTCC               GATCTGATCATCGCCGCCAGTACCTCAACCTTCTCAATGACGCCTGTAAACCTCGGCGTC               CCGTATAACCTGGTCGGCATTCACAACCTGACCCGCGACGCGGGCTTCCACATTGTCAAA               GAGCTGATTTTTACCGCTTCGCCAATCACCGCCCAGCGCGCGCTGGCTGTCGGCATCCTC               AACCATGTTGTGGAAGTGGAAGAACTGGAAGATTTCACCTTACAAATGGCGCACCACATC               TCTGAGAAAGCGCCGTTAGCCATTGCCGTTATCAAAGAAGAGCTGCGTGTACTGGGCGAA               GCACACACCATGAACTCCGATGAATTTGAACGTATTCAGGGGATGCGCCGCGCGGTGTAT               GACAGCGAAGATTACCAGGAAGGGATGAACGCTTTCCTCGAAAAACGTAAACCTAATTTC               GTTGGTCATTAATCCCTGCGAACGAAGGAGTAAAAATGGAAACTCAGTGGACAAGGATGA               CCGCCAATGAAGCGGCAGAAATTATCCAGCATAACGACATGGTGGCATTTAGCGGCTTTA               CCCCGGCGGGTTCGCCGAAAGCCCTACCCACCGCGATTGCCCGCAGAGCTAACGAACAGC               ATGAGGCCAAAAAGCCGTATCAAATTCGCCTTCTGACGGGTGCGTCAATCAGCGCCGCCG               CTGACGATGTACTTTCTGACGCCGATGCTGTTTCCTGGCGTGCGCCATATCAAACATCGT               CCGGTTTACGTAAAAAGATCAATCAGGGCGCGGTGAGTTTCGTTGACCTGCATTTGAGCG               AAGTGGCGCAAATGGTCAATTACGGTTTCTTCGGCGACATTGATGTTGCCGTCATTGAAG               CATCGGCACTGGCACCGGATGGTCGAGTCTGGTTAACCAGCGGGATCGGTAATGCGCCGA               CCTGGCTGCTGCGGGCGAAGAAAGTGATCATTGAACTCAATCACTATCACGATCCGCGCG               TTGCAGAACTGGCGGATATTGTGATTCCTGGCGCGCCACCGCGGCGCAATAGCGTGTCGA               TCTTCCATGCAATGGATCGCGTCGGTACCCGCTATGTGCAAATCGATCCGAAAAAGATTG               TCGCCGTCGTGGAAACCAACTTGCCCGACGCCGGTAATATGCTGGATAAGCAAAATCCCA               TGTGCCAGCAGATTGCCGATAACGTGGTCACGTTCTTATTGCAGGAAATGGCGCATGGGC               GTATTCCGCCGGAATTTCTGCCGCTGCAAAGTGGCGTGGGCAATATCAATAATGCGGTAA               TGGCGCGTCTGGGGGAAAACCCGGTAATTCCTCCGTTTATGATGTATTCGGAAGTGCTAC               AGGAATCGGTGGTGCATTTACTGGAAACCGGCAAAATCAGCGGGGCCAGCGCCTCCAGCC               TGACAATCTCGGCCGATTCCCTGCGCAAGATTTACGACAATATGGATTACTTTGCCAGCC               GCATTGTGTTGCGTCCGCAGGAGATTTCCAATAACCCGGAAATCATCCGTCGTCTGGGCG               TCATCGCTCTGAACGTCGGCCTGGAGTTTGATATTTACGGGCATGCCAACTCAACACACG               TAGCCGGGGTCGATCTGATGAACGGCATCGGCGGCAGCGGTGATTTTGAACGCAACGCGT               ATCTGTCGATCTTTATGGCCCCGTCGATTGCTAAAGAAGGCAAGATCTCAACCGTCGTGC               CAATGTGCAGCCATGTTGATCACAGCGAACACAGCGTCAAAGTGATCATCACCGAACAAG               GGATCGCCGATCTGCGCGGTCTTTCCCCGCTTCAACGCGCCCGCACTATCATTGATAATT               GTGCACATCCTATGTATCGGGATTATCTGCATCGCTATCTGGAAAATGCGCCTGGCGGAC               ATATTCACCACGATCTTAGCCACGTCTTCGACTTACACCGTAATTTAATTGCAACCGGCT               CGATGCTGGGTTAA                      
Next, this strain was tested for propionate production.
 
     Briefly, 3 mil 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. 20B  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 27. 
     Example 25. 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 26. 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 , was synthesized by Genewiz and placed in a high copy plasmid (Logic051). 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. 
     
       
         
           
               
            
               
                   
               
               
                 Table 46 of Exemplary Propionate Cassette Sequences 
               
            
           
           
               
               
            
               
                 Description and SEQ ID NO 
                 Sequence 
               
               
                   
               
               
                 Ptet-pct-lcdABC-acrABC; Ptet: 
                 ttaagacccactttcacatttaagttgatttctaatccgcatatgatcaattcaaggccg 
               
               
                 lower case; tertR/tetA promoter 
                 aataagaaggctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaataat 
               
               
                 within Ptet: lower case bold, 
                 ggcggcatactatcagtagtaggtgtttccctttcttctttagcgacttgatgctcttga 
               
               
                 with tet operator: lower case 
                 tatccaatacgcaacctaaagtaaaatgccccacagcgctgagtgcatataatgcattct 
               
               
                 bold underlined; ribosome 
                 ctagtgaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcatactgat 
               
               
                 binding site and leader: lowe 
                 ttctgtaggccgtgtacctaaatgtacttttgctccatcgcgatgacttagtaaagcaca 
               
               
                 case italic; ribosome binding 
                 tctaaaacttttagcgttattacgtaaaaaatcttgccagattcccatctaaagggcaaa 
               
               
                 sites: lower case underlined; 
                 agtgagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagcccgcttat 
               
               
                 coding regions: upper case; 
                 tttttacatgccaatacaatgtaggctgctctacacctagcttctgggcgagtttacggg 
               
               
                 (SEQ ID NO: 219) 
                 ttgttaaaccttcgattccgacctcattaagcagctctaatgcgctgttaatcactttac 
               
               
                   
                 ttttatctaatctagacatcattaattcctaattttta gttgac   
               
               
                   
                     tattttaccac       aa aagtgaa ct   
               
               
                   
                   ctagaaataattttgtttaactttaa     gaaggagatatacat   ATGCGCAAAGTGCCGATTA 
               
               
                   
                 TCACGGCTGACGAGGCCGCAAAACTGATCAAGGACGGCGACACCGTGACAACTAGCGGCT 
               
               
                   
                 TTGTGGGTAACGCGATCCCTGAGGCCCTTGACCGTGCAGTCGAAAAGCGTTTCCTGGAAA 
               
               
                   
                 CGGGCGAACCGAAGAACATTACTTATGTATATTGCGGCAGTCAGGGCAATCGCGACGGTC 
               
               
                   
                 GTGGCGCAGAACATTTCGCGCATGAAGGCCTGCTGAAACGTTATATCGCTGGCCATTGGG 
               
               
                   
                 CGACCGTCCCGGCGTTAGGGAAAATGGCCATGGAGAATAAAATGGAGGCCTACAATGTCT 
               
               
                   
                 CTCAGGGCGCCTTGTGTCATCTCTTTCGCGATATTGCGAGCCATAAACCGGGTGTGTTCA 
               
               
                   
                 CGAAAGTAGGAATCGGCACCTTCATTGATCCACGTAACGGTGGTGGGAAGGTCAACGATA 
               
               
                   
                 TTACCAAGGAAGATATCGTAGAACTGGTGGAAATTAAAGGGCAGGAATACCTGTTTTATC 
               
               
                   
                 CGGCGTTCCCGATCCATGTCGCGCTGATTCGTGGCACCTATGCGGACGAGAGTGGTAACA 
               
               
                   
                 TCACCTTTGAAAAAGAGGTAGCGCCTTTGGAAGGGACTTCTGTCTGTCAAGCGGTGAAGA 
               
               
                   
                 ACTCGGGTGGCATTGTCGTGGTTCAGGTTGAGCGTGTCGTCAAAGCAGGCACGCTGGATC 
               
               
                   
                 CGCGCCATGTGAAAGTTCCGGGTATCTATGTAGATTACGTAGTCGTCGCGGATCCGGAGG 
               
               
                   
                 ACCATCAACAGTCCCTTGACTGCGAATATGATCCTGCCCTTAGTGGAGAGCACCGTCGTC 
               
               
                   
                 CGGAGGTGGTGGGTGAACCACTGCCTTTATCCGCGAAGAAAGTCATCGGCCGCCGTGGCG 
               
               
                   
                 CGATTGAGCTCGAGAAAGACGTTGCAGTGAACCTTGGGGTAGGTGCACCTGAGTATGTGG 
               
               
                   
                 CCTCCGTGGCCGATGAAGAAGGCATTGTGGATTTTATGACTCTCACAGCGGAGTCCGGCG 
               
               
                   
                 CTATCGGTGGCGTTCCAGCCGGCGGTGTTCGCTTTGGGGCGAGCTACAATGCTGACGCCT 
               
               
                   
                 TGATCGACCAGGGCTACCAATTTGATTATTACGACGGTGGGGGTCTGGATCTTTGTTACC 
               
               
                   
                 TGGGTTTAGCTGAATGCGACGAAAAGGGTAATATCAATGTTAGCCGCTTCGGTCCTCGTA 
               
               
                   
                 TCGCTGGGTGCGGCGGATTCATTAACATTACCCAAAACACGCCGAAAGTCTTCTTTTGTG 
               
               
                   
                 GGACCTTTACAGCCGGGGGGCTGAAAGTGAAAATTGAAGATGGTAAGGTGATTATCGTTC 
               
               
                   
                 AGGAAGGGAAACAGAAGAAATTCCTTAAGGCAGTGGAGCAAATCACCTTTAATGGAGACG 
               
               
                   
                 TGGCCTTAGCGAACAAGCAACAAGTTACCTACATCACGGAGCGTTGCGTCTTCCTCCTCA 
               
               
                   
                 AAGAAGACGGTTTACACCTTTCGGAAATCGCGCCAGGCATCGATCTGCAGACCCAGATTT 
               
               
                   
                 TGGATGTTATGGACTTTGCCCCGATCATTGATCGTGACGCAAACGGGCAGATTAAACTGA 
               
               
                   
                 TGGACGCGGCGTTATTCGCAGAAGGGCTGATGGGCTTGAAAGAAATGAAGTCTTGAtaa g   
               
               
                   
                   aaggagatatacat ATGAGCTTAACCCAAGGCATGAAAGCTAAACAACTGTTAGCATACT 
               
               
                   
                 TTCAGGGTAAAGCCGATCAGGATGCACGTGAAGCGAAAGCCCGCGGTGAGCTGGTCTGCT 
               
               
                   
                 GGTCGGCGTCAGTCGCGCCGCCGGAATTTTGCGTAACAATGGGCATTGCCATGATCTACC 
               
               
                   
                 CGGAGACTCATGCAGCGGGCATCGGTGCCCGCAAAGGTGCGATGGACATGCTGGAAGTTG 
               
               
                   
                 CGGACCGCAAAGGCTACAACGTGGATTGTTGTTCCTACGGCCGTGTAAATATGGGTTACA 
               
               
                   
                 TGGAATGTTTAAAAGAAGCCGCCATCACGGGCGTCAAGCCGGAAGTTTTGGTTAATTCCC 
               
               
                   
                 CTGCTGCTGACGTTCCGCTTCCCGATTTGGTGATTACGTGTAATAATATCTGTAACACGC 
               
               
                   
                 TGCTGAAATGGTACGAAAACTTAGCAGCAGAACTCGATATTCCTTGCATCGTGATCGACG 
               
               
                   
                 TACCGTTTAATCATACCATGCCGATTCCGGAATATGCCAAGGCCTACATCGCGGACCAGT 
               
               
                   
                 TCCGCAATGCAATTTCTCAGCTGGAAGTTATTTGTGGCCGTCCGTTCGATTGGAAGAAAT 
               
               
                   
                 TTAAGGAGGTCAAAGATCAGACCCAGCGTAGCGTATACCACTGGAACCGCATTGCCGAGA 
               
               
                   
                 TGGCGAAATACAAGCCTAGCCCGCTGAACGGCTTCGATCTGTTCAATTACATGGCGTTAA 
               
               
                   
                 TCGTGGCGTGCCGCAGCCTGGATTATGCAGAAATTACCTTTAAAGCGTTCGCGGACGAAT 
               
               
                   
                 TAGAAGAGAATTTGAAGGCGGGTATCTACGCCTTTAAAGGTGCGGAAAAAACGCGCTTTC 
               
               
                   
                 AATGGGAAGGTATCGCGGTGTGGCCACATTTAGGTCACACGTTTAAATCTATGAAGAATC 
               
               
                   
                 TGAATTCGATTATGACCGGTACGGCATACCCCGCCCTTTGGGACCTGCACTATGACGCTA 
               
               
                   
                 ACGACGAATCTATGCACTCTATGGCTGAAGCGTACACCCGTATTTATATTAATACTTGTC 
               
               
                   
                 TGCAGAACAAAGTAGAGGTCCTGCTTGGGATCATGGAAAAAGGCCAGGTGGATGGTACCG 
               
               
                   
                 TATATCATCTGAATCGCAGCTGCAAACTGATGAGTTTCCTGAACGTGGAAACGGCTGAAA 
               
               
                   
                 TTATTAAAGAGAAGAACGGTCTTCCTTACGTCTCCATTGATGGCGATCAGACCGATCCTC 
               
               
                   
                 GCGTTTTTTCTCCGGCCCAGTTTGATACCCGTGTTCAGGCCCTGGTTGAGATGATGGAGG 
               
               
                   
                 CCAATATGGCGGCAGCGGAATAAtaagaaggagatatacatATGTCACGCGTGGAGGCAA 
               
               
                   
                 TCCTGTCGCAGCTGAAAGATGTCGCCGCGAATCCGAAAAAAGCCATGGATGACTATAAAG 
               
               
                   
                 CTGAAACAGGTAAGGGCGCGGTTGGTATCATGCCGATCTACAGCCCCGAAGAAATGGTAC 
               
               
                   
                 ACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGCGCCCAGGGCAAAACGATTAGTAAAG 
               
               
                   
                 CGCGCACCTATCTGCCTGCTTTTGCCTGCAGCGTAATGCAGCAGGTTATGGAATTACAGT 
               
               
                   
                 GCGAGGGCGCGTATGATGACCTGTCCGCAGTTATTTTTAGCGTACCGTGCGACACTCTCA 
               
               
                   
                 AATGTCTTAGCCAGAAATGGAAAGGTACGTCCCCAGTGATTGTATTTACGCATCCGCAGA 
               
               
                   
                 ACCGCGGATTAGAAGCGGCGAACCAATTCTTGGTTACCGAGTATGAACTGGTAAAAGCAC 
               
               
                   
                 AACTGGAATCAGTTCTGGGTGTGAAAATTTCAAACGCCGCCCTGGAAAATTCGATTGCAA 
               
               
                   
                 TTTATAACGAGAATCGTGCCGTGATGCGTGAGTTCGTGAAAGTGGCAGCGGACTATCCTC 
               
               
                   
                 AAGTCATTGACGCAGTGAGCCGCCACGCGGTTTTTAAAGCGCGCCAGTTTATGCTTAAGG 
               
               
                   
                 AAAAACATACCGCACTTGTGAAAGAACTGATCGCTGAGATTAAAGCAACGCCAGTCCAGC 
               
               
                   
                 CGTGGGACGGAAAAAAGGTTGTAGTGACGGGCATTCTGTTGGAACCGAATGAGTTATTAG 
               
               
                   
                 ATATCTTTAATGAGTTTAAGATCGCGATTGTTGATGATGATTTAGCGCAGGAAAGCCGTC 
               
               
                   
                 AGATCCGTGTTGACGTTCTGGACGGAGAAGGCGGACCGCTCTACCGTATGGCTAAAGCGT 
               
               
                   
                 GGCAGCAAATGTATGGCTGCTCGCTGGCAACCGACACCAAGAAGGGTCGCGGCCGTATGT 
               
               
                   
                 TAATTAACAAAACGATTCAGACCGGTGCGGACGCTATCGTAGTTGCAATGATGAAGTTTT 
               
               
                   
                 GCGACCCAGAAGAATGGGATTATCCGGTAATGTACCGTGAATTTGAAGAAAAAGGGGTCA 
               
               
                   
                 AATCACTTATGATTGAGGTGGATCAGGAAGTATCGTCTTTCGAACAGATTAAAACCCGTC 
               
               
                   
                 TGCAGTCATTCGTCGAAATGCTTTAAtaag aaggagatatacat ATGTATACCTTGGGGA 
               
               
                   
                 TTGATGTCGGTTCTGCCTCTAGTAAAGCGGTGATTCTGAAAGATGGAAAAGATATTGTCG 
               
               
                   
                 CTGCCGAGGTTGTCCAAGTCGGTACCGGCTCCTCGGGTCCCCAACGCGCACTGGACAAAG 
               
               
                   
                 CCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACATCAGCTACACAGTAGCTACGGGCTATG 
               
               
                   
                 GGCGCTTCAATTTTAGCGACGCGGATAAACAGATTTCGGAAATTAGCTGTCATGCCAAAG 
               
               
                   
                 GCATTTATTTCTTAGTACCAACTGCGCGCACTATTATTGACATTGGCGGCCAAGATGCGA 
               
               
                   
                 AAGCCATCCGCCTGGACGACAAGGGGGGTATTAAGCAATTCTTCATGAATGATAAATGCG 
               
               
                   
                 CGGCGGGCACGGGGCGTTTCCTGGAAGTCATGGCTCGCGTACTTGAAACCACCCTGGATG 
               
               
                   
                 AAATGGCTGAACTGGATGAACAGGCGACTGACACCGCTCCCATTTCAAGCACCTGCACGG 
               
               
                   
                 TTTTCGCCGAAAGCGAAGTAATTAGCCAATTGAGCAATGGTGTCTCACGCAACAACATCA 
               
               
                   
                 TTAAAGGTGTCCATCTGAGCGTTGCGTCACGTGCGTGTGGTCTGGCGTATCGCGGCGGTT 
               
               
                   
                 TGGAGAAAGATGTTGTTATGACAGGTGGCGTGGCAAAAAATGCAGGGGTGGTGCGCGCGG 
               
               
                   
                 TGGCGGGCGTTCTGAAGACCGATGTTATCGTTGCTCCGAATCCTCAGACGACCGGTGCAC 
               
               
                   
                 TGGGGGCAGCGCTGTATGCTTATGAGGCCGCCCAGAAGAAGTAAtaa gaaggagatatac   
               
               
                   
                   at ATGGCCTTCAATAGCGCAGATATTAATTCTTTCCGCGATATTTGGGTGTTTTGTGAAC 
               
               
                   
                 AGCGTGAGGGCAAACTGATTAACACCGATTTCGAATTAATTAGCGAAGGTCGTAAACTGG 
               
               
                   
                 CTGACGAACGCGGAAGCAAACTGGTTGGAATTTTGCTGGGGCACGAAGTTGAAGAAATCG 
               
               
                   
                 CAAAAGAATTAGGCGGCTATGGTGCGGACAAGGTAATTGTGTGCGATCATCCGGAACTTA 
               
               
                   
                 AATTTTACACTACGGATGCTTATGCCAAAGTTTTATGTGACGTCGTGATGGAAGAGAAAC 
               
               
                   
                 CGGAGGTAATTTTGATCGGTGCCACCAACATTGGCCGTGATCTCGGACCGCGTTGTGCTG 
               
               
                   
                 CACGCTTGCACACGGGGCTGACGGCTGATTGCACGCACCTGGATATTGATATGAATAAAT 
               
               
                   
                 ATGTGGACTTTCTTAGCACCAGTAGCACCTTGGATATCTCGTCGATGACTTTCCCTATGG 
               
               
                   
                 AAGATACAAACCTTAAAATGACGCGCCCTGCATTTGGCGGACATCTGATGGCAACGATCA 
               
               
                   
                 TTTGTCCACGCTTCCGTCCCTGTATGAGCACAGTGCGCCCCGGAGTGATGAAGAAAGCGG 
               
               
                   
                 AGTTCTCGCAGGAGATGGCGCAAGCATGTCAAGTAGTGACCCGTCACGTAAATTTGTCGG 
               
               
                   
                 ATGAAGACCTTAAAACTAAAGTAATTAATATCGTGAAGGAAACGAAAAAGATTGTGGATC 
               
               
                   
                 TGATCGGCGCAGAAATTATTGTGTCAGTTGGTCGTGGTATCTCGAAAGATGTCCAAGGTG 
               
               
                   
                 GAATTGCACTGGCTGAAAAACTTGCGGACGCATTTGGTAACGGTGTCGTGGGCGGCTCGC 
               
               
                   
                 GCGCAGTGATTGATTCCGGCTGGTTACCTGCGGATCATCAGGTTGGACAAACCGGTAAGA 
               
               
                   
                 CCGTGCACCCGAAAGTCTACGTGGCGCTGGGTATTAGTGGGGCTATCCAGCATAAGGCTG 
               
               
                   
                 GGATGCAAGACTCTGAACTGATCATTGCCGTCAACAAAGACGAAACGGCGCCTATCTTCG 
               
               
                   
                 ACTGCGCCGATTATGGCATCACCGGTGATTTATTTAAAATCGTACCGATGATGATCGACG 
               
               
                   
                 CGATCAAAGAGGGTAAAAACGCATGAtaa gaaggagatatacat ATGCGCATCTATGTGT 
               
               
                   
                 GTGTGAAACAAGTCCCAGATACGAGCGGCAAGGTGGCCGTTAACCCTGATGGGACCCTTA 
               
               
                   
                 ACCGTGCCTCAATGGCAGCGATTATTAACCCGGACGATATGTCCGCGATCGAACAGGCAT 
               
               
                   
                 TAAAACTGAAAGATGAAACCGGATGCCAGGTTACGGCGCTTACGATGGGTCCTCCTCCTG 
               
               
                   
                 CCGAGGGCATGTTGCGCGAAATTATTGCAATGGGGGCCGACGATGGTGTGCTGATTTCGG 
               
               
                   
                 CCCGTGAATTTGGGGGGTCCGATACCTTCGCAACCAGTCAAATTATTAGCGCGGCAATCC 
               
               
                   
                 ATAAATTAGGCTTAAGCAATGAAGACATGATCTTTTGCGGTCGTCAGGCCATTGACGGTG 
               
               
                   
                 ATACGGCCCAAGTCGGCCCTCAAATTGCCGAAAAACTGAGCATCCCACAGGTAACCTATG 
               
               
                   
                 GCGCAGGAATCAAAAAATCTGGTGATTTAGTGCTGGTGAAGCGTATGTTGGAGGATGGTT 
               
               
                   
                 ATATGATGATCGAAGTCGAAACTCCATGTCTGATTACCTGCATTCAGGATAAAGCGGTAA 
               
               
                   
                 AACCACGTTACATGACTCTCAACGGTATTATGGAATGCTACTCCAAGCCGCTCCTCGTTC 
               
               
                   
                 TCGATTACGAAGCACTGAAAGATGAACCGCTGATCGAACTTGATACCATTGGGCTTAAAG 
               
               
                   
                 GCTCCCCGACGAATATCTTTAAATCGTTTACGCCGCCTCAGAAAGGCGTTGGTGTCATGC 
               
               
                   
                 TCCAAGGCACCGATAAGGAAAAAGTCGAGGATCTGGTGGATAAGCTGATGCAGAAACATG 
               
               
                   
                 TCATCTAAtaa gaaggagatatacat ATGTTCTTACTGAAGATTAAAAAAGAACGTATGA 
               
               
                   
                 AACGCATGGACTTTAGTTTAACGCGTGAACAGGAGATGTTAAAAAAACTGGCGCGTCAGT 
               
               
                   
                 TTGCTGAGATCGAGCTGGAACCGGTGGCCGAAGAGATTGATCGTGAGCACGTTTTTCCTG 
               
               
                   
                 CAGAAAACTTTAAGAAGATGGCGGAAATTGGCTTAACCGGCATTGGTATCCCGAAAGAAT 
               
               
                   
                 TTGGTGGCTCCGGTGGAGGCACCCTGGAGAAGGTCATTGCCGTGTCAGAATTCGGCAAAA 
               
               
                   
                 AGTGTATGGCCTCAGCTTCCATTTTAAGCATTCATCTTATCGCGCCGCAGGCAATCTACA 
               
               
                   
                 AATATGGGACCAAAGAACAGAAAGAGACGTACCTGCCGCGTCTTACCAAAGGTGGTGAAC 
               
               
                   
                 TGGGCGCCTTTGCGCTGACAGAACCAAACGCCGGAAGCGATGCCGGCGCGGTAAAAACGA 
               
               
                   
                 CCGCGATTCTGGACAGCCAGACAAACGAGTACGTGCTGAATGGCACCAAATGCTTTATCA 
               
               
                   
                 GCGGGGGCGGGCGCGCGGGTGTTCTTGTAATTTTTGCGCTTACTGAACCGAAAAAAGGTC 
               
               
                   
                 TGAAAGGGATGAGCGCGATTATCGTGGAGAAAGGGACCCCGGGCTTCAGCATCGGCAAGG 
               
               
                   
                 TGGAGAGCAAGATGGGGATCGCAGGTTCGGAAACCGCGGAACTTATCTTCGAAGATTGTC 
               
               
                   
                 GCGTTCCGGCTGCCAACCTTTTAGGTAAAGAAGGCAAAGGCTTTAAAATTGCTATGGAAG 
               
               
                   
                 CCCTGGATGGCGCCCGTATTGGCGTGGGCGCTCAAGCAATCGGAATTGCCGAGGGGGCGA 
               
               
                   
                 TCGACCTGAGTGTGAAGTACGTTCACGAGCGCATTCAATTTGGTAAACCGATCGCGAATC 
               
               
                   
                 TGCAGGGAATTCAATGGTATATCGCGGATATGGCGACCAAAACCGCCGCGGCACGCGCAC 
               
               
                   
                 TTGTTGAGTTTGCAGCGTATCTTGAAGACGCGGGTAAACCGTTCACAAAGGAATCTGCTA 
               
               
                   
                 TGTGCAAGCTGAACGCCTCCGAAAACGCGCGTTTTGTGACAAATTTAGCTCTGCAGATTC 
               
               
                   
                 ACGGGGGTTACGGTTATATGAAAGATTATCCGTTAGAGCGTATGTATCGCGATGCTAAGA 
               
               
                   
                 TTACGGAAATTTACGAGGGGACATCAGAAATCCATAAGGTGGTGATTGCGCGTGAAGTAA 
               
               
                   
                 TGAAACGCTAA 
               
               
                   
               
               
                 pct-lcdABC-acrABC 
                 ATGCGCAAAGTGCCGATTATCACGGCTGACGAGGCCGCAAAACTGATCAAGGACGGCGAC 
               
               
                 (ribosome binding sites: lower 
                 ACCGTGACAACTAGCGGCTTTGTGGGTAACGCGATCCCTGAGGCCCTTGACCGTGCAGTC 
               
               
                 case underlined; coding regions: 
                 GAAAAGCGTTTCCTGGAAACGGGCGAACCGAAGAACATTACTTATGTATATTGCGGCAGT 
               
               
                 upper case) 
                 CAGGGCAATCGCGACGGTCGTGGCGCAGAACATTTCGCGCATGAAGGCCTGCTGAAACGT 
               
               
                 (SEQ ID NO: 220) 
                 TATATCGCTGGCCATTGGGCGACCGTCCCGGCGTTAGGGAAAATGGCCATGGAGAATAAA 
               
               
                   
                 ATGGAGGCCTACAATGTCTCTCAGGGCGCCTTGTGTCATCTCTTTCGCGATATTGCGAGC 
               
               
                   
                 CATAAACCGGGTGTGTTCACGAAAGTAGGAATCGGCACCTTCATTGATCCACGTAACGGT 
               
               
                   
                 GGTGGGAAGGTCAACGATATTACCAAGGAAGATATCGTAGAACTGGTGGAAATTAAAGGG 
               
               
                   
                 CAGGAATACCTGTTTTATCCGGCGTTCCCGATCCATGTCGCGCTGATTCGTGGCACCTAT 
               
               
                   
                 GCGGACGAGAGTGGTAACATCACCTTTGAAAAAGAGGTAGCGCCTTTGGAAGGGACTTCT 
               
               
                   
                 GTCTGTCAAGCGGTGAAGAACTCGGGTGGCATTGTCGTGGTTCAGGTTGAGCGTGTCGTC 
               
               
                   
                 AAAGCAGGCACGCTGGATCCGCGCCATGTGAAAGTTCCGGGTATCTATGTAGATTACGTA 
               
               
                   
                 GTCGTCGCGGATCCGGAGGACCATCAACAGTCCCTTGACTGCGAATATGATCCTGCCCTT 
               
               
                   
                 AGTGGAGAGCACCGTCGTCCGGAGGTGGTGGGTGAACCACTGCCTTTATCCGCGAAGAAA 
               
               
                   
                 GTCATCGGCCGCCGTGGCGCGATTGAGCTCGAGAAAGACGTTGCAGTGAACCTTGGGGTA 
               
               
                   
                 GGTGCACCTGAGTATGTGGCCTCCGTGGCCGATGAAGAAGGCATTGTGGATTTTATGACT 
               
               
                   
                 CTCACAGCGGAGTCCGGCGCTATCGGTGGCGTTCCAGCCGGCGGTGTTCGCTTTGGGGCG 
               
               
                   
                 AGCTACAATGCTGACGCCTTGATCGACCAGGGCTACCAATTTGATTATTACGACGGTGGG 
               
               
                   
                 GGTCTGGATCTTTGTTACCTGGGTTTAGCTGAATGCGACGAAAAGGGTAATATCAATGTT 
               
               
                   
                 AGCCGCTTCGGTCCTCGTATCGCTGGGTGCGGCGGATTCATTAACATTACCCAAAACACG 
               
               
                   
                 CCGAAAGTCTTCTTTTGTGGGACCTTTACAGCCGGGGGGCTGAAAGTGAAAATTGAAGAT 
               
               
                   
                 GGTAAGGTGATTATCGTTCAGGAAGGGAAACAGAAGAAATTCCTTAAGGCAGTGGAGCAA 
               
               
                   
                 ATCACCTTTAATGGAGACGTGGCCTTAGCGAACAAGCAACAAGTTACCTACATCACGGAG 
               
               
                   
                 CGTTGCGTCTTCCTCCTCAAAGAAGACGGTTTACACCTTTCGGAAATCGCGCCAGGCATC 
               
               
                   
                 GATCTGCAGACCCAGATTTTGGATGTTATGGACTTTGCCCCGATCATTGATCGTGACGCA 
               
               
                   
                 AACGGGCAGATTAAACTGATGGACGCGGCGTTATTCGCAGAAGGGCTGATGGGCTTGAAA 
               
               
                   
                 GAAATGAAGTCTTGAtaa gaaggagatatacat ATGAGCTTAACCCAAGGCATGAAAGCT 
               
               
                   
                 AAACAACTGTTAGCATACTTTCAGGGTAAAGCCGATCAGGATGCACGTGAAGCGAAAGCC 
               
               
                   
                 CGCGGTGAGCTGGTCTGCTGGTCGGCGTCAGTCGCGCCGCCGGAATTTTGCGTAACAATG 
               
               
                   
                 GGCATTGCCATGATCTACCCGGAGACTCATGCAGCGGGCATCGGTGCCCGCAAAGGTGCG 
               
               
                   
                 ATGGACATGCTGGAAGTTGCGGACCGCAAAGGCTACAACGTGGATTGTTGTTCCTACGGC 
               
               
                   
                 CGTGTAAATATGGGTTACATGGAATGTTTAAAAGAAGCCGCCATCACGGGCGTCAAGCCG 
               
               
                   
                 GAAGTTTTGGTTAATTCCCCTGCTGCTGACGTTCCGCTTCCCGATTTGGTGATTACGTGT 
               
               
                   
                 AATAATATCTGTAACACGCTGCTGAAATGGTACGAAAACTTAGCAGCAGAACTCGATATT 
               
               
                   
                 CCTTGCATCGTGATCGACGTACCGTTTAATCATACCATGCCGATTCCGGAATATGCCAAG 
               
               
                   
                 GCCTACATCGCGGACCAGTTCCGCAATGCAATTTCTCAGCTGGAAGTTATTTGTGGCCGT 
               
               
                   
                 CCGTTCGATTGGAAGAAATTTAAGGAGGTCAAAGATCAGACCCAGCGTAGCGTATACCAC 
               
               
                   
                 TGGAACCGCATTGCCGAGATGGCGAAATACAAGCCTAGCCCGCTGAACGGCTTCGATCTG 
               
               
                   
                 TTCAATTACATGGCGTTAATCGTGGCGTGCCGCAGCCTGGATTATGCAGAAATTACCTTT 
               
               
                   
                 AAAGCGTTCGCGGACGAATTAGAAGAGAATTTGAAGGCGGGTATCTACGCCTTTAAAGGT 
               
               
                   
                 GCGGAAAAAACGCGCTTTCAATGGGAAGGTATCGCGGTGTGGCCACATTTAGGTCACACG 
               
               
                   
                 TTTAAATCTATGAAGAATCTGAATTCGATTATGACCGGTACGGCATACCCCGCCCTTTGG 
               
               
                   
                 GACCTGCACTATGACGCTAACGACGAATCTATGCACTCTATGGCTGAAGCGTACACCCGT 
               
               
                   
                 ATTTATATTAATACTTGTCTGCAGAACAAAGTAGAGGTCCTGCTTGGGATCATGGAAAAA 
               
               
                   
                 GGCCAGGTGGATGGTACCGTATATCATCTGAATCGCAGCTGCAAACTGATGAGTTTCCTG 
               
               
                   
                 AACGTGGAAACGGCTGAAATTATTAAAGAGAAGAACGGTCTTCCTTACGTCTCCATTGAT 
               
               
                   
                 GGCGATCAGACCGATCCTCGCGTTTTTTCTCCGGCCCAGTTTGATACCCGTGTTCAGGCC 
               
               
                   
                 CTGGTTGAGATGATGGAGGCCAATATGGCGGCAGCGGAATAAtaa gaaggagatatacat   
               
               
                   
                 ATGTCACGCGTGGAGGCAATCCTGTCGCAGCTGAAAGATGTCGCCGCGAATCCGAAAAAA 
               
               
                   
                 GCCATGGATGACTATAAAGCTGAAACAGGTAAGGGCGCGGTTGGTATCATGCCGATCTAC 
               
               
                   
                 AGCCCCGAAGAAATGGTACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGGCGCCCAG 
               
               
                   
                 GGCAAAACGATTAGTAAAGCGCGCACCTATCTGCCTGCTTTTGCCTGCAGCGTAATGCAG 
               
               
                   
                 CAGGTTATGGAATTACAGTGCGAGGGCGCGTATGATGACCTGTCCGCAGTTATTTTTAGC 
               
               
                   
                 GTACCGTGCGACACTCTCAAATGTCTTAGCCAGAAATGGAAAGGTACGTCCCCAGTGATT 
               
               
                   
                 GTATTTACGCATCCGCAGAACCGCGGATTAGAAGCGGCGAACCAATTCTTGGTTACCGAG 
               
               
                   
                 TATGAACTGGTAAAAGCACAACTGGAATCAGTTCTGGGTGTGAAAATTTCAAACGCCGCC 
               
               
                   
                 CTGGAAAATTCGATTGCAATTTATAACGAGAATCGTGCCGTGATGCGTGAGTTCGTGAAA 
               
               
                   
                 GTGGCAGCGGACTATCCTCAAGTCATTGACGCAGTGAGCCGCCACGCGGTTTTTAAAGCG 
               
               
                   
                 CGCCAGTTTATGCTTAAGGAAAAACATACCGCACTTGTGAAAGAACTGATCGCTGAGATT 
               
               
                   
                 AAAGCAACGCCAGTCCAGCCGTGGGACGGAAAAAAGGTTGTAGTGACGGGCATTCTGTTG 
               
               
                   
                 GAACCGAATGAGTTATTAGATATCTTTAATGAGTTTAAGATCGCGATTGTTGATGATGAT 
               
               
                   
                 TTAGCGCAGGAAAGCCGTCAGATCCGTGTTGACGTTCTGGACGGAGAAGGCGGACCGCTC 
               
               
                   
                 TACCGTATGGCTAAAGCGTGGCAGCAAATGTATGGCTGCTCGCTGGCAACCGACACCAAG 
               
               
                   
                 AAGGGTCGCGGCCGTATGTTAATTAACAAAACGATTCAGACCGGTGCGGACGCTATCGTA 
               
               
                   
                 GTTGCAATGATGAAGTTTTGCGACCCAGAAGAATGGGATTATCCGGTAATGTACCGTGAA 
               
               
                   
                 TTTGAAGAAAAAGGGGTCAAATCACTTATGATTGAGGTGGATCAGGAAGTATCGTCTTTC 
               
               
                   
                 GAACAGATTAAAACCCGTCTGCAGTCATTCGTCGAAATGCTTTAAtaa gaaggagatata   
               
               
                   
                   cat ATGTATACCTTGGGGATTGATGTCGGTTCTGCCTCTAGTAAAGCGGTGATTCTGAAA 
               
               
                   
                 GATGGAAAAGATATTGTCGCTGCCGAGGTTGTCCAAGTCGGTACCGGCTCCTCGGGTCCC 
               
               
                   
                 CAACGCGCACTGGACAAAGCCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACATCAGCTAC 
               
               
                   
                 ACAGTAGCTACGGGCTATGGGCGCTTCAATTTTAGCGACGCGGATAAACAGATTTCGGAA 
               
               
                   
                 ATTAGCTGTCATGCCAAAGGCATTTATTTCTTAGTACCAACTGCGCGCACTATTATTGAC 
               
               
                   
                 ATTGGCGGCCAAGATGCGAAAGCCATCCGCCTGGACGACAAGGGGGGTATTAAGCAATTC 
               
               
                   
                 TTCATGAATGATAAATGCGCGGCGGGCACGGGGCGTTTCCTGGAAGTCATGGCTCGCGTA 
               
               
                   
                 CTTGAAACCACCCTGGATGAAATGGCTGAACTGGATGAACAGGCGACTGACACCGCTCCC 
               
               
                   
                 ATTTCAAGCACCTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAATTGAGCAATGGT 
               
               
                   
                 GTCTCACGCAACAACATCATTAAAGGTGTCCATCTGAGCGTTGCGTCACGTGCGTGTGGT 
               
               
                   
                 CTGGCGTATCGCGGCGGTTTGGAGAAAGATGTTGTTATGACAGGTGGCGTGGCAAAAAAT 
               
               
                   
                 GCAGGGGTGGTGCGCGCGGTGGCGGGCGTTCTGAAGACCGATGTTATCGTTGCTCCGAAT 
               
               
                   
                 CCTCAGACGACCGGTGCACTGGGGGCAGCGCTGTATGCTTATGAGGCCGCCCAGAAGAAG 
               
               
                   
                 TAAtaa gaaggagatatacat ATGGCCTTCAATAGCGCAGATATTAATTCTTTCCGCGAT 
               
               
                   
                 ATTTGGGTGTTTTGTGAACAGCGTGAGGGCAAACTGATTAACACCGATTTCGAATTAATT 
               
               
                   
                 AGCGAAGGTCGTAAACTGGCTGACGAACGCGGAAGCAAACTGGTTGGAATTTTGCTGGGG 
               
               
                   
                 CACGAAGTTGAAGAAATCGCAAAAGAATTAGGCGGCTATGGTGCGGACAAGGTAATTGTG 
               
               
                   
                 TGCGATCATCCGGAACTTAAATTTTACACTACGGATGCTTATGCCAAAGTTTTATGTGAC 
               
               
                   
                 GTCGTGATGGAAGAGAAACCGGAGGTAATTTTGATCGGTGCCACCAACATTGGCCGTGAT 
               
               
                   
                 CTCGGACCGCGTTGTGCTGCACGCTTGCACACGGGGCTGACGGCTGATTGCACGCACCTG 
               
               
                   
                 GATATTGATATGAATAAATATGTGGACTTTCTTAGCACCAGTAGCACCTTGGATATCTCG 
               
               
                   
                 TCGATGACTTTCCCTATGGAAGATACAAACCTTAAAATGACGCGCCCTGCATTTGGCGGA 
               
               
                   
                 CATCTGATGGCAACGATCATTTGTCCACGCTTCCGTCCCTGTATGAGCACAGTGCGCCCC 
               
               
                   
                 GGAGTGATGAAGAAAGCGGAGTTCTCGCAGGAGATGGCGCAAGCATGTCAAGTAGTGACC 
               
               
                   
                 CGTCACGTAAATTTGTCGGATGAAGACCTTAAAACTAAAGTAATTAATATCGTGAAGGAA 
               
               
                   
                 ACGAAAAAGATTGTGGATCTGATCGGCGCAGAAATTATTGTGTCAGTTGGTCGTGGTATC 
               
               
                   
                 TCGAAAGATGTCCAAGGTGGAATTGCACTGGCTGAAAAACTTGCGGACGCATTTGGTAAC 
               
               
                   
                 GGTGTCGTGGGCGGCTCGCGCGCAGTGATTGATTCCGGCTGGTTACCTGCGGATCATCAG 
               
               
                   
                 GTTGGACAAACCGGTAAGACCGTGCACCCGAAAGTCTACGTGGCGCTGGGTATTAGTGGG 
               
               
                   
                 GCTATCCAGCATAAGGCTGGGATGCAAGACTCTGAACTGATCATTGCCGTCAACAAAGAC 
               
               
                   
                 GAAACGGCGCCTATCTTCGACTGCGCCGATTATGGCATCACCGGTGATTTATTTAAAATC 
               
               
                   
                 GTACCGATGATGATCGACGCGATCAAAGAGGGTAAAAACGCATGAtaa gaaggagatata   
               
               
                   
                   cat ATGCGCATCTATGTGTGTGTGAAACAAGTCCCAGATACGAGCGGCAAGGTGGCCGTT 
               
               
                   
                 AACCCTGATGGGACCCTTAACCGTGCCTCAATGGCAGCGATTATTAACCCGGACGATATG 
               
               
                   
                 TCCGCGATCGAACAGGCATTAAAACTGAAAGATGAAACCGGATGCCAGGTTACGGCGCTT 
               
               
                   
                 ACGATGGGTCCTCCTCCTGCCGAGGGCATGTTGCGCGAAATTATTGCAATGGGGGCCGAC 
               
               
                   
                 GATGGTGTGCTGATTTCGGCCCGTGAATTTGGGGGGTCCGATACCTTCGCAACCAGTCAA 
               
               
                   
                 ATTATTAGCGCGGCAATCCATAAATTAGGCTTAAGCAATGAAGACATGATCTTTTGCGGT 
               
               
                   
                 CGTCAGGCCATTGACGGTGATACGGCCCAAGTCGGCCCTCAAATTGCCGAAAAACTGAGC 
               
               
                   
                 ATCCCACAGGTAACCTATGGCGCAGGAATCAAAAAATCTGGTGATTTAGTGCTGGTGAAG 
               
               
                   
                 CGTATGTTGGAGGATGGTTATATGATGATCGAAGTCGAAACTCCATGTCTGATTACCTGC 
               
               
                   
                 ATTCAGGATAAAGCGGTAAAACCACGTTACATGACTCTCAACGGTATTATGGAATGCTAC 
               
               
                   
                 TCCAAGCCGCTCCTCGTTCTCGATTACGAAGCACTGAAAGATGAACCGCTGATCGAACTT 
               
               
                   
                 GATACCATTGGGCTTAAAGGCTCCCCGACGAATATCTTTAAATCGTTTACGCCGCCTCAG 
               
               
                   
                 AAAGGCGTTGGTGTCATGCTCCAAGGCACCGATAAGGAAAAAGTCGAGGATCTGGTGGAT 
               
               
                   
                 AAGCTGATGCAGAAACATGTCATCTAAtaa gaaggagatatacat ATGTTCTTACTGAAG 
               
               
                   
                 ATTAAAAAAGAACGTATGAAACGCATGGACTTTAGTTTAACGCGTGAACAGGAGATGTTA 
               
               
                   
                 AAAAAACTGGCGCGTCAGTTTGCTGAGATCGAGCTGGAACCGGTGGCCGAAGAGATTGAT 
               
               
                   
                 CGTGAGCACGTTTTTCCTGCAGAAAACTTTAAGAAGATGGCGGAAATTGGCTTAACCGGC 
               
               
                   
                 ATTGGTATCCCGAAAGAATTTGGTGGCTCCGGTGGAGGCACCCTGGAGAAGGTCATTGCC 
               
               
                   
                 GTGTCAGAATTCGGCAAAAAGTGTATGGCCTCAGCTTCCATTTTAAGCATTCATCTTATC 
               
               
                   
                 GCGCCGCAGGCAATCTACAAATATGGGACCAAAGAACAGAAAGAGACGTACCTGCCGCGT 
               
               
                   
                 CTTACCAAAGGTGGTGAACTGGGCGCCTTTGCGCTGACAGAACCAAACGCCGGAAGCGAT 
               
               
                   
                 GCCGGCGCGGTAAAAACGACCGCGATTCTGGACAGCCAGACAAACGAGTACGTGCTGAAT 
               
               
                   
                 GGCACCAAATGCTTTATCAGCGGGGGCGGGCGCGCGGGTGTTCTTGTAATTTTTGCGCTT 
               
               
                   
                 ACTGAACCGAAAAAAGGTCTGAAAGGGATGAGCGCGATTATCGTGGAGAAAGGGACCCCG 
               
               
                   
                 GGCTTCAGCATCGGCAAGGTGGAGAGCAAGATGGGGATCGCAGGTTCGGAAACCGCGGAA 
               
               
                   
                 CTTATCTTCGAAGATTGTCGCGTTCCGGCTGCCAACCTTTTAGGTAAAGAAGGCAAAGGC 
               
               
                   
                 TTTAAAATTGCTATGGAAGCCCTGGATGGCGCCCGTATTGGCGTGGGCGCTCAAGCAATC 
               
               
                   
                 GGAATTGCCGAGGGGGCGATCGACCTGAGTGTGAAGTACGTTCACGAGCGCATTCAATTT 
               
               
                   
                 GGTAAACCGATCGCGAATCTGCAGGGAATTCAATGGTATATCGCGGATATGGCGACCAAA 
               
               
                   
                 ACCGCCGCGGCACGCGCACTTGTTGAGTTTGCAGCGTATCTTGAAGACGCGGGTAAACCG 
               
               
                   
                 TTCACAAAGGAATCTGCTATGTGCAAGCTGAACGCCTCCGAAAACGCGCGTTTTGTGACA 
               
               
                   
                 AATTTAGCTCTGCAGATTCACGGGGGTTACGGTTATATGAAAGATTATCCGTTAGAGCGT 
               
               
                   
                 ATGTATCGCGATGCTAAGATTACGGAAATTTACGAGGGGACATCAGAAATCCATAAGGTG 
               
               
                   
                 GTGATTGCGCGTGAAGTAATGAAACGCTAA 
               
               
                   
               
               
                 Ptet-acuI-pct-lcdABC 
                 caactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagg 
               
               
                 (Ptet: lower case; tetA/R 
                 gggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttg 
               
               
                 promoter within Ptet: lower case 
                 taaaacgacggccagtgaattgacgcgtattgggatgtaaaacgacggccagtgaattcg 
               
               
                 bold, with tet operator 
                 ttaagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaaggcc 
               
               
                 underlined; RBS and leader region 
                 gaataagaaggctggctctgcaccttggtgatcaaataattcgatagcttgtcgtaataa 
               
               
                 lower case italic; ribosome 
                 tggcggcatactatcagtagtaggtgtttccctttcttctttagcgacttgatgctcttg 
               
               
                 binding site: lower case 
                 atcttccaatacgcaacctaaagtaaaatgccccacagcgctgagtgcatataatgcatt 
               
               
                 underlined italic; coding region: 
                 ctctagtgaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcatactg 
               
               
                 upper case, rrnB T1 and T2 
                 tttttctgtaggccgtgtacctaaatgtacttttgctccatcgcgatgacttagtaaagc 
               
               
                 terminators: lower case bold 
                 acatctaaaacttttagcgttattacgtaaaaaatcttgccagctttccccttctaaagg 
               
               
                 underline italics) 
                 gcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagcccg 
               
               
                 (SEQ ID NO: 221) 
                 cttattttttacatgccaatacaatgtaggctgctctacacctagcttctgggcgagttt 
               
               
                   
                 acgggttgttaaaccttcgattccgacctcattaagcagctctaatgcgctgttaatcac 
               
               
                   
                 tttacttttatctaatctagacatcattaattcctaattttt gttgac     
               
               
                   
                     tattttaccac    aaagtgaa ctctagaaataat   
               
               
                   
                   tttgtttaactttaa     gaaggagatatacat   ATGCGTGCGGTACTGATCGAGAAGTCCGAT 
               
               
                   
                 GATACACAGTCCGTCTCTGTCACCGAACTGGCTGAAGATCAACTGCCGGAAGGCGACGTT 
               
               
                   
                 TTGGTAGATGTTGCTTATTCAACACTGAACTACAAAGACGCCCTGGCAATTACCGGTAAA 
               
               
                   
                 GCCCCCGTCGTTCGTCGTTTTCCGATGGTACCTGGAATCGACTTTACGGGTACCGTGGCC 
               
               
                   
                 CAGTCTTCCCACGCCGACTTCAAGCCAGGTGATCGCGTAATCCTGAATGGTTGGGGTGTG 
               
               
                   
                 GGGGAAAAACATTGGGGCGGTTTAGCGGAGCGCGCTCGCGTGCGCGGAGACTGGCTTGTT 
               
               
                   
                 CCCTTGCCAGCCCCCCTGGACTTACGCCAAGCGGCCATGATCGGTACAGCAGGATACACG 
               
               
                   
                 GCGATGTTGTGCGTTCTGGCGCTTGAACGTCACGGAGTGGTGCCGGGTAATGGGGAAATC 
               
               
                   
                 GTGGTGTCCGGTGCAGCAGGCGGCGTCGGCTCCGTTGCGACGACCCTTCTTGCCGCTAAG 
               
               
                   
                 GGCTATGAGGTAGCGGCAGTGACTGGACGTGCGTCCGAAGCAGAATATCTGCGCGGTTTG 
               
               
                   
                 GGGGCGGCGAGCGTAATTGATCGTAACGAATTAACGGGGAAGGTACGCCCGCTGGGTCAG 
               
               
                   
                 GAGCGTTGGGCTGGCGGGATTGACGTGGCGGGATCAACCGTGCTTGCGAACATGCTTTCT 
               
               
                   
                 ATGATGAAGTATCGCGGGGTAGTCGCTGCGTGTGGCCTGGCCGCGGGCATGGATCTGCCC 
               
               
                   
                 GCGTCTGTCGCGCCCTTTATTCTTCGTGGGATGACGCTGGCAGGGGTGGATAGCGTTATG 
               
               
                   
                 TGCCCAAAGACAGATCGTTTAGCAGCGTGGGCCCGTTTGGCGTCAGATCTTGACCCTGCC 
               
               
                   
                 AAGCTGGAGGAGATGACTACAGAGTTGCCGTTTAGTGAAGTAATCGAGACAGCACCCAAA 
               
               
                   
                 TTCTTGGACGGGACGGTTCGTGGCCGCATTGTTATCCCCGTAACGCCCTAAgaa ctctag   
               
               
                   
                   aaataattttgtttaactttaa     gaaggagatatacat   ATGCGCAAAGTGCCGATTATCAC 
               
               
                   
                 GGCTGACGAGGCCGCAAAACTGATCAAGGACGGCGACACCGTGACAACTAGCGGCTTTGT 
               
               
                   
                 GGGTAACGCGATCCCTGAGGCCCTTGACCGTGCAGTCGAAAAGCGTTTCCTGGAAACGGG 
               
               
                   
                 CGAACCGAAGAACATTACTTATGTATATTGCGGCAGTCAGGGCAATCGCGACGGTCGTGG 
               
               
                   
                 CGCAGAACATTTCGCGCATGAAGGCCTGCTGAAACGTTATATCGCTGGCCATTGGGCGAC 
               
               
                   
                 CGTCCCGGCGTTAGGGAAAATGGCCATGGAGAATAAAATGGAGGCCTACAATGTCTCTCA 
               
               
                   
                 GGGCGCCTTGTGTCATCTCTTTCGCGATATTGCGAGCCATAAACCGGGTGTGTTCACGAA 
               
               
                   
                 AGTAGGAATCGGCACCTTCATTGATCCACGTAACGGTGGTGGGAAGGTCAACGATATTAC 
               
               
                   
                 CAAGGAAGATATCGTAGAACTGGTGGAAATTAAAGGGCAGGAATACCTGTTTTATCCGGC 
               
               
                   
                 GTTCCCGATCCATGTCGCGCTGATTCGTGGCACCTATGCGGACGAGAGTGGTAACATCAC 
               
               
                   
                 CTTTGAAAAAGAGGTAGCGCCTTTGGAAGGGACTTCTGTCTGTCAAGCGGTGAAGAACTC 
               
               
                   
                 GGGTGGCATTGTCGTGGTTCAGGTTGAGCGTGTCGTCAAAGCAGGCACGCTGGATCCGCG 
               
               
                   
                 CCATGTGAAAGTTCCGGGTATCTATGTAGATTACGTAGTCGTCGCGGATCCGGAGGACCA 
               
               
                   
                 TCAACAGTCCCTTGACTGCGAATATGATCCTGCCCTTAGTGGAGAGCACCGTCGTCCGGA 
               
               
                   
                 GGTGGTGGGTGAACCACTGCCTTTATCCGCGAAGAAAGTCATCGGCCGCCGTGGCGCGAT 
               
               
                   
                 TGAGCTCGAGAAAGACGTTGCAGTGAACCTTGGGGTAGGTGCACCTGAGTATGTGGCCTC 
               
               
                   
                 CGTGGCCGATGAAGAAGGCATTGTGGATTTTATGACTCTCACAGCGGAGTCCGGCGCTAT 
               
               
                   
                 CGGTGGCGTTCCAGCCGGCGGTGTTCGCTTTGGGGCGAGCTACAATGCTGACGCCTTGAT 
               
               
                   
                 CGACCAGGGCTACCAATTTGATTATTACGACGGTGGGGGTCTGGATCTTTGTTACCTGGG 
               
               
                   
                 TTTAGCTGAATGCGACGAAAAGGGTAATATCAATGTTAGCCGCTTCGGTCCTCGTATCGC 
               
               
                   
                 TGGGTGCGGCGGATTCATTAACATTACCCAAAACACGCCGAAAGTCTTCTTTTGTGGGAC 
               
               
                   
                 CTTTACAGCCGGGGGGCTGAAAGTGAAAATTGAAGATGGTAAGGTGATTATCGTTCAGGA 
               
               
                   
                 AGGGAAACAGAAGAAATTCCTTAAGGCAGTGGAGCAAATCACCTTTAATGGAGACGTGGC 
               
               
                   
                 CTTAGCGAACAAGCAACAAGTTACCTACATCACGGAGCGTTGCGTCTTCCTCCTCAAAGA 
               
               
                   
                 AGACGGTTTACACCTTTCGGAAATCGCGCCAGGCATCGATCTGCAGACCCAGATTTTGGA 
               
               
                   
                 TGTTATGGACTTTGCCCCGATCATTGATCGTGACGCAAACGGGCAGATTAAACTGATGGA 
               
               
                   
                 CGCGGCGTTATTCGCAGAAGGGCTGATGGGCTTGAAAGAAATGAAGTCTTGAtaa   gaagg     
               
               
                   
                     agatatacat   ATGAGCTTAACCCAAGGCATGAAAGCTAAACAACTGTTAGCATACTTTCA 
               
               
                   
                 GGGTAAAGCCGATCAGGATGCACGTGAAGCGAAAGCCCGCGGTGAGCTGGTCTGCTGGTC 
               
               
                   
                 GGCGTCAGTCGCGCCGCCGGAATTTTGCGTAACAATGGGCATTGCCATGATCTACCCGGA 
               
               
                   
                 GACTCATGCAGCGGGCATCGGTGCCCGCAAAGGTGCGATGGACATGCTGGAAGTTGCGGA 
               
               
                   
                 CCGCAAAGGCTACAACGTGGATTGTTGTTCCTACGGCCGTGTAAATATGGGTTACATGGA 
               
               
                   
                 ATGTTTAAAAGAAGCCGCCATCACGGGCGTCAAGCCGGAAGTTTTGGTTAATTCCCCTGC 
               
               
                   
                 TGCTGACGTTCCGCTTCCCGATTTGGTGATTACGTGTAATAATATCTGTAACACGCTGCT 
               
               
                   
                 GAAATGGTACGAAAACTTAGCAGCAGAACTCGATATTCCTTGCATCGTGATCGACGTACC 
               
               
                   
                 GTTTAATCATACCATGCCGATTCCGGAATATGCCAAGGCCTACATCGCGGACCAGTTCCG 
               
               
                   
                 CAATGCAATTTCTCAGCTGGAAGTTATTTGTGGCCGTCCGTTCGATTGGAAGAAATTTAA 
               
               
                   
                 GGAGGTCAAAGATCAGACCCAGCGTAGCGTATACCACTGGAACCGCATTGCCGAGATGGC 
               
               
                   
                 GAAATACAAGCCTAGCCCGCTGAACGGCTTCGATCTGTTCAATTACATGGCGTTAATCGT 
               
               
                   
                 GGCGTGCCGCAGCCTGGATTATGCAGAAATTACCTTTAAAGCGTTCGCGGACGAATTAGA 
               
               
                   
                 AGAGAATTTGAAGGCGGGTATCTACGCCTTTAAAGGTGCGGAAAAAACGCGCTTTCAATG 
               
               
                   
                 GGAAGGTATCGCGGTGTGGCCACATTTAGGTCACACGTTTAAATCTATGAAGAATCTGAA 
               
               
                   
                 TTCGATTATGACCGGTACGGCATACCCCGCCCTTTGGGACCTGCACTATGACGCTAACGA 
               
               
                   
                 CGAATCTATGCACTCTATGGCTGAAGCGTACACCCGTATTTATATTAATACTTGTCTGCA 
               
               
                   
                 GAACAAAGTAGAGGTCCTGCTTGGGATCATGGAAAAAGGCCAGGTGGATGGTACCGTATA 
               
               
                   
                 TCATCTGAATCGCAGCTGCAAACTGATGAGTTTCCTGAACGTGGAAACGGCTGAAATTAT 
               
               
                   
                 TAAAGAGAAGAACGGTCTTCCTTACGTCTCCATTGATGGCGATCAGACCGATCCTCGCGT 
               
               
                   
                 TTTTTCTCCGGCCCAGTTTGATACCCGTGTTCAGGCCCTGGTTGAGATGATGGAGGCCAA 
               
               
                   
                 TATGGCGGCAGCGGAATAAtaa   gaaggagatatacat   ATGTCACGCGTGGAGGCAATCCT 
               
               
                   
                 GTCGCAGCTGAAAGATGTCGCCGCGAATCCGAAAAAAGCCATGGATGACTATAAAGCTGA 
               
               
                   
                 AACAGGTAAGGGCGCGGTTGGTATCATGCCGATCTACAGCCCCGAAGAAATGGTACACGC 
               
               
                   
                 CGCTGGCTATTTGCCGATGGGAATCTGGGGCGCCCAGGGCAAAACGATTAGTAAAGCGCG 
               
               
                   
                 CACCTATCTGCCTGCTTTTGCCTGCAGCGTAATGCAGCAGGTTATGGAATTACAGTGCGA 
               
               
                   
                 GGGCGCGTATGATGACCTGTCCGCAGTTATTTTTAGCGTACCGTGCGACACTCTCAAATG 
               
               
                   
                 TCTTAGCCAGAAATGGAAAGGTACGTCCCCAGTGATTGTATTTACGCATCCGCAGAACCG 
               
               
                   
                 CGGATTAGAAGCGGCGAACCAATTCTTGGTTACCGAGTATGAACTGGTAAAAGCACAACT 
               
               
                   
                 GGAATCAGTTCTGGGTGTGAAAATTTCAAACGCCGCCCTGGAAAATTCGATTGCAATTTA 
               
               
                   
                 TAACGAGAATCGTGCCGTGATGCGTGAGTTCGTGAAAGTGGCAGCGGACTATCCTCAAGT 
               
               
                   
                 CATTGACGCAGTGAGCCGCCACGCGGTTTTTAAAGCGCGCCAGTTTATGCTTAAGGAAAA 
               
               
                   
                 ACATACCGCACTTGTGAAAGAACTGATCGCTGAGATTAAAGCAACGCCAGTCCAGCCGTG 
               
               
                   
                 GGACGGAAAAAAGGTTGTAGTGACGGGCATTCTGTTGGAACCGAATGAGTTATTAGATAT 
               
               
                   
                 CTTTAATGAGTTTAAGATCGCGATTGTTGATGATGATTTAGCGCAGGAAAGCCGTCGGAT 
               
               
                   
                 CCGTGTTGACGTTCTGGACGGAGAAGGCGGACCGCTCTACCGTATGGCTAAAGCGTGGCA 
               
               
                   
                 GCAAATGTATGGCTGCTCGCTGGCAACCGACACCAAGAAGGGTCGCGGCCGTATGTTAAT 
               
               
                   
                 TAACAAAACGATTCAGACCGGTGCGGACGCTATCGTAGTTGCAATGATGAAGTTTTGCGA 
               
               
                   
                 CCCAGAAGAATGGGATTATCCGGTAATGTACCGTGAATTTGAAGAAAAAGGGGTCAAATC 
               
               
                   
                 ACTTATGATTGAGGTGGATCAGGAAGTATCGTCTTTCGAACAGATTAAAACCCGTCTGCA 
               
               
                   
                 GTCATTCGTCGAAATGCTTTAAtaa   gaaggagatatacat   ATGTATACCTTGGGGATTGA 
               
               
                   
                 TGTCGGTTCTGCCTCTAGTAAAGCGGTGATTCTGAAAGATGGAAAAGATATTGTCGCTGC 
               
               
                   
                 CGAGGTTGTCCAAGTCGGTACCGGCTCCTCGGGTCCCCAACGCGCACTGGACAAAGCCTT 
               
               
                   
                 TGAAGTCTCTGGCTTAAAAAAGGAAGACATCAGCTACACAGTAGCTACGGGCTATGGGCG 
               
               
                   
                 CTTCAATTTTAGCGACGCGGATAAACAGATTTCGGAAATTAGCTGTCATGCCAAAGGCAT 
               
               
                   
                 TTATTTCTTAGTACCAACTGCGCGCACTATTATTGACATTGGCGGCCAAGATGCGAAAGC 
               
               
                   
                 CATCCGCCTGGACGACAAGGGGGGTATTAAGCAATTCTTCATGAATGATAAATGCGCGGC 
               
               
                   
                 GGGCACGGGGCGTTTCCTGGAAGTCATGGCTCGCGTACTTGAAACCACCCTGGATGAAAT 
               
               
                   
                 GGCTGAACTGGATGAACAGGCGACTGACACCGCTCCCATTTCAAGCACCTGCACGGTTTT 
               
               
                   
                 CGCCGAAAGCGAAGTAATTAGCCAATTGAGCAATGGTGTCTCACGCAACAACATCATTAA 
               
               
                   
                 AGGTGTCCATCTGAGCGTTGCGTCACGTGCGTGTGGTCTGGCGTATCGCGGCGGTTTGGA 
               
               
                   
                 GAAAGATGTTGTTATGACAGGTGGCGTGGCAAAAAATGCAGGGGTGGTGCGCGCGGTGGC 
               
               
                   
                 GGGCGTTCTGAAGACCGATGTTATCGTTGCTCCGAATCCTCAGACGACCGGTGCACTGGG 
               
               
                   
                 GGCAGCGCTGTATGCTTATGAGGCCGCCCAGAAGAAGTAgatggtagtgtggggtctccc 
               
               
                   
                 catgcgagagtagggaactgccaggcat   
               
               
                   
                 
                   
                 
               
               
                   
                    ccgccgggagcggatttgaacgagcgaagcaacggccc 
               
               
                   
                 ggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagc 
               
               
                   
                 
                   
                 
               
               
                   
               
               
                 acuI-pct-lcdABC 
                 ATGCGTGCGGTACTGATCGAGAAGTCCGATGATACACAGTCCGTCTCTGTCACCGAACTG 
               
               
                 (SEQ ID NO: 222) 
                 GCTGAAGATCAACTGCCGGAAGGCGACGTTTTGGTAGATGTTGCTTATTCAACACTGAAC 
               
               
                   
                 TACAAAGACGCCCTGGCAATTACCGGTAAAGCCCCCGTCGTTCGTCGTTTTCCGATGGTA 
               
               
                   
                 CCTGGAATCGACTTTACGGGTACCGTGGCCCAGTCTTCCCACGCCGACTTCAAGCCAGGT 
               
               
                   
                 GATCGCGTAATCCTGAATGGTTGGGGTGTGGGGGAAAAACATTGGGGCGGTTTAGCGGAG 
               
               
                   
                 CGCGCTCGCGTGCGCGGAGACTGGCTTGTTCCCTTGCCAGCCCCCCTGGACTTACGCCAA 
               
               
                   
                 GCGGCCATGATCGGTACAGCAGGATACACGGCGATGTTGTGCGTTCTGGCGCTTGAACGT 
               
               
                   
                 CACGGAGTGGTGCCGGGTAATGGGGAAATCGTGGTGTCCGGTGCAGCAGGCGGCGTCGGC 
               
               
                   
                 TCCGTTGCGACGACCCTTCTTGCCGCTAAGGGCTATGAGGTAGCGGCAGTGACTGGACGT 
               
               
                   
                 GCGTCCGAAGCAGAATATCTGCGCGGTTTGGGGGCGGCGAGCGTAATTGATCGTAACGAA 
               
               
                   
                 TTAACGGGGAAGGTACGCCCGCTGGGTCAGGAGCGTTGGGCTGGCGGGATTGACGTGGCG 
               
               
                   
                 GGATCAACCGTGCTTGCGAACATGCTTTCTATGATGAAGTATCGCGGGGTAGTCGCTGCG 
               
               
                   
                 TGTGGCCTGGCCGCGGGCATGGATCTGCCCGCGTCTGTCGCGCCCTTTATTCTTCGTGGG 
               
               
                   
                 ATGACGCTGGCAGGGGTGGATAGCGTTATGTGCCCAAAGACAGATCGTTTAGCAGCGTGG 
               
               
                   
                 GCCCGTTTGGCGTCAGATCTTGACCCTGCCAAGCTGGAGGAGATGACTACAGAGTTGCCG 
               
               
                   
                 TTTAGTGAAGTAATCGAGACAGCACCCAAATTCTTGGACGGGACGGTTCGTGGCCGCATT 
               
               
                   
                 GTTATCCCCGTAACGCCCTAAgaa ctctagaaataattttgtttaactttaa     gaaggaga     
               
               
                   
                     tatacat   ATGCGCAAAGTGCCGATTATCACGGCTGACGAGGCCGCAAAACTGATCAAGGA 
               
               
                   
                 CGGCGACACCGTGACAACTAGCGGCTTTGTGGGTAACGCGATCCCTGAGGCCCTTGACCG 
               
               
                   
                 TGCAGTCGAAAAGCGTTTCCTGGAAACGGGCGAACCGAAGAACATTACTTATGTATATTG 
               
               
                   
                 CGGCAGTCAGGGCAATCGCGACGGTCGTGGCGCAGAACATTTCGCGCATGAAGGCCTGCT 
               
               
                   
                 GAAACGTTATATCGCTGGCCATTGGGCGACCGTCCCGGCGTTAGGGAAAATGGCCATGGA 
               
               
                   
                 GAATAAAATGGAGGCCTACAATGTCTCTCAGGGCGCCTTGTGTCATCTCTTTCGCGATAT 
               
               
                   
                 TGCGAGCCATAAACCGGGTGTGTTCACGAAAGTAGGAATCGGCACCTTCATTGATCCACG 
               
               
                   
                 TAACGGTGGTGGGAAGGTCAACGATATTACCAAGGAAGATATCGTAGAACTGGTGGAAAT 
               
               
                   
                 TAAAGGGCAGGAATACCTGTTTTATCCGGCGTTCCCGATCCATGTCGCGCTGATTCGTGG 
               
               
                   
                 CACCTATGCGGACGAGAGTGGTAACATCACCTTTGAAAAAGAGGTAGCGCCTTTGGAAGG 
               
               
                   
                 GACTTCTGTCTGTCAAGCGGTGAAGAACTCGGGTGGCATTGTCGTGGTTCAGGTTGAGCG 
               
               
                   
                 TGTCGTCAAAGCAGGCACGCTGGATCCGCGCCATGTGAAAGTTCCGGGTATCTATGTAGA 
               
               
                   
                 TTACGTAGTCGTCGCGGATCCGGAGGACCATCAACAGTCCCTTGACTGCGAATATGATCC 
               
               
                   
                 TGCCCTTAGTGGAGAGCACCGTCGTCCGGAGGTGGTGGGTGAACCACTGCCTTTATCCGC 
               
               
                   
                 GAAGAAAGTCATCGGCCGCCGTGGCGCGATTGAGCTCGAGAAAGACGTTGCAGTGAACCT 
               
               
                   
                 TGGGGTAGGTGCACCTGAGTATGTGGCCTCCGTGGCCGATGAAGAAGGCATTGTGGATTT 
               
               
                   
                 TATGACTCTCACAGCGGAGTCCGGCGCTATCGGTGGCGTTCCAGCCGGCGGTGTTCGCTT 
               
               
                   
                 TGGGGCGAGCTACAATGCTGACGCCTTGATCGACCAGGGCTACCAATTTGATTATTACGA 
               
               
                   
                 CGGTGGGGGTCTGGATCTTTGTTACCTGGGTTTAGCTGAATGCGACGAAAAGGGTAATAT 
               
               
                   
                 CAATGTTAGCCGCTTCGGTCCTCGTATCGCTGGGTGCGGCGGATTCATTAACATTACCCA 
               
               
                   
                 AAACACGCCGAAAGTCTTCTTTTGTGGGACCTTTACAGCCGGGGGGCTGAAAGTGAAAAT 
               
               
                   
                 TGAAGATGGTAAGGTGATTATCGTTCAGGAAGGGAAACAGAAGAAATTCCTTAAGGCAGT 
               
               
                   
                 GGAGCAAATCACCTTTAATGGAGACGTGGCCTTAGCGAACAAGCAACAAGTTACCTACAT 
               
               
                   
                 CACGGAGCGTTGCGTCTTCCTCCTCAAAGAAGACGGTTTACACCTTTCGGAAATCGCGCC 
               
               
                   
                 AGGCATCGATCTGCAGACCCAGATTTTGGATGTTATGGACTTTGCCCCGATCATTGATCG 
               
               
                   
                 TGACGCAAACGGGCAGATTAAACTGATGGACGCGGCGTTATTCGCAGAAGGGCTGATGGG 
               
               
                   
                 CTTGAAAGAAATGAAGTCTTGAtaa   gaaggagatatacat   ATGAGCTTAACCCAAGGCAT 
               
               
                   
                 GAAAGCTAAACAACTGTTAGCATACTTTCAGGGTAAAGCCGATCAGGATGCACGTGAAGC 
               
               
                   
                 GAAAGCCCGCGGTGAGCTGGTCTGCTGGTCGGCGTCAGTCGCGCCGCCGGAATTTTGCGT 
               
               
                   
                 AACAATGGGCATTGCCATGATCTACCCGGAGACTCATGCAGCGGGCATCGGTGCCCGCAA 
               
               
                   
                 AGGTGCGATGGACATGCTGGAAGTTGCGGACCGCAAAGGCTACAACGTGGATTGTTGTTC 
               
               
                   
                 CTACGGCCGTGTAAATATGGGTTACATGGAATGTTTAAAAGAAGCCGCCATCACGGGCGT 
               
               
                   
                 CAAGCCGGAAGTTTTGGTTAATTCCCCTGCTGCTGACGTTCCGCTTCCCGATTTGGTGAT 
               
               
                   
                 TACGTGTAATAATATCTGTAACACGCTGCTGAAATGGTACGAAAACTTAGCAGCAGAACT 
               
               
                   
                 CGATATTCCTTGCATCGTGATCGACGTACCGTTTAATCATACCATGCCGATTCCGGAATA 
               
               
                   
                 TGCCAAGGCCTACATCGCGGACCAGTTCCGCAATGCAATTTCTCAGCTGGAAGTTATTTG 
               
               
                   
                 TGGCCGTCCGTTCGATTGGAAGAAATTTAAGGAGGTCAAAGATCAGACCCAGCGTAGCGT 
               
               
                   
                 ATACCACTGGAACCGCATTGCCGAGATGGCGAAATACAAGCCTAGCCCGCTGAACGGCTT 
               
               
                   
                 CGATCTGTTCAATTACATGGCGTTAATCGTGGCGTGCCGCAGCCTGGATTATGCAGAAAT 
               
               
                   
                 TACCTTTAAAGCGTTCGCGGACGAATTAGAAGAGAATTTGAAGGCGGGTATCTACGCCTT 
               
               
                   
                 TAAAGGTGCGGAAAAAACGCGCTTTCAATGGGAAGGTATCGCGGTGTGGCCACATTTAGG 
               
               
                   
                 TCACACGTTTAAATCTATGAAGAATCTGAATTCGATTATGACCGGTACGGCATACCCCGC 
               
               
                   
                 CCTTTGGGACCTGCACTATGACGCTAACGACGAATCTATGCACTCTATGGCTGAAGCGTA 
               
               
                   
                 CACCCGTATTTATATTAATACTTGTCTGCAGAACAAAGTAGAGGTCCTGCTTGGGATCAT 
               
               
                   
                 GGAAAAAGGCCAGGTGGATGGTACCGTATATCATCTGAATCGCAGCTGCAAACTGATGAG 
               
               
                   
                 TTTCCTGAACGTGGAAACGGCTGAAATTATTAAAGAGAAGAACGGTCTTCCTTACGTCTC 
               
               
                   
                 CATTGATGGCGATCAGACCGATCCTCGCGTTTTTTCTCCGGCCCAGTTTGATACCCGTGT 
               
               
                   
                 TCAGGCCCTGGTTGAGATGATGGAGGCCAATATGGCGGCAGCGGAATAAtaa   gaaggaga     
               
               
                   
                     tatacat   ATGTCACGCGTGGAGGCAATCCTGTCGCAGCTGAAAGATGTCGCCGCGAATCC 
               
               
                   
                 GAAAAAAGCCATGGATGACTATAAAGCTGAAACAGGTAAGGGCGCGGTTGGTATCATGCC 
               
               
                   
                 GATCTACAGCCCCGAAGAAATGGTACACGCCGCTGGCTATTTGCCGATGGGAATCTGGGG 
               
               
                   
                 CGCCCAGGGCAAAACGATTAGTAAAGCGCGCACCTATCTGCCTGCTTTTGCCTGCAGCGT 
               
               
                   
                 AATGCAGCAGGTTATGGAATTACAGTGCGAGGGCGCGTATGATGACCTGTCCGCAGTTAT 
               
               
                   
                 TTTTAGCGTACCGTGCGACACTCTCAAATGTCTTAGCCAGAAATGGAAAGGTACGTCCCC 
               
               
                   
                 AGTGATTGTATTTACGCATCCGCAGAACCGCGGATTAGAAGCGGCGAACCAATTCTTGGT 
               
               
                   
                 TACCGAGTATGAACTGGTAAAAGCACAACTGGAATCAGTTCTGGGTGTGAAAATTTCAAA 
               
               
                   
                 CGCCGCCCTGGAAAATTCGATTGCAATTTATAACGAGAATCGTGCCGTGATGCGTGAGTT 
               
               
                   
                 CGTGAAAGTGGCAGCGGACTATCCTCAAGTCATTGACGCAGTGAGCCGCCACGCGGTTTT 
               
               
                   
                 TAAAGCGCGCCAGTTTATGCTTAAGGAAAAACATACCGCACTTGTGAAAGAACTGATCGC 
               
               
                   
                 TGAGATTAAAGCAACGCCAGTCCAGCCGTGGGACGGAAAAAAGGTTGTAGTGACGGGCAT 
               
               
                   
                 TCTGTTGGAACCGAATGAGTTATTAGATATCTTTAATGAGTTTAAGATCGCGATTGTTGA 
               
               
                   
                 TGATGATTTAGCGCAGGAAAGCCGTCGGATCCGTGTTGACGTTCTGGACGGAGAAGGCGG 
               
               
                   
                 ACCGCTCTACCGTATGGCTAAAGCGTGGCAGCAAATGTATGGCTGCTCGCTGGCAACCGA 
               
               
                   
                 CACCAAGAAGGGTCGCGGCCGTATGTTAATTAACAAAACGATTCAGACCGGTGCGGACGC 
               
               
                   
                 TATCGTAGTTGCAATGATGAAGTTTTGCGACCCAGAAGAATGGGATTATCCGGTAATGTA 
               
               
                   
                 CCGTGAATTTGAAGAAAAAGGGGTCAAATCACTTATGATTGAGGTGGATCAGGAAGTATC 
               
               
                   
                 GTCTTTCGAACAGATTAAAACCCGTCTGCAGTCATTCGTCGAAATGCTTTAAtaa   gaagg     
               
               
                   
                     agatatacat   ATGTATACCTTGGGGATTGATGTCGGTTCTGCCTCTAGTAAAGCGGTGAT 
               
               
                   
                 TCTGAAAGATGGAAAAGATATTGTCGCTGCCGAGGTTGTCCAAGTCGGTACCGGCTCCTC 
               
               
                   
                 GGGTCCCCAACGCGCACTGGACAAAGCCTTTGAAGTCTCTGGCTTAAAAAAGGAAGACAT 
               
               
                   
                 CAGCTACACAGTAGCTACGGGCTATGGGCGCTTCAATTTTAGCGACGCGGATAAACAGAT 
               
               
                   
                 TTCGGAAATTAGCTGTCATGCCAAAGGCATTTATTTCTTAGTACCAACTGCGCGCACTAT 
               
               
                   
                 TATTGACATTGGCGGCCAAGATGCGAAAGCCATCCGCCTGGACGACAAGGGGGGTATTAA 
               
               
                   
                 GCAATTCTTCATGAATGATAAATGCGCGGCGGGCACGGGGCGTTTCCTGGAAGTCATGGC 
               
               
                   
                 TCGCGTACTTGAAACCACCCTGGATGAAATGGCTGAACTGGATGAACAGGCGACTGACAC 
               
               
                   
                 CGCTCCCATTTCAAGCACCTGCACGGTTTTCGCCGAAAGCGAAGTAATTAGCCAATTGAG 
               
               
                   
                 CAATGGTGTCTCACGCAACAACATCATTAAAGGTGTCCATCTGAGCGTTGCGTCACGTGC 
               
               
                   
                 GTGTGGTCTGGCGTATCGCGGCGGTTTGGAGAAAGATGTTGTTATGACAGGTGGCGTGGC 
               
               
                   
                 AAAAAATGCAGGGGTGGTGCGCGCGGTGGCGGGCGTTCTGAAGACCGATGTTATCGTTGC 
               
               
                   
                 TCCGAATCCTCAGACGACCGGTGCACTGGGGGCAGCGCTGTATGCTTATGAGGCCGCCCA 
               
               
                   
                 GAAGAAGTA 
               
               
                   
               
            
           
         
       
     
     Example 27. 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 47 and Table 48. Tandem Mass Spectrometry details are found in Table 49. 
     
       
         
           
               
             
               
                 TABLE 47 
               
             
            
               
                   
               
               
                 HPLC Details 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Thermo Aquasil C18 
               
               
                   
                   
                   
                 column, 5 μm (50 × 2.1 
               
               
                   
                   
                 Column 
                 mm) 
               
               
                   
                   
               
               
                   
                   
                 Mobile 
                 100% H2O, 0.1% Formic 
               
               
                   
                   
                 Phase A 
                 Acid 
               
               
                   
                   
                 Mobile 
                 100% ACN, 0.1% Formic 
               
               
                   
                   
                 Phase B 
                 Acid 
               
               
                   
                   
                 Injection 
                 10 uL 
               
               
                   
                   
                 volume 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 48 
               
             
            
               
                   
               
               
                 HPLC Method 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 Total 
                 Flow 
                   
                   
               
               
                   
                   
                 Time 
                 Rate 
                   
                   
               
               
                   
                   
                 (min) 
                 (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 49 
               
               
                   
               
               
                 Tandem Mass Spectrometry Details 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Ion Source  
                 HESI-II 
               
               
                   
                   
                 Polarity 
                 Positive 
               
               
                   
                   
                 SRM 
                 Propionate 
               
               
                   
                   
                 transitions 
                 156.2/57.1, 
               
               
                   
                   
                   
                 Propionate-d5 
               
               
                   
                   
                   
                 161/62.1 
               
               
                   
                   
               
            
           
         
       
     
     Example 28. GLP-1 Production from Genetically Engineered Bacteria and Activity Measurements 
     To determine whether GLP-1 can be expressed by the genetically engineered bacteria, a construct expressing GLP-1 in conjunction with a modified flagellar type III secretion system shown in  FIG. 51  was generated and integrated into the  E coli  Nissle chromosome. The construct comprises GLP-1 under control of the native FliC promoter and 5′UTR (untranslated region containing the N-terminal flagellar secretion signal) with an optimized ribosome binding site  FIG. 21  and Table 50). 
     
       
         
           
               
             
               
                 TABLE 50 
               
             
            
               
                   
               
               
                 GLP-1 construct sequences 
               
            
           
           
               
               
            
               
                 Description and SEQ ID NO 
                 Sequence 
               
               
                   
               
               
                 GLP-1 under control of the native 
                 ttaaccacgacctttaaccagccaagcaataaactctttcgca 
               
               
                 FliC promoter and 5′UTR with an 
                 gcctggccctccaaatagctagaaacatcagaagtgaaagtt 
               
               
                 optimized ribosome binding site (in 
                 ccctccgcgtggcgttcgaactcgtccatattacctcctgactgt 
               
               
                 reverse orientation) 
                 gtctacttcgttgattacgttttgggtttccacccgtcggctcaatc 
               
               
                 (SEQ ID NO: 223) 
                 gccgtca 
               
               
                   
               
               
                 GLP-1 (in reverse orientation) 
                 ttaaccacgacctttaaccagccaagcaataaactctttcgca 
               
               
                 (SEQ ID NO: 224) 
                 gcctggccctccaaatagctagaaacatcagaagtgaaagtt 
               
               
                   
                 ccctccgcgtggcgttcgaactcgtccat 
               
               
                   
               
               
                 FliC 5′ UTR (in reverse orientation) 
                 attacctcctgactgtgtctacttcgttgattacgttttgggtttcca 
               
               
                 (SEQ ID NO: 225) 
                 cccgtcggctcaatcgccgtca 
               
               
                   
               
               
                 Optimized RBS (in reverse 
                 attacctcctgactgtgtctacttc 
               
               
                 orientation) 
                   
               
               
                 (SEQ ID NO: 226) 
                   
               
               
                   
               
               
                 Putative terminator 
                 gggcagaaaaaaccccgccgttggcggggaagcacgttgc 
               
               
                 (SEQ ID NO: 227) 
                   
               
               
                   
               
               
                 GLP-1 construct comprising 
                 
                   Gggcagaaaaaaccccgccgttggcggggaagcacgttg 
                 
               
               
                 terminator (lower case italic) GLP-1 
                   c tggcaaattaccattcatgttgccggatgcggcgtaaacgcc 
               
               
                 (lower case bold) under control of 
                 ttatccggcctacaaaaatgtgcaaattcaataaattgcaattc 
               
               
                 the native FliC promoter and 5′UTR 
                 cccttgtaggcctgataagcgcagcgcatcaggcaatttggc 
               
               
                 (upper case bold, with optimized 
                 gttgccgtcagtctcagttaatcaggttacggcga ttaaccac   
               
               
                 RBS underlined) and a 
                 
                   gacctttaaccagccaagcaataaactctttcgcagcctg 
                 
               
               
                 chloramphenicol resistance gene 
                 
                   gccctccaaatagctagaaacatcagaagtgaaagttcc 
                 
               
               
                 under the control of the cat promoter 
                 
                   ctccgcgtggcgttcgaactcgtccat 
                   ATTACCTCCT 
                 
               
               
                 (upper case italic bold), frt homology 
                   GACTGTGTCTACTTC GTTGATTACGTTTTG 
               
               
                 (upper case underlined) 
                 GGTTTCCACCCGTCGGCTCAATCGCCGTC 
               
               
                 (SEQ ID NO: 228) 
                 AACCCTGTTATCGTCTGTCGTAAAACAACC 
               
               
                   
                 TTTAGAATTTTTTTCACAAACAGCCATTTTT 
               
               
                   
                 TGTTAGTCGACGAAATACTCTTTTCTCTGC 
               
               
                   
                 CCCTTATTCCCGCTATTAAAAAAAACAATT 
               
               
                   
                 AAACGTAAACTTTGCGCAATTCAGGCCGA 
               
               
                   
                 TAACCCCGGTATTCGTTTTACGTGTCGAAA 
               
               
                   
                 GATAAA CGAAGTTCCTATACTTTCTAGAGA   
               
               
                   
                   ATAGGAACTTCGGAATAGGAACTTC ATTTC 
               
               
                   
                 TCGTTCGCTGCCACCTAAGAATACTCTAC 
               
               
                   
                 GGTCACATACAAATGGCGCGCCTTACGCC 
               
               
                   
                 CCGCCCTGCCA   
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                 
                   
                 
               
               
                   
                    ACGTCTCATTTTCGCCAAAAGTTG 
               
               
                   
                 GCCCAGGGCTTCCCGGTATCAACAGGGA 
               
               
                   
                 CACCAGGATTTATTTATTCTGCGAAGTGAT 
               
               
                   
                 CTTCCGTCACAGGTAGGCGCGC CGAAGTT   
               
               
                   
                 
                   CCTATACTTTCTAGAGAATAGGAACTTCGG 
                 
               
               
                   
                 
                   AATAGGAACT 
                 
               
               
                   
               
            
           
         
       
     
     Cultures (the genetically engineered bacteria comprising the GLP-1 construct or streptomycin resistant control Nissle) are grown overnight in F-12K medium (Mediatech, Manassas, Va.) without glucose (containing selective antibiotics (chloramphenicol or streptomycin) and then diluted 1:200. The cells are grown with shaking at 250 rpm, and at indicated times (0, 3, 6, and 12 h), the supernatant aliquots are collected for GLP-1 quantification. 
     Additionally, bacteria are pelleted, washed, and harvested, resuspended in 25 mL sonication buffer (50 mM Tris-HCl, 30 mM NaCl, pH 8.0) with protease inhibitors, and lysed by sonication on ice. Unsoluble debris is spun down twice for 20 min at 12,000 rpm at 4° C. to detect any intracellular recombinant protein. 
     To generate cell free medium, the supernatant is centrifuged, and filtered through a 0.2 microm filter to remove any remaining bacteria. The cell-free culture medium (CFM) is diluted to OD600=1 with F-12K, and 10 ng/ml leupeptin, 200 μM PMSF and 5 ng/mL aproitinin was added to the CFM to inhibit proteases prior storage at 4° C. 
     Western Blotting 
     The cell-free culture medium (CFM) was diluted to the same OD600 with F-12K, and 10 ng/ml leupeptin, PMSF and 5 ng/mL aprotinin was added to inhibit proteases. Clarified supernant (14 ml) is precipitated with 10% trichloroacetic acid (TCA, VWR) for 30 min on ice, and the pellet was washed twice in ice-cold ethanol/ether (1:1). The supernatant pellet is dried under vacuum, dissolved in 50 μl sample buffer (2% SDS, 50 mM Tris, pH 6.8, 20% glycerol, 10% mercaptoethanol, bromophenol blue) and boiled for 5 min at 95° C. The cell pellet is resuspended (From 14 ml culture) in room temperature BugBuster Master Mix by gentle vortexing, using 500 μl BugBuster Master Mix with protease inhibitors (10 ng/ml Leupeptin, 200 μM PMSF and 5 ng/mL aprotinin). The cell suspension is incubated on a shaking platform (VWR, Bristol, Conn.) at a slow setting for 10-20 min at room temperature. 125 μl 5× sample buffer is added to each sample before and boiling for 10 min at 95° C. 
     Protein concentration is determined by BCA protein assay, and isolated proteins are analyzed by Western blot. Proteins are transferred onto PVDF membranes are detected with an HRP-conjugated Glucagon Antibody (24HCLC), ABfinity™ Rabbit Oligoclonal, Thermo Fisher. 
     Co-Culture with Caco-2 Cells and ELISA for Insulin 
     To determine whether the GLP-1 expressed by the genetically engineered bacteria is functional, a co-culture experiment is conducted in which the bacterial supernatant containing GLP-1 is added to the growth medium of a mammalian intestinal cell line, Caco-2. Caco-2 cells are an intestinal cell line derived from a human colorectal carcinoma that spontaneously differentiates under standard culture conditions, and which lends itself to the in vitro study of human gut. The ability of the Caco-2 cells to produce insulin upon exposure to the bacterial cell free supernatant is measured. 
     Caco-2 epithelial cells (ATCC #CRL-2102, Manassas, Va.) are maintained in Dulbecco&#39;s Modified Eagle Media (DMEM, Cellgro, Herndon, Va.) plus 10% FBS (Cellgro) at 37° C. in a humidified incubator supplemented with 5% CO2. For co-culture experiments, Caco-2 cells are grown in F-12K supplemented with 10% FBS at 37° C. in a humidified incubator supplemented with 5% CO2. All co-culture experiments are performed in F-12K plus 10% FBS with Caco-2 cells in passages between 15 and 22. 
     Approximately 80% confluent monolayers of Caco-2 cells in 12-well plates are washed with fresh F-12K plus 10% FBS once and covered with 1 mL 50% CFM in F-12K with 10% FBS and incubated at 37° C. with 5% CO 2 . 200 nM. As a control, the same volume of recombinant GLP-1 (200 nM) in F-12K with 10% FBS is added as a positive control in separate wells. Following a 16 h incubation, an additional 1 mL of 50% CFM in F-12K with 10% FBS or GLP-1 is added to the cells, supplemented with 0.4% Glucose or 0.4% Glycerol before incubation for an additional 2 h. The media is removed from the cells, supplemented with Leupeptin (10 ng/mL), 0.2 mM PMSF and aprotinin (10 ng/mL), centrifuged (12,000 x rpm), and kept briefly at 4° C. prior to ELISA analysis for insulin expression (see “Immuno-blot and ELISA” section). 
     In order to estimate the amount of insulin secreted from Caco-2 cells activated by Glp-1, cell free supernatants are assayed using standard ELISA procedures using the Insulin ELISA Kit, Human (KAQ125, Thermo Fisher), according to manufacturer&#39;s instructions. 
     Example 30. In Vivo NASH Studies 
     For in vivo studies, a mouse model is used to study the effects of liver steatosis and hepatic inflammation (Jun Jin, et al., Brit. J. Nutrition, 114:145-1755 (2015)). To briefly summarize, female C57BL/6J mice are fasted and fed either a standard liquid diet of carbohydrates, fat, and protein; or a liquid Western style diet (WSD) fortified with fructose, fat, cholesterol, and a sodium butyrate supplement for six weeks. Butyrate is a short chain fatty acid naturally produced by intestinal bacteria effective in maintaining intestinal homoeostasis. Body weight and plasma samples can be taken throughout the duration of the study. Upon conclusion of the study, the mice can be killed, and the liver and intestine can be removed and assayed. A decrease in liver damage after treatment with the engineered bacterial cells indicates that the engineered bacterial cells described herein are effective for treating nonalcoholic steatohepatitis (NASH). 
     Additionally, throughout the study, phenotypes of the mice can also be analyzed. A decrease in the number of symptoms associated with nonalcoholic steatohepatitis (NASH), for example, weight loss, further indicates the efficacy of the engineered bacterial cells described herein for treating nonalcoholic steatohepatitis (NASH). 
     Example 31. Construction of Plasmids Encoding Bile Salt Hydrolase Enzymes 
     The bile salt hydrolase genes from  Lactobacillus plantarum  (SEQ ID NO:1) is synthesized (Genewiz), fused to the Tet promoter, cloned into the high-copy plasmid pUC57-Kan by Gibson assembly, and transformed into  E. coli  DH5a as described herein to generate the plasmid pTet-BSH. 
     Example 32. Generation of Recombinant Bacteria Comprising a Bile Salt 
     Hydrolase Enzyme 
     The pTet-BSH plasmid described above is transformed into  E. coli  Nissle, DH5α, or PIR1. All tubes, solutions, and cuvettes are pre-chilled to 4° C. An overnight culture of  E. coli  (Nissle, DH5α or PIR1) is diluted 1:100 in 4 mL of LB and grown until it reaches an OD 600  of 0.4-0.6. 1 mL of the culture is then centrifuged at 13,000 rpm for 1 min in a 1.5 mL microcentrifuge tube and the supernatant is removed. The cells are then washed three times in pre-chilled 10% glycerol and resuspended in 40 uL pre-chilled 10% glycerol. The electroporator is set to 1.8 kV. 1 uL of a pTet-BSH miniprep is added to the cells, mixed by pipetting, and pipetted into a sterile, chilled 1 mm cuvette. The dry cuvette is placed into the sample chamber, and the electric pulse is applied. 500 uL 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 50 ug/mL Kanamycin for pTet-BSH. 
     Example 33. Functional Assay Demonstrating that the Recombinant Bacterial Cells Decrease Bile Salt Concentration 
     For in vitro studies, all incubations will be performed at 37° C. Cultures of  E. coli  Nissle containing pTet-BSH are grown overnight in LB and then diluted 1:100 in LB. The cells are grown with shaking (250 rpm) to early log phase with the appropriate antibiotics. Anhydrous tetracycline (ATC) is added to cultures at a concentration of 100 ng/mL to induce expression of bile salt hydrolase, and bacteria are grown for another 3 hours. Culture broths are then inoculated at 20% in flasks containing fresh LB culture media containing excess bile salts (either 0.5% (wt/vol) TDCA, 0.5% (wt/vol) GDCA, or 3% (vol/vol) human bile) and grown for 16 hours with shaking (250 rpm). A “medium blank” for each culture condition broth is also prepared whereby the “medium blank” is not inoculated with bacteria but treated under the same conditions as the inoculated broths. Following the 16 hour incubation period, broth cultures are pasteurized at 90° C. for 15 minutes, centrifuged at 5,000 rpm for 10 minutes, and supernatants filtered with a 0.45 micron filter. 
     Bile salt levels and activity in the supernatants is determined. Briefly, bile salt hydrolase activity can be assessed using a plate assay as described in Dashkevicz and Feighner,  Applied Environ. Microbiol.,  55:11-16 (1989) and Christiaens et al.,  Appl. Environ. Microbiol.,  58:3792-3798 (1992). BSH activity can also be indicated by halos of precipitated deconjugated bile acids (see, also, Jones et al., PNAS, 105(36):13580-13585 (2008)). A ninhydrine assay for free taurine has also been described (see, for example, Clarke et al.,  Gut Microbes,  3(3):186-202 (2012)). 
     Example 34. In Vivo Studies Demonstrating that the Recombinant Bacterial Cells Decrease Bile Salt Concentration 
     For in vivo studies, a mouse model of weight gain and lipid metabolism (as described by Joyce et al.,  PNAS,  111(20):7421-7426 (2014)) is used. To briefly summarize, C57BL/6J mice and germ-free Swiss Webster mice can be fasted and fed either a normal low-fat diet or a high-fat diet for ten weeks. After ten weeks, the mice can be inoculated with recombinant bacteria comprising a bile salt hydrolase enzyme (as described herein) or control bacteria. Body weight, plasma samples, and fecal samples can be taken throughout the duration of the study. Upon conclusion of the study, the mice can be killed, and internal organs (liver, spleen, intestines) and fat pads can be removed and assayed. Treatment efficacy is determined, for example, by measuring levels of bile salts and bile acids. A decrease in levels of bile salts after treatment with the recombinant bacterial cells indicates that the recombinant bacterial cells described herein are effective for treating disorders associated with bile salts. 
     Additionally, throughout the study, phenotypes of the mice can also be analyzed. A decrease in the number of symptoms associated with disorders associated with bile salts, for example, weight loss, further indicates the efficacy of the recombinant bacterial cells described herein for treating disorders associated with bile salts. 
     Example 35. Generation of  E. Coli  Mutants with Ability to Consume L-Kynurenine and Produce Tryptophan from Kyrurenine 
       E. coli  Nissle can be engineered to efficiently import KYN and convert it to TRP. A strain was constructed (tryptophan auxotroph) that also expresses exogenous  Pseudomonas fluorescens  kynureninase mutiation, with the goal of generating a strain that is capable of converting L-kynurenine to anthranilate. Anthranilate can then be converted tryptophan through the enzymes of the tryptophan biosynthetic pathway. 
       E. coli  naturally utilizes anthranilate in its TRP biosynthetic pathway. Briefly, the TrpE (in complex with TrpD) enzyme converts chorismate into anthranilate. TrpD, TrpC, TrpA and TrpB then catalyze a five-step reaction ending with the condensation of an indole with serine to form tryptophan. Next, the kynureninase si introduced into a strain which harbors ΔtrpE (trypophan auxotrophy) deletion. By deleting the TrpE enzyme via lambda-RED recombineering, the subsequent strain of Nissle (ΔtrpE::Cm) is an auxotroph unable to grow in minimal media without supplementation of TRP or anthranilate. By expressing kynureninase in ΔtrpE::Cm (KYNase-trpE), this auxotrophy should alternatively rescued by providing KYN. 
     Indeed, as a proof of concept, we were able to show that—while Nissle does not typically utilize KYN—by introducing the Kynureninase (KYNase) from Pseudomonasfluorescens (kynU) on a medium-copy plasmid under the control of the tetracycline promoter (Ptet) a new strain with this plasmid (Ptet-KYNase) was able to convert L-kynurenine into anthranilate in the presence of a Tet inducer. 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 51 
               
               
                   
               
               
                   
                   
                   
                   
                   
                 Min + 
               
               
                   
                   
                 Rich  
                 Min  
                 Min + 
                 KYNU + 
               
               
                   
                 STRAIN 
                 Media 
                 Media 
                 Anthranilate  
                 aTc 
               
               
                   
               
             
            
               
                   
                 Wild type 
                 + 
                 + 
                 + 
                 + 
               
               
                   
                 Nissle 
                   
                   
                   
                   
               
               
                   
                 tipE 
                 + 
                 − 
                 + 
                 − 
               
               
                   
                 tipE 
                 + 
                 − 
                 + 
                 + 
               
               
                   
                 pseudoKYNase 
                   
                   
                   
                   
               
               
                   
                 tipE hKYNase 
                 + 
                 − 
                 + 
                 − 
               
               
                   
               
            
           
         
       
     
     In a preliminary assay (Table 51), wildtype Nissle (SYN094), Nissle with a deletion of trpE, and trpE mutants expressing either the human kynureninase (hKYNase) or the  Pseudomonas fluorescens  kynureninase (pseudoKYNase) from a Ptet promoter on a medium-copy plasmid were grown in either rich media, minimal media (min media), minimal media with 5 mM anthranilate (Min+anthranilate) or minimal media with 10 mM kynurenine and 100 ng/uL aTc (Min+KYNU+aTc). These were grown in 1 mL of media in a deep well plate with shaking at 37° C. A positive for growth (+) in Table 51 indicates a change in optical density of &gt;5-fold from inoculation. 
     The results show that in a mutant trpE (which is typically used in the tryptophan biosynthetic pathway to convert chorismate into anthranilate) background, Nissle is unable to grow in minimal media without supplementation with anthranilate (or tryptophan). When minimal media was supplemented with KYNU, the trpE mutant was also unable to grow. However, when the pseudoKYNase was expressed in the trpE tryptophan-auxotroph the cells were able to grow in Min+KYNU. This indicates that Nissle is able to import L-kynurenine from the media and convert it into anthranilate using the pseudoKYNase. The hKYNase homolog was unable to support growth on M9+KYNU, most likely due to differences in substrate specificity as it has been documented that the human kynureninase prefers 3-hydroxykynurenine as a substrate (Phillips, Structure and mechanism of kynureninase. Arch Biochem Biophys. 2014 Feb. 15; 544:69-74). 
     Example 36. Generation of  E. Coli  Mutants with Enhanced Ability to Consume L-Kynurenine and Produce Tryptophan from Kyrurenine 
     Adaptive Laboratory Evolution was used to produce mutant bacterial strains with improved Kynurenine consumption and reduced tryptophan uptake. First a lower limit of KYN concentration was established and mutants were evolved by passaging in lowering concentrations of KYN. 
     While this can select for mutants capable of increasing KYN import, the bacterial cells still prefer to utilize free, exogenous TRP. In the tumor environment, dual-therapeutic functions can be provided by depletion of KYN and increasing local concentrations of TRP. Therefore, to evolve a strain which prefers KYN over TRP, a toxic analogue of TRP—5-fluoro-L-tryptophan (ToxTRP)—can be incorporated into the ALE experiment. The resulting best performing strain is then whole genome sequenced in order to deconvolute the contributing mutations. Lambda-RED can be performed in order to reintroduce TrpE, to inactivate Trp regulation (trpR, tyrR, transcriptional attenuators) to up-regulate TrpABCDE expression and increase chorismate production. The resulting strain prefers external KYN over to external TRP, efficiently converts KYN into TRP, and also now overproduces TRP. 
     Moving forward with the knowledge that Nissle is able to grow on KYNU supplemented minimal media in a trpE auxotroph by importing and converting kynurenine, the next step was to establish the minimal concentrations of kynurenine capable of supporting growth. Additionally, in our selection experiment if 5-fluoro-L-tryptophan (ToxTrp) was employed the concentrations of both KYNU and ToxTrp capable of still sustaining growth. 
     A growth assay was performed in 96-well plates using streptomycin resistant Nissle, trpE and trpE pseudoKYNase with and without induction of pseudoKYNase expression using 100 ng/uL aTc. These strains were inoculated at very dilute concentrations into M9 minimal media with varying concentrations of KYNU across columns (2-fold dilutions starting at 2000 ug/mL) and varying concentrations of ToxTrp across rows (2-fold dilutions starting at 200 ug/mL). On a separate plate, the strains were grown in M9+KYNU (at the same concentrations) in the absence of ToxTrp, as described in Example 15. 
     The results of the initial checkerboard assay are displayed in  FIG. 7-9  as a function of optical density at 600 nm (normalized to a media blank). In  FIGS. 7 and 8 , the X-axis shows decreasing KYNU concentration from left-to-right, while the Z-axis shows decreasing ToxTrp concentration from front-to-back with the very back row representing media with no ToxTrp. In  FIG. 9 . the control sand trpE strains are shown in M9+KYNU without any ToxTrp, as there was no growth detected from either strain at any concentration of ToxTrp. The results of the assay show that expression of the pseudoKYNase provides protection against toxicity of ToxTrp. More importantly, growth is permitted between 250-62.5 ug/mL of KYNU and 6.3-1.55 ug/mL of ToxTrp. 
     Together these experiments establish that expression of the Pseudomonasfluorescens kynureninase is sufficient to rescue a trpE auxotrophy in the presence of kynurenine, as the strain ia able to consume KYN into anthranilate, and upstream metabolite in the TRP biosynthetic pathway. In addition, the KYNase is also capable of providing increased resistance to the toxic tryptophan, 5-fluoro-L-tryptophan. Using the information attained here it is possible to proceed to an adapative laboratory evolution experiment to select for mutants with highly efficient and selective conversion of kynurenine to tryptophan. 
     Example 37. Checkerboard Assay and ALE Parameters 
     To establish the minimum concentration of L-kynurenine and maximum concentration of 5-fluoro-L-tryptophan (ToxTrp) capable of sustaining growth of the KYNase strain, using a checkerboard assay, the following protocol was used. Using a 96-well plate with M9 minimal media with glucose, KYN is supplemented decreasing across columns in 2-fold dilutions from 2000 ug/mL down to ˜1 ug/mL. In the rows, ToxTrp concentration decreases by 2-fold from 200 ug/mL down to ˜1.5 ug/mL. In one plate, Anhydrous Tetracycline (aTc) was added to a final concentration of 100 ng/uL to induce production of the KYNase. From an overnight culture cells were diluted to an OD600=0.5 in 12 mL of TB (plus appropriate antibiotics and inducers, where applicable) and grown for 4 hours. 100 uL of cells were spun down and resuspended to an OD600=1.0. These were diluted 2000-fold and 25 uL was added to each well to bring the final volumes in each well to 100 uL. Cells were grown for roughly 20 hours with static incubation at 37 C then growth was assessed by OD600, making sure readings fell within linear range (0.05-1.0). 
     Once identified, the highest concentrations of ToxTrp and lowest concentration of kynurenine capable of supporting growth becomes the starting point for ALE. The ALE parental strain was chosen by culturing the KYNase strain on M9 minimal media supplemented with glucose and L-kynurenine (referred to as M9+KYNU from here on). A single colony was selected, resuspended in 20 uL of sterile phosphate-buffered saline solution. This colony was then used to inoculate three cultures of M9+KYNU, grown into late-logarithmic phase and optical density determined at 600 nm. These cultures were then diluted to 10 1  in 4 rows of a 96-well deep-well plate with 1 mL of M9+KYNU. Each one of the four rows has a different ToxTrp (increasing 2-fold), while each column has decreasing concentrations of KYNU (by 2-fold). Each morning and evening this plate is diluted back to 10 1  using the well in which the culture has grown to just below saturation so that the culture is always in logarithmic growth. This process is repeated until a change in growth rate is no longer detected. Once no growth rate increases are detected (usually around 10 11  Cumulative Cell Divisions) the culture is plated onto M9+KYNU (Lee, et al., Cumulative Number of Cell Divisions as a Meaningful Timescale for Adaptive Laboratory Evolution of  Escherichia coli . PLoS ONE 6, e26172; 2011). Individual colonies are selected and screened in M9+KYNU+ToxTrp media to confirm increased growth rate phenotype. Once mutants with significantly increased growth rate on M9+KYNU are isolated, genomic DNA can be isolated and sent for whole genome sequencing to reveal the mutations responsible for phenotype. All culturing is done shaking at 350 RPM at 37° C. 
     Example 38. 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 ( FIG. 28 ); 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  . 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. 64  shows 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.  FIG. 65  shows NsrR-regulated promoters are induced in DSS-treated mice, but not in untreated mice. 
     Example 39. 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 Tables 52-56 ((SEQ ID NO: 228-229). 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  FIG. 62 . 
     Table 52 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, P fnr1  (SEQ ID NO: 228). 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 53 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, Pfnr2 ((SEQ ID NO: 229). The construct comprises a translational fusion of the Nissle ydjZ 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 Pfnr2 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 54 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, P fnr3  ((SEQ ID NO: 230). 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 fcr2  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 55 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, P fnr4  ((SEQ ID NO: 2318). The construct comprises a transcriptional fusion of the Nissle ydjZ 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 56 shows the nucleotide sequence of an exemplary construct comprising a gene encoding lacZ, and an exemplary FNR promoter, PfnrS ((SEQ ID NO: 232). 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 IacZ 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 52 
               
               
                   
               
               
                 Pfnr1-lacZ construct Sequences 
               
               
                 Nucleotide sequences of Pfnr1-lacZ construct, 
               
               
                 low-copy (SEQ ID NO: 228) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 GGTACC gtcagcataacaccctgacctctcattaattgttcat   
               
               
                   
                   
               
               
                   
                 
                   gccgggcggcactatcgtcgtccggccttttcctctcttactc 
                 
               
               
                   
                   
               
               
                   
                 
                   tgctacgtacatctatttctataaatccgttcaatttgtctgt 
                 
               
               
                   
                   
               
               
                   
                 
                   tttttgcacaaacatgaaatatcagacaattccgtgacttaag 
                 
               
               
                   
                   
               
               
                   
                 
                   aaaatttatacaaatcagcaatataccccttaaggagtatata 
                 
               
               
                   
                   
               
               
                   
                 
                   aaggtgaatttgatttacatcaataagcggggttgctgaatcg 
                 
               
               
                   
                   
               
               
                   
                 
                   ttaaggtaggcggtaatagaaaagaaatcgaggcaaaaATGag 
                 
               
               
                   
                   
               
               
                   
                   caaagtcagactcgcaattat GGATCCTCTGGCCGTCGTATTA 
               
               
                   
                   
               
               
                   
                 CAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATC 
               
               
                   
                   
               
               
                   
                 GCCTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGA 
               
               
                   
                   
               
               
                   
                 AGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTG 
               
               
                   
                   
               
               
                   
                 AATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGG 
               
               
                   
                   
               
               
                   
                 TGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATAC 
               
               
                   
                   
               
               
                   
                 TGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCG 
               
               
                   
                   
               
               
                   
                 CCTATCTACACCAACGTGACCTATCCCATTACGGTCAATCCGC 
               
               
                   
                   
               
               
                   
                 CGTTTGTTCCCGCGGAGAATCCGACAGGTTGTTACTCGCTCAC 
               
               
                   
                   
               
               
                   
                 ATTTAATATTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGA 
               
               
                   
                   
               
               
                   
                 ATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCA 
               
               
                   
                   
               
               
                   
                 ACGGGCGCTGGGTCGGTTACGGCCAGGACAGCCGTTTGCCGTC 
               
               
                   
                   
               
               
                   
                 TGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGC 
               
               
                   
                   
               
               
                   
                 CTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGG 
               
               
                   
                   
               
               
                   
                 AAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGT 
               
               
                   
                   
               
               
                   
                 CTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATTTCCAA 
               
               
                   
                   
               
               
                   
                 GTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTACTGG 
               
               
                   
                   
               
               
                   
                 AGGCAGAAGTTCAGATGTACGGCGAGCTGCGCGATGAACTGCG 
               
               
                   
                   
               
               
                   
                 GGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCAGC 
               
               
                   
                   
               
               
                   
                 GGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGCG 
               
               
                   
                   
               
               
                   
                 GTTATGCCGATCGCGTCACACTACGCCTGAACGTTGAAAATCC 
               
               
                   
                   
               
               
                   
                 GGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCAGTG 
               
               
                   
                   
               
               
                   
                 GTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAG 
               
               
                   
                   
               
               
                   
                 CCTGCGACGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCT 
               
               
                   
                   
               
               
                   
                 GCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGCGGCGTTAAC 
               
               
                   
                   
               
               
                   
                 CGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGC 
               
               
                   
                   
               
               
                   
                 AGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTT 
               
               
                   
                   
               
               
                   
                 TAACGCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGG 
               
               
                   
                   
               
               
                   
                 TACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAG 
               
               
                   
                   
               
               
                   
                 CCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGAC 
               
               
                   
                   
               
               
                   
                 CGATGATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACG 
               
               
                   
                   
               
               
                   
                 CGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCT 
               
               
                   
                   
               
               
                   
                 GGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGC 
               
               
                   
                   
               
               
                   
                 GCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTA 
               
               
                   
                   
               
               
                   
                 CAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTA 
               
               
                   
                   
               
               
                   
                 TTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCC 
               
               
                   
                   
               
               
                   
                 GGCGGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTGCCT 
               
               
                   
                   
               
               
                   
                 GGAGAAATGCGCCCGCTGATCCTTTGCGAATATGCCCACGCGA 
               
               
                   
                   
               
               
                   
                 TGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAGGCGTT 
               
               
                   
                   
               
               
                   
                 TCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGG 
               
               
                   
                   
               
               
                   
                 GTGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGT 
               
               
                   
                   
               
               
                   
                 GGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCG 
               
               
                   
                   
               
               
                   
                 CCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCG 
               
               
                   
                   
               
               
                   
                 CATCCGGCGCTGACGGAAGCAAAACACCAACAGCAGTATTTCC 
               
               
                   
                   
               
               
                   
                 AGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCGAATA 
               
               
                   
                   
               
               
                   
                 CCTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTG 
               
               
                   
                   
               
               
                   
                 GCACTGGATGGCAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGG 
               
               
                   
                   
               
               
                   
                 ATGTTGGCCCGCAAGGTAAGCAGTTGATTGAACTGCCTGAACT 
               
               
                   
                   
               
               
                   
                 GCCGCAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGC 
               
               
                   
                   
               
               
                   
                 GTAGTGCAACCAAACGCGACCGCATGGTCAGAAGCCGGACACA 
               
               
                   
                   
               
               
                   
                 TCAGCGCCTGGCAGCAATGGCGTCTGGCGGAAAACCTCAGCGT 
               
               
                   
                   
               
               
                   
                 GACACTCCCCTCCGCGTCCCACGCCATCCCTCAACTGACCACC 
               
               
                   
                   
               
               
                   
                 AGCGGAACGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGC 
               
               
                   
                   
               
               
                   
                 AATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGG 
               
               
                   
                   
               
               
                   
                 CGATGAAAAACAACTGCTGACCCCGCTGCGCGATCAGTTCACC 
               
               
                   
                   
               
               
                   
                 CGTGCGCCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCC 
               
               
                   
                   
               
               
                   
                 GCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGG 
               
               
                   
                   
               
               
                   
                 CCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCACGGCAGAT 
               
               
                   
                   
               
               
                   
                 ACACTTGCCGACGCGGTGCTGATTACAACCGCCCACGCGTGGC 
               
               
                   
                   
               
               
                   
                 AGCATCAGGGGAAAACCTTATTTATCAGCCGGAAAACCTACCG 
               
               
                   
                   
               
               
                   
                 GATTGATGGGCACGGTGAGATGGTCATCAATGTGGATGTTGCG 
               
               
                   
                   
               
               
                   
                 GTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGACCT 
               
               
                   
                   
               
               
                   
                 GCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGCCT 
               
               
                   
                   
               
               
                   
                 GGGGCCGCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTGT 
               
               
                   
                   
               
               
                   
                 TTTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGT 
               
               
                   
                   
               
               
                   
                 ACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGA 
               
               
                   
                   
               
               
                   
                 ATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTC 
               
               
                   
                   
               
               
                   
                 AACATCAGCCGCTACAGCCAACAACAACTGATGGAAACCAGCC 
               
               
                   
                   
               
               
                   
                 ATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATAT 
               
               
                   
                   
               
               
                   
                 CGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGC 
               
               
                   
                   
               
               
                   
                 CCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACC 
               
               
                   
                   
               
               
                   
                 ATTACCAGTTGGTCTGGTGTCAAAAATAA 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 53 
               
               
                   
               
               
                 Pfnr2-lacZ construct sequences 
               
               
                 Nucleotide sequences of Pfnr2-lacZ construct, 
               
               
                 low-copy (SEQ ID NO: 229) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 GGTACC catttcctctcatcccatccggggtgagagtcttttcc   
               
               
                   
                   
               
               
                   
                 
                   cccgacttatggctcatgcatgcatcaaaaaagatgtgagcttg 
                 
               
               
                   
                   
               
               
                   
                 
                   atcaaaaacaaaaaatatttcactcgacaggagtatttatattg 
                 
               
               
                   
                   
               
               
                   
                 
                   cgcccgttacgtgggcttcgactgtaaatcagaaaggagaaaac 
                 
               
               
                   
                   
               
               
                   
                   acctATGacgacctacgatcg GGATCCTCTGGCCGTCGTATTAC 
               
               
                   
                   
               
               
                   
                 AACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGC 
               
               
                   
                   
               
               
                   
                 CTTGCGGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGA 
               
               
                   
                   
               
               
                   
                 GGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATG 
               
               
                   
                   
               
               
                   
                 GCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCG 
               
               
                   
                   
               
               
                   
                 GAAAGCTGGCTGGAGTGCGATCTTCCTGACGCCGATACTGTCGT 
               
               
                   
                   
               
               
                   
                 CGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCTATCT 
               
               
                   
                   
               
               
                   
                 ACACCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTT 
               
               
                   
                   
               
               
                   
                 CCCGCGGAGAATCCGACAGGTTGTTACTCGCTCACATTTAATAT 
               
               
                   
                   
               
               
                   
                 TGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTG 
               
               
                   
                   
               
               
                   
                 ATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGG 
               
               
                   
                   
               
               
                   
                 GTCGGTTACGGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCT 
               
               
                   
                   
               
               
                   
                 GAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGG 
               
               
                   
                   
               
               
                   
                 TGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATG 
               
               
                   
                   
               
               
                   
                 TGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAA 
               
               
                   
                   
               
               
                   
                 ACCGACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTA 
               
               
                   
                   
               
               
                   
                 ATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGATG 
               
               
                   
                   
               
               
                   
                 TACGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTTTGTG 
               
               
                   
                   
               
               
                   
                 GCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCG 
               
               
                   
                   
               
               
                   
                 GTGAAATTATCGATGAGCGTGGCGGTTATGCCGATCGCGTCACA 
               
               
                   
                   
               
               
                   
                 CTACGCCTGAACGTTGAAAATCCGGAACTGTGGAGCGCCGAAAT 
               
               
                   
                   
               
               
                   
                 CCCGAATCTCTATCGTGCAGTGGTTGAACTGCACACCGCCGACG 
               
               
                   
                   
               
               
                   
                 GCACGCTGATTGAAGCAGAAGCCTGCGACGTCGGTTTCCGCGAG 
               
               
                   
                   
               
               
                   
                 GTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTT 
               
               
                   
                   
               
               
                   
                 GCTGATTCGCGGCGTTAACCGTCACGAGCATCATCCTCTGCATG 
               
               
                   
                   
               
               
                   
                 GTCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTG 
               
               
                   
                   
               
               
                   
                 ATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATTATCC 
               
               
                   
                   
               
               
                   
                 GAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGT 
               
               
                   
                   
               
               
                   
                 ATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCA 
               
               
                   
                   
               
               
                   
                 ATGAATCGTCTGACCGATGATCCGCGCTGGCTACCCGCGATGAG 
               
               
                   
                   
               
               
                   
                 CGAACGCGTAACGCGGATGGTGCAGCGCGATCGTAATCACCCGA 
               
               
                   
                   
               
               
                   
                 GTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCT 
               
               
                   
                   
               
               
                   
                 AATCACGACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTC 
               
               
                   
                   
               
               
                   
                 CCGCCCGGTACAGTATGAAGGCGGCGGAGCCGACACCACGGCCA 
               
               
                   
                   
               
               
                   
                 CCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAG 
               
               
                   
                   
               
               
                   
                 CCCTTCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGCTTTC 
               
               
                   
                   
               
               
                   
                 GCTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATATGCCC 
               
               
                   
                   
               
               
                   
                 ACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGCAG 
               
               
                   
                   
               
               
                   
                 GCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCTGGGA 
               
               
                   
                   
               
               
                   
                 CTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACC 
               
               
                   
                   
               
               
                   
                 CGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGAT 
               
               
                   
                   
               
               
                   
                 CGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCC 
               
               
                   
                   
               
               
                   
                 GCATCCGGCGCTGACGGAAGCAAAACACCAACAGCAGTATTTCC 
               
               
                   
                   
               
               
                   
                 AGTTCCGTTTATCCGGGCGAACCATCGAAGTGACCAGCGAATAC 
               
               
                   
                   
               
               
                   
                 CTGTTCCGTCATAGCGATAACGAGTTCCTGCACTGGATGGTGGC 
               
               
                   
                   
               
               
                   
                 ACTGGATGGCAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATG 
               
               
                   
                   
               
               
                   
                 TTGGCCCGCAAGGTAAGCAGTTGATTGAACTGCCTGAACTGCCG 
               
               
                   
                   
               
               
                   
                 CAGCCGGAGAGCGCCGGACAACTCTGGCTAACGGTACGCGTAGT 
               
               
                   
                   
               
               
                   
                 GCAACCAAACGCGACCGCATGGTCAGAAGCCGGACACATCAGCG 
               
               
                   
                   
               
               
                   
                 CCTGGCAGCAATGGCGTCTGGCGGAAAACCTCAGCGTGACACTC 
               
               
                   
                   
               
               
                   
                 CCCTCCGCGTCCCACGCCATCCCTCAACTGACCACCAGCGGAAC 
               
               
                   
                   
               
               
                   
                 GGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACC 
               
               
                   
                   
               
               
                   
                 GCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATGAAAAA 
               
               
                   
                   
               
               
                   
                 CAACTGCTGACCCCGCTGCGCGATCAGTTCACCCGTGCGCCGCT 
               
               
                   
                   
               
               
                   
                 GGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTA 
               
               
                   
                   
               
               
                   
                 ACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCC 
               
               
                   
                   
               
               
                   
                 GAAGCGGCGTTGTTGCAGTGCACGGCAGATACACTTGCCGACGC 
               
               
                   
                   
               
               
                   
                 GGTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAAA 
               
               
                   
                   
               
               
                   
                 CCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACGGT 
               
               
                   
                   
               
               
                   
                 GAGATGGTCATCAATGTGGATGTTGCGGTGGCAAGCGATACACC 
               
               
                   
                   
               
               
                   
                 GCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGGCGCAGGTCT 
               
               
                   
                   
               
               
                   
                 CAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAGAAAACTAT 
               
               
                   
                   
               
               
                   
                 CCCGACCGCCTTACTGCAGCCTGTTTTGACCGCTGGGATCTGCC 
               
               
                   
                   
               
               
                   
                 ATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACG 
               
               
                   
                   
               
               
                   
                 GTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAG 
               
               
                   
                   
               
               
                   
                 TGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGCCAACA 
               
               
                   
                   
               
               
                   
                 ACAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAG 
               
               
                   
                   
               
               
                   
                 AAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGT 
               
               
                   
                   
               
               
                   
                 GGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCT 
               
               
                   
                   
               
               
                   
                 GAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAAT 
               
               
                   
                   
               
               
                   
                 AA 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 54 
               
               
                   
               
               
                 Pfnr3-lacZ construct Sequences 
               
               
                 Nucleotide sequences of Pfnr3-lacZ construct, 
               
               
                 low-copy (SEQ ID NO: 230) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 GGTACC gtcagcataacaccctgacctctcattaattgttca   
               
               
                   
                   
               
               
                   
                 
                   tgccgggcggcactatcgtcgtccggccttttcctctcttac 
                 
               
               
                   
                   
               
               
                   
                 
                   tctgctacgtacatctatttctataaatccgttcaatttgtc 
                 
               
               
                   
                   
               
               
                   
                 
                   tgttttttgcacaaacatgaaatatcagacaattccgtgact 
                 
               
               
                   
                   
               
               
                   
                 
                   taagaaaatttatacaaatcagcaatataccccttaaggagt 
                 
               
               
                   
                   
               
               
                   
                 
                   atataaaggtgaatttgatttacatcaataagcggggttgct 
                 
               
               
                   
                   
               
               
                   
                   gaatcgttaa GGATCC ctctagaaataattttgtttaacttt   
               
               
                   
                   
               
               
                   
                   aagaaggagatatacatATG ACTATGATTACGGATTCTCTGG 
               
               
                   
                   
               
               
                   
                 CCGTCGTATTACAACGTCGTGACTGGGAAAACCCTGGCGTTA 
               
               
                   
                   
               
               
                   
                 CCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGCT 
               
               
                   
                   
               
               
                   
                 GGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAAC 
               
               
                   
                   
               
               
                   
                 AGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTC 
               
               
                   
                   
               
               
                   
                 CGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATC 
               
               
                   
                   
               
               
                   
                 TTCCTGACGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGA 
               
               
                   
                   
               
               
                   
                 TGCACGGTTACGATGCGCCTATCTACACCAACGTGACCTATC 
               
               
                   
                   
               
               
                   
                 CCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGA 
               
               
                   
                   
               
               
                   
                 CAGGTTGTTACTCGCTCACATTTAATATTGATGAAAGCTGGC 
               
               
                   
                   
               
               
                   
                 TACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACT 
               
               
                   
                   
               
               
                   
                 CGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACG 
               
               
                   
                   
               
               
                   
                 GCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGCGCAT 
               
               
                   
                   
               
               
                   
                 TTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGC 
               
               
                   
                   
               
               
                   
                 GCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGC 
               
               
                   
                   
               
               
                   
                 GGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAAC 
               
               
                   
                   
               
               
                   
                 CGACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTTA 
               
               
                   
                   
               
               
                   
                 ATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAGA 
               
               
                   
                   
               
               
                   
                 TGTACGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCTT 
               
               
                   
                   
               
               
                   
                 TGTGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTT 
               
               
                   
                   
               
               
                   
                 TCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGATC 
               
               
                   
                   
               
               
                   
                 GCGTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGGA 
               
               
                   
                   
               
               
                   
                 GCGCCGAAATCCCGAATCTCTATCGTGCAGTGGTTGAACTGC 
               
               
                   
                   
               
               
                   
                 ACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGACG 
               
               
                   
                   
               
               
                   
                 TCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGC 
               
               
                   
                   
               
               
                   
                 TGAACGGCAAGCCGTTGCTGATTCGCGGCGTTAACCGTCACG 
               
               
                   
                   
               
               
                   
                 AGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGA 
               
               
                   
                   
               
               
                   
                 TGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACG 
               
               
                   
                   
               
               
                   
                 CCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACA 
               
               
                   
                   
               
               
                   
                 CGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCA 
               
               
                   
                   
               
               
                   
                 ATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCG 
               
               
                   
                   
               
               
                   
                 ATGATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACGC 
               
               
                   
                   
               
               
                   
                 GGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCT 
               
               
                   
                   
               
               
                   
                 GGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACG 
               
               
                   
                   
               
               
                   
                 CGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGG 
               
               
                   
                   
               
               
                   
                 TACAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGATA 
               
               
                   
                   
               
               
                   
                 TTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCT 
               
               
                   
                   
               
               
                   
                 TCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGC 
               
               
                   
                   
               
               
                   
                 TGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATATGCCC 
               
               
                   
                   
               
               
                   
                 ACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGGC 
               
               
                   
                   
               
               
                   
                 AGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTCT 
               
               
                   
                   
               
               
                   
                 GGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACG 
               
               
                   
                   
               
               
                   
                 GCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGC 
               
               
                   
                   
               
               
                   
                 CGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCG 
               
               
                   
                   
               
               
                   
                 ACCGCACGCCGCATCCGGCGCTGACGGAAGCAAAACACCAAC 
               
               
                   
                   
               
               
                   
                 AGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAAG 
               
               
                   
                   
               
               
                   
                 TGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGTTCC 
               
               
                   
                   
               
               
                   
                 TGCACTGGATGGTGGCACTGGATGGCAAGCCGCTGGCAAGCG 
               
               
                   
                   
               
               
                   
                 GTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTGA 
               
               
                   
                   
               
               
                   
                 TTGAACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAAC 
               
               
                   
                   
               
               
                   
                 TCTGGCTAACGGTACGCGTAGTGCAACCAAACGCGACCGCAT 
               
               
                   
                   
               
               
                   
                 GGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTC 
               
               
                   
                   
               
               
                   
                 TGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACG 
               
               
                   
                   
               
               
                   
                 CCATCCCTCAACTGACCACCAGCGGAACGGATTTTTGCATCG 
               
               
                   
                   
               
               
                   
                 AGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCT 
               
               
                   
                   
               
               
                   
                 TTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTGA 
               
               
                   
                   
               
               
                   
                 CCCCGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGATAACG 
               
               
                   
                   
               
               
                   
                 ACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCT 
               
               
                   
                   
               
               
                   
                 GGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAG 
               
               
                   
                   
               
               
                   
                 CGGCGTTGTTGCAGTGCACGGCAGATACACTTGCCGACGCGG 
               
               
                   
                   
               
               
                   
                 TGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAAA 
               
               
                   
                   
               
               
                   
                 CCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCACG 
               
               
                   
                   
               
               
                   
                 GTGAGATGGTCATCAATGTGGATGTTGCGGTGGCAAGCGATA 
               
               
                   
                   
               
               
                   
                 CACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGGCGC 
               
               
                   
                   
               
               
                   
                 AGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAAG 
               
               
                   
                   
               
               
                   
                 AAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGCT 
               
               
                   
                   
               
               
                   
                 GGGATCTGCCATTGTCAGACATGTATACCCCGTACGTCTTCC 
               
               
                   
                   
               
               
                   
                 CGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATT 
               
               
                   
                   
               
               
                   
                 ATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCA 
               
               
                   
                   
               
               
                   
                 GCCGCTACAGCCAACAACAACTGATGGAAACCAGCCATCGCC 
               
               
                   
                   
               
               
                   
                 ATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACG 
               
               
                   
                   
               
               
                   
                 GTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGT 
               
               
                   
                   
               
               
                   
                 CAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATT 
               
               
                   
                   
               
               
                   
                 ACCAGTTGGTCTGGTGTCAAAAATAA 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 55 
               
               
                   
               
               
                 Pfnr4-lacZ construct Sequences 
               
               
                 Nucleotide sequences of Pfnr4-lacZ construct, 
               
               
                 low-copy (SEQ ID NO: 231) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 GGTACC catttcctctcatcccatccggggtgagagtctttt   
               
               
                   
                   
               
               
                   
                 
                   cccccgacttatggctcatgcatgcatcaaaaaagatgtgag 
                 
               
               
                   
                   
               
               
                   
                 
                   cttgatcaaaaacaaaaaatatttcactcgacaggagtattt 
                 
               
               
                   
                   
               
               
                   
                   atattgcgccc GGATCC ctctagaaataattttgtttaactt   
               
               
                   
                   
               
               
                   
                   taagaaggagatatacatATG ACTATGATTACGGATTCTCTG 
               
               
                   
                   
               
               
                   
                 GCCGTCGTATTACAACGTCGTGACTGGGAAAACCCTGGCGTT 
               
               
                   
                   
               
               
                   
                 ACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGCCAGC 
               
               
                   
                   
               
               
                   
                 TGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAA 
               
               
                   
                   
               
               
                   
                 CAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTT 
               
               
                   
                   
               
               
                   
                 CCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGAT 
               
               
                   
                   
               
               
                   
                 CTTCCTGACGCCGATACTGTCGTCGTCCCCTCAAACTGGCAG 
               
               
                   
                   
               
               
                   
                 ATGCACGGTTACGATGCGCCTATCTACACCAACGTGACCTAT 
               
               
                   
                   
               
               
                   
                 CCCATTACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCG 
               
               
                   
                   
               
               
                   
                 ACAGGTTGTTACTCGCTCACATTTAATATTGATGAAAGCTGG 
               
               
                   
                   
               
               
                   
                 CTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAAC 
               
               
                   
                   
               
               
                   
                 TCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTAC 
               
               
                   
                   
               
               
                   
                 GGCCAGGACAGCCGTTTGCCGTCTGAATTTGACCTGAGCGCA 
               
               
                   
                   
               
               
                   
                 TTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTG 
               
               
                   
                   
               
               
                   
                 CGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGG 
               
               
                   
                   
               
               
                   
                 CGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAA 
               
               
                   
                   
               
               
                   
                 CCGACCACGCAAATCAGCGATTTCCAAGTTACCACTCTCTTT 
               
               
                   
                   
               
               
                   
                 AATGATGATTTCAGCCGCGCGGTACTGGAGGCAGAAGTTCAG 
               
               
                   
                   
               
               
                   
                 ATGTACGGCGAGCTGCGCGATGAACTGCGGGTGACGGTTTCT 
               
               
                   
                   
               
               
                   
                 TTGTGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCT 
               
               
                   
                   
               
               
                   
                 TTCGGCGGTGAAATTATCGATGAGCGTGGCGGTTATGCCGAT 
               
               
                   
                   
               
               
                   
                 CGCGTCACACTACGCCTGAACGTTGAAAATCCGGAACTGTGG 
               
               
                   
                   
               
               
                   
                 AGCGCCGAAATCCCGAATCTCTATCGTGCAGTGGTTGAACTG 
               
               
                   
                   
               
               
                   
                 CACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGAC 
               
               
                   
                   
               
               
                   
                 GTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTG 
               
               
                   
                   
               
               
                   
                 CTGAACGGCAAGCCGTTGCTGATTCGCGGCGTTAACCGTCAC 
               
               
                   
                   
               
               
                   
                 GAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACG 
               
               
                   
                   
               
               
                   
                 ATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAAC 
               
               
                   
                   
               
               
                   
                 GCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTAC 
               
               
                   
                   
               
               
                   
                 ACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCC 
               
               
                   
                   
               
               
                   
                 AATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACC 
               
               
                   
                   
               
               
                   
                 GATGATCCGCGCTGGCTACCCGCGATGAGCGAACGCGTAACG 
               
               
                   
                   
               
               
                   
                 CGGATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATC 
               
               
                   
                   
               
               
                   
                 TGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGAC 
               
               
                   
                   
               
               
                   
                 GCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCG 
               
               
                   
                   
               
               
                   
                 GTACAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGAT 
               
               
                   
                   
               
               
                   
                 ATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCC 
               
               
                   
                   
               
               
                   
                 TTCCCGGCGGTGCCGAAATGGTCCATCAAAAAATGGCTTTCG 
               
               
                   
                   
               
               
                   
                 CTGCCTGGAGAAATGCGCCCGCTGATCCTTTGCGAATATGCC 
               
               
                   
                   
               
               
                   
                 CACGCGATGGGTAACAGTCTTGGCGGCTTCGCTAAATACTGG 
               
               
                   
                   
               
               
                   
                 CAGGCGTTTCGTCAGTACCCCCGTTTACAGGGCGGCTTCGTC 
               
               
                   
                   
               
               
                   
                 TGGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAAC 
               
               
                   
                   
               
               
                   
                 GGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACG 
               
               
                   
                   
               
               
                   
                 CCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCC 
               
               
                   
                   
               
               
                   
                 GACCGCACGCCGCATCCGGCGCTGACGGAAGCAAAACACCAA 
               
               
                   
                   
               
               
                   
                 CAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACCATCGAA 
               
               
                   
                   
               
               
                   
                 GTGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGTTC 
               
               
                   
                   
               
               
                   
                 CTGCACTGGATGGTGGCACTGGATGGCAAGCCGCTGGCAAGC 
               
               
                   
                   
               
               
                   
                 GGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTG 
               
               
                   
                   
               
               
                   
                 ATTGAACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAA 
               
               
                   
                   
               
               
                   
                 CTCTGGCTAACGGTACGCGTAGTGCAACCAAACGCGACCGCA 
               
               
                   
                   
               
               
                   
                 TGGTCAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGT 
               
               
                   
                   
               
               
                   
                 CTGGCGGAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCAC 
               
               
                   
                   
               
               
                   
                 GCCATCCCTCAACTGACCACCAGCGGAACGGATTTTTGCATC 
               
               
                   
                   
               
               
                   
                 GAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGC 
               
               
                   
                   
               
               
                   
                 TTTCTTTCACAGATGTGGATTGGCGATGAAAAACAACTGCTG 
               
               
                   
                   
               
               
                   
                 ACCCCGCTGCGCGATCAGTTCACCCGTGCGCCGCTGGATAAC 
               
               
                   
                   
               
               
                   
                 GACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCC 
               
               
                   
                   
               
               
                   
                 TGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAA 
               
               
                   
                   
               
               
                   
                 GCGGCGTTGTTGCAGTGCACGGCAGATACACTTGCCGACGCG 
               
               
                   
                   
               
               
                   
                 GTGCTGATTACAACCGCCCACGCGTGGCAGCATCAGGGGAAA 
               
               
                   
                   
               
               
                   
                 ACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGGCAC 
               
               
                   
                   
               
               
                   
                 GGTGAGATGGTCATCAATGTGGATGTTGCGGTGGCAAGCGAT 
               
               
                   
                   
               
               
                   
                 ACACCGCATCCGGCGCGGATTGGCCTGACCTGCCAGCTGGCG 
               
               
                   
                   
               
               
                   
                 CAGGTCTCAGAGCGGGTAAACTGGCTCGGCCTGGGGCCGCAA 
               
               
                   
                   
               
               
                   
                 GAAAACTATCCCGACCGCCTTACTGCAGCCTGTTTTGACCGC 
               
               
                   
                   
               
               
                   
                 TGGGATCTGCCATTGTCAGACATGTATACCCCGTACGTCTTC 
               
               
                   
                   
               
               
                   
                 CCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAAT 
               
               
                   
                   
               
               
                   
                 TATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATC 
               
               
                   
                   
               
               
                   
                 AGCCGCTACAGCCAACAACAACTGATGGAAACCAGCCATCGC 
               
               
                   
                   
               
               
                   
                 CATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGAC 
               
               
                   
                   
               
               
                   
                 GGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCG 
               
               
                   
                   
               
               
                   
                 TCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCAT 
               
               
                   
                   
               
               
                   
                 TACCAGTTGGTCTGGTGTCAAAAATAA  
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 56 
               
               
                   
               
               
                 Pfnrs-lacZ construct Sequences 
               
               
                 Nucleotide sequences of Pfnrs-lacZ construct, 
               
               
                 low-copy (SEQ ID NO: 232) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 GGTACC agttgttcttattggtggtgttgctttatggttgcatc   
               
               
                   
                   
               
               
                   
                 
                   gtagtaaatggttgtaacaaaagcaatttttccggctgtctgta 
                 
               
               
                   
                   
               
               
                   
                 
                   tacaaaaacgccgtaaagtttgagcgaagtcaataaactctcta 
                 
               
               
                   
                   
               
               
                   
                   cccattcagggcaatatctctctt GGATCC ctctagaaataatt   
               
               
                   
                   
               
               
                   
                   ttgtttaactttaagaaggagatatacatATG CTATGATTACGG 
               
               
                   
                   
               
               
                   
                 ATTCTCTGGCCGTCGTATTACAACGTCGTGACTGGGAAAACCCT 
               
               
                   
                   
               
               
                   
                 GGCGTTACCCAACTTAATCGCCTTGCGGCACATCCCCCTTTCGC 
               
               
                   
                   
               
               
                   
                 CAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCC 
               
               
                   
                   
               
               
                   
                 AACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTT 
               
               
                   
                   
               
               
                   
                 CCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCT 
               
               
                   
                   
               
               
                   
                 TCCTGACGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGC 
               
               
                   
                   
               
               
                   
                 ACGGTTACGATGCGCCTATCTACACCAACGTGACCTATCCCATT 
               
               
                   
                   
               
               
                   
                 ACGGTCAATCCGCCGTTTGTTCCCGCGGAGAATCCGACAGGTTG 
               
               
                   
                   
               
               
                   
                 TTACTCGCTCACATTTAATATTGATGAAAGCTGGCTACAGGAAG 
               
               
                   
                   
               
               
                   
                 GCCAGACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCAT 
               
               
                   
                   
               
               
                   
                 CTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGCCG 
               
               
                   
                   
               
               
                   
                 TTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAG 
               
               
                   
                   
               
               
                   
                 AAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGT 
               
               
                   
                   
               
               
                   
                 TATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCG 
               
               
                   
                   
               
               
                   
                 TGACGTCTCGTTGCTGCATAAACCGACCACGCAAATCAGCGATT 
               
               
                   
                   
               
               
                   
                 TCCAAGTTACCACTCTCTTTAATGATGATTTCAGCCGCGCGGTA 
               
               
                   
                   
               
               
                   
                 CTGGAGGCAGAAGTTCAGATGTACGGCGAGCTGCGCGATGAACT 
               
               
                   
                   
               
               
                   
                 GCGGGTGACGGTTTCTTTGTGGCAGGGTGAAACGCAGGTCGCCA 
               
               
                   
                   
               
               
                   
                 GCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGC 
               
               
                   
                   
               
               
                   
                 GGTTATGCCGATCGCGTCACACTACGCCTGAACGTTGAAAATCC 
               
               
                   
                   
               
               
                   
                 GGAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCAGTGG 
               
               
                   
                   
               
               
                   
                 TTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCC 
               
               
                   
                   
               
               
                   
                 TGCGACGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCT 
               
               
                   
                   
               
               
                   
                 GCTGCTGAACGGCAAGCCGTTGCTGATTCGCGGCGTTAACCGTC 
               
               
                   
                   
               
               
                   
                 ACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACG 
               
               
                   
                   
               
               
                   
                 ATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGC 
               
               
                   
                   
               
               
                   
                 CGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGC 
               
               
                   
                   
               
               
                   
                 TGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCAATATT 
               
               
                   
                   
               
               
                   
                 GAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCC 
               
               
                   
                   
               
               
                   
                 GCGCTGGCTACCCGCGATGAGCGAACGCGTAACGCGGATGGTGC 
               
               
                   
                   
               
               
                   
                 AGCGCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGG 
               
               
                   
                   
               
               
                   
                 AATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTG 
               
               
                   
                   
               
               
                   
                 GATCAAATCTGTCGATCCTTCCCGCCCGGTACAGTATGAAGGCG 
               
               
                   
                   
               
               
                   
                 GCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTAC 
               
               
                   
                   
               
               
                   
                 GCGCGCGTGGATGAAGACCAGCCCTTCCCGGCGGTGCCGAAATG 
               
               
                   
                   
               
               
                   
                 GTCCATCAAAAAATGGCTTTCGCTGCCTGGAGAAATGCGCCCGC 
               
               
                   
                   
               
               
                   
                 TGATCCTTTGCGAATATGCCCACGCGATGGGTAACAGTCTTGGC 
               
               
                   
                   
               
               
                   
                 GGCTTCGCTAAATACTGGCAGGCGTTTCGTCAGTACCCCCGTTT 
               
               
                   
                   
               
               
                   
                 ACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTA 
               
               
                   
                   
               
               
                   
                 AATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGAT 
               
               
                   
                   
               
               
                   
                 TTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCT 
               
               
                   
                   
               
               
                   
                 GGTCTTTGCCGACCGCACGCCGCATCCGGCGCTGACGGAAGCAA 
               
               
                   
                   
               
               
                   
                 AACACCAACAGCAGTATTTCCAGTTCCGTTTATCCGGGCGAACC 
               
               
                   
                   
               
               
                   
                 ATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGATAACGA 
               
               
                   
                   
               
               
                   
                 GTTCCTGCACTGGATGGTGGCACTGGATGGCAAGCCGCTGGCAA 
               
               
                   
                   
               
               
                   
                 GCGGTGAAGTGCCTCTGGATGTTGGCCCGCAAGGTAAGCAGTTG 
               
               
                   
                   
               
               
                   
                 ATTGAACTGCCTGAACTGCCGCAGCCGGAGAGCGCCGGACAACT 
               
               
                   
                   
               
               
                   
                 CTGGCTAACGGTACGCGTAGTGCAACCAAACGCGACCGCATGGT 
               
               
                   
                   
               
               
                   
                 CAGAAGCCGGACACATCAGCGCCTGGCAGCAATGGCGTCTGGCG 
               
               
                   
                   
               
               
                   
                 GAAAACCTCAGCGTGACACTCCCCTCCGCGTCCCACGCCATCCC 
               
               
                   
                   
               
               
                   
                 TCAACTGACCACCAGCGGAACGGATTTTTGCATCGAGCTGGGTA 
               
               
                   
                   
               
               
                   
                 ATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACAG 
               
               
                   
                   
               
               
                   
                 ATGTGGATTGGCGATGAAAAACAACTGCTGACCCCGCTGCGCGA 
               
               
                   
                   
               
               
                   
                 TCAGTTCACCCGTGCGCCGCTGGATAACGACATTGGCGTAAGTG 
               
               
                   
                   
               
               
                   
                 AAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAG 
               
               
                   
                   
               
               
                   
                 GCGGCGGGCCATTACCAGGCCGAAGCGGCGTTGTTGCAGTGCAC 
               
               
                   
                   
               
               
                   
                 GGCAGATACACTTGCCGACGCGGTGCTGATTACAACCGCCCACG 
               
               
                   
                   
               
               
                   
                 CGTGGCAGCATCAGGGGAAAACCTTATTTATCAGCCGGAAAACC 
               
               
                   
                   
               
               
                   
                 TACCGGATTGATGGGCACGGTGAGATGGTCATCAATGTGGATGT 
               
               
                   
                   
               
               
                   
                 TGCGGTGGCAAGCGATACACCGCATCCGGCGCGGATTGGCCTGA 
               
               
                   
                   
               
               
                   
                 CCTGCCAGCTGGCGCAGGTCTCAGAGCGGGTAAACTGGCTCGGC 
               
               
                   
                   
               
               
                   
                 CTGGGGCCGCAAGAAAACTATCCCGACCGCCTTACTGCAGCCTG 
               
               
                   
                   
               
               
                   
                 TTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGT 
               
               
                   
                   
               
               
                   
                 ACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAA 
               
               
                   
                   
               
               
                   
                 TTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAA 
               
               
                   
                   
               
               
                   
                 CATCAGCCGCTACAGCCAACAACAACTGATGGAAACCAGCCATC 
               
               
                   
                   
               
               
                   
                 GCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGAC 
               
               
                   
                   
               
               
                   
                 GGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGTC 
               
               
                   
                   
               
               
                   
                 AGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATTACC 
               
               
                   
                   
               
               
                   
                 AGTTGGTCTGGTGTCAAAAATAA 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 57 
               
               
                   
               
               
                 Example 40. 
               
               
                 Other Sequences of interest 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Wild-type clbA 
                 caaatatcacataatcttaacatatcaataaacacagtaaagtttcatgtgaaaaacat 
               
               
                 (SEQ ID NO: 233) 
                 caaacataaaatacaagctcggaatacgaatcacgctatacacattgctaacagga 
               
               
                   
                 atgagattatctaaatgaggattgatatattaattggacatactagtttttttcatcaaac 
               
               
                   
                 cagtagagataacttccttcactatctcaatgaggaagaaataaaacgctatgatca 
               
               
                   
                 gtttcattttgtgagtgataaagaactctatattttaagccgtatcctgctcaaaacagc 
               
               
                   
                 actaaaaagatatcaacctgatgtctcattacaatcatggcaatttagtacgtgcaaat 
               
               
                   
                 atggcaaaccatttatagtttttcctcagttggcaaaaaagattttttttaacctttcccat 
               
               
                   
                 actatagatacagtagccgttgctattagttctcactgcgagcttggtgtcgatattga 
               
               
                   
                 acaaataagagatttagacaactcttatctgaatatcagtcagcatttttttactccaca 
               
               
                   
                 ggaagctactaacatagtttcacttcctcgttatgaaggtcaattacttttttggaaaat 
               
               
                   
                 gtggacgctcaaagaagcttacatcaaatatcgaggtaaaggcctatctttaggact 
               
               
                   
                 ggattgtattgaatttcatttaacaaataaaaaactaacttcaaaatatagaggttcacc 
               
               
                   
                 tgtttatttctctcaatggaaaatatgtaactcatttctcgcattagcctctccactcatca 
               
               
                   
                 cccctaaaataactattgagctatttcctatgcagtcccaactttatcaccacgactatc 
               
               
                   
                 agctaattcattcgtcaaatgggcagaattgaatcgccacggataatctagacacttc 
               
               
                   
                 tgagccgtcgataatattgattttcatattccgtcggtggtgtaagtatcccgcataatc 
               
               
                   
                 gtgccattcacatttag 
               
               
                   
               
               
                 clbA knock-out 
                 ggatggggggaaacatggataagttcaaagaaaaaaacccgttatctctgcgtgaaa 
               
               
                 (SEQ ID NO: 234) 
                 gacaagtattgcgcatgctggcacaaggtgatgagtactctcaaatatcacataatctt 
               
               
                   
                 aacatatcaataaacacagtaaagtttcatgtgaaaaacatcaaacataaaatacaagc 
               
               
                   
                 tcggaatacgaatcacgctatacacattgctaacaggaatgagattatctaaatgagga 
               
               
                   
                 ttgaTGTGTAGGCTGGAGCTGCTTCGAAGTTCCTATAC 
               
               
                   
                 TTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCG 
               
               
                   
                 GAATAGGAACTAAGGAGGATATTCATATGtcgtcaaatggg 
               
               
                   
                 cagaattgaatcgccacggataatctagacacttctgagccgtcgataatattgattttc 
               
               
                   
                 atattccgtcggtgg