Patent Publication Number: US-2023139445-A1

Title: Bacterial cells with improved tolerance to diacids

Description:
FIELD OF THE INVENTION 
     The present invention relates to bacterial cells genetically modified to improve their tolerance to certain commodity chemicals, such as dicarboxylic acids (herein referred to as “diacids”) and other polycarboxylic acids, and to methods of preparing and using such bacterial cells for production of diacids and other compounds. 
     BACKGROUND OF THE INVENTION 
     Aliphatic diacids are commonly used as precursors for nylon polymers (polyamides), typically prepared by condensing diamines with diacids. Diacids are also used as monomers for various other polymers and copolymers including polyurethanes. Different chain lengths and the presence of unsaturated bonds or branched chains within the constituent diacids imparts different physical properties to the polymer. 
     There has been significant recent interest in producing diacids biologically, i.e., in microbial cells. For example, as reported on their respective websites (accessed in October 2016), Myriant Corporation and BioAmber Inc. have both begun biological production of succinic acid, as a replacement molecule for petrochemical-derived adipic acid, Verdezyne Inc. is developing a process to produce adipic acid in yeast, and one of the major worldwide manufacturers of nylons, INVISTA™, is actively seeking the development of biologically produced precursors through collaborations with external parties. The production of diacids in metabolically engineered microbial cells have been reviewed and described in several publications such as, e.g., Polen et al., 2013; Adkins et al., 2013; Park et al., 2013; Yu et al., 2014; Cheong et al., 2016; Deng and Mao, 2015; WO 2011/003034 A2 (Verdezyne); Curran et al., 2013; Sengupta et al., 2015; and Zhang et al., 2015. 
     For production of bulk chemicals from renewable plant-based carbon feedstocks, high product titers are essential in order to minimize capital equipment and downstream separations costs for product purification. At the high titers required for economical fermentation processes, however, most chemicals exhibit significant toxicity that reduces yields and productivities by negatively affecting microbial growth (Van Dien, 2013; Zingaro et al., 2013).  Escherichia coli  being a suitable host for industrial applications, there has been some interest in developing  E. coli  strains with improved tolerance to chemicals of interest for production, such as, e.g., n-butanol, ethanol and isobutanol, or to stress conditions present during fermentation (see, e.g., Haft et al, 2014; Sandberg et al., 2014; Lennen and Herrgard, 2014; Tenaillon et al., 2012; Minty et al., 2011; Dragosits et al., 2013a,b; Winkler et al., 2014; Wu et al., 2014; LaCroix et al., 2015; Jensen et al., 2015 and 2016; Doukyu et al., 2012; Shenhar et al., 2012; and Rath and Jawali, 2006). 
     In addition, Byrne et al., 2012, describes computational modelling of microorganisms such as  E. coli , proposing combinations of medium compositions and gene-deletion strains for six industrially important byproducts, e.g., succinate. WO 01/05959 (Ajinomoto K K) relates to production of a target substance such as glutamic acid in, e.g.,  E. coli  strains. Finally, WO 2016/162442 (Metabolic Explorer) relates to a recombinant microorganism capable of producing 2,4-dihydroxybutyrate, which is characterized by an increased cellular export, and preferably by a decreased cellular import, of 2,4-dihydroxybutyrate. 
     Despite these and other advances in the art, there is still a need for bacterial cells with improved tolerance to chemicals of interest for bio-based production, such as aliphatic diacids and other compounds. It is an object of the invention to provide such bacterial cells. 
     SUMMARY OF THE INVENTION 
     It has been found by the present inventors that certain genetic modifications unexpectedly improve the tolerance of bacterial cells, such as those of, e.g., the  Escherichia  genera, to certain chemical compounds, particularly aliphatic diacids (herein also referred to as “aliphatic dicarboxylic acids”). 
     Accordingly, the invention provides bacterial cells with improved tolerance to at least one aliphatic diacid, as well as bacterial cells which are capable of producing an aliphatic diacid and have improved tolerance to the aliphatic diacid. Particularly contemplated are glutaric acid, adipic acid, succinic acid, muconic acid, fumaric acid, itaconic acid, malic acid, malonic acid, maleic acid, glucaric acid, pimelic acid, suberic acid, sebacic acid, 2,5-furandicarboxylic acid, terephthalic acid, azelaic acid, mesaconic acid, citraconic acid, tartaric acid, tartronic acid, diaminopimelic acid, and glutaconic acid. 
     The invention also relates to compositions comprising such bacterial cells and one or more aliphatic diacids, methods of preparing or screening for such bacterial cells, and methods of producing aliphatic diacids using such bacterial cells. 
     These and other aspects and embodiments are described in more detail below. 
    
    
     DETAILED DISCLOSURE OF THE INVENTION 
     In the present work, glutaric acid and adipic acid were selected for performing adaptive laboratory evolutions. Based on the findings reported herein, various aspects of the invention provide for genetically modified bacterial host cells with a higher tolerance to one or more diacids or other compounds. When transformed with a recombinant biosynthetic pathway for producing the diacid from a carbon source, the genetically modified bacterial host cells of the invention result in improved production of the diacid from carbon feedstock, since they maintain robust metabolic activity in the presence of higher concentrations of the diacid than the parent cells. For example, it was found that a reduced expression of kgtP improved tolerance to glutaric acid, implicating KgtP, an α-ketoglutarate importer, as being a direct importer for glutarate. 
     So, in a first aspect, a bacterial cell is provided, comprising a biosynthetic pathway for producing an aliphatic dicarboxylic acid and at least one genetic modification which reduces expression of an endogenous gene selected from the group consisting of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR, or a combination of any thereof. In one preferred embodiment, the genetic modification reduces the expression of kgtP. In another preferred embodiment, the at least one genetic modification reduces the expression of ybjL, proV, proW, proX, sspA or a combination of any thereof. For example, the bacterial cell may comprise genetic modifications which reduce the expression of kgtP, proV and ybjL; kgtP and proV; kgtP and ybjL; or kgtP and sspA. Non-limiting examples of genetic modifications include a knock-down or knock-out of the endogenous gene. In a particular embodiment, the genetic modification is a knock-out. The genetic modification may, for example, provide for an increased growth rate, a reduced lag time, or both, of the cell in the presence of at least one of glutaric acid and adipic acid as compared to a control, e.g., the bacterial cell without the genetic modification. 
     In a second aspect, a bacterial cell is provided, genetically modified from a parent bacterial cell so as to comprise one or more of
     (a) a mutant SpoT, comprising at least one mutation in the threonyl-tRNA synthetase GTPase and SpoT (TGS) domain corresponding to amino acid residues 1388 to T447 and/or the linker segment between the TGS and the aspartokinase, chorismate mutase and TyrA (ACT) domain corresponding to amino acid residues A448 to T621, optionally in one or more amino acid residues selected from A451, R236, V422, W457, N454, D580, M247, T442, S434, N601, 1602 and R603;   (b) a mutant PolB, comprising a mutation in amino acid residue R477;   (c) a mutant RpoC, comprising a mutation in at least one of the amino acid residues corresponding to H419 and P64;   (d) a mutant RpoB, comprising a mutation in an amino acid residue corresponding to K203;   (e) a mutant Rnt, comprising a mutation in at least one of the amino acid residues corresponding to Q179, A27, F194 and A180;   (f) a mutant SapC, comprising a mutation in the amino acid residue corresponding to G79; and   (g) increased expression of PyrE as compared to the parent bacterial cell;
 
optionally in combination with a knock-down or knock-out of (i) at least one endogenous gene selected from the group consisting of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR; or (ii) a combination of two or more endogenous genes selected from kgtP, proV, ybjL and sspA, such as kgtP, proV and ybjL; kgtP and proV; kgtP and ybjL; and kgtP and sspA,
 
wherein the genetic modification provides for an increased growth rate, a reduced lag time, or both, in the presence of at least one of glutaric acid and adipic acid as compared to the parent bacterial cell.
   

     The bacterial cell, may, for example, comprise (a) at least one mutant protein selected from the group consisting of SpoT-V422A, SpoT-A451D, SpoT-A451V, SpoT-W457C, SpoT-N454H, SpoT-D580Y, SpoT-R236L, SpoT-R236S, SpoT-M247K, SpoT-NIR(601-603)S, SpoT-T442I, SpoT-S434L, PolB-R477G, RpoC-H419P, RpoC-P64L, RpoB-K203T, Rnt-Q179P, Rnt-A27T, Rnt-F194L, Rnt-A180T, SapC-G79W; and/or (b) a mutation in rph or the pyrE/rph intergenic region which increases the expression of PyrE. 
     The bacterial cell of any aspect or embodiment may further comprise a recombinant biosynthetic pathway for producing at least one of glutaric acid, adipic acid, succinic acid, muconic acid, fumaric acid, itaconic acid, malic acid, malonic acid, maleic acid, glucaric acid, pimelic acid, suberic acid, sebacic acid, 2,5-furandicarboxylic acid, terephthalic acid, or azelaic acid, mesaconic acid, citraconic acid, tartaric acid, tartronic acid, diaminopimelic acid and glutaconic acid. 
     Also provided is a process for preparing a recombinant bacterial cell for producing an aliphatic dicarboxylic acid, the process comprising genetically modifying an  E. coli  cell to (a) introduce a recombinant biosynthetic pathway for producing the aliphatic dicarboxylic acid, and (b) knock-down or knock-out at least one endogenous gene selected from the group consisting of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR; such as a combination of two or more endogenous genes selected from kgtP, proV, ybjL and sspA; such as a combination selected from kgtP, proV and ybjL; kgtP and proV; kgtP and ybjL and kgtP and sspA, and/or (c) express a mutant of at least one of SpoT, PolB, RpoC, RpoB, Rnt and SapC and/or increase the expression of PyrE; wherein steps (a), (b) and (c) can be performed in any order. 
     Also provided is a process for improving the tolerance of a bacterial cell to an aliphatic dicarboxylic acid comprising genetically modifying the bacterial cell to (a) knock-down or knock-out at least one endogenous gene selected from the group consisting of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR; such as a combination of two or more endogenous genes selected from kgtP, proV, ybjL and sspA; such as a combination selected from kgtP, proV and ybjL; kgtP and proV; kgtP and ybjL and kgtP and sspA; and/or (b) express a mutant of at least one of SpoT, PolB, RpoC, RpoB, Rnt and SapC and/or increase the expression of PyrE, wherein steps (a) and (b) are performed in any order. 
     The bacterial cell may, for example, be derived from the  Escherichia, Lactobacillus, Lactococcus, Bacillus, Pseudomonas, Corynebacterium, Deinococcus  or  Ralstonia  species, such as the  Escherichia coli  species. 
     Also provided is a method for producing an aliphatic dicarboxylic acid, comprising culturing such genetically modified bacterial cells in the presence of a carbon source, and, optionally, isolating the aliphatic dicarboxylic acid. 
     Also provided is a composition comprising glutaric acid or adipic acid at a concentration of at least 5 g/L and a plurality of bacterial cells of the  Escherichia  genus genetically modified to (a) knock-down or knock-out at least one endogenous gene selected from the group consisting of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR; such as a combination of two or more endogenous genes selected from kgtP, proV, ybjL and sspA; such as a combination selected from kgtP, proV and ybjL; kgtP and proV; kgtP and ybjL and kgtP and sspA; and/or (b) express a mutant of at least one of SpoT, PolB, RpoC, RpoB, Rnt and SapC and/or increase expression of PyrE. 
     In embodiments where the bacterial cell comprises a biosynthetic pathway for producing an aliphatic dicarboxylic acid, the pathway may, for example, comprise
     (a) a lysine monooxygenase, a 5-aminovaleramidase, a 5-aminovalerate transaminase, and a glutaraldehyde semialdehyde dehydrogenase;   (b) a reversible 3-oxoadipyl-CoA thiolase, a 3-hydroxyacyl-CoA dehydrogenase, an enoyl-CoA hydratase, an enoyl-CoA reductase, and either a terminal carboxyacyl-CoA thioesterase, or a terminal carboxyacyl-CoA phosphotransferase and a reversible alkyl-1,n-dicarboxylate kinase, where n is the carbon chain length of the product; and, optionally, a malonyl-CoA or glutaryl-CoA transferase; or   (c) a 2-dehydro-3-deoxy-D-arabinoheptonate-7-phosphate synthase, a 3-dehydroquinate synthase, a 3-dehydroxyquinate dehydratase, a dehydroshikimic acid dehydratase, a protocatechuate decarboxylase, and a catechol 1,2-dioxygenase.   

     Definitions 
     Unless otherwise indicated or contradicted by context, a “diacid” as used herein is an aliphatic dicarboxylic acid of the general formula COOH—R—COOH (I), where R is an alkyl chain. An “aliphatic diacid” or “aliphatic dicarboxylic acid” herein refers to an organic compound comprising an aliphatic carbon chain to which two or more carboxyl (—COOH) groups are attached, and includes linear aliphatic diacids, as well as derivatives thereof. Aliphatic diacids suitable for production in bacteria typically comprise from 3 to 12 carbon atoms, preferably 3 to 10 carbon atoms, more preferably 3 to 8 carbon atoms, even more preferably, 4 to 7 or 5 to 8 carbon atoms, and, most preferably, 5 to 7 carbon atoms, and optionally comprises one or more heteroatoms or other substituents. Examples of heteroatoms include oxygen (e.g., in the form of an oxo group, a.k.a. keto group), nitrogen, sulphur and halogens. Examples of other substituents include hydroxyl groups, amino groups, carboxyl groups, and alkyl groups. Preferred aliphatic diacids include, but are not limited to, glutaric acid, adipic acid, succinic acid, muconic acid, fumaric acid, itaconic acid, malic acid, malonic acid, maleic acid, glucaric acid, pimelic acid, suberic acid, sebacic acid, 2,5-furandicarboxylic acid, terephthalic acid, or azelaic acid, mesaconic acid, citraconic acid, tartaric acid, tartronic acid, diaminopimelic acid and glutaconic acid. In some embodiments, the aliphatic diacid does not comprise any heteroatom substituents. In some embodiments, the aliphatic diacid does not comprise any substituents. Glutaric, adipic, pimelic and sebacic acid are particularly preferred. 
     As used herein, a “recombinant biosynthetic pathway” for a compound of interest refers to an enzymatic pathway resulting in the production of a compound of interest in a host cell, wherein at least one of the enzymes is expressed from a transgene, i.e., a gene added to the host cell genome by transformation. In some cases, the recombinant biosynthetic pathway also comprises a deletion of one or more native genes in the host cell. The compound of interest is typically a diacid, and may be the actual end product or a precursor or intermediate in the production of another end product. 
     The terms “tolerant” or “improved tolerance”, when used to describe a genetically modified bacterial cell of the invention or a strain derived therefrom, refers to a genetically modified bacterial cell or strain that shows a reduced lag time, an improved growth rate, or both, in the presence of a diacid than the parent bacterial cell or strain from which it is derived, typically at concentrations of 1 g/L, such as at least 2 g/L or higher, such as at least 5 g/L or higher, such as at least 10 g/L or higher, such as at least 20 g/L or higher, such as at least 40 g/L or higher, such as at least 75 g/L or higher, such as at least 100 g/L or higher. An improved growth rate is at least 5%, such as at least 10%, such as at least 20%, such as at least 50%, such as at least 75% higher than that of a control, typically the parent cell or strain. A reduced lag time is at least 10%, such as at least 20%, such as at least 50%, such as at least 75%, such as at least 90% shorter than that of a control, typically the parent cell or strain. 
     The term “gene” refers to a nucleic acid sequence that encodes a cellular function, such as a protein, optionally including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. An “endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “transgene” is a gene, native or heterologous, that has been introduced into the genome by a transformation procedure. Genes names are herein set forth in italicised text with a lower-case first letter (e.g., metJ) whereas protein names are set forth in normal text with a capital first letter (e.g., MetJ). 
     As used herein the term “coding sequence” refers to a DNA sequence that encodes a specific amino acid sequence. 
     The term “native”, when used to characterize a gene or a protein herein with respect to a host cell, refers to a gene or protein having the nucleic acid or amino acid sequence as found in the host cell. 
     The term “heterologous”, when used to characterize a gene or protein with respect to a host cell, refers to a gene or protein which has a nucleic acid or amino acid sequence not normally found in the host cell. 
     As used herein the term “transformation” refers to the transfer of a nucleic acid fragment, such as a gene, into a host cell. Host cells containing a gene introduced by transformation or a “transgene” are referred to as “transgenic” or “recombinant” or “transformed” cells. 
     As used herein, a “genetic modification” or “genetically modified” refers to the introduction a genetically inherited change in the host cell genome. Examples of changes include mutations in genes and regulatory sequences, coding and non-coding DNA sequences. “Mutations” include deletions, substitutions and insertions of one or more nucleotides or nucleic acid sequences in the genome. Other genetic modifications include the introduction of heterologous genes or coding DNA sequences on a plasmid and/or into a chromosome by recombinant techniques. In one embodiment, the genetic modification is in a chromosome. 
     The term “expression”, as used herein, refers to the process in which a gene is transcribed into mRNA, and may optionally include the subsequent translation of the mRNA into an amino acid sequence, i.e., a protein or polypeptide. 
     As used herein, “reduced expression” or “downregulation” of an endogenous gene in a host cell means that the levels of the mRNA, protein and/or protein activity encoded by the gene are significantly reduced in the host cell, typically by at least 25%, such as at least 50%, such as at least 75%, such as at least 90%, such as at least 95%, as compared to a control. Typically, when the reduced expression is obtained by a genetic modification in the host cell, the control is the unmodified host cell. Sometimes, e.g., in the case of gene knock-out, the reduction of native rnRNA and functional protein encoded by the gene is higher, such as 99% or greater. 
     “Increased expression”, “upregulation”, “overexpressing” or the like, when used in the context of a protein or activity described herein, means increasing the protein level or activity within a bacterial cell. An up-regulation of an activity can occur through, e.g., increased activity of a protein, increased potency of a protein or increased expression of a protein. The protein with increased activity, potency or expression can be encoded by genes disclosed herein. 
     Genetic modifications resulting in a reduced expression of a target gene/protein can include, e.g., knock-down of the gene (e.g., a mutation in a promoter or other expression control sequence that results in decreased gene expression), a knock-out or disruption of the gene (e.g., a mutation or deletion of the gene that results in 99 percent or greater decrease in gene expression), a mutation or deletion in the coding sequence which results in the expression of non-functional protein, and/or the introduction of a nucleic acid sequence that reduces the expression of the target gene, e.g. a repressor that inhibits expression of the target or inhibitory nucleic acids (e.g. CRISPR/dCas9, antisense RNA, etc.) that reduces the expression of the target gene. 
     Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 4 th  ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 2012; and by Silhavy, T. J., Berman, M. L. and Enquist, L. W. Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1984; and by Ausubel, F. M. et al., In Current Protocols in Molecular Biology, published by John Wiley &amp; Sons (1995); and by Datsenko and Wanner, 2000; and by Baba et al., 2006; and by Thomason et al., 2007. 
     A “conservative” amino acid substitution in a protein is one that does not negatively influence protein activity. Typically, a conservative substitution can be made within groups of amino acids sharing physicochemical properties, such as, e.g., basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagines), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, and threonine). Most commonly, substitutions can be made between Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly. Other preferred substitutions are set out in Table 1 below. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Examples of amino acid substitutions. 
               
            
           
           
               
               
               
            
               
                 Original 
                   
                 Preferred 
               
               
                 amino acid 
                 Examples of substitutions 
                 substitution 
               
               
                   
               
               
                 Ala (A) 
                 val; leu; ile 
                 Val 
               
               
                 Arg (R) 
                 lys; gln; asn 
                 Lys 
               
               
                 Asn (N) 
                 gln; his; asp, lys; arg 
                 Gln 
               
               
                 Asp (D) 
                 glu; asn 
                 Glu 
               
               
                 Cys (C) 
                 ser; ala 
                 Ser 
               
               
                 Gln (Q) 
                 asn; glu 
                 Asn 
               
               
                 Glu (E) 
                 asp; gln 
                 Asp 
               
               
                 Gly (G) 
                 Ala 
                 Ala 
               
               
                 His (H) 
                 asn; gln; lys; arg 
                 Arg 
               
               
                 Ile (I) 
                 leu; val; met; ala; phe; norleucine 
                 Leu 
               
               
                 Leu (L) 
                 norleucine; ile; val; met; ala; phe 
                 Ile 
               
               
                 Lys (K) 
                 arg; gln; asn 
                 Arg 
               
               
                 Met (M) 
                 leu; phe; ile 
                 Leu 
               
               
                 Phe (F) 
                 leu; val; ile; ala; tyr 
                 Tyr 
               
               
                 Pro (P) 
                 Ala 
                 Ala 
               
               
                 Ser (S) 
                 thr 
                 Thr 
               
               
                 Thr (T) 
                 Ser 
                 Ser 
               
               
                 Trp (W) 
                 tyr; phe 
                 Tyr 
               
               
                 Tyr (Y) 
                 trp; phe; thr; ser 
                 Phe 
               
               
                 Val (V) 
                 ile; leu; met; phe; ala; norleucine 
                 Leu 
               
               
                   
               
            
           
         
       
     
     Specific Embodiments of the Invention 
     As described herein, the invention provides bacterial cells with improved tolerance to one or more diacids, as well as related processes and materials for producing and using such bacterial cells. 
     1) Genetic Modifications 
     The genetic modifications according to the invention include those resulting in reduced expression of genes, e.g., by gene knock-down or knock-out, herein referred to as “Group 1 modifications”; as well as silent mutations in coding or non-coding regions and non-silent (i.e., coding) mutations in coding regions, herein referred to as “Group 2 modifications”; and combinations thereof. 
     In a preferred embodiment, the one or more genetic modifications provide for an increased growth rate, a reduced lag time, or both, of the bacterial cell in the presence of at least one of glutaric and adipic acid as compared to the bacterial cell without the genetic modification, e.g., the parent or wild-type bacterial cell. The glutaric and/or adipic acid may be present in the growth medium at, e.g., a concentration of at least about 1 g/L, such as at least about 2 g/L, such as at least about 5 g/L, such as at least about 10 g/L, such as at least about 20 g/L. 
     a) Group 1 Modifications 
     In one aspect, the bacterial cell has at least one genetic modification which reduces expression an endogenous gene selected from the group consisting of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR. 
     For example, in one embodiment, the expression of one or more of kgtP, ybjL, proV, proW, proX, proQ and sspA, such as kgtP, ybjL, proV or sspA, is reduced. In one specific embodiment, the expression of kgtP is reduced, optionally wherein the expression of lysP is not reduced. In one specific embodiment, the expression of ybjL is reduced. In one specific embodiment, the expression of sspA is reduced. In one specific embodiment, the expression of proV, proW, proX or proQ, such as e.g. proV, is reduced, optionally wherein the expression of marR is not reduced. In another specific embodiment, the expression of cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, or yeaR is reduced. 
     In another aspect, there is provided a bacterial cell which comprises genetic modifications reducing the expression of two or more endogenous genes, wherein at least one gene is selected from the group consisting of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR. In one embodiment, the bacterial cell comprises a genetic modification reducing the expression of kgtP but no genetic modification which reduces the expression of lysP. In one embodiment, the bacterial cell comprises a genetic modification reducing the expression of proV but no genetic modification which reduces the expression of marR. In one embodiment, the bacterial cell comprises genetic modifications which reduce the expression of at least two genes selected from the group consisting of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR. 
     In one embodiment, the genetic modifications reduce the expression of kgtP and one or more other endogenous genes, optionally selected from ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG and yeaR, such as from ybjL, proV, proW, proX, and sspA, optionally wherein the other endogenous genes do not comprise lysP. In separate and specific embodiments, the bacterial cell comprises genetic modifications which reduce the expression of kgtP and ybjL, kgtP and proV, proV and ybjL, or kgtP and sspA. 
     In one embodiment, the genetic modifications reduce the expression of kgtP and two or more endogenous genes selected from ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG and yeaR, wherein at least one endogenous gene is selected from ybjL, proV, proW, proX, and sspA. In one specific embodiment, the bacterial cell comprises genetic modifications which reduce the expression of kgtP, proV and ybjL. 
     In other separate and specific embodiments, the bacterial cell comprises:
         a first genetic modification which reduces the expression of ybjL, and a second genetic modification which reduces the expression of a gene selected from kgtP, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR;   a first genetic modification which reduces the expression of proV and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR;   a first genetic modification which reduces the expression of prow and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR;   a first genetic modification which reduces the expression of proX and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proW, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR;   a first genetic modification which reduces the expression of proQ and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proW, proX, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR;   a first genetic modification which reduces the expression of cspE and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proW, proX, proQ, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR;   a first genetic modification which reduces the expression of rfaE and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proW, proX, proQ, cspE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR;   a first genetic modification which reduces the expression of yfbP and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR;   a first genetic modification which reduces the expression of yfjM and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR;   a first genetic modification which reduces the expression of pstS and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proW, proX, proQ cspE, rfaE, yfbP, yfjM, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR;   a first genetic modification which reduces the expression of pstA and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR;   a first genetic modification which reduces the expression of pstB and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR;   a first genetic modification which reduces the expression of pstC and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR;   a first genetic modification which reduces the expression of rph and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proW, proX, proQ cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rpoS, sspA, tdk, uvrB, ycjG, and yeaR;   a first genetic modification which reduces the expression of rpoS and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proW, proX, proQ cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, sspA, tdk, uvrB, ycjG, and yeaR;   a first genetic modification which reduces the expression of sspA and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, tdk, uvrB, ycjG, and yeaR;   a first genetic modification which reduces the expression of tdk and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, uvrB, ycjG, and yeaR;   a first genetic modification which reduces the expression of uvrB and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, ycjG, and yeaR;   a first genetic modification which reduces the expression of ycjG and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, and yeaR; or   a first genetic modification which reduces the expression of yeaR and a second genetic modification which reduces the expression of a gene selected from of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC rph, rpoS, sspA, tdk, uvrB, and ycjG.       

     In one specific embodiment, either one or both of the first and second genetic modifications is a knock-out of the gene, optionally a deletion. In an alternative embodiment at least one of the first and second genetic modifications is a knock-down of the gene. 
     In one aspect, there is provided a bacterial cell according to any one of the preceding aspects and embodiments, wherein the genetic modification is a knock-down of the one or more endogenous genes, resulting in at least 25%, such as at least 50%, such as at least 75%, such as at least 90%, such as at least 95%, reduction in the level of mRNA encoded by the gene. 
     In one aspect, there is provided a bacterial cell according to any one of the preceding aspects and embodiments, wherein the genetic modification is a knock-down of the one or more endogenous genes, resulting in at least 25%, such as at least 50%, such as at least 75%, such as at least 90%, such as at least 95%, reduction in the level of protein encoded by the gene. 
     In one aspect, there is provided a bacterial cell according to any one of the preceding aspects and embodiments, wherein the genetic modification is a knock-out of the one or more endogenous genes. 
     Knock-down or knock-out of a gene can be accomplished by any method known in the art for bacterial cells, and include, e.g., lambda Red mediated recombination, P1 phage transduction, and single-stranded oligonucleotide recombineering/MAGE technologies (see, e.g., Datsenko and Wanner, 2000; Thomason et al., 2007a,b; Wang et al., 2009). Typically, a knock-down of a gene can be accomplished by, for example, a mutation in the promoter region resulting in decreased transcription, a deletion or mutation in the coding region of the gene resulting in a reduced or fully or substantially eliminated activity of the protein, or by the presence of antisense sequences that interfere with transcription or translation of the gene, resulting in reduced expression of the protein. Preferably, the knocking-down of a gene results in at least 20% reduction in the expression level of the gene product in the bacterial cell, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95% or higher. 
     A knock-out of a gene includes elimination of a gene&#39;s expression, such as by introducing a mutation in the coding sequence and/or promoter so that at least a portion (up to and including all) of the coding sequence and/or promoter is disrupted, shifted or deleted, resulting in loss of expression of the protein, or expression only of a non-functional mutant or non-functional fragment of the endogenous protein. As used herein, the symbol “A”, i.e., the greek uppercase letter for “delta”, denotes a deletion of an endogenous gene. Preferably, a knock-out of a gene results in 1% or less of the native gene product being detectable, such as no detectable gene product. 
     b) Group 2 Modifications 
     In certain embodiments, a mutant protein is expressed in the bacterial cell, e.g., from a mutated version of an endogenous gene, or from a transgene encoding the mutant protein. 
     In one aspect, the bacterial cell comprises a Group 2 modification, e.g., a mutation in one or more of SpoT, PolB, RpoC, RpoB, Rnt and SapC; an increased expression of one or more of SpoT, PolB, RpoC, RpoB, Rnt and SapC; and/or a mutation in rph or the pyrE/rph intergenic region which increases the expression of pyrE, wherein the one or more mutations improve tolerance to at least one aliphatic diacid such as, e.g. glutaric or adipic acid. Preferably, the bacterial cell further comprises a Group 1 modification according to any aspect or embodiment herein. 
     In one embodiment, the Group 2 modification comprises a mutant SpoT, comprising one or more mutations. The mutations may be located, for example, in the threonyl-tRNA synthetase GTPase and SpoT (TGS) domain corresponding to amino acid residues 1388 to T447, or the linker region between the TGS domain and the aspartokinase, chorismate mutase and TyrA (ACT) domain corresponding to A448 to T621 in  E. coli  SpoT. Without being limited to theory, since the mutations identified are near the TGS domain which is involved in nucleotide binding, at least some of the mutations may decrease the ppGpp synthetase activity of SpoT, e.g., by decreasing its binding affinity to substrates such as ATP or (p)ppGpp, or increasing its binding affinity to products such as GTP, AMP, or GDP. This may, in turn, reduce sensitivity of the cells to accumulating ppGpp and delay the onset of the stringent response. The stringent response may be activated under general stress conditions such as in high concentrations of diacids and might prevent growth under such conditions. In one embodiment, the SpoT mutant comprises a mutation in one or more amino acid residues selected from those corresponding to R236, M247, V422, S434, T442, A451, N454, W457, D580, N601, 1602 and R603 in  E. coli  SpoT. In one embodiment, the mutant SpoT comprises at least one amino acid substitution selected from V422A, A451D, A451V, W457C, N454H, D580Y, R236L, R236S, M247K, NIR(601-603)S, T442I, and S434L or a conservative substitution of any thereof. In a specific embodiment, the mutant SpoT comprises a mutation in A451 or R236, e.g., an amino acid substitution selected from A451D, A451V, R236L and R236S, or a conservative substitution thereof, e.g., selected from A451E, A451N, A451G, A451A, A451L, A451I, R236I, R236V, R236T, R236A, R236N and R236G. 
     In one embodiment, the Group 2 modification comprises a mutant PolB comprising one or more mutations. The mutation may be located, e.g., in the residue corresponding to R477 in  E. coli  PolB, and may be an amino acid substitution such as R477G or a conservative substitution thereof, e.g., R477A, R477D or R477S. 
     In one embodiment, the Group 2 modification comprises a mutant RpoC comprising one or more mutations. The mutation may be located in, e.g., the residue corresponding to H419 and/or P64 in  E. coli  RpoC, and may be an amino acid substitution such as H419P, P64L, or a conservative substitution thereof, e.g., H419A, P64I, P64V, P64M, P64A or P64F. Without being limited to theory, since some of these residues (e.g., H419) are close to residues involved in ppGpp-binding, at least some of them may decrease ppGpp binding to RpoC which in turn may reduce sensitivity of the cells to accumulating ppGpp and delay the onset of the stringent response. 
     In one embodiment, the Group 2 modification comprises a mutant RpoB comprising one or more mutations. The mutation may be located in, e.g., the residue corresponding to K203 in  E. coli  RpoB, and may be an amino acid substitution such as K203T or a conservative substitution thereof, e.g., K203S or K203A. Without being limited to theory, since residue K203 is near the entrance for dsDNA, it may interact with phosphate on dsDNA, possibly reducing premature transcription termination. 
     In one embodiment, the Group 2 modification comprises a mutant Rnt comprising one or more mutations. The mutations may be located, for example, in or close to catalytic residues of conserved exonuclease motifs corresponding to positions 23, 25, 181, and 186 in  E. coli  Rnt. In one embodiment, the Rnt mutant comprises a mutation in one or more amino acid residues selected from those corresponding to Q179, A27, F194 and A180. In one embodiment, the mutant Rnt comprises at least one amino acid substitution selected from Q179P, A27T, F194L and A180T or a conservative substitution of any thereof, e.g., Q179A, A27S, A27G, F1951, F195V, F195T, F195A, A180S and A180G. 
     In one embodiment, the Group 2 modification comprises a mutant SapC comprising one or more mutations. The mutation may be located in, e.g., the residue corresponding to G79 in  E. coli  SapC, and may be an amino acid substitution such as G79W or a conservative substitution thereof, e.g., G79Y or G79F. 
     In one embodiment, the bacterial cell comprises one or more mutations which increase(s) the expression level or activity of PyrE, optionally in combination with a Group 1 modification.  E. coli  K-12 MG1655 and W3110, plus their common ancestor strain W1485, are known to exhibit pyrimidine starvation in minimal media due to the presence a frameshift mutation occurring in rph relative to other  E. coli  strains (Jensen et al., 1993). This mutation disrupts the transcriptional/translational coupling required for efficient translation of pyrE, encoding orotate phosphoribosyltransferase in the pyrimidine biosynthesis pathway. Compensatory mutations that correct this deficiency are well-known in the art. One of these mutations is an 82 bp deletion near the 3′ terminus of rph, due to presence of two homologous GCAGAAGGC sequences flanking this 82 bp region (Conrad et al., 2009). In addition to the 82 bp deletion, a 1 bp deletion at coordinate 3815809 in the pyrE/rph intergenic region has previously been encountered in strains evolved for growth on a minimal glucose medium (LaCroix et al., 2015), and a wide array of other frameshift mutations, substitutions, and coding mutations near the 3′ terminus of rph were encountered in a short-term selection/evolution of combinatorial mutant libraries in minimal medium at an elevated temperature of 42° C. (Sandberg et al., 2014). Without being limited to theory, all of these mutations can serve the same function of increasing expression of PyrE, with the selective pressure for these mutations being even stronger in minimal media with particular imposed stresses (certain chemicals or heat) than in minimal media alone. In one embodiment, the bacterial cell comprises mutations in rph or the pyrE/rph intergenic region, such as, e.g., the 82 bp deletion near the 3′ terminus of rph, the 1 bp deletion in the intergenic region between pyrE and rph, or both. In one embodiment, increased expression of PyrE is achieved by transforming the bacterial cell with a transgene expressing the endogenous protein. Increased expression may be obtained by causing an up-regulation through increased expression of a protein, the copy number of a gene or genes encoding the protein may be increased. Alternatively, a strong and/or inducible promoter can be used to direct the expression of the gene, the gene being expressed either as a transient expression vehicle or homologously or heterologously incorporated into the bacterial genome. In another embodiment, the promoter, regulatory region and/or the ribosome binding site upstream of the gene can be altered to achieve the over-expression. The expression can also be enhanced by increasing the relative half-life of the messenger or other forms of RNA. Any one or a combination of these approaches can be used to effect upregulation of a desired target protein as needed. 
     In a specific embodiment, the bacterial cell comprises at least one Group 1 modification and at least one Group 2 modification. Non-limiting examples of Group 1 modifications for combination with any one or more of the overexpressed or mutant SpoT, PolB, RpoC, RpoB, Rnt, SapC and pyrE/rph include knock-down or knock-out of
         i) at least one endogenous gene selected from the group consisting of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR; and   ii) a combination of two or more endogenous genes selected from kgtP, proV, ybjL and sspA, such as kgtP, proV and ybjL; kgtP and proV; kgtP and ybjL; and kgtP and sspA.       

     In separate and specific embodiments, the bacterial cell comprises:
         a mutation selected from SpoT-V422A, SpoT-A451D, SpoT-A451V, SpoT-W457C, SpoT-N454H, SpoT-D580Y, SpoT-R236L, SpoT-R236S, SpoT-M247K, SpoT-NIR(601-603)S, SpoT-T442I, SpoT-S434L, PolB-R477G, RpoC-H419P, RpoC-P64L, RpoB-K203T, Rnt-Q179P, Rnt-A27T, Rnt-F194L, Rnt-A180T, and SapC-G79W   a mutation selected from SpoT-V422A, SpoT-A451D, SpoT-A451V, SpoT-W457C, SpoT-N454H, SpoT-D580Y, SpoT-R236L, SpoT-R236S, SpoT-M247K, SpoT-NIR(601-603)S, SpoT-T442I, SpoT-S434L, PolB-R477G, RpoC-H419P, RpoC-P64L, RpoB-K203T, Rnt-Q179P, Rnt-A27T, Rnt-F194L, Rnt-A180T, and SapC-G79W, and a knock-out or knockdown of at least one of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, or yeaR, such as kgtP.   a mutation selected from SpoT-V422A, SpoT-A451D, SpoT-A451V, SpoT-W457C, SpoT-N454H, SpoT-D580Y, SpoT-R236L, SpoT-R236S, SpoT-M247K, SpoT-NIR(601-603)S, SpoT-T442I, SpoT-S434L, PolB-R477G, RpoC-H419P, RpoC-P64L, RpoB-K203T, Rnt-Q179P, Rnt-A27T, Rnt-F194L, Rnt-A180T, and SapC-G79W, and a knock-out or knockdown of at least two of kgtP, ybjL, and proV (or genes encoding other subunits in the same protein complex, which are proX and proW), such as kgtP and proV.   a mutation selected from SpoT-V422A, SpoT-A451D, SpoT-A451V, SpoT-W457C, SpoT-N454H, SpoT-D580Y, SpoT-R236L, SpoT-R236S, SpoT-M247K, SpoT-NIR(601-603)S, SpoT-T442I, SpoT-S434L, PolB-R477G, RpoC-H419P, RpoC-P64L, RpoB-1K203T, Rnt-Q179P, Rnt-A27T, Rnt-F194L, Rnt-A180T, and SapC-G79W, and a knock-out or knockdown of kgtP, ybjL, and proV (or genes encoding other subunits in the same protein complex, which are proX and proW) in combination, such as kgtP, proV, and ybjL.   a mutation increasing the expression of pyrE and a knock-out or knockdown of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, or yeaR, such as kgtP.   a mutation increasing the expression of pyrE and a knock-out or knockdown of at least two of kgtP, ybjL, and proV (or genes encoding other subunits in the same protein complex, which are proX and proW), such as kgtP and proV.   a mutation increasing the expression of pyrE and a knock-out or knockdown of kgtP, ybjL, and proV (or genes encoding other subunits in the same protein complex, which are proX and proW) in combination, such as kgtP, proV, and ybjL.       

     In other separate and specific embodiments, the bacterial cell comprises:
         a combination of mutations SpoT-V422A and RpoC-H419P, and a knock-out or knockdown of kgtP.   a mutation SpoT-V422A, and a knock-out or knockdown of kgtP and at least one of proV, proW and proX, and a knock-out, knockdown, or reduction of function mutation in sspA.   a mutation SpoT-A451D, and a knock-out or knockdown of kgtP.   a mutation SpoT-A451D, and a knock-out or knockdown of kgtP and at least one of proV, proW and proX.   a combination of mutations SpoT-A451D and RpoC-H419P, and a knock-out or knockdown of kgtP and at least one of proV, proW and proX.   a combination of mutations SpoT-W457C and RpoC-H419P, and a knock-out or knockdown of kgtP.   a mutation SpoT-D580Y, and a knock-out or knockdown of kgtP and at least one of proV, proW and proX.   a combination of mutations SpoT-R236L and RpoB-K203T, and a knock-out or knockdown of kgtP and sspA.   a combination of mutations PolB-R477G and RpoC-P64L, and a knock-out or knockdown of kgtP and at least one of proV, proW and proX.   a knock-out or knockdown of kgtP and ybjL, and a mutation that increases the expression of PyrE.   a knock-out or knockdown of kgtP and a mutation that increases the expression of PyrE.   a knock-out of knockdown of kgtP, ybjL, and at least one of nagA or nagC.   a mutation SpoT-S434L, and a knock-out of knockdown of kgtP; at least one of proV, proW and proX; ybjL; and at least one of nagC and nagA.   a combination of mutations SpoT-S434L and ProQ-R80C, and a knock-out of knockdown of kgtP; at least one of proV, proW and proX; ybjL, and at least one of nagC and nagA.   a mutation SpoT-S434L, and a knock-out or knockdown of kgtP; at least one of proV, proW and proX; ybjL; and at least one of nagC and nagA; and proQ.   a knock-out of knockdown of kgtP and one of pstS, tdk, and rpoS, or any combination thereof.   a knock-out of knockdown of kgtP; ybjL; at least one of proV, proW and proX; and sspA.   a knock-out of knockdown of kgtP, tdk, and pstS.   2) Production Pathways       

     In some aspects, the bacterial cell comprises a recombinant pathway for producing an aliphatic diacid of interest, optionally providing for a production level of at least about 5 g/L of the aliphatic diacid over a predetermined period of time, e.g., about 200h, about 100h, about 72h, about 48h or about 24h. A recombinant pathway can, for example, be added to introduce the capability to produce the diacid in a bacterial cell which does not have a native pathway to do so, typically by transforming the cell with one or more heterologous enzymes catalyzing the desired reaction(s). Alternatively, in cases where the bacterial cell has a native pathway for production of the diacid of interest, a recombinant pathway can nonetheless be introduced in order to increase the production yield, e.g., by overexpressing one or more native enzymes or transforming the cell with heterologous enzymes. In separate and specific embodiments, the recombinant pathway provides for a production level of at least 5 g/L, such as at least 10 g/L or higher, such as at least 20 g/L or higher, such as at least 40 g/L or higher, such as at least 45 g/L or higher, such as at least 75 g/L or higher, such as at least 100 g/L or higher. 
     So, in one aspect, there is provided a bacterial cell with improved tolerance to at least one aliphatic diacid according to any aspect or embodiment described herein, wherein the bacterial cell further comprises a recombinant biosynthetic pathway for producing an aliphatic diacid of interest, such as, e.g., glutaric acid, adipic acid, succinic acid, muconic acid, fumaric acid, itaconic acid, malic acid, malonic acid, maleic acid, glucaric acid, pimelic acid, suberic acid, sebacic acid, 2,5-furandicarboxylic acid, terephthalic acid, or azelaic acid, mesaconic acid, citraconic acid, tartaric acid, tartronic acid, diaminopimelic acid or glutaconic acid. In principle, any such recombinant biosynthetic pathway which is known in the art can be introduced into the cell by standard recombinant technologies. Some specific, preferred pathways are, however, exemplified below and in Example 1—see the section entitled “Biological production of diacids” and references cited therein. 
     It is to be understood that, when a specific enzyme of these biosynthetic pathways is mentioned by name such as, e.g., “lysine monooxygenase”, the enzyme may be any characterized and sequenced enzyme, from any species, that have been reported in the literature so long as it provides the desired activity. In some embodiments, the enzyme is an overexpressed gene which is native to the host cell used. In some embodiments, the enzyme is a functionally active fragment or variant of an enzyme which is heterologous or native to the host cell. Also, in some embodiments, the recombinant biosynthetic pathway comprises a knock-down or a knock-out of one or more genes, typically for the purpose of avoiding competing reactions reducing the yield of the desired aliphatic diacid. 
     So, in one embodiment, the biosynthetic pathway is for producing glutaric acid from glucose, and comprises genes, optionally overexpressed and/or heterologous, encoding:
         a lysine monooxygenase;   a 5-aminovaleramidase;   a 5-aminovalerate transaminase, and   a glutarate semialdehyde dehydrogenase.       

     The bacterial cell may further be modified by one or more of
         (i) introducing feedback resistance mutations in native genes corresponding to DapA (4-hydroxytetrahydrodipicolinate synthase) and LysC (asparate kinase III), optionally also overexpressing the modified proteins; and   (ii) knockdown or knock-out of native genes corresponding to cadA and/dcC;   (iii) constitutive overexpression of lysine biosynthesis, e.g., via a dapA promoter replacement; and   (iv) knock-down or knock-out of native genes corresponding to speE, speG, patA and puuPA.       

     In one embodiment, the biosynthetic pathway is for producing adipic acid and comprises
         a reversible 3-oxoadipyl-CoA thiolase (e.g., PaaJ from  E. coli ),   a 3-hydroxyacyl-CoA dehydrogenase (e.g., PaaH1 from  Ralstonia eutropha ),   an enoyl-CoA hydratase (e.g., h16_AA307 gene product from  Ralstonia eutropha  H16),   a trans-enoyl-CoA reductase (e.g., Ter from  Euglena gracilis ),   a phosphate butyryltransferase (e.g., Ptb from  Clostridium acetobutylicum ), and   a butyryl kinase (e.g., Buk1 from  Clostridium acetobutylicum )       

     The bacterial cell may further be modified to knock-down or knock-out one or more native genes corresponding to ptsG, poxB, pta, sdhA, and iclR. 
     In one embodiment, the biosynthetic pathway is for producing adipic acid and comprises
         a 3-oxoadipyl-CoA thiolase (e.g., PaaJ from  E. coli ),   a 3-hydroxyacyl-CoA dehydrogenase (e.g. PaaH from  E. coli )   an enoyl-CoA hydratase (e.g. PaaF from  E. coli )   an enoyl-CoA reductase (e.g. Ter from  Treponema denticola )   an acyl-CoA thioesterase (e.g. Acot8 from  Mus musculus )       

     The bacterial cell may further be modified to knock-down or knock-out one or more native genes corresponding to pta, poxB, ldhA, and adhE. 
     In one embodiment, the biosynthetic pathway is for producing pimelic acid and comprises feeding glutaric acid and
         a glutaryl-CoA transferase (e.g., Cat1 from  Clostridium kluyveri ),   a 3-oxoadipyl-CoA thiolase (e.g., PaaJ from  E. coli ),   a 3-hydroxyacyl-CoA dehydrogenase (e.g. PaaH from  E. coli )   an enoyl-CoA hydratase (e.g. PaaF from  E. coli )   an enoyl-CoA reductase (e.g. Ter from  Treponema denticola )   an acyl-CoA thioesterase (e.g. Acot8 from  Mus musculus )       

     The bacterial cell may further be modified to knock-down or knock-out one or more native genes corresponding to pta, poxB, IdhA, adhE, and frdA. 
     In one embodiment, the biosynthetic pathway is for producing sebacic acid and comprises
         a glutaryl-CoA transferase (e.g., Cat1 from  Clostridium kluyveri ),   a 3-oxoadipyl-CoA thiolase (e.g., DcaF from  Acinetobacter  sp. ADP1),   a 3-hydroxyacyl-CoA dehydrogenase (e.g. DcaH from  Acinetobacter  sp. ADP1)   an enoyl-CoA hydratase (e.g. DcaE from  Acinetobacter  sp. ADP1)   an enoyl-CoA reductase (e.g. Ter from  Treponema denticola )   an acyl-CoA thioesterase (e.g. Acot8 from  Mus musculus )       

     The bacterial cell may further be modified to knock-down or knock-out one or more native genes corresponding to pta, poxB, ldhA, adhE, frdA, and native acyl-CoA thioesterases including yciA, ybgC, ydiI, tesA, fadM, and tesB. 
     In one embodiment, the biosynthetic pathway is for producing adipic acid and comprises
         a 3-oxoadipyl-CoA thiolase/p-ketothioase (e.g. from  Thermobifida fusca  B6),   a 3-hydroxyacyl-CoA dehydrogenase (e.g. from  Thermobifida fusca  B6)   an enoyl-CoA hydratase (e.g. from  Thermobifida fusca  B6)   an enoyl-CoA reductase (e.g. from  Thermobifida fusca  B6)   a succinyl-CoA synthetase (e.g. Tfu_2577 and Tfu_2576 from  Thermobifida fusca  B6)       

     The bacterial cell may further be modified to knock-down or knock-out one or more native genes corresponding to pta, poxB, ldhA, adhE, ptsG, sdhA, and iclR. 
     In one embodiment, the biosynthetic pathway is for producing adipic acid via whole cell bioconversion from supplied medium to long chain free fatty acids (C 12 -C 16 ) and comprises combinations of
         a heterologously expressed or increased native activity of a 6-oxohexanoic acid dehydrogenase   a heterologously expressed or increased native activity of an omega exo fatty acid dehydrogenase   a heterologously expressed or increased native activity of a 6-hydroxyhexanoic acid dehydrogenase   a heterologously expressed or increased native activity of an omega hydroxyl fatty acid dehydrogenase   a heterologously expressed or increased native activity of a hexanoate synthase   a heterologously expressed or increased native activity of a monooxygenase   a heterologously expressed or increased native activity of a monooxygenase reductase   a heterologously expressed or increased native activity of a fatty acid oxidase   a heterologously expressed or increased native activity of an acyl-CoA ligase   a heterologously expressed or increased native activity of an acyl-CoA oxidase   a heterologously expressed or increased native activity of an enoyl-CoA reductase   a heterologously expressed or increased native activity of a 3-L-hydroxyacyl-CoA dehydrogenase   a heterologously expressed or increased native activity of an acetyl-CoA C-acetyltransferase       

     In one embodiment, the biosynthetic pathway is for producing muconic acid and comprises
         a native or heterologously expressed pathway comprising a 2-dehydro-3-deoxy-D-arabinoheptonate 7-phosphate (DAHP) synthase, a 3-dehydroquinate synthase, and a 3-dehydroxyquinate dehydratase   a dehydroshikimic acid dehydratase (e.g. PobA from  Pseudomonas putida  KT2440)   a protocatechuate decarboxylase (e.g. AroY from  Klebsiella pneumoniae )   a catechol 1,2-dioxygenase (e.g. CatA from  Acinetobacter  sp. ADP1)       

     The bacterial cell may further be modified to increase the flux toward precursors for DAHP (erythrose 4-phosphate and phosphoenolpyruvate), such as by knock-down or knock-out of genes corresponding to  E. coli  ptsH, ptsI, crr, and pykF; by overexpressing genes corresponding to ubiC, aroF, aroE, and aroL (or feedback-resistant mutants thereof), or combinations thereof. 
     Some bacteria contain a native pathway for production of a diacid, avoiding the necessity for a recombinant pathway. These include, for example,  Actinobacillus succinogenes  (succinic acid),  Mannheimia succiniproducens  (succinic acid),  Thermobifida fusca  (malic acid), and  Escherichia coli  (fumaric and malic acids), among numerous others. 
     3) Processes 
     In one aspect, there is provided a process for preparing a recombinant bacterial cell, e.g., an  E. coli  cell. Also provided is a process for improving the tolerance of a bacterial cell, e.g., an  E. coli  cell, to a diacid. Also provided is a method of identifying a bacterial cell which is tolerant to at least one diacid. Also provided is a process for preparing a recombinant bacterial cell, e.g., an  E. coli  cell, for producing a diacid. 
     These processes may comprise one or more steps of genetically modifying a bacterial cell to knock-down or knock-out one or more endogenous genes of any aspect or embodiment of the Group 1 modifications and/or introducing one or more mutations in the endogenous protein(s) or gene(s) of any Group 2 aspect or embodiment. This can be achieved by, e.g., transforming the bacterial cell with genetic constructs, e.g., vectors, antisense nucleic acids or siRNA, which result, e.g., in the knock-out or knock-down of a gene, introduce a mutation into an endogenous gene, or which encode the mutated protein from a transgene. 
     The genetic constructs, particularly vectors, can also comprise suitable regulatory sequences, typically nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters (e.g., constitutive promoters or inducible promoters), translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures. 
     Alternatively, bacterial cells can be exposed to selection pressure (as described in the Examples) or to conditions which introduce random mutations in endogenous genes, and bacterial cells which comprise one or more Group 1 and/or Group 2 modifications according to any preceding aspects and embodiments can then be identified. Typically, this involves preparing a population of the genetically modified bacterial cell, having different Group 1 and/or Group 2 modifications, and then selecting from this population any bacterial cell which has an improved tolerance to a diacid at a predetermined concentration. 
     In one specific embodiment, the Group 1 modification is a knock-down or knock-out of one or more endogenous genes selected from kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR or, e.g., a knock-down or knock-out of kgtP in combination one or more other genes, e.g., ybjL, proV and/or sspA. In one specific embodiment, the Group 2 modification is a mutation in at least one endogenous protein or gene selected from SpoT, PolB, RpoC, RpoB, Rnt or SapC, such as e.g., at least one mutant protein selected from the group consisting of SpoT-V422A, SpoT-A451D, SpoT-A451V, SpoT-W457C, SpoT-N454H, SpoT-D580Y, SpoT-R236L, SpoT-R236S, SpoT-M247K, SpoT-NIR(601-603)S, SpoT-T442I, SpoT-S434L, PolB-R477G, RpoC-H419P, RpoC-P64L, RpoB-K203T, Rnt-Q179P, Rnt-A27T, Rnt-F194L, Rnt-A180T, SapC-G79W; and/or a mutation which increases the expression of PyrE, such as, e.g. a mutation in rph or the pyrE/rph intergenic region. 
     The processes may further comprise
         a step of selecting any bacterial cell which has an improved tolerance to a diacid at a predetermined concentration in the medium, such as at least 1 g/L, such as at least 2 g/L, or higher, such as at least 5 g/L or higher, such as at least 10 g/L or higher, such as at least 20 g/L or higher, such as at least 40 g/L or higher, such as at least 75 g/L or higher, such as at least 100 g/L or higher;   an optional step of introducing a recombinant biosynthetic pathway for producing the diacid; or   both of the above steps, in any order.       

     In one embodiment, the diacid is glutaric acid, and the predetermined concentration is at least 2 g/L or higher, such as at least 5 g/L or higher, such as at least 10 g/L or higher, such as at least 20 g/L or higher, such as at least 40 g/L or higher, such as at least 75 g/L or higher, such as at least 100 g/L or higher. In one embodiment, the diacid is adipic acid, and the predetermined concentration is at least 2 g/L or higher, such as at least 5 g/L or higher, such as at least 10 g/L or higher, such as at least 20 g/L or higher, such as at least 40 g/L or higher, such as at least 75 g/L or higher, such as at least 100 g/L or higher. In one embodiment, the diacid is pimelic acid, and the predetermined concentration is at least 2 g/L or higher, such as at least 5 g/L or higher, such as at least 10 g/L or higher, such as at least 20 g/L or higher, such as at least 30 g/L or higher, such as at least 45 g/L or higher, such as at least 75 g/L or higher. In one embodiment, the diacid is sebacic acid, and the predetermined concentration is at least 2 g/L or higher, such as at least 5 g/L or higher, such as at least 10 g/L or higher, such as at least 20 g/L or higher, such as at least 30 g/L or higher, such as at least 45 g/L or higher, such as at least 75 g/L or higher. 
     In a particular embodiment, the predetermined concentration is at most 20 g/L, such as at most 30 g/L, such as at most 50 g/L, such as at most 75 g/L, such as at most 100 g/L, such as at most 150 g/L. 
     Assays for assessing the tolerance of a modified bacterial cell to a diacid typically evaluate the growth rate, lag time, or both, of the bacterial cell at predetermined concentrations for the diacid in question, typically as compared to a control. Preferably, the control is the native or unmodified parent cell or strain, and an improved tolerance is identified as an improved growth rate, a reduced lag-time or both. For example, an improved growth rate can be at least 5%, such as at least 10%, such as at least 20%, such as at least 50%, such as at least 75% higher than that of the control, while a reduced lag time can be at least 10%, such as at least 20%, such as at least 50%, such as at least 75%, such as at least 90% shorter than that of the control. Specific assays are described, in detail, in the Examples. 
     Also provided is a method of producing a diacd, comprising culturing the bacterial cell obtained by any one of these methods, or the bacterial cell of any preceding aspect or embodiment, under conditions where the diacid is produced. Typically, these conditions include the presence of a suitable carbon source or mixes of different suitable carbon sources. Non-limiting examples of suitable carbon sources include, e.g., sucrose, D-glucose, D-xylose, L-arabinose, glycerol; raw carbon feedstocks such as crude glycerol and cane syrup; as well as hydrolysates produced from cellulosic or lignocellulosic materials. For further details see, e.g., Adkins et al., 2013; Park et al., 2013; Yu et al., 2014; Cheong et al., 2016; Deng and Mao, 2015; WO 2011/003034 A2 (Verdezyne); Curran et al., 2013; Sengupta et al., 2015; and Zhang et al., 2015. 
     4) Compositions 
     A bacterial cell which has an increased tolerance to a diacid can be useful as a production host for the diacid. Bacterial cells according to the invention may have an increased growth rate, a decreased lag time, or both, in the diacid. For example, the bacterial cell may have Group 1 and/or Group 2 modifications providing for an increased growth rate, a reduced lag time, or both, of the cell in at least one diacid, e.g., in glutaric acid, adipic acid, succinic acid, muconic acid, fumaric acid, itaconic acid, malic acid, malonic acid, maleic acid, glucaric acid, pimelic acid, suberic acid, sebacic acid, 2,5-furandicarboxylic acid, terephthalic acid, or azelaic acid, mesaconic acid, citraconic acid, tartaric acid, tartronic acid, diaminopimelic acid and/or glutaconic acid. 
     In one aspect, there is provided a composition comprising a plurality of bacterial cells according to any aspect or embodiment described herein, e.g., an in vitro culture of such bacterial cells, optionally in a suitable culture medium and/or a chemically-defined medium comprising a carbon source. In one embodiment, the composition is substantially homogenous with respect to the bacterial cells. 
     In one aspect, there is provided a composition comprising a plurality of bacterial cells according to any preceding aspect or embodiment and a diacid. In one embodiment, the diacid is present at a concentration at which the genetic modification(s) and/or mutant(s) comprised in the bacterial cells results in an improved tolerance as compared to the parent bacterial cells, e.g., wild-type or native bacterial cells. The concentrations at which bacterial cells according to the invention have improved tolerance are shown in Example 1, e.g., in “Cross-compound tolerance testing”. Typically, the concentration of the diacid is at least 1 g/L, such as at least 2 g/L, or higher, such as at least 5 g/L or higher, such as at least 10 g/L or higher, such as at least 20 g/L or higher, such as at least 40 g/L or higher, such as at least 45 g/L or higher, such as at least 75 g/L or higher, such as at least 100 g/L or higher. 
     In one aspect, there is provided a composition comprising
         a diacid at a concentration of at least 1 g/L, such as at least 2 g/L, or higher, such as at least 5 g/L or higher, such as at least 10 g/L or higher, such as at least 20 g/L or higher, such as at least 40 g/L or higher, such as at least 45 g/L or higher, such as at least 75 g/L or higher, such as at least 100 g/L or higher; and   a plurality of bacterial cells according to any preceding aspect or embodiment.       

     In separate and specific embodiments, the diacid is glutaric acid, adipic acid, pimelic acid and sebacic acid, respectively. In other specific embodiments, the diacid present in the composition is at least 45 g/L fumaric acid, at least 45 g/L itaconic acid, at least 55 g/L malic acid, at least 50 g/L succinic acid, at least 45 g/L pimelic acid, and at least 38 g/L sebacic acid, respectively. 
     As described in Example 1; “Cross-compound tolerance testing,” genetic modifications according to the invention also conferred tolerance to other chemicals, such as to other carboxylic acids (glutarate and adipate; hexanoate, octanoate, isobutyrate, glutarate and p-coumarate), to diamines (e.g., HMDA, putrescine) and diols (2,3-butanediol, 1,2-propanediol). Accordingly, in one embodiment, there is provided a composition comprising a plurality of bacterial cells according to any preceding aspect or embodiment, and a chemical selected from the following, at at least the indicated concentration: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 butanol 
                 1.4% 
                 v/v 
               
               
                   
                 glutarate 
                 40 
                 g/L 
               
               
                   
                 p-coumarate 
                 7.5 
                 g/L 
               
               
                   
                 putrescine 
                 32 
                 g/L 
               
               
                   
                 HMDA 
                 32 
                 g/L 
               
               
                   
                 adipate 
                 45 
                 g/L 
               
               
                   
                 isobutyrate 
                 7.5 
                 g/L 
               
               
                   
                 hexanoate 
                 3 
                 g/L 
               
               
                   
                 octanoate 
                 8 
                 g/L 
               
               
                   
                 2,3-butanediol 
                 6% 
                 v/v 
               
               
                   
                 1,2-propanediol 
                 6% 
                 v/v 
               
            
           
           
               
               
               
            
               
                   
                 sodium chloride 
                 0.6M 
               
               
                   
                   
               
            
           
         
       
     
     Preferably, the bacterial cells are of the  Escherichia, Lactobacillus, Lactococcus, Bacillus, Pseudomonas, Corynebacterium, Deinococcus  or  Ralstonia  genera, such as, e.g.,  E. coli  cells, and comprise
         a) at least one genetic modification which reduces expression of an endogenous gene selected From the group consisting of kgtP, ybjL, proV, proW, proX, proQ, cspE, rfaE, yfbP, yfjM, pstS, pstA, pstB, pstC, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR, or a combination of any thereof;   b) a mutation in one or more of SpoT, PolB, RpoC, RpoB, Rnt and SapC; an increased expression of one or more of SpoT, PolB, RpoC, RpoB, Rnt and SapC; and/or a mutation in rph or the pyrE/rph intergenic region which increases the expression of pyrE, or   c) a combination of (a) and (b).       

     Assays for assessing the tolerance of a modified bacterial cell to a selected diacid typically evaluate the growth rate, lag time, or both, of the bacterial cell at one or more predetermined concentrations of the compound, typically as compared to a control (e.g., no compound). The predetermined concentrations(s) could be, for example, 1, 2, 5, 10, 20, 40, 45, 75, or 100 g/L. Preferably, the control is the native or unmodified parent cell or strain, and an improved tolerance is identified as an improved growth rate, a reduced lag-time or both. For example, an improved growth rate can be at least 5%, such as at least 10%, such as at least 20%, such as at least 50%, such as at least 75%, such as at least 100%, such as at least 200%, such as at least 300%, such as at least 500%, such as at least 1000%, such as at least 10000% higher than that of the control; while a reduced lag time can be at least 10%, such as at least 20%, such as at least 50%, such as at least 75%, such as at least 90% shorter than that of the control. Indeed, in some cases the native or unmodified parent cell cannot grow at all in a concentration of the diacid that the modified bacterial cell can grow in. Specific assays are described, in detail, in the Examples. 
     5) Bacterial Cells 
     Also provided are strains, clones and other progeny of the bacterial cells of these and other aspects and embodiments, as well as cell cultures of such bacterial cells or strains. Typically, as used herein, a “strain” typically refers to a group of cells which are descendants of a initial single colony of parent cells whereas a “clone” is a group of cells which are the descendants of an initial genetically modified single parent cell. 
     Non-limiting examples of bacterial cells suitable for modification according to any one of the aspects and embodiments described herein include bacteria of the  Escherichia, Lactobaccillus, Lactococcus, Corynebacterium, Bacillus, Ralstonia, Clostridia, Deinococcus  or  Pseudomonas  genera, such as from the  Escherichia, Lactobacillus, Lactococcus, Bacillus, Pseudomonas, Corynebacterium, Deinococcus  or  Ralstonia  genera. In one embodiment, the bacterial cell is an  E. coli  cell, such as a cell of the commercially available and/or fully characterized strains K-12 MG1655, BW25113, BL21, BL21(DE3), K-12 W3110, W, JM109, or Crooks (ATCC 8739). In a specific embodiment, the bacterial cell is derived from an  E. coli  K12 strain. In another embodiment, the bacterial cell is a  Lactobacillus  cell, such as a cell of the commercially available and/or fully characterized strains  Lactobacillus plantarum  JDM1,  Lactobacillus plantarum  WCFS1, and  Lactobacillus plantarum  NCIMB 8826. In another embodiment, the bacterial cell is a  Lactococcus  cell, such as a cell of the commercially available and/or fully characterized strains  Lactococcus lactis lactis  CV56,  Lactococcus lactis lactis  NIZO B40, and  Lactococcus lactis cremoris  NZ9000. In another embodiment, the bacterial cell is a  Bacillus  cell, such as a cell of the commercially available and/or fully characterized strains  Bacillus subtilis  168 and  Bacillus subtilis  PY79. In one embodiment, the bacterial cell is a  Pseudomonas  cell, such as a cell of the commercially available and/or fully characterized strain  Pseudomonas putida  KT2440. In another embodiment, the bacterial cell is a  Ralstonia  cell, such as a cell of the commercially available and/or fully characterized strains  Ralstonia eutropha  H16 and  Ralstonia eutropha  JMP134. In another embodiment, the bacterial cell is a  Corynebacterium  cell, such as a cell of the commercially available and/or fully characterized strains 534 (ATCC 13032), K051, MB001, R, SCgG1, and SCgG2. In another embodiment, the bacterial cell is a  Deinococcus  cell, such as a  D. radiodurans  or  D. geothermalis  cell, such as a cell of the commercially available and/or fully characterized strain  D. radiodurans  R1. 
     While aspect and embodiments relating to bacterial cells herein typically refer to genes or proteins according to their designation in  E. coli , for bacterial cells of another family or species, it is within the level of skill in the art to identify the corresponding gene or protein, i.e., the ortholog and/or paralog, in the other family or species, typically by identifying sequences having moderate or high homology to the  E. coli  sequence, optionally taking the function of the protein expressed by the gene and/or the locus of the gene in the genome into account, Table 2 sets out the function of the protein encoded by each specific gene, the corresponding E.C. number (if applicable), its locus in the  E. coli  K-12 MG1655 genome and the SEQ ID number of the coding or non-coding sequence and, where applicable, the encoded amino acid sequence. 
     Tables 3 and 4 set out some examples of homologs or orthologs in selected organisms, identified in a preliminary and non-limiting analysis. Indeed, homologs or orthologs of these proteins exist also in other bacteria, and other homologs or orthologs not identified in this preliminary search can exist in the species listed in Table 3. The skilled person is well-familiar with different searching and/or screening methods for identifying homologs or orthologs across different species. To briefly summarize some of the preliminary findings in Table 3:
         KgtP, ProV, ProW, CspE, RfaE, PstS, pstA, pstB, pstC, UvrB, SpoT, RpoB, RpoC, SapC, and PyrE are widely conserved and were identified in all organisms. ProW and ProX appear to be fused in  Lactococcus lactis  and  Lactobacillus plantarum.      Two YbjL homologs or orthologs were identified in  Corynebacterium glutamicum.      Rph was found to be conserved in all organisms with the exception of  Lactococcus lactis.      ProQ was only found in  Pseudomonas putida , indicating it is likely only conserved in the Gammaproteobacteria.   RpoS is likely only conserved as an alternative sigma factor to RpoD in Gram-negative bacteria.   SspA was found to be conserved in Gram-negative organisms.   Tdk was found to be conserved between  E. coli  and only certain Gram-positive organisms in Table 3 ( B. subtilis, L. lactis, L. plantarum )   YcjG has a conserved annotated function in  Bacillus subtilis  and has homology to annotated muconate/chloromuconate cycloisomerases, which may be another activity of the enzyme, in the Gram-negative organisms ( P. putida, R. eutropha ) and  C. glutamicum.      PolB is conserved in the Gram-negative organisms ( P. putida, R. eutropha )   Rnt has a high degree of identity and with similar annotated functions to a protein in  P. putida , and is a homolog of annotated DNA polymerases in  B. subtilis, L. plantarum, L. lactis , and  R. eutropha .       

     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Protein function and Locus IDs 
               
            
           
           
               
               
               
               
               
            
               
                   E. coli  gene 
                   
                 E.C. 
                 Locus 
                   
               
               
                 designation 
                 Protein function 
                 number 
                 ID 
                 SEQ ID NO: 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 kgtP 
                 α-ketoglutarate:H +  symporter 
                 N/A 
                 b2587 
                 1 
               
               
                 ybjL 
                 inner membrane protein YbjL 
                 N/A 
                 b0847 
                 2 
               
               
                 proV 
                 glycine betaine/proline ABC 
                 3.6.3.32 
                 b2677 
                 3 
               
               
                   
                 transporter - ATP binding 
               
               
                   
                 subunit 
               
               
                 proW 
                 glycine betaine/proline ABC 
                 3.6.3.32 
                 b2678 
                 4 
               
               
                   
                 transporter - membrane 
               
               
                   
                 subunit 
               
               
                 proX 
                 glycine betaine/proline ABC 
                 3.6.3.32 
                 b2679 
                 5 
               
               
                   
                 transporter - periplasmic 
               
               
                   
                 binding protein 
               
               
                 proQ 
                 RNA chaperone, involved in 
                 N/A 
                 b1831 
                 6 
               
               
                   
                 posttranscriptional control of 
               
               
                   
                 ProP levels 
               
               
                 cspE 
                 transcription antiterminator and 
                 N/A 
                 b0623 
                 7 
               
               
                   
                 regulator of RNA stability 
               
               
                 rfaE 
                 fused heptose-7-phosphate 
                 2.7.7.70, 
                 b3052 
                 8 
               
               
                   
                 kinase/heptose-1-phosphate 
                 2.7.1.167 
               
               
                   
                 adenyltransferase 
               
               
                 yfbP 
                 predicted protein 
                 N/A 
                 b2275 
                 9 
               
               
                 yfjM 
                 CP4-57 prophage; predicted 
                 N/A 
                 b2629 
                 10 
               
               
                   
                 protein 
               
               
                 pstS 
                 phosphate ABC transporter - 
                 3.6.3.27 
                 b3728 
                 11 
               
               
                   
                 periplasmic binding protein 
               
               
                 pstA 
                 phosphate ABC transporter - 
                 3.6.3.27 
                 b3726 
                 34 
               
               
                   
                 membrane subunit 
               
               
                 pstB 
                 phosphate ABC transporter - 
                 3.6.3.27 
                 b3725 
                 35 
               
               
                   
                 ATP binding subunit 
               
               
                 pstC 
                 phosphate ABC transporter - 
                 3.6.3.27 
                 b3727 
                 36 
               
               
                   
                 membrane subunit 
               
               
                 rph 
                 RNAse PH 
                 2.7.7.56 
                 b3643 
                 12 
               
               
                 rpoS 
                 RNA polymerase, sigma S 
                 N/A 
                 b2741 
                 13 
               
               
                   
                 (sigma 38) factor 
               
               
                 sspA 
                 stringent starvation protein A 
                 N/A 
                 b3229 
                 14 
               
               
                 tdk 
                 thymidine kinase/deoxyuridine 
                 2.7.1.145, 
                 b1238 
                 15 
               
               
                   
                 kinase 
                 2.7.1.21 
               
               
                 uvrB 
                 DNA repair; excision nuclease 
                 3.1.25.— 
                 b0779 
                 16 
               
               
                   
                 subunit B 
               
               
                 ycjG 
                 L-Ala-D/L-Glu epimerase 
                 5.1.1.20 
                 b1325 
                 17 
               
               
                 yeaR 
                 conserved protein 
                 N/A 
                 b1797 
                 18 
               
               
                 spoT 
                 guanosine 3′-diphosphate 5′- 
                 3.1.7.2 
                 b3650 
                 19 (DNA) 
               
               
                   
                 triphosphate 3′-diphosphatase 
                   
                   
                 20 (protein) 
               
               
                   
                 [multifunctional] 
               
               
                 polB 
                 DNA polymerase II 
                 3.1.11.—, 
                 b0060 
                 21 (DNA) 
               
               
                   
                   
                 2.7.7.7 
                   
                 22 (protein) 
               
               
                 rpoB 
                 RNA polymerase, β subunit 
                 2.7.7.6 
                 b3987 
                 23 (DNA) 
               
               
                   
                   
                   
                   
                 24 (protein) 
               
               
                 rpoC 
                 RNA polymerase, β′ subunit 
                 2.7.7.6 
                 b3988 
                 25 (DNA) 
               
               
                   
                   
                   
                   
                 26 (protein) 
               
               
                 rnt 
                 RNase T 
                 3.1.13.— 
                 b1652 
                 27 (DNA) 
               
               
                   
                   
                   
                   
                 28 (protein) 
               
               
                 sapC 
                 integral membrane protein 
                 N/A 
                 b1292 
                 29 (DNA) 
               
               
                   
                 SapC of predicted ABC 
                   
                   
                 30 (protein) 
               
               
                   
                 transporter 
               
               
                 pyrE 
                 Orotate 
                 2.4.2.10 
                 b3642 
                 31(DNA) 
               
               
                   
                 phosphoribosyltransferase 
                   
                   
                 32 (protein) 
               
               
                 pyrE/rph 
                 — 
                 — 
                 — 
                 33 
               
               
                 intergenic 
               
               
                 region 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Homologs or orthologs identified by protein BLAST (BLASTP) of  E. coli  K-12 MG1655 proteins against protein databases from selected 
               
               
                 reference organisms. Hits with the largest e-value are shown, and hits are only shown when the e-value &lt; 1.0. Hit proteins with 
               
               
                 e-value &lt; 0.1 (non-italicized) are deemed more probable of having the same or similar function as the  E. coli  protein. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Protein 
                   
                   
                   
                   
                 
                   Ralstonia 
                 
                 
                   Corynebacterium 
                 
               
               
                 (# of 
                 
                   B. subtilis 
                 
                 
                   P. putida 
                 
                 
                   L. plantarum 
                 
                 
                   L. lactis 
                 
                 
                   eutropha 
                 
                 
                   glutamicum 
                 
               
               
                 residues) 
                 168 
                 KT2440 
                 JDM1 
                 KF147 
                 H16 
                 ATCC 13032 
               
               
                   
               
               
                 KgtP 
                 24-27% identity 
                 56-70% identity 
                 23-28% identity 
                 23% identity 
                 33-37% identity 
                 28-33% identity 
               
               
                 (432 aa) 
                 (339-420 aa) 
                 (415-416 aa) 
                 (176-355 aa) 
                 (347 aa) 
                 (337-381 aa) 
                 (392-443 aa) 
               
               
                   
                 “metabolite 
                 “major facilitator 
                 “arabinose 
                 “arabinose- 
                 “major facilitator 
                 “integral 
               
               
                   
                 transporter” 
                 superfamily 
                 transport protein” 
                 proton symporter” 
                 superfamily 
                 membrane 
               
               
                   
                 (NP_388707.1), 
                 metabolite/H+ 
                 (YP_003064425.1), 
                 (NP_003354045.1) 
                 transporter MHS 
                 transport 
               
               
                   
                 “metabolite 
                 symporter” 
                 “transport 
                   
                 family protein” 
                 protein” 
               
               
                   
                 transport 
                 (NP_743537,1, 
                 protein” 
                   
                 (YP_725964.1, 
                 (NP_602106.1). 
               
               
                   
                 protein YwtG” 
                 NP_743559.1) 
                 (YP_003063609.1), 
                   
                 YP_725210.1), 
                 “proline- 
               
               
                   
                 (NP_391464.2), 
                   
                 “sugar transport 
                   
                 “MFS family 
                 betaine 
               
               
                   
                 “metabolite 
                   
                 protein” 
                   
                 transporter” 
                 transporter” 
               
               
                   
                 transport 
                   
                 (YP_003064460.1) 
                   
                 (YP_725057.1) 
                 (NP_602258.1), 
               
               
                   
                 protein YncC” 
                   
                   
                   
                   
                 “major 
               
               
                   
                 (NP_389645.2), 
                   
                   
                   
                   
                 facilitator 
               
               
                   
                 “major myo- 
                   
                   
                   
                   
                 superfamily 
               
               
                   
                 inositol 
                   
                   
                   
                   
                 permease” 
               
               
                   
                 transporter 
                   
                   
                   
                   
                 (NP_599668.1, 
               
               
                   
                 IolT” 
                   
                   
                   
                   
                 NP_599535.2, 
               
               
                   
                 (NP_388504.1) 
                   
                   
                   
                   
                 NP_600684.1) 
               
               
                 YbjL 
                   28 %  identity   
                 31% identity 
                   34 %  identity   
                   25 %  identity   
                 31% identity 
                 25% identity 
               
               
                 (561 aa) 
                   (116 αα ) 
                 (123 aa) 
                 ( 68 αα ) 
                 ( 76 αα ) 
                 (566 aa) 
                 (548 aa) 
               
               
                   
                 
                   “sulfate 
                 
                 “potassium/ 
                 
                   “sugar 
                 
                 
                   “chloride 
                 
                 “aspartate: 
                 “permease” 
               
               
                   
                 
                   transporter 
                 
                 proton 
                 
                   transport protein” 
                 
                 
                   channel protein” 
                 
                 alanine 
                 (NP_601414.1); 
               
               
                   
                 Y vdB”   
                 antiporter” 
                 (YP_003064460.1) 
                 (YP_003354224.1) 
                 antiporter” 
                 25% identity 
               
               
                   
                 (NP_391346.1) 
                 (NP_747167.2) 
                   
                   
                 (YP_726110.1); 
                 (550 aa) 
               
               
                   
                   
                   
                   
                   
                 31% identity 
                 “hypothetical 
               
               
                   
                   
                   
                   
                   
                 (550 aa) 
                 protein 
               
               
                   
                   
                   
                   
                   
                 “permease” 
                 NCgl0565” 
               
               
                   
                   
                   
                   
                   
                 (YP_725451.1) 
                 (NP_599826.1) 
               
               
                 ProV 
                 51% identity 
                 36-51% identity 
                 34-44% identity 
                 39-48% identity 
                 33-44% identity 
                 35-40% identity 
               
               
                 (400 aa) 
                 (390 aa) 
                 (222-352 aa) 
                 (224-361 aa) 
                 (247-392 aa) 
                 (195-354 aa) 
                 (226-283 aa) 
               
               
                   
                 “glycine/ 
                 “glycine 
                 “glycine/betaine/ 
                 “glycine 
                 “ABC transporter 
                 “ABC transporter 
               
               
                   
                 betaine ABC 
                 betaine/L-proline 
                 carnitine/choline 
                 betaine/carnitine/ 
                 ATPase” 
                 ATPase” 
               
               
                   
                 transporter 
                 ABC transporter 
                 ABC transporter 
                 choline ABC 
                 (YP_724876.1, 
                 (NP_599870.1, 
               
               
                   
                 ATP-binding 
                 ATP-binding 
                 ATP-binding 
                 transporter ATP- 
                 YP_726702.1, 
                 NP_601662.1, 
               
               
                   
                 protein” 
                 subunit” 
                 protein” 
                 binding protein” 
                 YP_725457.1, 
                 NP_599673.1, 
               
               
                   
                 (NP_388180.2) 
                 (NP_742461.1), 
                 (YP_003062931.1, 
                 (YP_003353988.1), 
                 YP_725326.1, 
                 NP_599959.1, 
               
               
                   
                   
                 “glycine 
                 YP_003061916.1) 
                 “glycine/betaine 
                 YP_726845.1, 
                 NP_600605.1, 
               
               
                   
                   
                 betaine/L-proline 
                   
                 ABC transporter 
                 YP_724565.1, 
                 NP_600550.1), 
               
               
                   
                   
                 ABC transporter 
                   
                 ATP-binding 
                 YP_727745.1, 
                 “glutamate ABC 
               
               
                   
                   
                 ATPase/permease 
                   
                 protein” 
                 YP_727463.1, 
                 transporter 
               
               
                   
                   
                 fusion protein” 
                   
                 (YP_003353316.1) 
                 YP_725974.1, 
                 ATPase” 
               
               
                   
                   
                 (NP_744918.1), 
                   
                   
                 YP_724993.1, 
                 (NP_601157.1), 
               
               
                   
                   
                 “glycine 
                   
                   
                 YP_727203.1, 
                 “ABC 
               
               
                   
                   
                 betaine/L-proline 
                   
                   
                 YP_725812.1, 
                 transporter 
               
               
                   
                   
                 ABC transporter 
                   
                   
                 YP_726707.1), 
                 duplicated 
               
               
                   
                   
                 ATPase” 
                   
                   
                 “ABC-type 
                 ATPase” 
               
               
                   
                   
                 (NP_743029.1) 
                   
                   
                 transporter, 
                 (NP_601199.1) 
               
               
                   
                   
                   
                   
                   
                 ATPase 
               
               
                   
                   
                   
                   
                   
                 component” 
               
               
                   
                   
                   
                   
                   
                 (YP_727764.1) 
               
               
                 ProW 
                 48% identity 
                 40-53% identity 
                 34-40% identity 
                 35-47% identity 
                 27-34% identity 
                 25-30% identity 
               
               
                 (354 aa) 
                 (275 aa) 
                 (206-265 aa) 
                 (161-169 aa) 
                 (155-284 aa) 
                 (152-194 aa) 
                 (157-232 aa) 
               
               
                   
                 “glycine 
                 “glycine 
                 “glycine 
                 “glycine 
                 “ABC 
                 “ABC 
               
               
                   
                 betaine 
                 betalne/L-proline 
                 betaine/carnitine/ 
                 betaine/carnitine/ 
                 transporter 
                 transporter 
               
               
                   
                 transport 
                 ABC transporter 
                 choline ABC 
                 choline ABC 
                 permease” 
                 permease” 
               
               
                   
                 system 
                 permease” 
                 transporter, 
                 transporter 
                 (YP_725456.1, 
                 (NP_600676.1, 
               
               
                   
                 permease 
                 (NP_742462.1), 
                 substrate binding 
                 permease/substrate- 
                 YP_725454.1, 
                 NP_600445.1) 
               
               
                   
                 protein OpuAB” 
                 “binding protein- 
                 and permease 
                 binding protein” 
                 YP_726844.1, 
               
               
                   
                 (NP_388181.1) 
                 dependent 
                 protein” 
                 (YP_003353987.1), 
                 YP_724987.1), 
               
               
                   
                   
                 transport system 
                 (YP_003061915.1), 
                 “glycine betaine 
                 “ABC-type 
               
               
                   
                   
                 inner membrane 
                 “glycine 
                 ABC transporter 
                 transporter, fused 
               
               
                   
                   
                 protein” 
                 betaine/carnitine/ 
                 permease/substrate- 
                 periplasmic and 
               
               
                   
                   
                 (NP_745696.1), 
                 choline ABC 
                 binding protein 
                 permease 
               
               
                   
                   
                 “glycine 
                 transporter, 
                 (YP_003353317.1) 
                 components” 
               
               
                   
                   
                 betaine/L-proline 
                 permease protein” 
                   
                 (YP_726088.1) 
               
               
                   
                   
                 ABC transporter 
                 (YP_003062932.1) 
               
               
                   
                   
                 ATPase/permease 
               
               
                   
                   
                 fusion protein” 
               
               
                   
                   
                 (NP_744918.1) 
               
               
                 ProX 
                 25% identity 
                 21-27% identity 
                 Not found 
                 22% identity 
                   29 %  identity   
                 Not found 
               
               
                 (330 aa) 
                 (124 aa) 
                 (155-327 aa) 
                   
                 (279 aa) 
                 ( 75 αα ) 
               
               
                   
                 “glycine 
                 “glycine betaine 
                   
                 “glycine 
                 
                   “RND 
                 
               
               
                   
                 betaine-binding 
                 ABC transporter 
                   
                 betaine/carnitine/ 
                 
                   superfamily 
                 
               
               
                   
                 protein OpuAC” 
                 substrate- 
                   
                 choline ABC 
                 
                   exporter” 
                 
               
               
                   
                 (NP_388182.1) 
                 binding protein” 
                   
                 transporter 
                 (YP_725351.1) 
               
               
                   
                   
                 (NP_745695.1, 
                   
                 permease/ 
               
               
                   
                   
                 NP_744919.1), 
                   
                 substrate-binding 
               
               
                   
                   
                 “glycine/betaine- 
                   
                 protein” 
               
               
                   
                   
                 binding protein” 
                   
                 (YP_003353987.1) 
               
               
                   
                   
                 (NP_742246.1) 
               
               
                 ProQ 
                   24 %  identity   
                 27% identity 
                 Not found 
                 Not found 
                   29 %  identity   
                   33 %  identity   
               
               
                 (232 aa) 
                 ( 128 αα ) 
                 (115 aa) 
                   
                   
                 ( 76 αα ) 
                 ( 66 αα ) 
               
               
                   
                 
                   “hypothetical 
                 
                 “ProQ 
                   
                   
                 
                   “dehydrogenase” 
                 
                 
                   “elongation 
                 
               
               
                   
                 
                   protein 
                 
                 activator of 
                   
                   
                 (YP_724959.1) 
                 
                   factor Ts” 
                 
               
               
                   
                 
                   BSU32070” 
                 
                 osmoprotectant 
                   
                   
                   
                 (NP_601230.1) 
               
               
                   
                 (NP_391087.1) 
                 transporter 
               
               
                   
                   
                 ProP” 
               
               
                   
                   
                 (NP_744331.1) 
               
               
                 CspE 
                 61-68% identity 
                 54-62% identity 
                 65-69% identity 
                 62-66% identity 
                 62% identity 
                 63-65% identity 
               
               
                 (69 aa) 
                 (62-64 aa) 
                 (61-68 aa) “cold 
                 (61-62 aa) 
                 (61-62 aa) 
                 (61 aa) 
                 (65 aa) 
               
               
                   
                 “cold shock 
                 shock protein 
                 “cold shock 
                 “cold-shock 
                 “cold shock 
                 “cold shock 
               
               
                   
                 protein 
                 CspA” 
                 protein 
                 protein” 
                 protein, DNA- 
                 protein” 
               
               
                   
                 CspB” 
                 (NP_743679.1), 
                 CspP” 
                 (YP_003352748.1, 
                 binding” 
                 (NP_599426.1, 
               
               
                   
                 (NP_388791.1), 
                 “cold-shock 
                 (YP_003062538.1), 
                 YP_003353646.1, 
                 (YP_727497.1) 
                 NP_599560.1) 
               
               
                   
                 “cold shock 
                 domain-contain 
                 “cold shock 
                 YP_003352635.1, 
               
               
                   
                 protein CspC” 
                 protein” 
                 protein CspL” 
                 YP_003354678.1) 
               
               
                   
                 (NP_388393.1), 
                 (NP_743146.1, 
                 (YP_003061635.1, 
               
               
                   
                 “cold shock 
                 NP_743260.1), 
                 YP_003061614.1), 
               
               
                   
                 protein CspD” 
                 “cold shock 
                 “cold shock 
               
               
                   
                 (NP_390076.1) 
                 protein CspA” 
                 protein CspC” 
               
               
                   
                   
                 (NP_744611.1), 
                 (YP_003062410.1) 
               
               
                   
                   
                 “cold-shock 
               
               
                   
                   
                 domain-contain 
               
               
                   
                   
                 protein, partial” 
               
               
                   
                   
                 (NP_743369.1), 
               
               
                   
                   
                 “cold-shock 
               
               
                   
                   
                 protein CspD” 
               
               
                   
                   
                 (NP_746140.1) 
               
               
                 RfaE 
                 32% identity 
                 57% identity 
                 34% identity 
                 35% identity 
                 49% identity 
                 28% identity 
               
               
                 (477 aa) 
                 (133 aa) 
                 (474 aa) 
                 (128 aa) 
                 (98 aa) 
                 (312 aa) 
                 (120 aa) 
               
               
                   
                 “glycerol-3- 
                 “bifunctional 
                 “glycerol-3- 
                 “glycerol-3- 
                 “D-beta-D- 
                 “ribokinase 
               
               
                   
                 phosphate 
                 heptose-7- 
                 phosphate 
                 phosphate 
                 heptose 7- 
                 sugar kinase” 
               
               
                   
                 cytidylyltrans- 
                 phosphate 
                 cytidylyltransferase” 
                 cytidylyltransferase” 
                 phophosphate 
                 (NP_599410.1) 
               
               
                   
                 ferase”, 24-26% 
                 kinase/heptose- 
                 (YP_003062640.1), 
                 (YP_003352686.1); 
                 kinase” 
               
               
                   
                 identity 
                 1-phosphate 
                 24% identity 
                 33% identity 
                 (YP_725318.1) 
               
               
                   
                 (228-327 aa) 
                 adenyltransferase” 
                 (240-309 aa) 
                 (147 aa) 
               
               
                   
                 “ribokinase” 
                 (NP_747037.1) 
                 “ribokinase” 
                 “glycerol-3- 
               
               
                   
                 (NP_391473.1), 
                   
                 (YP_003064511.1, 
                 phosphate 
               
               
                   
                 “sugar kinase 
                   
                 YP_003063470.1) 
                 cytidyltransferase” 
               
               
                   
                 YdjE” 
                   
                   
                 (YP_003353407.1) 
               
               
                   
                 (NP_388498.1), 
               
               
                   
                 “fructosamine 
               
               
                   
                 kinase FrlD” 
               
               
                   
                 (NP_391137.1) 
               
               
                 YfbP 
                   28 %  identity   
                   24 %  identity   
                 Not found 
                   24 %  identity   
                   29 %  identity   
                 33% identity 
               
               
                 (282 aa) 
                 ( 68 αα ) 
                 ( 148 αα ) 
                   
                 ( 106 αα ) 
                 ( 106 αα ) 
                 (39 aa) 
               
               
                   
                 
                   “sensor 
                 
                 
                   “tryptophan 
                 
                   
                 
                   “multimodular 
                 
                   “O - linked N - 
                 “hypothetical 
               
               
                   
                 
                   histidine 
                 
                 
                   synthase subunit 
                 
                   
                   transpeptidase - 
                 
                   acetylglucosamine 
                 
                 protein 
               
               
                   
                 
                   kinase” 
                 
                 
                   alpha” 
                 
                   
                 
                   transglycosylase 
                 
                 
                   transferase OGT” 
                 
                 NCgl0374” 
               
               
                   
                 (NP_391844.1) 
                 (NP_742252.1) 
                   
                 
                   Pbp2A” 
                 
                 (YP_724894.1) 
                 (NP_599633.1) 
               
               
                   
                   
                   
                   
                 (YP_003354748.1) 
               
               
                 YfjM 
                 Not found 
                   30 %  identity   
                   26 %  identity   
                 Not found 
                 Not found 
                 Not found 
               
               
                 (87 aa) 
                   
                 ( 54 αα ) 
                 ( 53 αα ) 
               
               
                   
                   
                 
                   “ABC transporter 
                 
                 
                   “small heat 
                 
               
               
                   
                   
                   substrate - 
                 
                   shock protein” 
                 
               
               
                   
                   
                 
                   binding protein” 
                 
                 (YP_003064271.1) 
               
               
                   
                   
                 (NP_744413.1) 
               
               
                 PstS 
                 26% identity 
                 39% identity 
                 27-28% identity 
                 26% identity 
                 64% identity 
                 30% identity 
               
               
                 (346 aa) 
                 (229 aa) 
                 (339 aa) 
                 (224-318 aa) 
                 (232-251 aa) 
                 (347 aa) 
                 (282 aa) 
               
               
                   
                 “phosphate- 
                 “phosphate 
                 “phosphate 
                 “phosphate 
                 “ABC 
                 “ABC 
               
               
                   
                 binding protein 
                 ABC transporter 
                 ABC transporter 
                 ABC transporter 
                 transporter 
                 transporter 
               
               
                   
                 PstS” 
                 substrate- 
                 substrate-binding 
                 substrate-binding 
                 periplasmic 
                 periplasmic 
               
               
                   
                 (NP_390378.1) 
                 binding protein” 
                 protein” 
                 protein” 
                 protein” 
                 component” 
               
               
                   
                   
                 (NP_744800.1) 
                 (YP_003062200.1, 
                 (YP_003354292.1, 
                 (YP_726901.1) 
                 (NP_601773.1) 
               
               
                   
                   
                   
                 YP_003062190.1) 
                 YP_003354291.1) 
               
               
                 Rph 
                 58% identity 
                 69% identity 
                 Not found 
                 24-27% identity 
                 62% identity 
                 59% identity 
               
               
                 (228 aa) 
                 (222 aa) 
                 (228 aa) 
                   
                 (59-207 aa in 
                 (221 aa) 
                 (217 aa) 
               
               
                   
                 “ribonuclease 
                 “ribonuclease 
                   
                 stretches) 
                 “ribonuclease 
                 “ribonuclease 
               
               
                   
                 PH” 
                 PH” 
                   
                 “polyribo- 
                 PH” 
                 PH” 
               
               
                   
                 (NP_390715.1) 
                 (NP_747395.1) 
                   
                 nucleotide 
                 (YP_725462.1) 
                 (NP_601703.2) 
               
               
                   
                   
                   
                   
                 nucleotidyltrans- 
               
               
                   
                   
                   
                   
                 ferase” 
               
               
                   
                   
                   
                   
                 (YP_003354448.1) 
               
               
                 RpoS 
                 43% identity 
                 76% identity 
                 44% identity 
                 40% identity 
                 52% identity 
                 39% identity 
               
               
                 (330 aa) 
                 (321 aa) “RNA 
                 (277 aa) “RNA 
                 (271 aa) “RNA 
                 (288 aa) “RNA 
                 (280 aa) “RNA 
                 (316 aa) “RNA 
               
               
                   
                 polymerase 
                 polymerase 
                 polymerase 
                 polymerase 
                 polymerase 
                 polymerase 
               
               
                   
                 sigma factor 
                 sigma factor 
                 sigma factor 
                 sigma factor 
                 sigma factor 
                 sigma factor 
               
               
                   
                 RpoD” 
                 RpoS” 
                 RpoD” 
                 RpoD” 
                 RpoS” 
                 SigB” 
               
               
                   
                 (NP_390399.2) 
                 (NP_743780.1) 
                 (YP_003063237.1) 
                 (YP_003352999.1) 
                 (YP_726836.1) 
                 (NP_601125.1), 
               
               
                   
                   
                   
                   
                   
                   
                 36% identity 
               
               
                   
                   
                   
                   
                   
                   
                 (304 aa) “RNA 
               
               
                   
                   
                   
                   
                   
                   
                 polymerase 
               
               
                   
                   
                   
                   
                   
                   
                 sigma factor” 
               
               
                   
                   
                   
                   
                   
                   
                 (NP_601117.2) 
               
               
                 SspA 
                 Not found 
                 57% identity 
                   45 %  identity   
                 Not found 
                 46% identity 
                   56 %  identity   
               
               
                 (212 aa) 
                   
                 (200 aa) 
                 ( 22 αα ) 
                   
                 (203 aa) 
                 ( 16 αα ) 
               
               
                   
                   
                 “stringent 
                 
                   “hypothetical 
                 
                   
                 “stringent 
                 
                   “hypothetical 
                 
               
               
                   
                   
                 starvation 
                 
                   protein 
                 
                   
                 starvation protein 
                 
                   protein 
                 
               
               
                   
                   
                 protein A” 
                 
                   JDM1 
                   — 
                   0823” 
                 
                   
                 A” (YP_727831.1) 
                 
                   NCgl2333” 
                 
               
               
                   
                   
                 (NP_743480.1) 
                 (YP_003062407.1) 
                   
                   
                 (NP_601617.1) 
               
               
                 Tdk 
                 30% identity 
                 Not found 
                 45% identity 
                 49% identity 
                   25 %  identity   
                 Not found 
               
               
                 (205 aa) 
                 (184 aa) 
                   
                 (152 aa) 
                 (187 aa) 
                 ( 132 αα ) 
               
               
                   
                 “thymidine 
                   
                 “thymidine 
                 “thymidine 
                   “prolyl - tRNA   
               
               
                   
                 kinase” 
                   
                 kinase” 
                 kinase” 
                 
                   synthetase” 
                 
               
               
                   
                 (NP_391587.1) 
                   
                 (YP_003063573.1) 
                 (YP_003353039.1) 
                 (YP_727689.1) 
               
               
                 UvrB 
                 59% identity 
                 69% identity 
                 59% identity 
                 55% identity 
                 66% identity 
                 56% identity 
               
               
                 (673 aa) 
                 (666 aa) 
                 (670 aa) 
                 (663 aa) 
                 (693 aa) 
                 (673 aa) 
                 (668 aa) 
               
               
                   
                 “UvrABC 
                 “excinuclease 
                 “exonuclease 
                 “excinuclease 
                 “excinuclease 
                 “excinuclease 
               
               
                   
                 system protein 
                 ABC subunit B” 
                 ABC subunit B” 
                 ABC subunit B” 
                 ABC subunit B” 
                 ABC subunit B” 
               
               
                   
                 B” 
                 (NP_744125.1) 
                 (YP_003062224.1) 
                 (YP_003353008.1) 
                 (YP_725661.1) 
                 (NP_600587.1) 
               
               
                   
                 (NP_391397.1) 
               
               
                 YcjG 
                 31% identity 
                 26% identity 
                   31 %  identity   
                 24% identity 
                 26% identity 
                 27% identity 
               
               
                 (321 aa) 
                 (322 aa) 
                 (337 aa) 
                 ( 91 αα ) 
                 (338 aa) “O- 
                 (324 aa) 
                 (290 aa) 
               
               
                   
                 “L-Ala- 
                 “muconate and 
                 
                   “phosphopyruvate 
                 
                 succinylbenzoate 
                 “muconate 
                 “chloromuconate 
               
               
                   
                 D/L-Glu 
                 chloromuconate 
                 
                   hydratase” 
                 
                 synthase” 
                 cyclo-isomerase” 
                 cycloisomerase” 
               
               
                   
                 epimerase” 
                 cycloisomerase” 
                 (YP_003063198.1) 
                 (YP_003353203.1) 
                 (YP_726435.1) 
                 (NP_601602.2) 
               
               
                   
                 (NP_389181.1) 
                 (NP_745848.1) 
               
               
                 YeaR 
                   26 %  identity   
                   24 %  identity   
                 Not found 
                 Not found 
                 Not found 
                 Not found 
               
               
                 (119 aa) 
                 ( 91 αα ) 
                 ( 41 αα ) 
               
               
                   
                 
                   “hypothetical 
                 
                 
                   “30S ribosomal 
                 
               
               
                   
                 
                   protein 
                 
                 
                   protein S12” 
                 
               
               
                   
                 
                   BSU13060” 
                 
                 (NP_742615.1) 
               
               
                   
                 (NP_389189.1) 
               
               
                 SpoT 
                 40% identity 
                 37-55% identity 
                 38% identity 
                 40% identity 
                 36-47% identity 
                 38% identity 
               
               
                 (702 aa) 
                 (719 aa) “GTP 
                 (681-701 aa) 
                 (741 aa) “GTP 
                 (725 aa) “GTP 
                 (674-720 aa) “GTP 
                 (723 aa) 
               
               
                   
                 pyrophosphokinase” 
                 “(p)ppGpp 
                 pyrophosphokinase” 
                 pyrophosphokinase/ 
                 pyrophosphokinase” 
                 “guanosine 
               
               
                   
                 (NP_390638.2) 
                 synthetase I 
                 (YP_003063260.1) 
                 guanosine-3,5- 
                 (YP_725468.1, 
                 polyphosphate 
               
               
                   
                   
                 SpoT/RelA” 
                   
                 bis(diphosphate) 3- 
                 YP_725845.1) 
                 pyrophospho- 
               
               
                   
                   
                 (NP_747403.1, 
                   
                 pyrophospho- 
                   
                 hydrolase/ 
               
               
                   
                   
                 NP_743813.1) 
                   
                 hydrolase” 
                   
                 synthetase” 
               
               
                   
                   
                   
                   
                 (YP_003352549.1) 
                   
                 (NP_600866.1) 
               
               
                 PolB 
                 Not found 
                 68% identity 
                 Not found 
                   32 %  identity   
                 68% identity 
                   35 %  identity   
               
               
                 (783 aa) 
                   
                 (784 aa) “DNA 
                   
                 ( 63 αα ) 
                 (784 aa) “DNA 
                 ( 55 αα ) 
               
               
                   
                   
                 polymerase II” 
                   
                 
                   “O-succinylbenzoate 
                 
                 polymerase II” 
                   “rhodanese - 
               
               
                   
                   
                 (NP_744541.1) 
                   
                 
                   synthase” 
                 
                 (YP_726151.1) 
                 
                   related 
                 
               
               
                   
                   
                   
                   
                 (YP_003353203.1) 
                   
                 
                   sulfurtransferase” 
                 
               
               
                   
                   
                   
                   
                   
                   
                 (NP_599306.1) 
               
               
                 RpoB 
                 59% identity 
                 72% identity 
                 47-52% identity 
                 46-47% identity 
                 66% identity 
                 41-56% identity 
               
               
                 (1342 aa) 
                 (533 aa) “DNA- 
                 (1360 aa) “DNA- 
                 (304-953 aa in 
                 (307-951 aa in 
                 (1370 aa) 
                 (238-616 aa in 
               
               
                   
                 directed RNA 
                 directed RNA 
                 stretches) “DNA- 
                 stretches) “DNA- 
                 “DNA-directed 
                 stretches) 
               
               
                   
                 polymerase 
                 polymerase 
                 directed RNA 
                 directed RNA 
                 RNA polymerase 
                 “DNA-directed 
               
               
                   
                 subunit beta” 
                 subunit beta” 
                 polymerase 
                 polymerase 
                 subunit beta” 
                 RNA 
               
               
                   
                 (NP_387988.2) 
                 (NP_742613.1) 
                 subunit beta” 
                 subunit beta” 
                 (YP_727933.1) 
                 polymerase 
               
               
                   
                   
                   
                 (YP_003062426.1) 
                 (YP_003354373.1) 
                   
                 subunit beta” 
               
               
                   
                   
                   
                   
                   
                   
                 (NP_599733.1) 
               
               
                 RpoC 
                 50% identity 
                 75% identity 
                 44-51% identity 
                 48-51% identity 
                 67% identity 
                 46-50% identity 
               
               
                 (1407 aa) 
                 (1134 aa) 
                 (1399 aa) “DNA- 
                 (235-1061 aa in 
                 (238-1043 aa in 
                 (1397 aa) 
                 (206-819 aa in 
               
               
                   
                 “DNA-directed 
                 directed RNA 
                 stretches) “DNA- 
                 stretches) “DNA- 
                 “DNA-directed 
                 stretches) 
               
               
                   
                 RNA 
                 polymerase 
                 directed RNA 
                 directed RNA 
                 RNA polymerase 
                 “DNA-directed 
               
               
                   
                 polymerase 
                 subunit beta&#39;” 
                 polymerase 
                 polymerase 
                 subunit beta&#39;” 
                 RNA 
               
               
                   
                 subunit beta&#39;” 
                 (NP_742614.1) 
                 subunit beta&#39;” 
                 subunit beta&#39;” 
                 (YP_727932.1) 
                 polymerase 
               
               
                   
                 (NP_387989.2) 
                   
                 (YP_003062427.1) 
                 (YP_003354372.1) 
                   
                 subunit beta&#39;” 
               
               
                   
                   
                   
                   
                   
                   
                 (NP_599734.1) 
               
               
                 Rnt 
                 27% identity 
                 63% identity 
                 28% identity 
                 29% identity 
                 27% identity 
                 Not found 
               
               
                 (215 aa) 
                 (191 aa) “DNA 
                 (198 aa) 
                 (180 aa) “DNA 
                 (206 aa) “DNA 
                 (180 aa) “DNA 
               
               
                   
                 polymerase III 
                 “Ribonuclease T” 
                 polymerase III 
                 polymerase III 
                 polymerase III 
               
               
                   
                 PolC-type” 
                 (NP_743246.1) 
                 PolC” 
                 subunit alpha” 
                 subunit epsilon” 
               
               
                   
                 (NP_389540.1) 
                   
                 (YP_003063293.1) 
                 (YP_003354765.1) 
                 (YP_726924.1) 
               
               
                 SapC 
                 28-30% identity 
                 42% identity 
                 25% identity 
                 23-27% identity 
                 34-37% identity 
                 31-38% identity 
               
               
                 (296 aa) 
                 (286-288 aa) 
                 (281 aa) 
                 (302 aa) 
                 (219-306 aa) 
                 (216-238 aa) 
                 (215-230 aa) 
               
               
                   
                 “oligopeptide 
                 “binding- 
                 “peptide ABC 
                 “peptide ABC 
                 “ABC 
                 “ABC 
               
               
                   
                 transport system 
                 protein- 
                 transporter 
                 transporter 
                 transporter 
                 transporter 
               
               
                   
                 permease 
                 dependent 
                 permease” 
                 permease” 
                 permease” 
                 permease” 
               
               
                   
                 protein AppC” 
                 transport system 
                 (YP_003062653.1) 
                 (YP_003354430.1, 
                 (YP_725975.1, 
                 (NP_601522.1, 
               
               
                   
                 (NP_389022.1), 
                 inner membrane 
                   
                 YP_003352873.1) 
                 YP_726565.1, 
                 NP_601635.1, 
               
               
                   
                 “dipeptide 
                 protein” 
                   
                   
                 YP_726551.1, 
                 NP_601198.1) 
               
               
                   
                 transport system 
                 (NP_743041.1) 
                   
                   
                 YP_727395.1) 
               
               
                   
                 permease 
               
               
                   
                 protein DppC” 
               
               
                   
                 (NP_389177.1), 
               
               
                   
                 “oligopeptide 
               
               
                   
                 transport system 
               
               
                   
                 permease 
               
               
                   
                 protein OppC” 
               
               
                   
                 (NP_389027.1) 
               
               
                 PyrE 
                 25-34% identity 
                 67% identity 
                 29% identity 
                 30% identity 
                 56% identity 
                 29% identity 
               
               
                 (213 aa) 
                 (in stretches) 
                 (213 aa) 
                 (138 aa) 
                 (131 aa) 
                 (215 aa) 
                 (139 aa) 
               
               
                   
                 “orotate 
                 “orotate 
                 “orotate 
                 “orotate 
                 “orotate 
                 “orotate 
               
               
                   
                 phosphoribosyl- 
                 phosphoribosyl- 
                 phosphoribosyl- 
                 phosphoribosyl- 
                 phosphoribosyl- 
                 phosphoribosyl- 
               
               
                   
                 transferase” 
                 transferase” 
                 transferase” 
                 transferase” 
                 transferase” 
                 transferase” 
               
               
                   
                 (NP_389439.1) 
                 (NP_747392.1) 
                 (YP_003063746.1) 
                 (YP_003353548.1) 
                 (YP_724744.1) 
                 (NP_601967.1) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Homologs or orthologs identified by protein BLAST (BLASTP) of  E. coli  K-12 MG1655 proteins against 
               
               
                 protein databases from the two chromosomes and two endogenous plasmids of  Deinococcus radiodurans   
               
               
                 R1. Hits with the largest e-values are shown, and hits are only shown when the e-value &lt; 1.0. 
               
            
           
           
               
               
               
               
               
            
               
                 Protein 
                   D. radiodurans  R1 
                   D. radiodurans  R1 
                   D. radiodurans  R1 
                   D. radiodurans  R1 
               
               
                 (# of 
                 chromosome 1 
                 chromosome 2 
                 circular plasmid 1 
                 megaplasmid 1 
               
               
                 residues) 
                 (NC_001263) 
                 (NC_001264) 
                 (NC_000959) 
                 (NC_000958) 
               
               
                   
               
               
                 KgtP 
                 36% identity (56 aa) 
                 30% identity (107 aa) 
                 32% identity (73 aa) 
                 58% identity (17 aa) 
               
               
                 (432 aa) 
                 “hypothetical protein 
                 “sugar transporter 
                 “hypothetical protein 
                 “hypothetical protein 
               
               
                   
                 DR_1056” 
                 putative” 
                 DR_C0021” 
                 DR_B0098” 
               
               
                   
                 (NP_294780.1) 
                 (NP_285594.1) 
                 (NP_051691.1) 
                 (NP_051631.1) 
               
               
                 YbjL 
                 24% identity (169 aa) 
                 33% identity (52 aa) 
                 Not found 
                 Not found 
               
               
                 (561 aa) 
                 “sodium/sulfate symporter 
                 “transcriptional 
               
               
                   
                 family protein” (NP_295134.1) 
                 regulator” (NP_285659.1) 
               
               
                 ProV 
                 35-41% identity (196-265 aa) 
                 27-38% identity (215-317 aa) 
                 36% identity (28 aa) 
                 32% identity (186-231 aa) 
               
               
                 (400 aa) 
                 “spermidine/putrescine ABC 
                 “amino acid ABC 
                 “transposase-related” 
                 “iron ABC transporter 
               
               
                   
                 transporter ATP-binding 
                 transporter ATP-binding 
                 (NP_051690.1) 
                 ATP-binding protein” 
               
               
                   
                 protein” (NP_295026.1), “ABC 
                 protein” (NP_285461.1), 
                   
                 (NP_051651.1), “ABC 
               
               
                   
                 transporter ATP-binding 
                 “phosphate ABC 
                   
                 transporter, ATP-binding 
               
               
                   
                 protein” (NP_295079.1, 
                 transporter ATP-binding 
                   
                 protein” (NP_051588.1) 
               
               
                   
                 NP_295920.1, NP_293788.1), 
                 protein” (NP_285484.1), 
               
               
                   
                 “amino acid ABC transporter 
                 “branched-chain amino 
               
               
                   
                 ATP-binding protein” 
                 acid ABC transporter 
               
               
                   
                 (NP_295371.1), “sugar ABC 
                 ATP-binding protein” 
               
               
                   
                 transporter, ATP-binding 
                 (NP_285584.1), “ABC 
               
               
                   
                 protein” (NP_295876.1), 
                 transporter ATP-binding 
               
               
                   
                 “peptide ABC transporter 
                 protein” (NP_285331.1, 
               
               
                   
                 ATP-binding protein” 
                 NP_285672.1), 
               
               
                   
                 (NP_295290.1) 
                 “urea/short-chain amide 
               
               
                   
                   
                 ABC transporter 
               
               
                   
                   
                 ATP-binding protein” 
               
               
                   
                   
                 (NP_285647.1) 
               
               
                 ProW 
                 26-31% identity (116-183 aa) 
                 30-35% identity (114-158 aa) 
                 29% identity (62 aa) 
                 28% identity (57 aa) 
               
               
                 (354 aa) 
                 “ABC transporter ATP-binding 
                 “amino acid ABC 
                 “transposase, putative” 
                 “iron ABC transporter 
               
               
                   
                 protein” (NP_294234.1), “ABC 
                 transporter permease” 
                 (NP_051699.1) 
                 permease” 
               
               
                   
                 transporter permease” 
                 (NP_285462.1, 
                   
                 (NP_051652.1) 
               
               
                   
                 (NP_295919.1) 
                 NP_285460.1) 
               
               
                 ProX 
                 Not found 
                 29% identity (68 aa) 
                 29% identity (41 aa) 
                 65% identity (17 aa) 
               
               
                 (330 aa) 
                   
                 “putative FAD-binding 
                 “phosphoenolpyruvate 
                 “hypothetical protein 
               
               
                   
                   
                 dehydrogenase” 
                 synthase-related protein” 
                 DR_B0023” 
               
               
                   
                   
                 (NP_285561.1) 
                 (NP_051677.1) 
                 (NP_051564.1) 
               
               
                 ProQ 
                 31% identity (58 aa) 
                 Not found 
                 Not found 
                 Not found 
               
               
                 (232 aa) 
                 “hypothetical protein 
               
               
                   
                 DR_2622” (NP_296341.1) 
               
               
                 CspE 
                 59% identity (64 aa) “CSD 
                 36-39% identity (31-36 aa) 
                 22-33% identity (45-50 aa) 
                 Not found 
               
               
                 (69 aa) 
                 family cold shock protein” 
                 “methyl-accepting 
                 “hypothetical protein 
               
               
                   
                 (NP_294631.1) 
                 chemotaxis-like protein” 
                 DR_C0022” 
               
               
                   
                   
                 (NP_285676.1), “methyl- 
                 (NP_051692.1), 
               
               
                   
                   
                 accepting chemotaxis 
                 “nodulation protein- 
               
               
                   
                   
                 protein” (NP_285677.1) 
                 related protein” 
               
               
                   
                   
                   
                 (NP_051705.1) 
               
               
                 RfaE 
                 26% identity (274 aa) 
                 33% identity (78 aa) 
                 30% identity (54 aa) 
                 26% identity (248 aa) 
               
               
                 (477 aa) 
                 “carbohydrate kinase” 
                 “ribokinase” 
                 “coenzyme PQQ synthesis 
                 “1-phospho- 
               
               
                   
                 (NP_296273.1) 
                 (NP_285378.1) 
                 protein, putative” 
                 fructokinase” 
               
               
                   
                   
                   
                 (NP_051702.1) 
                 (NP_051610.1) 
               
               
                 YfbP 
                 Not found 
                 39% identity (61 aa) 
                 21% identity (85 aa) 
                 Not found 
               
               
                 (282 aa) 
                   
                 “hypothetical protein 
                 “oxidative cyclase, 
               
               
                   
                   
                 DR_A0109” 
                 putative” 
               
               
                   
                   
                 (NP_285432.1) 
                 (NP_051704.1) 
               
               
                 YfjM 
                 Not found 
                 Not found 
                 Not found 
                 Not found 
               
               
                 (87 aa) 
               
               
                 PstS 
                 Not found 
                 44% identity (336 aa) 
                 Not found 
                 36% identity (47 aa) 
               
               
                 (346 aa) 
                   
                 “phosphate ABC 
                   
                 “hypothetical protein 
               
               
                   
                   
                 transporter periplasmic 
                   
                 DR_B0023” 
               
               
                   
                   
                 phosphate-binding 
                   
                 (NP_051564.1) 
               
               
                   
                   
                 protein” (NP_285481.1) 
               
               
                 Rph 
                 44% identity (215 aa) 
                 33% identity (62 aa) 
                 Not found 
                 35% identity (31 aa) “iron- 
               
               
                 (228 aa) 
                 “ribonuclease PH” 
                 “uroporphyrin-III C- 
                   
                 chelator utilization 
               
               
                   
                 (NP_295308.1) 
                 methyltransferase/ 
                   
                 protein” 
               
               
                   
                   
                 uroporphyrinogen-III 
                   
                 (NP_051560.1) 
               
               
                   
                   
                 synthase (NP_285335.1) 
               
               
                 RpoS 
                 42% identity (288 aa) “RNA 
                 30% identity (47 aa) 
                 34% identity (50 aa) 
                 Not found 
               
               
                 (330 aa) 
                 polymerase sigma-A factor” 
                 “hypothetical protein 
                 “hypothetical protein 
               
               
                   
                 (NP_294640.1) 
                 DR_A0192” 
                 DR_C0027” 
               
               
                   
                   
                 (NP_285515.1) 
                 (NP_051697.1) 
               
               
                 SspA 
                 24-29% identity (48-99 aa) 
                 35% identity (46 aa) “P49 
                 24% identity (71 aa) 
                 Not found 
               
               
                 (212 aa) 
                 “glutaredoxin” (NP_295808.1), 
                 secreted protein” 
                 “hypothetical protein 
               
               
                   
                 “hypothetical protein 
                 (NP_285686.1) 
                 DR_C0009” 
               
               
                   
                 DR_0390” (NP_294113.1) 
                   
                 (NP_051682.1) 
               
               
                 Tdk 
                 27% identity (191 aa) 
                 24% identity (80 aa) 
                 27% identity (52 aa) 
                 38% Identity (50 aa) 
               
               
                 (205 aa) 
                 “thymidine kinase” 
                 “urea/short-chain amide 
                 “putative transposase” 
                 “ABC transporter, 
               
               
                   
                 (NP_295707.1) 
                 ABC transporter 
                 (NP_277100.1) 
                 ATP-binding 
               
               
                   
                   
                 periplasmic urea/short- 
                   
                 protein” 
               
               
                   
                   
                 chain amide-binding 
                   
                 (NP_051588.1) 
               
               
                   
                   
                 protein” (NP_285643.1) 
               
               
                 UvrB 
                 56% identity (661 aa) 
                 33% identity (96 aa) 
                 Not found 
                 Not found 
               
               
                 (673 aa) 
                 “excinuclease ABC subunit B” 
                 “hypothetical protein 
               
               
                   
                 (NP_295996.1) 
                 DR_A0131” 
               
               
                   
                   
                 (NP_285455.1) 
               
               
                 YcjG 
                 24-30% identity (285-306 aa) 
                 Not found 
                 Not found 
                 29% identity (201 aa) 
               
               
                 (321 aa) 
                 “chloromuconate 
                   
                   
                 “N-acylamino acid 
               
               
                   
                 cyclosisomerase” 
                   
                   
                 racemase” 
               
               
                   
                 (NP_295594.1), “N-acylamino 
                   
                   
                 (NP_051613.1) 
               
               
                   
                 acid racemase” 
               
               
                   
                 (NP_293770.1) 
               
               
                 YeaR 
                 Not found 
                 37% identity (35 aa) 
                 29% identity (68 aa) 
                 30% identity (89 aa) 
               
               
                 (119 aa) 
                   
                 “exopolyphosphatase” 
                 “nodulation protein- 
                 “KdpD-related protein” 
               
               
                   
                   
                 (NP_285509.1) 
                 related protein” 
                 (NP_051621.1) 
               
               
                   
                   
                   
                 (NP_051705.1) 
               
               
                 SpoT 
                 38% identity (734 aa) “GTP 
                 30% identity (53 aa) 
                 Not found 
                 Not found 
               
               
                 (702 aa) 
                 pyrophosphokinase” 
                 “long-chain-fatty-acid- 
               
               
                   
                 (NP_295561.1) 
                 CoA ligase” 
               
               
                   
                   
                 (NP_296364.1) 
               
               
                 PolB 
                 24-32% identity (65-135 aa) 
                 34% identity (65 aa) 
                 54% identity (13 aa) 
                 30% identity (69 aa) 
               
               
                 (783 aa) 
                 “excinuclease ABC subunit A” 
                 “P49 secreted protein” 
                 “hypothetical protein 
                 “hypothetical protein 
               
               
                   
                 (NP_295494.1), “hypothetical 
                 (NP_285686.1) 
                 DR_C0014” 
                 DR_B0054” 
               
               
                   
                 protein DR_2521” 
                   
                 (NP_051686.1) 
                 (NP_051592.1) 
               
               
                   
                 (NP_296241.1) 
               
               
                 RpoB 
                 40-53% identity (in stretches, 
                 29% identity (63 aa) 
                 33% identity (40 aa) 
                 34% identity (44 aa) 
               
               
                 (1342 aa) 
                 227-620 aa) “DNA-directed 
                 “hypothetical protein 
                 “modification methylase, 
                 “hypothetical protein 
               
               
                   
                 RNA polymerase subunit beta” 
                 DR_A0017” 
                 putative” 
                 DR_B0144” 
               
               
                   
                 (NP_ 294636.1) 
                 (NP_285341.1) 
                 (NP_277101.1) 
                 (NP_051673.1) 
               
               
                 RpoC 
                 45-58% identity (in stretches, 
                 37% identity (30 aa) 
                 48% identity (27 aa) 
                 32% identity (62 aa) 
               
               
                 (1407 aa) 
                 156-937 aa) “DNA-directed 
                 “succinate-semialdehyde 
                 “putative transposase” 
                 “hypothetical protein 
               
               
                   
                 RNA polymerase subunit 
                 dehydrogenase” 
                 (NP_277100.1) 
                 DR_B0013” 
               
               
                   
                 beta&#39;” (NP_294635.1) 
                 (NP_285327.1) 
                   
                 (NP_051556.1) 
               
               
                 Rnt 
                 25% identity (192 aa) “DNA 
                 38% identity (37 aa) 
                 22% identity (79 aa) 
                 Not found 
               
               
                 (215 aa) 
                 polymerase III subunit 
                 “transcriptional 
                 “hypothetical protein 
               
               
                   
                 epsilon” 
                 regulator” 
                 DR_C0027” 
               
               
                   
                 (NP_294580.1) 
                 (NP_285659.1) 
                 (NP_051697.1) 
               
               
                 SapC 
                 26-39% identity (215-230 aa) 
                 32% identity (248 aa) 
                 Not found 
                 39% identity (23 aa) 
               
               
                 (296 aa) 
                 “peptide ABC transporter 
                 “peptide ABC transporter 
                   
                 “sensor histidine kinase, 
               
               
                   
                 permease” (NP_294682.1, 
                 permease” 
                   
                 copper metabolism” 
               
               
                   
                 NP_295292.1, NP_294088.1) 
                 (NP_285531.1) 
                   
                 (NP_051623.1) 
               
               
                 PyrE 
                 31% identity (154 aa) “orotate 
                 32% identity (86 aa) 
                 Not found 
                 38% identity (39 aa) 
               
               
                 (213 aa) 
                 phosphoribosyltransferase” 
                 “serine protease” 
                   
                 “hypothetical protein 
               
               
                   
                 (NP_294170.1) 
                 (NP_285606.1) 
                   
                 DR_B0068” (NP_051604.1) 
               
               
                   
               
            
           
         
       
     
     So, in one aspect, there is provided a bacterial cell according to any one of the preceding aspects and embodiments, wherein each recited gene is instead (i) a gene encoding the corresponding (homolog or ortholog) protein in Table 3 or 4 (ii) a gene located at the corresponding locus, or (iii) both. 
     Example 1 
     Methods 
     Screening for Tolerance in Wild-Type Cells 
       Escherichia coli  K-12 MG1655 was grown overnight in M9 minimal medium+1% glucose and subcultured the following morning to an initial OD 600  of 0.05 in M9+1% glucose. Cells were grown to mid-exponential phase (OD 600  0.7-1.0) and were back-diluted with fresh medium to an OD 600  of 0.7. The diluted cells were used to inoculate M9+1% glucose containing varying concentrations of glutaric acid or adipic acid which were neutralized to pH 7.0 with sodium hydroxide, and growth was measured in FlowerPlates in a Biolector microbioreactor system (m2p-labs) at 37° C. with 1000 rpm shaking. The culture volume in each well was 1.4 mL. 
     Adaptive Laboratory Evolution of Tolerant Strains 
     Based on the screening results,  E. coli  K-12 MG1655 was grown overnight in M9 minimal medium and 150 μL was transferred the next day into 8 tubes containing 15 mL of M9+1% glucose+20 g/L glutaric acid or 25 g/L adipic acid on a Tecan Evo robotic platform custom-designed for performing adaptive laboratory evolutions (ALE). Cells were cultured on a 37° C. heat block with stirring by magnetic stir bars. Culture OD 600  was monitored at times determined by a predictive custom script, and when the OD 600  reached approximately 0.3, 150 μL of culture was inoculated into a new tube with the same media concentration. Instrument downtime would occasionally result in cells overgrowing to saturation or an OD 600  greater than 0.3, and reinoculations were occasionally performed from cryogenic stocks of the population. When the growth rate was observed to substantially increase, the media concentration was changed. These concentration changes for glutaric acid were to 30 g/L, 40 g/L, and 45 g/L, and 47.5 g/L, while the changes adipic acid were to 35 g/L, 40 g/L, 45 g/L and 50 g/L. Approximately 100 μL of each population (8 per chemical) were plated on LB agar and incubated at 37° C. overnight. 
     Primary Screening of ALE Isolates 
     Five colonies from wild-type K-12 MG1655 and 10 individual colonies deriving from each population were inoculated into 300 μL M9+1% glucose in 96 well deepwell plates and incubated in a 300 rpm plate shaker at 37° C. The next day, cells were diluted 10× in M9+1% glucose and 30 μL was transferred into clear-bottomed 96 well half-deepwell plates (with rectangular wells) containing M9+1% glucose and M9+1% glucose+52.78 g/L glutaric acid or 55.56 g/L adipic acid, such that the final concentration of glutaric acid or adipic acid was 47.5 g/L or 50 g/L, respectively. In addition, cryogenic glycerol stocks of the overnight culture were saved in a 96 well plate format. Half deepwell plates were incubated at 37° C. with 225 rpm shaking in a Growth Profiler (Enzyscreen), with optical scans of the plates taken at 15 minute intervals. Green pixel values integrated over a 1 mm diameter circular area in each well were converted to OD 600  values using a previously determined calibration between OD 600  and green pixel values. Resulting growth curves were visually inspected for isolates exhibiting the most robust or unique growth patterns within each population. In general, it was attempted to select three isolates per population for further analysis, and all populations were represented in the resequenced isolates. 
     Secondary Screening of ALE Isolates 
     Selected isolates from the primary screen were restruck onto LB agar from the cryogenic stock made from the overnight culture plate for the primary screen. Five K-12 MG1655 colonies and three individual colonies from each isolate were inoculated as biological replicates into a new 96 well deepwell plate containing 300 μL of M9+1% glucose, and grown overnight as for the primary screen. The next day, a cryogenic stock and half deepwell plates containing M9+1% glucose with or without glutaric acid or adipic acid were inoculated using the plate of overnight cultures, and growth was measured as described for the primary screen. Resulting growth curves were visually inspected for isolates exhibiting robust and reproducible growth between replicates in high concentrations of glutaric acid or adipic acid. 
     Re-Sequencing of ALE Isolates 
     A total of 20 isolates were selected from the secondary screen for whole-genome resequencing. An individual colony was taken from the LB agar plates prepared following the primary screen, inoculated into 2 mL LB, and grown overnight at 37° C. in a 250 rpm shaker. The following morning, 0.5 mL of cells were transferred to microcentrifuge tubes and centrifuged at 16000×g for 2 minutes. The supernatant was removed and pellets were stored at −20° C. until further processing. Genomic DNA was extracted from thawed cell pellets using a PureLink genomic DNA extraction kit, with further concentration and purification performed by ethanol precipitation. To generate libraries for sequencing, the Illumina TruSeq Nano kit was used according to the manufacturers&#39; directions using an input quantity of 200 ng of genomic DNA from each isolate. Sequencing was performed on an Illumina MiSeq sequencer, with a minimum 20×average genomic coverage ensured for each isolate based on the number of reads. Fastq output files were analyzed for variants compared to the K-12 MG1655 reference genome (accession number NC_000913.3) using breseq (www-adress barricklab.org/twiki/bin/view/Lab/ToolsBacterialGenomeResequencing). 
     Sole Carbon Source Plate Growth Assay 
     M9 agar plates lacking glucose and instead containing 10 g/L of glutaric acid or 10 g/L of adipic acid (both neutralized to pH 7.0 with sodium hydroxide) were prepared, and strains were struck onto wedges of the plate from a colony on an LB plate. Plates were incubated for up to 4 weeks at 37° C. 
     Construction of Gene Knockouts 
     Probable important losses-of-function were determined by identifying genes across all isolates that harboured mutations, especially those occurring in multiple populations, and by the presence of at least one mutation that either generated a premature stop codon, a frameshift mutation, or the presence of an insertion element sequence within the gene. For those genes, the corresponding knockout strain from the Keio collection of single knockout mutants (where each gene is replaced with a cassette consisting of a kanamycin resistance gene flanked by FRT sites) was used as a donor strain for P1vir phage transduction. Briefly, the Keio strain was grown to early exponential phase in LB+5 mM CaCl 2  and 80 μL of a P1vir stock raised on K-12 MG1655 was added. After significant lysis was observed after 1.5 to 2 hours, the lysate was filter-sterilized to remove cells and stored at 4° C. Strain K-12 MG1655 was grown overnight in LB+5 mM CaCl 2 ) and 100 μL of the overnight culture was mixed with 100 μL of the P1vir lysate of the Keio collection mutant, and the mixture was incubated at 37° C. without shaking for 20 minutes. The entire mixture was then plated on LB agar containing 1.25 mM sodium pyrophosphate as a chelating agent and 25 μg/mL kanamycin. One colony was then restruck on LB+1.25 mM Na 2 P 4 O 7 +25 μg/mL kanamycin plate and analyzed for presence of the Keio cassette in place of the wild-type gene by colony PCR. When further knockouts were constructed in the same strain, the Keio cassette was flipped out to generate a scar sequence such that Kan R  marker could be recycled. This was performed by transforming with pCP20, which constitutively expresses a flippase recombinase, and plating cells on LB agar+100 μg/mL ampicillin and incubating at 30° C. The next day, one or more colonies was tested by colony PCR for loss of the Keio cassette, and successful mutants were then cured of pCP20 by elevated temperature curing at 40° C. Strains were verified to be cured of plasmid by plating on LB agar+100 μg/mL ampicillin and incubation at 30° C. P1vir transductions were then performed using these mutant strains as recipients. 
     Biolector Growth Screening of Evolved Isolates and Reconstructed Mutants 
     Biological triplicate cultures of each strain were grown to saturation overnight in 96 well deepwell plates containing 300 μL M9+1% glucose. The next day, cells were diluted 1:10 in deionized water in a clear 96 well plate and the OD 600  was measured on a BioTek plate reader. 48 well FlowerPlates containing a final volume of 1.4 mL of M9+1/o glucose (plus relevant chemical) were inoculated to OD 600  0.03 (with plate reader pathlength, 200 μL volume) with the overnight culture and sealed with Breathseal film. Light backscatter intensity was monitored in a Biolector microbioreactor system at 37° C. with 1000 rpm shaking. 
     Keio Collection Screening for Loss-of-Function Mutations 
     For primary screening, Keio collection mutants were inoculated directly from a cryogenic stock of the Keio collection into 300 μL LB medium containing 25 μg/mL kanamycin in 96 well deepwell plates and grown at 37° C. with 300 rpm shaking overnight. The Keio background strain, BW25113, was also inoculated into wells of this plate as a control. A cryogenic stock was made from each plate, and the cryogenic stock was replica plated into another 96 well deepwell plate containing 300 μL M9+1% glucose and grown overnight. The next day, cells were inoculated 1:100 into clear bottomed 96 well half-deepwell plates containing M9+1% glucose plus 40 g/L and 47.5 g/L putrescine, or 45 and 50 g/L adipic acid, and cultivated in a Growth Profiler as previously described for screening of ALE isolates. 
     As a secondary screen, promising Keio collection mutants were struck on LB+25 μg/mL kanamycin from the cryogenic stock plate prepared during primary screening above and biological triplicate colonies were inoculated into a 96 well deepwell plate containing 300 μL M9+1% glucose. The next day, cells were inoculated into plates for cultivation on the Growth Profiler as described above. 
     Cross-Compound Tolerance Screening 
     96 well deepwell plates containing 300 μL of M9+1% glucose were inoculated directly from cryogenic stocks made from precultures for the secondary screening of ALE isolates and were grown overnight at 37° C. with 300 rpm shaking. The next day, cells were diluted 1:100 into 96 well half-deepwell plates containing the following final concentrations of each chemical in M9+1% glucose: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 butanol 
                 1.4% 
                 v/v 
               
               
                   
                 glutarate 
                 40 
                 g/L 
               
               
                   
                 p-coumarate 
                 7.5 
                 g/L 
               
               
                   
                 putrescine 
                 32 
                 g/L 
               
               
                   
                 HMDA 
                 32 
                 g/L 
               
               
                   
                 adipate 
                 45 
                 g/L 
               
               
                   
                 isobutyrate 
                 7.5 
                 g/L 
               
               
                   
                 hexanoate 
                 3 
                 g/L 
               
               
                   
                 octanoate 
                 8 
                 g/L 
               
               
                   
                 2,3-butanediol 
                 6% 
                 v/v 
               
               
                   
                 1,2-propanediol 
                 6% 
                 v/v 
               
            
           
           
               
               
               
            
               
                   
                 sodium chloride 
                 0.6M 
               
               
                   
                   
               
            
           
         
       
     
     Plates were cultivated in a Growth Profiler for 48 hours as described for screening of ALE isolates. Green pixel integrated values from each well were converted to OD 600  values using a calibration curve and the resulting OD 600  vs. elapsed time data was processed using custom scripts to determine the time required for each culture to reach an OD of 1.0 (t OD1 ). This value is a combined measure of growth rate and lag time in each culture. The median value was taken for biological triplicates of each isolate and was normalized to the median t OD1  for K-12 MG1655 controls (5 replicates). The ratio of t OD1(evolved) /t OD1(wild-type)  is presented. 
     Results 
     Wild-Type Tolerance to Diacids 
     The maximum measured concentration of glutaric acid at which exponentially growing K-12 MG1655 can grow was found to be 50 g/L with severe inhibition (Table 5). Increasing inhibition of growth was observed from 10 to 50 g/L. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Growth of K-12 MG1655 in varying concentrations of 
               
               
                 glutaric acid (neutralized with sodium hydroxide). 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Mean 
                   
                 std. error 
                   
               
            
           
           
               
               
               
               
               
            
               
                 glutaric acid 
                 μ 
                 t lag   
                 μ 
                 t lag   
               
               
                 (g/L) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 0 
                 0.661 
                 0.7 
                 0.011 
                 0.1 
               
               
                 10 
                 0.526 
                 2.0 
                 0.019 
                 0.1 
               
               
                 20 
                 0.377 
                 1.9 
                 0.006 
                 0.3 
               
               
                 30 
                 0.234 
                 0.6 
                 0.034 
                 1.1 
               
               
                 40 
                 0.132 
                 0.7 
                 0.028 
                 1.7 
               
               
                 50 
                 0.096 
                 15.9 
                 0.062 
                 0.8 
               
               
                 75 
                 0.000 
                 — 
                 0.000 
                 — 
               
               
                   
               
            
           
         
       
     
     The maximum measured concentration of adipic acid at which exponentially growing K-12 MG1655 can grow was found to be 75 g/L with an extensive lag phase of 27 hours (Table 6). Growth rates dropped sharply as a function of concentration between 10 and 50 g/L. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Growth of K-12 MG1655 in varying concentrations of 
               
               
                 adipic acid (neutralized with sodium hydroxide). 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Mean 
                   
                 std. error 
                   
               
            
           
           
               
               
               
               
               
            
               
                 adipic acid 
                 μ 
                 t lag   
                 μ 
                 t lag   
               
               
                 (g/L) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 0 
                 0.653 
                 0.6 
                 0.007 
                 0.0 
               
               
                 10 
                 0.575 
                 1.0 
                 0.018 
                 0.1 
               
               
                 20 
                 0.494 
                 1.3 
                 0.017 
                 0.2 
               
               
                 30 
                 0.367 
                 1.8 
                 0.030 
                 0.7 
               
               
                 40 
                 0.263 
                 4.0 
                 0.012 
                 0.4 
               
               
                 50 
                 0.120 
                 2.4 
                 0.009 
                 1.6 
               
               
                 75 
                 0.203 
                 27.2 
                 0.082 
                 0.6 
               
               
                 100 
                 0.000 
                 — 
                 0.000 
                 — 
               
               
                   
               
            
           
         
       
     
     Growth was also tested in pimelic acid (C 7 ) and sebacic acid (C 10 ) (Table 7). Robust growth was still observed in pimelic acid at 45 g/L, however inhibition was observed as a function of increasing concentration. Sebacic acid was more toxic, with nearly no growth detected above 40 g/L concentration. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Growth of K-12 MG1655 in varying concentrations of pimelic 
               
               
                 and sebacic acids (neutralized with sodium hydroxide). 
               
            
           
           
               
               
               
            
               
                   
                 pimelic acid 
                 sebacic acid 
               
            
           
           
               
               
               
               
               
            
               
                   
                 mean 
                 std. error 
                 Mean 
                 std. error 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 concentration 
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
               
               
                 (g/L) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 0 
                 0.650 
                 0.5 
                 0.027 
                 0.2 
                 0.668 
                 0.5 
                 0.016 
                 0.0 
               
               
                 10 
                 0.694 
                 1.9 
                 0.021 
                 0.2 
                 0.707 
                 1.9 
                 0.048 
                 0.2 
               
               
                 20 
                 0.656 
                 2.4 
                 0.022 
                 0.1 
                 0.578 
                 2.7 
                 0.057 
                 0.3 
               
               
                 25 
                 0.572 
                 2.5 
                 0.021 
                 0.2 
                 0.413 
                 3.3 
                 0.003 
                 0.2 
               
               
                 30 
                 0.560 
                 3.3 
                 0.021 
                 0.2 
                 0.386 
                 5.7 
                 0.054 
                 0.2 
               
               
                 35 
                 0.534 
                 4.1 
                 0.006 
                 0.0 
                 0.306 
                 10.8 
                 0.026 
                 0.4 
               
               
                 40 
                 0.439 
                 4.3 
                 0.008 
                 0.3 
                 0.222 
                 22.5 
                 0.030 
                 0.6 
               
               
                 45 
                 0.280 
                 4.6 
                 0.007 
                 0.3 
                 0.051 
                 50.6 
                 0.020 
                 3.4 
               
               
                   
               
            
           
         
       
     
     Aiming for a starting growth rate between 0.3 to 0.4 h −1 , it was decided to begin evolutions at a concentration of 20 g/L glutaric acid and 25 g/L adipic acid. 
     Resequencing of Tolerant Isolates 
     Variants detected in glutaric and adipic acid evolved strains are presented in Tables 8 and 9. Each strain name corresponds to the chemical the strain was isolated from, the population the strain was isolated from, and the original number of the strain assigned during primary screening (e.g. GLUT1-3 is a glutaric acid-evolved strain isolated from population 1). In each table, strains are arranged such that all that were isolated from the same population are presented in the same rows. Strains with an asterisk (*) following their name are hypermutator strains, and only the mutation identified that can be associated with generating the hypermutator phenotype (here only in mutS or mutT in 1 isolate from 1 glutaric acid population and all isolates from 1 adipic acid population) and those mutations that are shared with other mutations in the same gene in other strains are shown. 
     Mutations that occur independently across multiple populations, or that appear fixed in a highly variable population are likely causative and of highest interest. For glutaric acid, these include mutations in kgtP or its promoter region (24 out of 24 isolates), spoT (all isolates except in population GLUT8), rpoC (9 isolates in 5 populations), proV (6 isolates in 3 populations) and proX (2 isolates in 1 population), rnt (5 isolates in 3 populations), nagC and nagA (4 isolates in 2 populations). In place of mutations in spoT, a coding mutation in polB was found in all 3 isolates of population GLUT8. Mutations in rpoB, encoding another subunit of RNA polymerase in addition to rpoC, were found in all 3 isolates from population GLUT5. Of these mutations, those of kgtP, proV and proX, and nagC are likely loss-of-function mutations, due to the presence of frameshift mutations, premature stop codons, or IS element insertions in at least one population of individual isolate that possesses mutations in that gene. Other mutations are likely gain-of-function or weakening of function, for example coding mutations in genes encoding subunits of RNA polymerase (RpoC and RpoB), SpoT (plus an in-frame deletion in one population), and PolB. 
     For adipic acid, mutated genes that occurred across multiple populations included those in kgtP (19 out of 19 isolates), ybjL (12 isolates in 6 populations), proV or its promoter region (11 isolates in 5 populations; plus 3 isolates in 1 population that possessed a large deletion spanning proV, proX, and proW plus other neighboring genes), sspA (7 isolates in 4 populations; also found in 1 isolate from glutaric acid), the intergenic region between pyrE and rph (6 isolates in 3 populations), nagC (5 isolates in 2 populations), yicC (5 isolates in 2 populations), spoT (4 isolates in 2 populations), and pstS or its promoter region (4 isolates in 2 populations). Notably lacking were mutations in any subunit of RNA polymerase. Of these mutations, those of kgtP, ybjL, proV, sspA, and nagC are likely loss-of-function mutations, due to the presence of frameshift mutations, premature stop codons, or IS element insertions in at least one population of individual isolate that possesses mutations in that gene. Coding mutations in SpoT are likely gain-of-function or weakening of function, as for glutaric acid. Mutations in sspA are either coding SNPs or an in-frame (21 bp) deletion, therefore it is unclear whether this mutation is a loss-of-function. PstS is one subunit of a transporter complex complex PstBACS which is involved in the import of inorganic phosphate under phosphate starvation conditions. 
     
       
         
           
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 Variants detected in glutaric acid-evolved isolates 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 coordinate 
                 gene 
                 change 
                 coordinate 
                 gene 
                 change 
                 coordinate 
                 gene 
                 change 
               
               
                   
               
            
           
           
               
               
               
            
               
                 GLUT1-3 
                 GLUT1-9 
                 GLUT1-10 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1347104 
                 rnb 
                 C603* (G→T) 
                 1347104 
                 rnb 
                 C603* (G→T) 
                 2725668 
                 kgtP 
                 1 bp deletion 
               
               
                 1630841 
                 ydfl 
                 V25F (C→A) 
                 2725668 
                 kgtP 
                 1 bp deletion 
                 2726161 
                 [rrfG] 
                 4703 bp deletion 
               
               
                   
                   
                   
                   
                   
                   
                   
                 [rrsG] 
               
               
                 2725668 
                 kgtP 
                 1 bp deletion 
                 2726129 
                 [rrfG] 
                 4742 bp deletion 
                 2805532 
                 proV 
                 1 bp insertion (→T) 
               
               
                   
                   
                   
                   
                 [rrsG] 
               
               
                 2726147 
                 [rrfG] 
                 4703 bp deletion 
                 3823664 
                 spoT 
                 V422A (T→C) 
                 3328463 
                 greA 
                 IS4 element insertion 
               
               
                   
                 [rrsG] 
               
               
                 3823664 
                 spoT 
                 V422A (T→C) 
                 4186605 
                 rpoC 
                 H419P (A→C) 
                 3377214 
                 sspA 
                 21 bp amplification 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 (X2) 
               
               
                 4186605 
                 rpoC 
                 H419P (A→C) 
                   
                   
                   
                 3522182 
                 hofM 
                 noncoding SNP 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 (C→A) 
               
               
                   
                   
                   
                   
                   
                   
                 3751884 
                 yiaT/yiaU 
                 IS5 element insertion 
               
               
                   
                   
                   
                   
                   
                   
                 3823664 
                 spoT 
                 V422A (T→C) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 GLUT2-1 
                 GLUT2-9 
                 GLUT2-10 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 481075 
                 tomB/acrB 
                 noncoding SNP 
                 1347882 
                 rnb 
                 1 bp deletion 
                 1654069 
                 rspA 
                 T358S (G→C) 
               
               
                   
                   
                 (A→G) 
               
               
                 2725642 
                 kgtP 
                 1 bp deletion 
                 2725642 
                 kgtP 
                 1 bp deletion 
                 2725642 
                 kgtP 
                 1 bp deletion 
               
               
                 2804858 
                 proV 
                 13 bp deletion 
                 3823751 
                 spoT 
                 A451D (C→A) 
                 2804858 
                 proV 
                 13 bp deletion 
               
               
                 3636414 
                 ygjP 
                 D152A (A→C) 
                   
                   
                   
                 3823751 
                 spoT 
                 A451D (C→A) 
               
               
                 3823751 
                 spoT 
                 A451D (C→A) 
                   
                   
                   
                 4186605 
                 rpoC 
                 H419P(A→C) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 GLUT3-5 
                 GLUT3-7 
                 GLUT3-9 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 2724971 
                 kgtP 
                 F259C (A→C) 
                 2724971 
                 kgtP 
                 F259C (A→C) 
                 318484 
                 ykgl 
                 noncoding SNP 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 (C→T) 
               
               
                 3823770 
                 spoT 
                 W457C (G→T) 
                 3823770 
                 spoT 
                 W457C (G→T) 
                 996768 
                 ssuA 
                 noncoding SNP 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 (G→A) 
               
               
                 4186605 
                 rpoC 
                 H419P (A→C) 
                 4186605 
                 rpoC 
                 H419P (A→C) 
                 1728882 
                 rnt 
                 Q179P (A→C) 
               
               
                   
                   
                   
                   
                   
                   
                 2725518 
                 kgtP 
                 1 bp insertion (→C) 
               
               
                   
                   
                   
                   
                   
                   
                 3823759 
                 spoT 
                 N454H (A→C) 
               
               
                   
                   
                   
                   
                   
                   
                 3969048 
                 wzzE 
                 1 bp insertion (→G) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 GLUT4-1 
                 GLUT4-4 
                 GLUT4-10 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 2724611 
                 kgtP 
                 A379V(G→A) 
                 2390019 
                 yfbP/nuoN 
                 G→A 
                 2724971 
                 kgtP 
                 F259C (A→C) 
               
               
                 2807193 
                 proX 
                 8 bp deletion 
                 2724611 
                 kgtP 
                 A379V (G→A) 
                 2788702 
                 ygaQ/csiD 
                 IS5 element insertion 
               
               
                 3824137 
                 spoT 
                 D580Y (G→T) 
                 2807193 
                 proX 
                 8 bp deletion 
                 3823751 
                 spoT 
                 A451V (C→T) 
               
               
                   
                   
                   
                 3195220 
                 ibsE/rfaE 
                 IS186 element insertion 
                 4186605 
                 rpoC 
                 H419P (A→C) 
               
               
                   
                   
                   
                 3824137 
                 spoT 
                 D580Y (G→T) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 GLUT5-4 
                 GLUT5-5 
                 GLUT5-9 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 303119 
                 yagU 
                 1 bp deletion 
                 2725374 
                 kgtP 
                 1 bp deletion 
                 2725374 
                 kgtP 
                 1 bp deletion 
               
               
                 2725374 
                 kgtP 
                 1 bp deletion 
                 3377491 
                 sspA/rpsI 
                 IS2 element insertion 
                 3377491 
                 sspA/rpsI 
                 IS2 element insertion 
               
               
                 3377359 
                 sspA 
                 18 bp deletion 
                 3823106 
                 spoT 
                 R236L (G→T) 
                 3823106 
                 spoT 
                 R236L (G→T) 
               
               
                 3823106 
                 spoT 
                 R236L (G→T) 
                 4181852 
                 rpoB 
                 K203T (A→C) 
                 4181852 
                 rpoB 
                 K203T (A→C) 
               
               
                 4181852 
                 rpoB 
                 K203T (A→C) 
                 4451123 
                 ytfR 
                 noncoding SNP 
                 4451123 
                 ytfR 
                 noncoding SNP 
               
               
                   
                   
                   
                   
                   
                 (C→A) 
                   
                   
                 (C→A) 
               
               
                 4451123 
                 ytfR 
                 noncoding SNP 
               
               
                   
                   
                 (C→A) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 GLUT6-4 
                 GLUT6-5 
                 GLUT6-10 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 657215 
                 pagP/cspE 
                 25 bp deletion 
                 657215 
                 pagP/cspE 
                 25 bp deletion 
                 1728425 
                 rnt 
                 A27T (G→A) 
               
               
                 700680 
                 nagC 
                 IS1 element insertion 
                 700680 
                 nagC 
                 IS1 element insertion 
                 2672970 
                 hcaD 
                 6 bp deletion 
               
               
                 2725370 
                 kgtP 
                 L126* (A→C) 
                 2725370 
                 kgtP 
                 L126* (A→C) 
                 2724725 
                 kgtP 
                 6 bp insertion 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 (→CAAAAG) 
               
               
                 2765412 
                 yfjL/yfjM 
                 8 bp deletion 
                 2765412 
                 yfjL/yfjM 
                 8 bp deletion 
                 3823139 
                 spoT 
                 M247K (T→A) 
               
               
                 3823105 
                 spoT 
                 R236S (C→A) 
                 3823105 
                 spoT 
                 R236S (C→A) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 GLUT7-2 
                 GLUT7-6 
                 GLUT7-7* 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 701396 
                 nagC 
                 Q67* (G→A) 
                 1636300 
                 ydfJ 
                 T→G 
                 701377 
                 nagC 
                 1 bp deletion 
               
               
                 1728926 
                 rnt 
                 F194L (T→C) 
                 1728926 
                 rnt 
                 F194L (T→C) 
                 1728884 
                 rnt 
                 A180T (G→A) 
               
               
                 2724848 
                 kgtP 
                 G300V (C→A) 
                 2724848 
                 kgtP 
                 G300V (C→A) 
                 2725668 
                 kgtP 
                 1 bp deletion 
               
               
                 3824201 
                 spoT 
                 6 bp detection 
                 3824201 
                 spoT 
                 6 bp deletion 
                 2859432 
                 mutS 
                 noncoding SNP 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 (C→T) 
               
               
                   
                   
                   
                   
                   
                   
                 3823724 
                 spoT 
                 T442I (C→T) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 GLUT8-5 
                 GLUT8-6 
                 GLUT8-9 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 64352 
                 polB 
                 R477G (G→C) 
                 64352 
                 polB 
                 R477G (G→C) 
                 64352 
                 polB 
                 R477G (G→C) 
               
               
                 2725818 
                 kgtP/rrfG 
                 noncoding SNP 
                 702331 
                 nagA 
                 1 bp insertion (→T) 
                 1354284 
                 sapC 
                 G79W (C→A) 
               
               
                   
                   
                 (A→G) 
               
               
                 2804858 
                 proV 
                 13 bp deletion 
                 1354284 
                 sapC 
                 G79W (C→A) 
                 1907448 
                 yobF 
                 IS5 element insertion 
               
               
                 2810987 
                 mprA 
                 IS1 element insertion 
                 2725232 
                 kgtP 
                 G172V (C→A) 
                 2725208 
                 kgtP 
                 9 bp deletion 
               
               
                 4185540 
                 rpoC 
                 P64L (C→T) 
                 2804858 
                 proV 
                 13 bp deletion 
                 2804921 
                 proV 
                 1 bp deletion 
               
               
                   
                   
                   
                 4185540 
                 ropC 
                 P64L (C→T) 
                 2927703 
                 ygdH/sdaC 
                 intergenic SNP 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 (G→A) 
               
               
                   
                   
                   
                   
                   
                   
                 4185540 
                 rpoC 
                 P64L (C→T) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
               
                   
               
               
                 Variants detected in adipic acid-evolved isolates 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 coordinate 
                 gene 
                 change 
                 coordinate 
                 gene 
                 change 
                 coordinate 
                 gene 
                 change 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 ADIP1-1 
                 ADIP1-9 
                   
                   
                   
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 814029 
                 uvrB 
                 D168E (T→G) 
                 546309 
                 allD 
                 A19T (C→T) 
                   
                   
                   
               
               
                 889562 
                 ybjL 
                 1 bp deletion 
                 702405 
                 nagA 
                 36 bp amplification 
               
               
                   
                   
                   
                   
                   
                 (X2) 
               
               
                 2725207 
                 kgtP 
                 IS1 element 
                 889562 
                 ybjL 
                 1 bp deletion 
               
               
                   
                   
                 insertion 
               
               
                 2804648 
                 nrdF/proV 
                 38 bp deletion 
                 2530235 
                 ligA/ZipA 
                 noncoding SNP 
               
               
                   
                   
                   
                   
                   
                 (G→T) 
               
               
                 3377068 
                 sspA 
                 21 bp deletion 
                 2725207 
                 kgtP 
                 IS1 element 
               
               
                   
                   
                   
                   
                   
                 insertion 
               
               
                 3815823 
                 pyrE/rph 
                 noncoding SNP 
                 2804648 
                 nrdF/proV 
                 38 bp deletion 
               
               
                   
                   
                 (C→A) 
               
               
                 3816848 
                 yicC 
                 T58M (C→T) 
                 3377068 
                 sspA 
                 21 bp deletion 
               
               
                 4294366 
                 nrfG/gltP 
                 noncoding SNP 
                 3815823 
                 pyrE/rph 
                 noncoding SNP 
               
               
                   
                   
                 (A→T) 
                   
                   
                 (C→A) 
               
               
                   
                   
                   
                 3816848 
                 yicC 
                 T58M (C→T) 
               
               
                   
                   
                   
                 4294366 
                 nrfG/gltP 
                 noncoding SNP 
               
               
                   
                   
                   
                   
                   
                 (A→T) 
               
            
           
           
               
               
               
            
               
                 ADIP2-5 
                 ADIP2-6 
                 ADIP2-10 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 362830 
                 lacY 
                 C117F (C→A) 
                 362830 
                 lacY 
                 C117F (C→A) 
                 700628 
                 nagC 
                 IS5 element 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 insertion 
               
               
                 700529 
                 nagC 
                 IS1 element 
                 2725613 
                 kgtP 
                 S45L (G→A) 
                 1530007 
                 ydcD 
                 S29* (C→A) 
               
               
                   
                   
                 insertion 
               
               
                 2725613 
                 kgtP 
                 S45L (G→A) 
                 2798606 
                 alaE-ygaY 
                 10942 bp deletion 
                 2725613 
                 kgtP 
                 S45L (G→A) 
               
               
                 2798606 
                 alaE-ygaY 
                 10942 bp deletion 
                 3815883 
                 rph 
                 2 bp deletion 
                 2798606 
                 alaE-ygaY 
                 10942 bp deletion 
               
               
                 3815883 
                 rph 
                 2 bp deletion 
                   
                   
                   
                 3815809 
                 pyrE/rph 
                 1 bp deletion 
               
            
           
           
               
               
               
            
               
                 ADIP3-2 
                 ADIP3-4 
                 ADIP3-8 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 889534 
                 ybjL 
                 IS5 element 
                 233954 
                 mltD 
                 2 bp insertion 
                 889534 
                 ybjL 
                 IS5 element 
               
               
                   
                   
                 insertion 
                   
                   
                 (→TG) 
                   
                   
                 insertion 
               
               
                 2614996 
                 yfgO 
                 noncoding SNP 
                 889534 
                 ybjL 
                 IS5 element 
                 2725207 
                 kgtP 
                 IS1 element 
               
               
                   
                   
                 (A→G) 
                   
                   
                 insertion 
                   
                   
                 insertion 
               
               
                 2725207 
                 kgtP 
                 IS1 element 
                 1879829 
                 yeaR 
                 IS186 element 
                 2804648 
                 nrdF/proV 
                 38 bp deletion 
               
               
                   
                   
                 insertion 
                   
                   
                 insertion 
               
               
                 2804648 
                 nrdF/proV 
                 38 bp deletion 
                 2725207 
                 kgtP 
                 IS1 element 
                 3377068 
                 sspA 
                 21 bp deletion 
               
               
                   
                   
                   
                   
                   
                 insertion 
               
               
                 3377068 
                 sspA 
                 21 bp deletion 
                 2804648 
                 nrdF/proV 
                 38 bp deletion 
                 3815823 
                 pyrE/rph 
                 noncoding SNP 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 (C→A) 
               
               
                 3815823 
                 pyrE/rph 
                 noncoding SNP 
                 3377068 
                 sspA 
                 21 bp deletion 
                 3816848 
                 yicC 
                 T58M (C→T) 
               
               
                   
                   
                 (C→A) 
               
               
                 3816848 
                 yicC 
                 T58M (C→T) 
                 3633911 
                 yhiL 
                 IS5 element 
               
               
                   
                   
                   
                   
                   
                 insertion 
               
               
                   
                   
                   
                 3815823 
                 pyrE/rph 
                 noncoding SNP 
               
               
                   
                   
                   
                   
                   
                 (A→C) 
               
               
                   
                   
                   
                 3816848 
                 yicC 
                 T58M (C→T) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 ADIP4-8 
                   
                   
                   
                   
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 702405 
                 nagA 
                 36 bp amplification 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                 (X2) 
               
               
                 889488 
                 ybjL 
                 IS1 element 
               
               
                   
                   
                 insertion 
               
               
                 1728708 
                 rnt 
                 N121S(A→G) 
               
               
                 2675452 
                 yphC 
                 P104A(G→C) 
               
               
                 2693818 
                 purL 
                 V576L(C→G) 
               
               
                 2701175 
                 pdxJ 
                 noncoding SNP 
               
               
                   
                   
                 (G→C) 
               
               
                 2713302 
                 srmB 
                 R136L(G→T) 
               
               
                 2724590 
                 kgtP 
                 G386D(C→T) 
               
               
                 3548179 
                 malQ 
                 A631D(G→T) 
               
               
                 4130167 
                 metL 
                 462 bp deletion 
               
            
           
           
               
               
               
               
               
            
               
                 ADIP5-2* 
                 ADIP5-6* 
                   
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 111305 
                 mutT 
                 E88* (G→T) 
                 111305 
                 mutT 
                 E88* (G→T) 
                   
                   
                   
               
               
                 888480 
                 ybjL 
                 F447C (A→C) 
                 888948 
                 ybjL 
                 E291A (T→G) 
               
               
                 2724576 
                 kgtP 
                 Y391D (A→C) 
                 2615642 
                 yfgO 
                 noncoding SNP 
               
               
                   
                   
                   
                   
                   
                 (T→G) 
               
               
                 2805674 
                 proV 
                 L287* (T→G) 
                 2724576 
                 kgtP 
                 Y391D (A→C) 
               
               
                 3549583 
                 malQ 
                 D163A (T→G) 
                 2805674 
                 proV 
                 L287* (T→G) 
               
               
                   
                   
                   
                 3377387 
                 sspA 
                 T12P (T→G) 
               
            
           
           
               
               
               
            
               
                 ADIP6-3 
                 ADIP6-9 
                 ADIP6-10 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 700928 
                 nagC 
                 G223* (C→A) 
                 700928 
                 nagC 
                 G223* (C→A) 
                 700928 
                 nagC 
                 G223* (C→A) 
               
               
                 889534 
                 ybjL 
                 IS5 element 
                 889534 
                 ybjL 
                 IS5 element 
                 889534 
                 ybjL 
                 IS5 element 
               
               
                   
                   
                 insertion 
                   
                   
                 insertion 
                   
                   
                 insertion 
               
               
                 1915297 
                 proQ 
                 R80C (G→A) 
                 1196319 
                 icd 
                 noncoding SNP 
                 1915297 
                 proQ 
                 R80C (G→A) 
               
               
                   
                   
                   
                   
                   
                 (C→A) 
               
               
                 2725155 
                 kgtP 
                 R198S (G→T) 
                 1389396 
                 ycjG 
                 L156P (T→C) 
                 2725155 
                 kgtP 
                 R198S (G→T) 
               
               
                 2804858 
                 proV 
                 13 bp deletion 
                 1915297 
                 proQ 
                 R80C (G→A) 
                 2805493 
                 proV 
                 E227* (G→T) 
               
               
                 3823700 
                 spoT 
                 S434L (C→T) 
                 2725155 
                 kgtP 
                 R198S (G→T) 
                 3823700 
                 spoT 
                 S434L (C→T) 
               
               
                 4019173 
                 ubiE 
                 K107E (A→G) 
                 2804831 
                 proV 
                 7 bp deletion 
               
               
                   
                   
                   
                 3823700 
                 spoT 
                 S434L (C→T) 
               
            
           
           
               
               
               
               
               
            
               
                 ADIP7-2 
                 ADIP7-5 
                   
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 293574 
                 yagL 
                 noncoding SNP 
                 889540 
                 ybjL 
                 IS5 element 
                   
                   
                   
               
               
                   
                   
                 (A→G) 
                   
                   
                 insertion 
               
               
                 1293038 
                 hns/tdk 
                 IS1 element 
                 2725642 
                 kgtP 
                 1 bp deletion 
               
               
                   
                   
                 insertion 
               
               
                 1598223 
                 yneO/lsrK 
                 IS5 element 
                 2804858 
                 proV 
                 13 bp deletion 
               
               
                   
                   
                 insertion 
               
               
                 2725329 
                 kgtP 
                 G140* (C→A) 
                 3377240 
                 sspA 
                 T61P (T→G) 
               
               
                 2867354 
                 rpoS 
                 9 bp deletion 
                 4490689 
                 idnR 
                 IS1 element 
               
               
                   
                   
                   
                   
                   
                 insertion 
               
               
                 3910996 
                 pstS 
                 4 bp insertion 
               
               
                   
                   
                 (→CTTT) 
               
            
           
           
               
               
               
            
               
                 ADIP8-3 
                 ADIP8-7 
                 ADIP8-10 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1293015 
                 hns/tdk 
                 IS1 element 
                 1293015 
                 hns/tdk 
                 IS1 element 
                 280003 
                 insl1 
                 IS5 element 
               
               
                   
                   
                 insertion 
                   
                   
                 insertion 
                   
                   
                 insertion 
               
               
                 2192447 
                 yehD/yehE 
                 1 bp deletion 
                 2192447 
                 yehD/yehE 
                 1 bp deletion 
                 2192447 
                 yehD/yehE 
                 1 bp deletion 
               
               
                 2724588 
                 kgtP 
                 G387S (C→T) 
                 2724588 
                 kgtP 
                 G387S (C→T) 
                 2724588 
                 kgtP 
                 G387S (C→T) 
               
               
                 3911563 
                 pstS/glmS 
                 IS1 element 
                 3911563 
                 pstS/glmS 
                 IS1 element 
                 3911563 
                 pstS/glmS 
                 IS1 element 
               
               
                   
                   
                 insertion 
                   
                   
                 insertion 
                   
                   
                 insertion 
               
               
                   
               
            
           
         
       
     
     Characterization of Selected Isolates 
     Each re-sequenced isolate was characterized using the Biolector system for growth at the screening concentration of chemical (47.5 g/L glutaric acid or 50 g/L adipic acid) in biological triplicates. The average growth rates with standard errors for the three replicates are shown in Tables 10 and 11. 
     Variations in growth behavior amongst evolved isolates can be noted. Better growing strains are defined by both the slope of the curve (higher growth rate) and at what time the cultures begin growing (reduced lag time). Some isolates exhibit poorer improvements in growth rates (e.g. ADIP7-2 and ADIP8 isolates) but especially reduced lag times. The phenotype to genotype relationship infers mutations that are of highest interest and those that are not of interest. For example, GLUT2-10 was the best performing isolate from population GLUT2, indicating that either the RpoC-H419P and/or RpsA-T358S mutations are causative for higher growth rate, or the lack of other mutations found in the other two isolates is beneficial. Another example of this would, for example, be when multiple isolates from one population are growing nearly identically (e.g. GLUT4-1 and GLUT4-4). This indicates that any differences in mutations between these two isolates are not important for tolerance, in this case the intergenic mutations between yfbP and nuoN, and between ibsE and rfaE, found in GLUT4-4. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Growth rates and lag times of re-sequenced glutaric 
               
               
                 acid evolved isolates in M9 + 47.5 g/L glutaric 
               
               
                 acid (neutralized with sodium hydroxide). 
               
            
           
           
               
               
               
               
               
            
               
                   
                 mean 
                   
                 std. error 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 μ 
                 t lag   
                 μ 
                 t lag   
               
               
                   
                 strain 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 MG1655 
                 0.103 
                 10.7 
                 0.020 
                 4.7 
               
               
                   
                 GLUT1-3 
                 0.304 
                 5.1 
                 0.012 
                 1.8 
               
               
                   
                 GLUT1-9 
                 0.319 
                 6.4 
                 0.012 
                 0.7 
               
               
                   
                 GLUT1-10 
                 0.458 
                 11.3 
                 0.085 
                 0.1 
               
               
                   
                 GLUT2-1 
                 0.276 
                 6.8 
                 0.077 
                 0.6 
               
               
                   
                 GLUT2-9 
                 0.298 
                 6.5 
                 0.014 
                 1.5 
               
               
                   
                 GLUT2-10 
                 0.378 
                 6.5 
                 0.051 
                 0.5 
               
               
                   
                 GLUT3-5 
                 0.284 
                 5.6 
                 0.021 
                 2.1 
               
               
                   
                 GLUT3-7 
                 0.277 
                 5.6 
                 0.044 
                 3.1 
               
               
                   
                 GLUT3-9 
                 0.324 
                 8.3 
                 0.014 
                 3.8 
               
               
                   
                 GLUT4-1 
                 0.279 
                 6.2 
                 0.007 
                 1.1 
               
               
                   
                 GLUT4-4 
                 0.287 
                 6.6 
                 0.015 
                 1.1 
               
               
                   
                 GLUT4-10 
                 0.297 
                 6.9 
                 0.020 
                 2.8 
               
               
                   
                 GLUT5-4 
                 0.438 
                 17.0 
                 0.098 
                 18.0 
               
               
                   
                 GLUT5-5 
                 0.349 
                 14.5 
                 0.054 
                 13.8 
               
               
                   
                 GLUT6-4 
                 0.277 
                 5.9 
                 0.013 
                 0.7 
               
               
                   
                 GLUT6-5 
                 0.300 
                 6.5 
                 0.025 
                 0.7 
               
               
                   
                 GLUT6-10 
                 0.341 
                 6.7 
                 0.093 
                 0.9 
               
               
                   
                 GLUT7-2 
                 0.318 
                 6.6 
                 0.027 
                 0.2 
               
               
                   
                 GLUT7-6 
                 0.318 
                 7.1 
                 0.010 
                 1.2 
               
               
                   
                 GLUT7-7 
                 0.283 
                 7.8 
                 0.023 
                 0.8 
               
               
                   
                 GLUT8-5 
                 0.322 
                 6.2 
                 0.018 
                 0.9 
               
               
                   
                 GLUT8-6 
                 0.347 
                 7.1 
                 0.013 
                 0.9 
               
               
                   
                 GLUT8-9 
                 0.345 
                 7.4 
                 0.042 
                 2.4 
               
               
                   
                   
               
            
           
         
       
     
                     TABLE 11                  Growth rates and lag times of re-sequenced adipic       acid evolved isolates in M9 + 50 g/L adipic       acid (neutralized with sodium hydroxide).                                     mean       std. error                                                 μ   t lag     μ   t lag             strain   (h −1 )   (h)   (h −1 )   (h)                                                     MG1655-1   0.133   21.4   0.017   2.1           ADIP1-1   0.315   16.6   0.037   5.6           ADIP1-9   0.274   5.8   0.013   1.4           ADIP2-5   0.332   20.1   0.019   2.4           ADIP2-6   0.337   22.1   0.038   2.0           ADIP2-10   0.326   20.8   0.007   1.6           ADIP3-2   0.295   16.6   0.011   4.1           ADIP3-4   0.305   23.3   0.017   11.5           ADIP3-8   0.295   20.1   0.032   4.7           ADIP4-8   0.332   6.8   0.022   0.7           ADIP5-2   0.401   4.7   0.015   1.3           ADIP5-6   0.370   14.4   0.130   7.9           ADIP6-3   0.280   6.5   0.003   0.4           ADIP6-9   0.279   6.8   0.014   0.5           ADIP6-10   0.284   7.1   0.023   0.2           ADIP7-2   0.189   7.6   0.024   4.4           ADIP7-5   0.292   19.3   0.006   12.7           ADIP8-3   0.201   6.0   0.011   1.4           ADIP8-7   0.206   7.2   0.011   0.5           ADIP8-10   0.200   6.8   0.016   1.9                        
Sole Carbon Source Plate Growth Assay Wild-type, glutaric acid, and adipic acid evolved strains were struck on M9 agar containing glutarate or adipate as a sole carbon source. No growth was observed on adipic acid plates, indicating that  E. coli  cannot utilize adipic acid as a sole carbon source. Robust but very slow growth of wild-type K-12 MG1655 was observed after a few weeks (Table 12), indicating the ability of  E. coli  to use glutarate as a sole carbon source, almost certainly through promiscuous activity of pathway enzymes (because glutarate is not a natural metabolite in  E. coli ). Growth on this compound as a sole carbon source has not been previously reported in the literature. Evolved isolates generally could not grow on glutaric acid, with the exception of weak growth exhibited by GLUT8-9. The only common genetic feature between all evolved isolates relative to the wild-type are probable loss-of-function mutations in kgtP, implicating KgtP, an α-ketoglutarate importer, as being a direct importer for glutarate. GLUT8-9 notably features an in-frame 9 bp deletion in kgtP that is unique among all the isolates. Without being limited to theory, this mutation may result in a reduced activity of KgtP, rather than a full loss-of-function.
 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Qualitative growth score of glutarate-evolved isolates 
               
               
                 on M9 agar plates containing 10 g/L glutaric acid 
               
               
                 (neutralized) as a sole carbon source. 
               
            
           
           
               
               
            
               
                   
                 growth score 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 MG1655 
                 +++ 
               
               
                   
                 GLUT1-3 
                 None 
               
               
                   
                 GLUT1-9 
                 None 
               
               
                   
                 GLUT1-10 
                 None 
               
               
                   
                 GLUT2-1 
                 None 
               
               
                   
                 GLUT2-9 
                 None 
               
               
                   
                 GLUT2-10 
                 None 
               
               
                   
                 GLUT3-5 
                 None 
               
               
                   
                 GLUT3-7 
                 None 
               
               
                   
                 GLUT3-9 
                 None 
               
               
                   
                 GLUT4-1 
                 None 
               
               
                   
                 GLUT4-4 
                 None 
               
               
                   
                 GLUT4-10 
                 None 
               
               
                   
                 GLUT5-4 
                 None 
               
               
                   
                 GLUT5-5 
                 None 
               
               
                   
                 GLUT6-4 
                 None 
               
               
                   
                 GLUT6-5 
                 None 
               
               
                   
                 GLUT6-10 
                 None 
               
               
                   
                 GLUT7-2 
                 None 
               
               
                   
                 GLUT7-6 
                 None 
               
               
                   
                 GLUT7-7 
                 None 
               
               
                   
                 GLUT8-5 
                 None 
               
               
                   
                 GLUT8-6 
                 None 
               
               
                   
                 GLUT8-9 
                 + 
               
               
                   
                   
               
            
           
         
       
     
     Knockout Strain Growth Performance 
     High Glutaric Acid Concentrations: 
     Probable loss-of-function mutations were identified from re-sequencing results as described in methods and in the results of the resequencing analysis. Because there was probable loss-of-function of kgtP in all resequenced isolates, this was the only single knockout tested, and additional knockouts were selected to be tested as double combinations together with kgtP (Table 13). Only the triple knockout in kgtP, proV, and nagC was tested initially due to one isolate (GLUT8-6) possessing probable loss-of-function mutations in kgtP, proV, and nagA, and due to previous studies indicating similar phenotypes and likely the same mechanism of action for improved tolerance in high osmotic pressures (or due to high Na +  concentrations) from both nagC and nagA knockouts (Lennen and Herrgard, 2014). All strains with kgtP knockouts exhibited higher growth rates than the wild-type in 23.8 g/L and 47.5 g/L glutaric acid, however none of the multiple knockout strains exhibited significantly improved growth relative to the single kgtP knockout strain alone. K-12 MG1655 ΔkgtP nagC::kan exhibited reduced growth relative to K-12 MG1655 kgtP::kan in 47.5 g/L glutaric acid. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Growth rates and lag times of preliminary selections of single, double, 
               
               
                 and triple gene knockout mutants in M9 + 23.8 g/L or 47.5 g/L glutaric 
               
               
                 acid (neutralized), as measured in the Growth Profiler testing format. 
               
            
           
           
               
               
               
            
               
                   
                 23.8 g/L glutarate 
                 47.5 g/L glutarate 
               
            
           
           
               
               
               
               
               
            
               
                   
                 mean 
                 std. error 
                 mean 
                 std. error 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
               
               
                 strain 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 MG1655 
                 0.466 
                 7.4 
                 0.010 
                 0.1 
                 0.101 
                 21.8 
                 0.032 
                 0.7 
               
               
                 MG1655 kgtP::kan 
                 0.487 
                 6.1 
                 0.019 
                 0.0 
                 0.191 
                 13.5 
                 0.019 
                 0.4 
               
               
                 MG1655 ΔkgtP proV::kan 
                 0.520 
                 6.2 
                 0.019 
                 0.1 
                 0.251 
                 13.0 
                 0.019 
                 0.2 
               
               
                 MG1655 ΔkgtP nagC::kan 
                 0.491 
                 6.7 
                 0.019 
                 0.1 
                 0.179 
                 15.0 
                 0.007 
                 0.5 
               
               
                 MG1655 ΔkgtP rnb::kan 
                 0.505 
                 6.3 
                 0.012 
                 0.0 
                 0.183 
                 13.6 
                 0.017 
                 0.8 
               
               
                 MG1655 ΔkgtP sspA::kan 
                 0.473 
                 6.1 
                 0.007 
                 0.1 
                 0.225 
                 12.2 
                 0.008 
                 0.4 
               
               
                 MG1655 ΔkgtP ΔproV nagC::kan 
                 0.541 
                 6.6 
                 0.006 
                 0.2 
                 0.215 
                 14.0 
                 0.016 
                 0.9 
               
               
                   
               
            
           
         
       
     
     A second group of selected single, double, and triple knockout mutants was tested in the Biolector testing format in M9+47.5 g/L glutaric acid (Table 14). The ybjL loss-of-function which had been identified from resequencing of adipic acid evolved isolates was included, to determine if that mutation would also confer tolerance toward glutaric acid. The proV and ybjL mutations did not increase growth rates alone, but in double combinations with the kgtP mutation, were found to increase the growth rate over that of the kgtP single mutant. K-12 MG1655 ΔkgtP sspA::kan additionally exhibited an increased growth rate over that of the kgtP single knockout mutant. The triple knockout mutant in kgtP, proV, and ybjL exhibited a growth rate higher than that of the tested double knockout combinations, with a growth rate nearly equivalent to two of the evolved isolates tested alongside in the same experiment (GLUT1-3 and GLUT4-1), and exceeding the growth rate of many other evolved isolates in Table 6. 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Growth rates and lag times of selected single, double, and 
               
               
                 triple gene knockout mutants in M9 + 47.5 g/L glutaric 
               
               
                 acid (neutralized), as measured in the Biolector testing format. 
               
            
           
           
               
               
               
            
               
                   
                 mean 
                 std. error 
               
            
           
           
               
               
               
               
               
            
               
                   
                 μ 
                 t lag   
                 μ 
                 t lag   
               
               
                 Strain 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 MG1655 
                 0.100 
                 10.4 
                 0.018 
                 2.8 
               
               
                 GLUT1-3 
                 0.278 
                 4.6 
                 0.014 
                 0.6 
               
               
                 GLUT1-10 
                 0.442 
                 10.8 
                 0.009 
                 0.1 
               
               
                 GLUT4-1 
                 0.312 
                 7.1 
                 0.009 
                 0.1 
               
               
                 GLUT8-6 
                 0.402 
                 7.8 
                 0.001 
                 0.2 
               
               
                 MG1655 kgtP::kan 
                 0.181 
                 5.8 
                 0.005 
                 0.6 
               
               
                 MG1655 proV::kan 
                 0.085 
                 0.6 
                 0.008 
                 2.4 
               
               
                 MG1655 ybjL::kan 
                 0.126 
                 12.8 
                 0.006 
                 0.7 
               
               
                 MG1655 ΔkgtP proV::kan 
                 0.244 
                 6.5 
                 0.000 
                 0.2 
               
               
                 MG1655 ΔkgtP sspA::kan 
                 0.256 
                 5.7 
                 0.007 
                 0.9 
               
               
                 MG1655 ΔkgtP ybjL::kan 
                 0.213 
                 6.0 
                 0.008 
                 0.3 
               
               
                 MG1655 ΔkgtP ΔproV ybjL::kan 
                 0.300 
                 7.0 
                 0.013 
                 0.2 
               
               
                   
               
            
           
         
       
     
     High Adipic Acid Concentrations 
     Probable loss-of-function mutations were identified as previously described. As for glutarate, probable loss-of-function of kgtP was identified in all resequenced isolates, therefore this single knockout was tested with additional double and triple combinations all containing the kgtP knockout. Of the tested strains, K-12 MG1655 kgtP::kan exhibited slightly improved tolerance in 25 g/L adipate, and a much larger improvement in growth in 50 g/L adipate (Table 15). The only tested combinatorial knockout with a higher growth rate than the kgtP single knockout strain was MG1655 ΔkgtP proV::kan. K-12 MG1655 ΔkgtP sspA::kan exhibited greatly reduced tolerance relative to the wild-type, indicating that the sspA mutations isolated in resequenced mutants are likely either gain-of-function mutations or that they only result in weakened activity of the gene product. 
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Growth rates and lag times of preliminary selections of single, double, 
               
               
                 and triple gene knockout mutants in M9 + 25 g/L or 50 g/L adipic 
               
               
                 acid (neutralized), as measured in the Growth Profiler testing format. 
               
            
           
           
               
               
               
            
               
                   
                 25 g/L adipate 
                 50 g/L adipate 
               
            
           
           
               
               
               
               
               
            
               
                   
                 mean 
                 std. error 
                 mean 
                 std. error 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
               
               
                 strain 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 MG1655 
                 0.467 
                 6.9 
                 0.015 
                 0.1 
                 0.115 
                 25.7 
                 0.004 
                 0.7 
               
               
                 MG1655 kgtP::kan 
                 0.534 
                 6.4 
                 0.003 
                 0.2 
                 0.192 
                 17.5 
                 0.002 
                 0.6 
               
               
                 MG1655 ΔkgtP proV::kan 
                 0.583 
                 6.2 
                 0.029 
                 0.2 
                 0.264 
                 16.5 
                 0.005 
                 0.2 
               
               
                 MG1655 ΔkgtP pstS::kan 
                 0.465 
                 7.6 
                 0.006 
                 0.2 
                 0.139 
                 21.3 
                 0.005 
                 0.3 
               
               
                 MG1655 ΔkgtP sspA::kan 
                 0.525 
                 6.8 
                 0.006 
                 0.4 
                 0.117 
                 49.2 
                 0.029 
                 12.4 
               
               
                 MG1655 ΔkgtP ΔproV nagC::kan 
                 0.501 
                 6.8 
                 0.008 
                 0.2 
                 0.213 
                 19.7 
                 0.009 
                 0.7 
               
               
                   
               
            
           
         
       
     
     In a second experiment, additional single, double, and triple knockout combinations were tested in 25 g/L and 50 g/L adipate (Table 16). 
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Growth rates and lag times of additional selections of single, double, 
               
               
                 and triple gene knockout mutants in M9 + 25 g/L or 50 g/L adipic 
               
               
                 acid (neutralized), as measured in the Growth Profiler testing format. 
               
            
           
           
               
               
               
            
               
                   
                 25 g/L adipate 
                 50 g/L adipate 
               
            
           
           
               
               
               
               
               
            
               
                   
                 mean 
                 std. error 
                 mean 
                 std. error 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
               
               
                 strain 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 MG1655 
                 0.558 
                 7.5 
                 0.030 
                 0.1 
                 0.080 
                 52.5 
                 0.024 
                 2.8 
               
               
                 MG1655 kgtP::kan 
                 0.575 
                 6.3 
                 0.017 
                 0.2 
                 0.189 
                 16.0 
                 0.002 
                 0.6 
               
               
                 MG1655 ΔkgtP proV::kan 
                 0.608 
                 6.2 
                 0.032 
                 0.2 
                 0.255 
                 15.1 
                 0.010 
                 0.7 
               
               
                 MG1655 ybjL::kan 
                 0.516 
                 7.4 
                 0.020 
                 0.1 
                 0.142 
                 26.1 
                 0.010 
                 1.0 
               
               
                 MG1655 ΔkgtP ybjL::kan 
                 0.552 
                 6.3 
                 0.029 
                 0.1 
                 0.254 
                 13.5 
                 0.006 
                 0.5 
               
               
                 MG1655 ΔkgtP ΔproV ybjL::kan 
                 0.586 
                 6.3 
                 0.011 
                 0.2 
                 0.326 
                 12.6 
                 0.019 
                 0.7 
               
               
                   
               
            
           
         
       
     
     The ybjL single knockout strain was found to moderately improve tolerance, but below the levels conferred by deletion of kgtP. K-12 MG1655 ΔkgtP ybjL::kan exhibited growth rates similar to K-12 MG1655 ΔkgtP proV::kan, and the triple knockout combination found in K-12 MG1655 ΔkgtP ΔproV ybjL::kan exhibited an improved growth rate over either double knockout combination. Selected single, double, and triple knockout strains were then tested in the Biolector testing format (Table 17). Results were similar to that observed in Table 16, with a higher growth rate observed for K-12 MG1655 ΔkgtP ΔproV ybjL::kan than for some ALE isolates. K-12 MG1655 ΔkgtP sspA::kan was additionally tested, however growth rates were not improved for media containing adipate as they were for glutarate. 
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Growth rates and lag times of selected single, double, and 
               
               
                 triple gene knockout mutants in M9 + 50 g/L adipic acid 
               
               
                 (neutralized), as measured in the Biolector testing format. 
               
            
           
           
               
               
               
            
               
                   
                 mean 
                 std. error 
               
            
           
           
               
               
               
               
               
            
               
                   
                 μ 
                 t lag   
                 μ 
                 t lag   
               
               
                 strain 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 MG1655 
                 0.188 
                 13.8 
                 0.030 
                 1.4 
               
               
                 ADIP1-9 
                 0.284 
                 8.1 
                 0.011 
                 9.0 
               
               
                 ADIP4-8 
                 0.351 
                 6.1 
                 0.006 
                 0.2 
               
               
                 ADIP6-3 
                 0.311 
                 6.3 
                 0.008 
                 0.2 
               
               
                 ADIP8-3 
                 0.208 
                 4.5 
                 0.004 
                 2.7 
               
               
                 MG1655 kgtP::kan 
                 0.225 
                 11.5 
                 0.004 
                 0.4 
               
               
                 MG1655 proV::kan 
                 0.153 
                 19.2 
                 0.003 
                 0.5 
               
               
                 MG1655 ybjL::kan 
                 0.195 
                 10.9 
                 0.005 
                 0.0 
               
               
                 MG1655 ΔkgtP proV::kan 
                 0.262 
                 11.0 
                 0.007 
                 0.1 
               
               
                 MG1655 ΔkgtP ybjL::kan 
                 0.229 
                 10.4 
                 0.007 
                 0.7 
               
               
                 MG1655 ΔkgtP sspA::kan 
                 0.164 
                 20.5 
                 0.056 
                 10.0 
               
               
                 MG1655 ΔkgtP ΔproV ybjL::kan 
                 0.298 
                 9.7 
                 0.034 
                 2.2 
               
               
                   
               
            
           
         
       
     
     Screening of a Single Deletion Mutant Collection for Diacid Tolerance 
     To determine if any additional single gene deletion candidates were overlooked, screening on elevated glutarate and adipate concentrations was also conducted using the Keio collection of gene knockouts, which is a commercial collection of knockouts in nearly all non-essential genes and ORFs in  E. coli  strain BW25113. This strain is a K-12 derivative and possesses known mutations relative to the K-12 MG1655 background. All Keio collection strains with knockouts in genes that were found to be mutated in Tables 8 and 9 were screened for growth against the BW25113 control in M9+40 g/L or 47.5 g/L glutaric acid, or M9+45 g/L or 50 g/L adipic acid (neutralized with sodium hydroxide) in the Growth Profiler screening format. Primary screening hits were measured again in a secondary screen in biological replicates, with averaged growth curves for 3 biological replicate cultures shown individually for each strain in Tables 18 and 19, For glutaric acid (Table 18), BW25113 cspE::kan and BW25113 proX::kan exhibited the largest increases in growth rate at 47.5 g/L glutarate, with small improvements also seen with 40 g/L glutarate. ProX is a subunit with ProV in the ProVWX ABC transporter. In M9+47.5 g/L glutarate, additional knockout strains with smaller improvements in growth rates were the rfaE, yfbP, and yfjM knockout strains. 
     For adipic acid (Table 19), a number of single deletion mutants exhibited moderate increases in growth rate in 50 g/L adipate. These were knockouts in proQ, pstS, rph, rpoS, sspA, tdk, uvrB, ycjG, and yeaR. In 47.5 g/L adipate, smaller percentage improvements in growth rate were observed, however all of these mutants similar exhibited significant increases in growth rate. 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Growth rates of Keio collection knockouts in M9 + 40 
               
               
                 g/L and 47.5 g/L glutaric acid (neutralized with sodium 
               
               
                 hydroxide) as measured in the Growth Profiler testing format. 
               
            
           
           
               
               
               
            
               
                   
                 40 g/L glutarate 
                 47.5 g/L glutarate 
               
            
           
           
               
               
               
               
               
            
               
                   
                 μ 
                 std. 
                 μ 
                 std. 
               
               
                 Strain 
                 (h −1 ) 
                 error 
                 (h −1 ) 
                 error 
               
               
                   
               
               
                 BW25113 
                 0.227 
                 0.010 
                 0.090 
                 0.011 
               
               
                 BW25113 kgtP::kan 
                 0.346 
                 — 
                 0.278 
                 — 
               
               
                 BW25113 cspE::kan 
                 0.248 
                 0.012 
                 0.138 
                 0.008 
               
               
                 BW25113 greA::kan 
                 0.228 
                 0.017 
                 0.094 
                 0.003 
               
               
                 BW25113 mprA::kan 
                 0.219 
                 0.017 
                 0.096 
                 0.015 
               
               
                 BW25113 polB::kan 
                 0.205 
                 0.012 
                 0.097 
                 0.005 
               
               
                 BW25113 proX::kan 
                 0.261 
                 0.005 
                 0.140 
                 0.011 
               
               
                 BW25113 rfaE::kan 
                 0.220 
                 0.006 
                 0.121 
                 0.007 
               
               
                 BW25113 ssuA::kan 
                 0.207 
                 0.007 
                 0.093 
                 0.005 
               
               
                 BW25113 yfbP::kan 
                 0.216 
                 0.009 
                 0.118 
                 0.009 
               
               
                 BW25113 yfjM::kan 
                 0.228 
                 0.006 
                 0.118 
                 0.004 
               
               
                 BW25113 ygdH::kan 
                 0.215 
                 0.014 
                 0.098 
                 0.007 
               
               
                 BW25113 yiaU::kan 
                 0.235 
                 0.005 
                 0.111 
                 0.009 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 Growth rates of Keio collection knockouts in M9 + 
               
               
                 45 g/L and 50 g/L adipic acid (neutralized with sodium 
               
               
                 hydroxide) as measured in the Growth Profiler testing format. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 45 g/L adipate 
                   
                 50 g/L adipate 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 μ 
                 std. 
                 μ 
                 std. 
               
               
                   
                 strain 
                 (h −1 ) 
                 error 
                 (h −1 ) 
                 error 
               
               
                   
                   
               
               
                   
                 BW25113 
                 0.158 
                 0.009 
                 0.072 
                 0.010 
               
               
                   
                 BW25113 kgtP::kan 
                 0.230 
                 — 
                 0.218 
                 — 
               
               
                   
                 BW25113 idnR::kan 
                 0.165 
                 0.014 
                 0.070 
                 0.001 
               
               
                   
                 BW25113 lsrK::kan 
                 0.154 
                 0.006 
                 0.070 
                 0.017 
               
               
                   
                 BW25113 malQ::kan 
                 0.162 
                 0.017 
                 0.077 
                 0.002 
               
               
                   
                 BW25113 metL::kan 
                 0.167 
                 0.003 
                 0.067 
                 0.036 
               
               
                   
                 BW25113 nrfG::kan 
                 0.174 
                 0.007 
                 0.064 
                 0.005 
               
               
                   
                 BW25113 proQ::kan 
                 0.189 
                 0.016 
                 0.096 
                 0.006 
               
               
                   
                 BW25113 pstS::kan 
                 0.201 
                 0.011 
                 0.107 
                 0.011 
               
               
                   
                 BW25113 rph::kan 
                 0.206 
                 0.005 
                 0.119 
                 0.012 
               
               
                   
                 BW25113 rpoS::kan 
                 0.189 
                 0.002 
                 0.113 
                 0.001 
               
               
                   
                 BW25113 sspA::kan 
                 0.186 
                 0.003 
                 0.116 
                 0.021 
               
               
                   
                 BW25113 tdk::kan 
                 0.205 
                 0.006 
                 0.113 
                 0.003 
               
               
                   
                 BW25113 uvrB::kan 
                 0.197 
                 0.016 
                 0.113 
                 0.019 
               
               
                   
                 BW25113 ycjG::kan 
                 0.195 
                 0.015 
                 0.127 
                 0.019 
               
               
                   
                 BW25113 yeaR::kan 
                 0.197 
                 0.011 
                 0.101 
                 0.023 
               
               
                   
                   
               
            
           
         
       
     
     A list of all gene disruption mutants in both the K-12 MG1655 and BW25113 background strains that exhibited increased tolerance to glutaric acid is shown in Table 20. A similar table for adipic acid is shown in Table 21. 
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 Summary of knockout strains with improved growth 
               
               
                 over the wild-type strain in glutaric acid 
               
            
           
           
               
               
               
            
               
                   
                 Growth rate 
                 Lag time 
               
               
                 Strain genotype 
                 improvement 
                 improvement 
               
               
                   
               
               
                 K-12 MG1655 kgtP::kan 
                 Moderate 
                 large 
               
               
                 K-12 MG1655 ybjL::kan 
                 Small 
                 none 
               
               
                 K-12 MG1655 ΔkgtP proV::kan 
                 Moderate 
                 large 
               
               
                 K-12 MG1655 ΔkgtP ybjL::kan 
                 Moderate 
                 large 
               
               
                 K-12 MG1655 ΔkgtP sspA::kan 
                 Moderate 
                 large 
               
               
                 K-12 MG1655 ΔkgtP ΔproV ybjL::kan 
                 Large 
                 large 
               
               
                 BW25113 kgtP::kan 
                 Large (40 and 47.5 g/L) 
                 not quantified 
               
               
                 BW25113 cspE::kan 
                 small (40 and 47.5 g/L) 
                 not quantified 
               
               
                 BW25113 proX::kan 
                 small (40 and 47.5 g/L) 
                 not quantified 
               
               
                 BW25113 rfaE::kan 
                 minor (47.5 g/L) 
                 not quantified 
               
               
                 BW25113 yfbP::kan 
                 minor (47.5 g/L) 
                 not quantified 
               
               
                 BW25113 yfjM::kan 
                 minor (47.5 g/L) 
                 not quantified 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 Summary of knockout strains with improved growth 
               
               
                 over the wild-type strain in adipic acid 
               
            
           
           
               
               
               
            
               
                   
                 Growth rate 
                 Lag time 
               
               
                 Strain genotype 
                 improvement 
                 improvement 
               
               
                   
               
               
                 K-12 MG1655 kgtP::kan 
                 Moderate 
                 large 
               
               
                 K-12 MG1655 ybjL::kan 
                 Small 
                 none 
               
               
                 K-12 MG1655 ΔkgtP proV::kan 
                 Moderate 
                 large 
               
               
                 K-12 MG1655 ΔkgtP ybjL::kan 
                 Moderate 
                 large 
               
               
                 K-12 MG1655 ΔkgtP ΔproV ybjL::kan 
                 Large 
                 large 
               
               
                 BW25113 kgtP::kan 
                 Large (45 and 50 g/L) 
                 not quantified 
               
               
                 BW25113 proQ::kan 
                 small (45 and 50 g/L) 
                 not quantified 
               
               
                 BW25113 pstS::kan 
                 moderate (45 and 50 g/L) 
                 not quantified 
               
               
                 BW25113 rph::kan 
                 moderate (45 and 50 g/L) 
                 not quantified 
               
               
                 BW25113 rpoS::kan 
                 small (45 and 50 g/L) 
                 not quantified 
               
               
                 BW25113 sspA::kan 
                 small (45 and 50 g/L) 
                 not quantified 
               
               
                 BW25113 tdk::kan 
                 small (45 and 50 g/L) 
                 not quantified 
               
               
                 BW25113 uvrB::kan 
                 small (45 and 50 g/L) 
                 not quantified 
               
               
                 BW25113 ycjG::kan 
                 small (45 g/L), moderate (50 g/L) 
                 not quantified 
               
               
                 BW25113 yeaR::kan 
                 small (45 and 50 g/L) 
                 not quantified 
               
               
                   
               
            
           
         
       
     
     Sole Carbon Source Plate Growth Assay of Knockout Strains 
     The single knockout strains in kgtP, proV, and ybjL, plus the double and triple combination knockout strains, were struck as previously described on glutarate as a sole carbon source, together with wild-type K-12 MG1655 and a selection of ALE evolved isolates as controls. Robust growth was again observed from K-12 MG1655 after a few weeks incubation, with reduced growth of GLUT8-9 and greatly reduced or no growth in other evolved isolates (Table 22). A larger inoculum was spread on the plates, which likely explains why very weak growth was observed for GLUT8-6 and GLUT1-10. K-12 MG1655 kgtP::kan exhibited no growth, indicating that loss-of-function of kgtP is explicitly responsible for the weak or absent growth of evolved strains on glutarate, and suggesting that KgtP is indeed a direct importer of glutrate. The proV and ybjL single knockout strains did not exhibit reduced growth relative to K-12 MG1655, and double and triple knockout combination strains with kgtP did not exhibit any growth, as would be expected from the loss of kgtP. 
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 Qualitative growth scores of selected controls (K-12 MG1655 and 
               
               
                 glutarate evolved isolates) plus selected single, double, and 
               
               
                 triple knockout mutants isolates on M9 agar plates containing 
               
               
                 10 g/L glutaric acid (neutralized) as a sole carbon source. 
               
            
           
           
               
               
               
            
               
                   
                 strain 
                 growth score 
               
               
                   
                   
               
               
                   
                 MG1655 
                 +++ 
               
               
                   
                 GLUT1-3 
                 none 
               
               
                   
                 GLUT1-10 
                 + 
               
               
                   
                 GLUT4-1 
                 none 
               
               
                   
                 GLUT8-6 
                 + 
               
               
                   
                 GLUT8-9 
                 + 
               
               
                   
                 MG1655 kgtP::kan 
                 none 
               
               
                   
                 MG1655 proV::kan 
                 +++ 
               
               
                   
                 MG1655 ybjL::kan 
                 +++ 
               
               
                   
                 MG1655 ΔkgtP proV::kan 
                 none 
               
               
                   
                 MG1655 ΔkgtP ybjL::kan 
                 none 
               
               
                   
                 MG1655 ΔkgtP ΔproV ybjL::kan 
                 none 
               
               
                   
                   
               
            
           
         
       
     
     Cross-Compound Tolerance Testing 
     Every secondary screened evolved isolate from the glutaric acid and adipic acid evolutions was grown in the presence of every other compound in the study as indicated in the Methods. The normalized t OD1(evolved strain) /t OD1(wild-type)  are shown in Tables 23 and 24 for the glutaric acid and adipic acid evolved isolates, respectively. Lower values are indicative a larger improvement in growth of the evolved isolate (left column) in that chemical condition (top row), whereas higher values are indicative of a lower improvement or decrease in growth compared to the wild-type. Averaged ratios across conditions and strains shown at the right and bottom of the plot allow for overall by-chemical and by-strain trends to be observed. Strain names that are followed by an asterisk (*) were not re-sequenced, and strain names in italics were found to be hypermutator strains. 
     The majority of glutaric acid-evolved isolates exhibit cross-tolerance to adipic acid (notable exceptions were isolates from the GLUT5 population, GLUT1-3, GLUT1-9, and GLUT2-10). Likewise, the majority of adipic acid evolved isolates exhibit cross-tolerance to glutaric acid (notable exceptions were most isolates in the ADIP1, ADIP2, and ADIP3 populations, plus a couple isolates from other populations (ADIP4-4, ADIP5-5, neither of which were resequenced). Isolates with the highest degree of cross-tolerance were GLUT4-10, GLUT8-6, and GLUT8-9. The GLUT8 population was notable for possessing coding mutations in polB and lacking coding mutations in spoT. Adipic acid evolved isolates exhibited a lower overall degree of cross tolerance, with the best performing isolate being ADIP6-9. This isolate most likely has loss-of-function of kgtP, proV, and ybjL, plus coding mutations in proQ (suggested to be loss-of-function from Keio mutant screen) and spoT. The ADIP6 population specifically exhibited a high level of cross-tolerance toward all other acid salts in the study (hexanoate, octanoate, isobutyrate, glutarate, and p-coumarate). Acid cross-tolerance was also evident from many glutaric acid evolved isolates, however high cross-tolerance toward the diamine HMDA and the diols 2,3-butanediol and 1,2-propanediol was evident in a number of isolates. Cross-tolerance toward HMDA in population GLUT8 can be inferred to be due to a mutation present in GLUT8-6 and GLUT8-9 that is not found in GLUT8-5, which in this case is a coding mutation in sapC that is likely causative for HMDA cross-tolerance. 
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 Normalized t OD1(evolved) /t OD1(wild-type)  values for glutaric acid-evolved isolates 
               
               
                 grown in the presence of inhibitory concentrations of 12 different chemicals. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Buta- 
                   
                   
                 2,3- 
                 putres- 
                   
                   
                 iso- 
                   
                 octa- 
                 1,2- 
                   
                   
               
               
                   
                 nol 
                 glutarate 
                 coumarate 
                 butanediol 
                 cine 
                 HMDA 
                 adipate 
                 butyrate 
                 hexanoate 
                 noate 
                 propanediol 
                 NaCl 
                 average 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 GLUT1-3 
                 1.31 
                 1.94 
                 2.24 
                 3.06 
                 4.02 
                 1.00 
                 1.97 
                 1.58 
                 0.78 
                 0.86 
                 2.03 
                 3.16 
                 2.00 
               
               
                 GLUT1-9 
                 1.31 
                 1.74 
                 2.24 
                 3.06 
                 4.02 
                 1.00 
                 1.75 
                 1.39 
                 0.92 
                 0.88 
                 1.97 
                 3.16 
                 1.96 
               
               
                 GLUT1-10 
                 1.31 
                 0.72 
                 1.4S 
                 0.65 
                 1.42 
                 1.00 
                 0.58 
                 2.92 
                 0.68 
                 0.79 
                 0.78 
                 1.39 
                 1.14 
               
               
                 GLUT2-1 
                 1.12 
                 0.68 
                 1.47 
                 1.32 
                 1.10 
                 0.99 
                 0.63 
                 1.61 
                 0.86 
                 0.92 
                 1.00 
                 1.54 
                 1.10 
               
               
                 GLUT2-9 
                 1.22 
                 0.68 
                 1.27 
                 1.24 
                 0.96 
                 1.00 
                 0.63 
                 1.47 
                 0.84 
                 0.91 
                 0.94 
                 1.08 
                 1.02 
               
               
                 GLUT2-10 
                 1.31 
                 1.42 
                 2.24 
                 3.06 
                 4.02 
                 1.00 
                 1.40 
                 1.29 
                 0.86 
                 0.86 
                 1.75 
                 3.16 
                 1.87 
               
               
                   GLUT3 - 5   
                 
                   1.31 
                 
                 
                   0.58 
                 
                 
                   1.05 
                 
                 
                   0.98 
                 
                 
                   0.90 
                 
                 
                   1.00 
                 
                 
                   0.53 
                 
                 
                   1.21 
                 
                 
                   0.70 
                 
                 
                   0.52 
                 
                 
                   0.81 
                 
                 
                   2.28 
                 
                 
                   0.99 
                 
               
               
                 GLUT3-7 
                 1.31 
                 0.58 
                 0.97 
                 1.05 
                 0.98 
                 0.96 
                 0.53 
                 1.34 
                 0.81 
                 0.59 
                 0.84 
                 1.28 
                 0.94 
               
               
                 GLUT3-9 
                 1.24 
                 0.68 
                 1.55 
                 0.76 
                 1.06 
                 1.00 
                 0.85 
                 1.63 
                 0.73 
                 0.77 
                 0.97 
                 2.80 
                 1.17 
               
               
                 GLUT4-1 
                 1.31 
                 0.76 
                 1.43 
                 3.06 
                 1.15 
                 0.53 
                 0.68 
                 1.34 
                 1.14 
                 1.34 
                 1.41 
                 0.92 
                 1.26 
               
               
                 GLUT4-4 
                 1.31 
                 0.76 
                 1.63 
                 3.06 
                 1.15 
                 0.52 
                 0.73 
                 1.32 
                 1.11 
                 1.51 
                 1.44 
                 1.00 
                 1.29 
               
               
                 GLUT4-10 
                 1.31 
                 0.56 
                 0.79 
                 0.76 
                 0.90 
                 0.73 
                 0.52 
                 1.37 
                 0.70 
                 0.45 
                 0.84 
                 1.07 
                 0.83 
               
               
                 GLUT5-4 
                 1.31 
                 2.82 
                 1.17 
                 3.06 
                 4.02 
                 1.00 
                 2.48 
                 5.08 
                 0.81 
                 0.96 
                 1.56 
                 3.16 
                 2.29 
               
               
                 GLUT5-5 
                 1.31 
                 2.48 
                 0.87 
                 3.06 
                 4.02 
                 1.00 
                 1.88 
                 2.82 
                 0.73 
                 0.64 
                 1.28 
                 3.16 
                 1.94 
               
               
                 GLUT5-9 
                 1.31 
                 2.78 
                 1.26 
                 3.06 
                 4.02 
                 1.00 
                 2.47 
                 5.08 
                 0.76 
                 0.41 
                 1.47 
                 3.16 
                 2.23 
               
               
                 GLUT6-4 
                 1.16 
                 0.70 
                 1.01 
                 1.14 
                 1.17 
                 1.00 
                 0.67 
                 1.37 
                 0.86 
                 0.81 
                 0.94 
                 1.70 
                 1.04 
               
               
                 GLUT6-5 
                 1.27 
                 0.74 
                 1.05 
                 1.38 
                 1.33 
                 0.90 
                 0.67 
                 1.39 
                 0.95 
                 0.83 
                 1.06 
                 1.82 
                 1.12 
               
               
                 GLUT6-10 
                 1.25 
                 0.68 
                 0.84 
                 1.51 
                 1.00 
                 1.00 
                 0.63 
                 1.29 
                 0.97 
                 0.81 
                 1.06 
                 0.98 
                 1.00 
               
               
                 GLUT7-2 
                 1.22 
                 0.64 
                 0.64 
                 1.48 
                 0.88 
                 0.60 
                 0.57 
                 1.21 
                 0.84 
                 0.79 
                 1.00 
                 0.85 
                 0.89 
               
               
                 GLUT7-6 
                 1.20 
                 0.62 
                 0.77 
                 1.03 
                 0.96 
                 0.39 
                 0.65 
                 1.29 
                 0.92 
                 0.70 
                 1.09 
                 1.00 
                 0.88 
               
               
                 GLUT7-7 
                 1.16 
                 0.62 
                 1.12 
                 0.83 
                 1.04 
                 0.75 
                 0.58 
                 1.71 
                 0.68 
                 0.55 
                 0.88 
                 1.13 
                 0.92 
               
               
                 GLUT8-5 
                 1.31 
                 0.94 
                 1.00 
                 1.03 
                 2.29 
                 1.00 
                 0.83 
                 0.92 
                 0.70 
                 0.80 
                 0.94 
                 3.05 
                 1.23 
               
               
                 GLUT8-6 
                 1.31 
                 0.60 
                 1.00 
                 1.21 
                 0.90 
                 0.33 
                 0.53 
                 1.18 
                 0.70 
                 0.48 
                 0.84 
                 0.90 
                 0.83 
               
               
                 GLUT8-9 
                 1.31 
                 0.60 
                 0.91 
                 0.94 
                 0.96 
                 0.34 
                 0.52 
                 1.03 
                 0.68 
                 0.48 
                 0.88 
                 0.95 
                 0.80 
               
               
                 average 
                 1.27 
                 1.06 
                 1.25 
                 1.74 
                 1.84 
                 0.83 
                 0.97 
                 1.79 
                 0.82 
                 0.78 
                 1.16 
                 1.86 
                 1.28 
               
               
                 # &gt; wt 
                 0 
                 18 
                 7 
                 6 
                 8 
                 11 
                 18 
                 1 
                 22 
                 22 
                 11 
                 5 
               
               
                 % &gt; wt 
                 0.0 
                 75.0 
                 29.2 
                 25.0 
                 33.3 
                 45.8 
                 75.0 
                 4.2 
                 91.7 
                 91.7 
                 45.8 
                 20.8 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 Normalized t OD1(evolved) /t OD1(wild-type)  values for adipic acid-evolved isolates 
               
               
                 grown in the presence of inhibitory concentrations of 12 different chemicals. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 buta- 
                   
                   
                 2,3- 
                 putres- 
                   
                   
                 iso- 
                   
                 octa- 
                 1,2- 
                   
                   
               
               
                   
                 nol 
                 glutarate 
                 courmarate 
                 butanediol 
                 cine 
                 HMDA 
                 Adipate 
                 butyrate 
                 hexanoate 
                 noate 
                 propanediol 
                 NaCl 
                 average 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 ADIP1-1 
                 1.22 
                 1.38 
                 1.75 
                 0.59 
                 3.22 
                 3.51 
                 1.37 
                 4.20 
                 0.74 
                 1.38 
                 5.68 
                 2.64 
                 2.31 
               
               
                 ADIP1-6* 
                 1.25 
                 0.96 
                 1.75 
                 0.59 
                 3.22 
                 3.51 
                 0.73 
                 3.13 
                 1.00 
                 1.24 
                 0.74 
                 2.64 
                 1.73 
               
               
                 ADIP1-9 
                 1.27 
                 1.32 
                 1.75 
                 0.58 
                 3.22 
                 3.51 
                 1.34 
                 4.20 
                 0.81 
                 1.38 
                 0.79 
                 2.64 
                 1.90 
               
               
                 ADIP2-5 
                 1.17 
                 1.09 
                 1.06 
                 0.87 
                 2.77 
                 3.51 
                 1.28 
                 0.87 
                 0.67 
                 1.38 
                 5.68 
                 2.64 
                 1.92 
               
               
                 ADIP2-6 
                 1.22 
                 0.95 
                 1.09 
                 0.71 
                 2.48 
                 3.51 
                 0.96 
                 0.87 
                 0.63 
                 1.38 
                 0.79 
                 2.64 
                 1.44 
               
               
                 ADIP2-10 
                 1.18 
                 1.32 
                 0.99 
                 1.10 
                 2.87 
                 3.51 
                 1.16 
                 0.91 
                 0.65 
                 1.38 
                 3.26 
                 2.64 
                 1.75 
               
               
                   ADIP3 - 2   
                 
                   1.27 
                 
                 
                   0.90 
                 
                 
                   1.42 
                 
                 
                   0.51 
                 
                 
                   3.22 
                 
                 
                   3.51 
                 
                 
                   0.94 
                 
                 
                   3.67 
                 
                 
                   0.74 
                 
                 
                   1.38 
                 
                 
                   0.68 
                 
                 
                   2.64 
                 
                 
                   1.74 
                 
               
               
                 ADIP3-4 
                 1.27 
                 1.35 
                 1.75 
                 0.59 
                 3.22 
                 3.51 
                 1.16 
                 4.20 
                 0.84 
                 1.38 
                 1.24 
                 2.64 
                 1.93 
               
               
                 ADIP3-8 
                 1.18 
                 0.85 
                 1.35 
                 0.50 
                 3.22 
                 3.51 
                 1.37 
                 3.91 
                 0.74 
                 1.38 
                 0.85 
                 2.64 
                 1.79 
               
               
                 ADIP4-1 
                 1.27 
                 0.50 
                 1.15 
                 0.73 
                 0.93 
                 1.00 
                 0.47 
                 1.30 
                 0.84 
                 0.94 
                 1.15 
                 0.95 
                 0.94 
               
               
                 ADIP4-4* 
                 1.27 
                 0.96 
                 0.83 
                 1.01 
                 1.02 
                 1.07 
                 0.71 
                 1.28 
                 1.05 
                 1.03 
                 1.79 
                 2.64 
                 1.22 
               
               
                 ADIP4-8 
                 1.26 
                 0.53 
                 0.76 
                 0.83 
                 1.00 
                 1.11 
                 1.80 
                 1.33 
                 0.88 
                 0.78 
                 1.03 
                 0.86 
                 1.01 
               
               
                 ADIP5-2 
                 1.12 
                 0.41 
                 0.96 
                 1.87 
                 0.75 
                 0.73 
                 0.32 
                 1.13 
                 0.91 
                 0.91 
                 5.29 
                 2.64 
                 1.42 
               
               
                 ADIP5-5* 
                 1.27 
                 1.06 
                 0.98 
                 1.87 
                 1.02 
                 1.11 
                 0.93 
                 1.15 
                 1.26 
                 0.89 
                 1.18 
                 1.22 
                 1.16 
               
               
                 ADIP5-6 
                 1.18 
                 1.37 
                 0.77 
                 0.77 
                 3.22 
                 3.51 
                 1.17 
                 2.07 
                 0.88 
                 0.81 
                 1.97 
                 2.64 
                 1.70 
               
               
                 ADIP6-3 
                 1.16 
                 0.42 
                 0.55 
                 1.87 
                 0.90 
                 1.27 
                 0.40 
                 1.91 
                 0.70 
                 0.53 
                 1.12 
                 0.79 
                 0.97 
               
               
                 ADIP6-9 
                 1.09 
                 0.45 
                 0.55 
                 0.62 
                 0.88 
                 1.11 
                 0.38 
                 1.30 
                 0.70 
                 0.56 
                 0.91 
                 0.84 
                 0.78 
               
               
                 ADIP6-10 
                 1.12 
                 0.58 
                 0.89 
                 0.68 
                 1.02 
                 1.11 
                 0.47 
                 3.35 
                 0.84 
                 0.89 
                 1.47 
                 1.42 
                 1.15 
               
               
                 ADIP7-2 
                 1.27 
                 0.53 
                 0.63 
                 0.73 
                 1.22 
                 1.55 
                 0.47 
                 0.91 
                 0.84 
                 1.38 
                 1.00 
                 1.07 
                 0.96 
               
               
                 ADIP7-5 
                 1.27 
                 0.68 
                 1.13 
                 0.59 
                 1.92 
                 1.93 
                 0.56 
                 3.93 
                 0.77 
                 1.06 
                 1.29 
                 2.47 
                 1.47 
               
               
                 ADIP7-10* 
                 1.27 
                 0.54 
                 0.89 
                 0.46 
                 2.12 
                 2.13 
                 0.53 
                 3.50 
                 0.72 
                 0.99 
                 0.71 
                 1.81 
                 1.30 
               
               
                 ADIP8-3 
                 1.27 
                 0.63 
                 0.55 
                 1.10 
                 1.35 
                 1.96 
                 0.56 
                 1.41 
                 1.00 
                 1.38 
                 1.38 
                 2.64 
                 1.27 
               
               
                 ADIP8-7 
                 1.27 
                 0.63 
                 0.46 
                 0.73 
                 1.02 
                 1.40 
                 0.47 
                 1.52 
                 0.88 
                 1.29 
                 1.97 
                 2.64 
                 1.19 
               
               
                 ADIP8-10 
                 1.27 
                 0.60 
                 0.48 
                 0.73 
                 1.42 
                 1.55 
                 0.45 
                 1.09 
                 0.81 
                 1.38 
                 1.06 
                 1.03 
                 0.99 
               
               
                 average 
                 1.22 
                 0.83 
                 1.02 
                 0.86 
                 1.97 
                 2.25 
                 0.83 
                 2.21 
                 0.83 
                 1.13 
                 1.79 
                 2.06 
                 1.42 
               
               
                 # &gt; wt 
                 0 
                 17 
                 14 
                 18 
                 4 
                 1 
                 16 
                 4 
                 20 
                 9 
                 7 
                 4 
               
               
                 % &gt; wt 
                 0.0 
                 70.8 
                 58.3 
                 75.0 
                 16.7 
                 4.2 
                 66.7 
                 16.7 
                 83.3 
                 37.5 
                 29.2 
                 16.7 
               
               
                   
               
            
           
         
       
     
     Additionally, each evolved isolate was tested for cross-tolerance toward other dicarboxylic acids of interest. First, K-12 MG1655 was tested in the Growth Profiler screening format for growth in the presence of a range of concentrations of each compound (note that this had been done in the Biolector format previously for pimelic acid and sebacic acid (Table 7) thus was not repeated here): fumaric acid, itaconic acid, malic acid, and succinic acid. All diacids tested were either the neutral sodium salts, or the free diacid was neutralized with sodium hydroxide to pH 7.0 for testing. Variable concentrations of these compounds elicited growth inhibition in  E. coli  K-12 MG1655 (Table 25). Based on these results, a screening concentration was selected for the evolved isolates for which wild-type cells could achieve at a growth rate of 0.15-0.3 h −1  (versus uninhibited growth at 0.7-0.9 h −1  in M9 glucose minimal medium). These concentrations were: 45 g/L fumaric acid, 45 g/L itaconic acid, 55 g/L malic acid, 50 g/L succinic acid, 45 g/L pimelic acid, and 38 g/L sebacic acid. The results of glutaric acid and adipic acid-evolved isolates grown in these concentrations of fumaric, itaconic, and malic acid are shown in are shown in Tables 26 and 27, and the same isolates grown at the selected concentration of succinic, pimelic, and sebacic acid (linear aliphatic diacids) are shown in Tables 28 and 29. A majority of evolved isolates exhibited increased growth rates and/or reductions in lag time in all tested diacids. These in particular included isolates from the ADIP1, ADIP2, ADIP3, and ADIP7 populations, ADIP5-6 (a hypermutator strain) for all diacids generally. ADIP7-2 and ADIP7-5 exhibited the highest growth rates in sebacic acid, with the ADIP2 population also exhibiting significantly improved growth. ADIP7-2 and ADIP7-5 possessed different sets of mutations but notably only possessed loss-of-function mutations in kgtP plus distinct modulatory mutations (probable reduction-of-function of rpoS based on the Keio collection screen and loss-of-function of pstS plus an intergenic insertion between hns and tdk in ADIP7-2, and loss-of-function of ybjL, proV, and probable reduction-of-function of sspA based on the Keio collection screen in ADIP7.5). The common features of ADIP2 isolates were possessing only loss-of-function mutations in kgtP, deletion of proVWX, and mutations that restore expression of PyrE. Glutaric acid evolved isolates tended to exhibit a specificity toward tolerance to particular diacids. Isolates from the GLUT5 population exhibited significantly improved growth rates in fumaric acid (these isolates exhibited probable reduction-of-function of sspA and the RpoB-K203T and SpoT-R236L mutations), whereas the GLUT1 and GLUT2 populations had some of the most improved growth rates and reduced lag times in itaconic acid (these isolates featured loss-of-function of kgtP and the SpoT-V422A and RpoC-H419P mutations in GLUT1-3 and GLUT1-9; loss-of-function of kgtP, proV, probable reduction-of-function of sspA, and the SpoT-V422A mutation in GLUT1-10; loss-of-function of kgtP and optionally proV, and the SpoT-A451D mutation in GLUT2-1 and GLUT2-9; and loss-of-function of kgtP and proV plus the SpoT-A451D and RpoC-H419P mutations in GLUT2-10). Malic acid cross-tolerance was weak across all glutaric acid evolved isolates. Isolates from the GLUT8 population had dramatically improved growth rates (as well as moderate reductions in lag time) toward sebacic acid (these isolates commonly featured loss-of-function of kgtP and proV, and the RpoC-P64L and PolB-R477G mutations, with the best performing isolates GLUT8-6 and GLUT8-9 additionally possessing the SapC-G79W mutation), with GLUT1-10 and GLUT2-9 also exhibiting significantly enhanced growth rates. Notably, the GLUT8 population had relatively poor cross-tolerance for most diacids, and all tested isolates featured the PolB-R477G mutation, whereas all other populations featured isolates with mutations in SpoT. 
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 Growth rates and lag times of K-12 MG1655 in varying concentrations of the sodium salts of fumaric, 
               
               
                 itaconic, malic, and succinic acid, as measured in the Growth Profiler testing format. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 fumaric acid 
                 itaconic acid 
                 malic acid 
                 succinic acid 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 mean 
                 std. error 
                 mean 
                 std. error 
                 mean 
                 std. error 
                 mean 
                 std. error 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 diacid 
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
               
               
                 (g/L) 
                 (h 1 ) 
                 (h) 
                 (h 1 ) 
                 (h) 
                 (h 1 ) 
                 (h) 
                 (h 1 ) 
                 (h) 
                 (h 1 ) 
                 (h) 
                 (h 1 ) 
                 (h) 
                 (h 1 ) 
                 (h) 
                 (h 1 ) 
                 (h) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 0 
                 0.747 
                 5.1 
                 0.030 
                 0.2 
                 0.747 
                 5.1 
                 0.030 
                 0.2 
                 0.747 
                 5.1 
                 0.030 
                 0.2 
                 0.747 
                 5.1 
                 0.030 
                 0.2 
               
               
                 10 
                 0.662 
                 5.8 
                 0.021 
                 0.0 
                 0.703 
                 5.8 
                 0.037 
                 0.1 
                 0.686 
                 5.9 
                 0.053 
                 0.0 
                 0.665 
                 5.4 
                 0.084 
                 0.0 
               
               
                 20 
                 0.615 
                 6.9 
                 0.019 
                 0.0 
                 0.603 
                 7.0 
                 0.036 
                 0.2 
                 0.639 
                 6.6 
                 0.024 
                 0.1 
                 0.610 
                 6.7 
                 0.024 
                 0.1 
               
               
                 30 
                 0.482 
                 9.5 
                 0.009 
                 0.1 
                 0.520 
                 9.4 
                 0.012 
                 0.2 
                 0.526 
                 8.0 
                 0.002 
                 0.2 
                 0.496 
                 8.9 
                 0.028 
                 0.1 
               
               
                 40 
                 0.285 
                 15.4 
                 0.005 
                 0.2 
                 0.320 
                 14.1 
                 0.014 
                 0.2 
                 0.410 
                 11.1 
                 0.005 
                 0.2 
                 0.345 
                 13.4 
                 0.014 
                 0.1 
               
               
                 50 
                 0.116 
                 30.2 
                 0.011 
                 1.0 
                 0.117 
                 29.9 
                 0.008 
                 0.7 
                 0.287 
                 16.9 
                 0.002 
                 0.2 
                 0.179 
                 22.4 
                 0.008 
                 0.0 
               
               
                 60 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 0.154 
                 28.3 
                 0.006 
                 0.3 
                 0.127 
                 42.5 
                 0.016 
                 1.6 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 Growth rates and lag times of K-12 MG1655 and glutaric acid-evolved isolates in specified 
               
               
                 inhibitory concentrations of fumaric, itaconic, and malic acid (sodium salts or neutralized 
               
               
                 with sodium hydroxide to pH 7.0), as measured in the Growth Profiler testing format. 
               
            
           
           
               
               
               
               
            
               
                   
                 45 g/L fumaric acid 
                 45 g/L itaconic acid 
                 55 g/L malic acid 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 mean 
                 std. error 
                 mean 
                 std. error 
                 mean 
                 std. error 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
               
               
                 strain 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 MG1655 
                 0.216 
                 16.6 
                 0.017 
                 0.3 
                 0.248 
                 13.4 
                 0.014 
                 0.4 
                 0.235 
                 17.1 
                 0.008 
                 0.3 
               
               
                 GLUT1-3 
                 0.293 
                 13.3 
                 0.019 
                 1.5 
                 0.454 
                 10.2 
                 0.039 
                 1.2 
                 0.294 
                 13.5 
                 0.044 
                 1.5 
               
               
                 GLUT1-9 
                 0.274 
                 14.9 
                 0.028 
                 1.2 
                 0.446 
                 10.4 
                 0.028 
                 0.4 
                 0.259 
                 15.3 
                 0.019 
                 0.9 
               
               
                 GLUT1-10 
                 0.289 
                 18.9 
                 0.015 
                 7.2 
                 0.446 
                 18.1 
                 0.028 
                 13.7 
                 0.286 
                 19.2 
                 0.027 
                 8.0 
               
               
                 GLUT2-1 
                 0.277 
                 13.5 
                 0.023 
                 0.7 
                 0.385 
                 10.8 
                 0.036 
                 0.3 
                 0.273 
                 13.7 
                 0.025 
                 0.4 
               
               
                 GLUT2-9 
                 0.267 
                 12.2 
                 0.007 
                 0.4 
                 0.415 
                 9.0 
                 0.028 
                 0.3 
                 0.270 
                 12.5 
                 0.021 
                 0.4 
               
               
                 GLUT2-10 
                 0.312 
                 12.3 
                 0.013 
                 1.0 
                 0.494 
                 8.8 
                 0.030 
                 0.4 
                 0.282 
                 12.2 
                 0.038 
                 0.9 
               
               
                 GLUT3-5 
                 0.263 
                 16.6 
                 0.014 
                 3.3 
                 0.310 
                 12.0 
                 0.058 
                 2.0 
                 0.257 
                 15.4 
                 0.014 
                 2.3 
               
               
                 GLUT3-7 
                 0.264 
                 16.2 
                 0.023 
                 3.3 
                 0.326 
                 11.7 
                 0.017 
                 1.7 
                 0.234 
                 15.5 
                 0.053 
                 3.5 
               
               
                 GLUT3-9 
                 0.305 
                 24.3 
                 0.012 
                 0.7 
                 0.257 
                 14.5 
                 0.094 
                 1.9 
                 0.252 
                 20.5 
                 0.015 
                 1.5 
               
               
                 GLUT4-1 
                 0.295 
                 14.6 
                 0.026 
                 0.7 
                 0.414 
                 11.7 
                 0.016 
                 0.9 
                 0.264 
                 15.9 
                 0.014 
                 0.7 
               
               
                 GLUT4-4 
                 0.287 
                 15.3 
                 0.015 
                 0.4 
                 0.339 
                 13.4 
                 0.110 
                 1.6 
                 0.264 
                 16.5 
                 0.012 
                 0.2 
               
               
                 GLUT4-10 
                 0.274 
                 16.3 
                 0.014 
                 1.6 
                 0.328 
                 13.5 
                 0.016 
                 3.2 
                 0.250 
                 15.6 
                 0.017 
                 1.5 
               
               
                 GLUT5-4 
                 0.386 
                 14.1 
                 0.016 
                 1.5 
                 0.398 
                 15.2 
                 0.056 
                 6.4 
                 0.272 
                 15.3 
                 0.022 
                 1.7 
               
               
                 GLUT5-5 
                 0.375 
                 15.1 
                 0.008 
                 0.2 
                 0.368 
                 11.4 
                 0.078 
                 0.8 
                 0.275 
                 16.6 
                 0.032 
                 0.5 
               
               
                 GLUT5-9 
                 0.370 
                 14.9 
                 0.013 
                 0.6 
                 0.438 
                 22.5 
                 0.026 
                 2.8 
                 0.246 
                 16.4 
                 0.026 
                 0.9 
               
               
                 GLUT6-4 
                 0.281 
                 13.8 
                 0.020 
                 0.6 
                 0.360 
                 11.3 
                 0.080 
                 1.0 
                 0.251 
                 14.5 
                 0.022 
                 0.7 
               
               
                 GLUT6-5 
                 0.279 
                 13.2 
                 0.007 
                 0.4 
                 0.420 
                 10.6 
                 0.007 
                 0.5 
                 0.241 
                 13.8 
                 0.014 
                 0.8 
               
               
                 GLUT6-10 
                 0.277 
                 14.2 
                 0.018 
                 0.8 
                 0.502 
                 10.5 
                 0.150 
                 0.7 
                 0.245 
                 14.8 
                 0.071 
                 1.8 
               
               
                 GLUT7-2 
                 0.304 
                 14.6 
                 0.008 
                 0.6 
                 0.441 
                 10.6 
                 0.025 
                 0.2 
                 0.275 
                 16.1 
                 0.035 
                 1.4 
               
               
                 GLUT7-6 
                 0.261 
                 19.5 
                 0.007 
                 1.1 
                 0.293 
                 13.7 
                 0.015 
                 0.3 
                 0.247 
                 21.9 
                 0.015 
                 5.5 
               
               
                 GLUT7-7 
                 0.193 
                 22.0 
                 0.076 
                 2.9 
                 0.239 
                 8.5 
                 0.207 
                 7.4 
                 0.185 
                 19.1 
                 0.022 
                 1.0 
               
               
                 GLUT8-5 
                 0.247 
                 14.4 
                 0.024 
                 0.6 
                 0.372 
                 17.3 
                 0.024 
                 8.5 
                 0.180 
                 15.8 
                 0.034 
                 0.9 
               
               
                 GLUT8-6 
                 0.272 
                 43.5 
                 0.107 
                 13.5 
                 0.236 
                 10.3 
                 0.206 
                 9.2 
                 0.208 
                 32.7 
                 0.047 
                 3.7 
               
               
                 GLUT8-9 
                 0.196 
                 54.3 
                 0.033 
                 9.0 
                 0.224 
                 12.0 
                 0.195 
                 10.4 
                 0.192 
                 47.7 
                 0.052 
                 15.3 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 27 
               
             
            
               
                   
               
               
                 Growth rates and lag times of K-12 MG1655 and adipic acid-evolved 
               
               
                 isolates in specified inhibitory concentrations of fumaric, itaconic, 
               
               
                 and malic acid (sodium salts or neutralized with sodium hydroxide 
               
               
                 to pH 7.0), as measured in the Growth Profiler testing format. 
               
            
           
           
               
               
               
               
            
               
                   
                 45 g/L fumaric acid 
                 45 g/L itaconic acid 
                 55 g/L malic acid 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 mean 
                 std. error 
                 mean 
                 std. error 
                 mean 
                 std. error 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
               
               
                 strain 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 MG1655 
                 0.212 
                 15.5 
                 0.012 
                 0.8 
                 0.257 
                 13.0 
                 0.016 
                 0.4 
                 0.222 
                 16.5 
                 0.031 
                 1.6 
               
               
                 ADIP1-1 
                 0.347 
                 10.2 
                 0.017 
                 0.0 
                 0.466 
                 8.3 
                 0.019 
                 0.2 
                 0.333 
                 12.8 
                 0.028 
                 1.6 
               
               
                 ADIP1-9 
                 0.328 
                 9.8 
                 0.001 
                 0.5 
                 0.434 
                 8.7 
                 0.007 
                 0.2 
                 0.336 
                 10.7 
                 0.015 
                 1.0 
               
               
                 ADIP2-5 
                 0.358 
                 11.4 
                 0.040 
                 0.3 
                 0.440 
                 8.3 
                 0.028 
                 0.3 
                 0.338 
                 17.9 
                 0.027 
                 1.1 
               
               
                 ADIP2-6 
                 0.380 
                 11.0 
                 0.006 
                 0.3 
                 0.470 
                 8.2 
                 0.009 
                 0.1 
                 0.348 
                 16.8 
                 0.007 
                 1.1 
               
               
                 ADIP2-10 
                 0.353 
                 11.0 
                 0.014 
                 0.6 
                 0.438 
                 8.3 
                 0.015 
                 0.4 
                 0.347 
                 16.8 
                 0.017 
                 1.8 
               
               
                 ADIP3-2 
                 0.327 
                 9.4 
                 0.011 
                 1.1 
                 0.459 
                 7.9 
                 0.004 
                 0.9 
                 0.318 
                 10.7 
                 0.016 
                 2.1 
               
               
                 ADIP3-4 
                 0.324 
                 9.7 
                 0.002 
                 0.6 
                 0.451 
                 8.4 
                 0.018 
                 0.5 
                 0.347 
                 9.9 
                 0.009 
                 0.9 
               
               
                 ADIP3-8 
                 0.343 
                 10.1 
                 0.008 
                 1.3 
                 0.433 
                 8.5 
                 0.023 
                 1.1 
                 0.329 
                 11.4 
                 0.007 
                 2.0 
               
               
                 ADIP4-1 
                 0.264 
                 11.9 
                 0.005 
                 0.4 
                 0.339 
                 11.2 
                 0.023 
                 0.6 
                 0.275 
                 12.1 
                 0.006 
                 0.6 
               
               
                 ADIP4-8 
                 0.254 
                 12.4 
                 0.009 
                 1.2 
                 0.326 
                 10.4 
                 0.029 
                 1.1 
                 0.238 
                 13.4 
                 0.016 
                 2.1 
               
               
                 ADIP5-2 
                 0.280 
                 10.6 
                 0.074 
                 3.5 
                 0.354 
                 9.4 
                 0.108 
                 2.7 
                 0.299 
                 10.1 
                 0.052 
                 2.1 
               
               
                 ADIP5-6 
                 0.444 
                 9.0 
                 0.017 
                 1.0 
                 0.527 
                 7.2 
                 0.020 
                 0.6 
                 0.404 
                 10.2 
                 0.018 
                 2.4 
               
               
                 ADIP6-3 
                 0.275 
                 11.3 
                 0.066 
                 1.2 
                 0.327 
                 9.9 
                 0.088 
                 0.9 
                 0.295 
                 11.6 
                 0.057 
                 1.6 
               
               
                 ADIP6-9 
                 0.250 
                 12.3 
                 0.047 
                 2.2 
                 0.337 
                 10.3 
                 0.057 
                 1.7 
                 0.250 
                 12.7 
                 0.073 
                 2.7 
               
               
                 ADIP6-10 
                 0.308 
                 10.0 
                 0.026 
                 0.2 
                 0.343 
                 9.1 
                 0.059 
                 0.6 
                 0.323 
                 10.0 
                 0.026 
                 0.6 
               
               
                 ADIP7-2 
                 0.312 
                 10.1 
                 0.011 
                 0.2 
                 0.342 
                 7.9 
                 0.122 
                 1.2 
                 0.329 
                 10.3 
                 0.006 
                 0.3 
               
               
                 ADIP7-5 
                 0.326 
                 10.0 
                 0.010 
                 0.7 
                 0.402 
                 8.6 
                 0.012 
                 0.4 
                 0.326 
                 10.2 
                 0.016 
                 0.6 
               
               
                 ADIP8-3 
                 0.202 
                 14.4 
                 0.003 
                 0.3 
                 0.308 
                 11.7 
                 0.006 
                 0.1 
                 0.194 
                 14.4 
                 0.038 
                 0.5 
               
               
                 ADIP8-7 
                 0.188 
                 16.1 
                 0.040 
                 2.4 
                 0.235 
                 12.5 
                 0.138 
                 1.2 
                 0.202 
                 14.9 
                 0.003 
                 0.3 
               
               
                 ADIP8-10 
                 0.221 
                 14.3 
                 0.015 
                 0.5 
                 0.290 
                 11.3 
                 0.025 
                 0.3 
                 0.202 
                 14.2 
                 0.006 
                 0.4 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 28 
               
             
            
               
                   
               
               
                 Growth rates and lag times of K-12 MG1655 and glutaric acid-evolved isolates in specified 
               
               
                 inhibitory concentrations of succinic, pimelic, and sebacic acid (sodium salts or neutralized 
               
               
                 with sodium hydroxide to pH 7.0), as measured in the Growth Profiler testing format. 
               
            
           
           
               
               
               
               
            
               
                   
                 50 g/L succinic acid 
                 45 g/L pimelic acid 
                 38 g/L sebacic acid 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 mean 
                 std. error 
                 mean 
                 std. error 
                 mean 
                 std. error 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
               
               
                 strain 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 MG1655 
                 0.169 
                 20.2 
                 0.006 
                 0.7 
                 0.275 
                 13.4 
                 0.019 
                 0.1 
                 0.167 
                 26.9 
                 0.008 
                 1.2 
               
               
                 GLUT1-3 
                 0.231 
                 14.5 
                 0.025 
                 1.8 
                 0.369 
                 10.4 
                 0.028 
                 0.8 
                 0.074 
                 34.9 
                 0.006 
                 5.4 
               
               
                 GLUT1-9 
                 0.194 
                 20.8 
                 0.013 
                 8.0 
                 0.337 
                 11.9 
                 0.022 
                 0.2 
                 0.043 
                 44.7 
                 0.024 
                 8.4 
               
               
                 GLUT1-10 
                 0.246 
                 20.0 
                 0.042 
                 8.2 
                 0.399 
                 13.1 
                 0.026 
                 3.3 
                 0.278 
                 20.6 
                 0.015 
                 2.8 
               
               
                 GLUT2-1 
                 0.232 
                 14.7 
                 0.016 
                 0.2 
                 0.362 
                 11.6 
                 0.002 
                 0.4 
                 0.153 
                 21.7 
                 0.005 
                 2.7 
               
               
                 GLUT2-9 
                 0.213 
                 14.4 
                 0.012 
                 0.3 
                 0.349 
                 10.3 
                 0.025 
                 0.3 
                 0.282 
                 12.5 
                 0.006 
                 1.0 
               
               
                 GLUT2-10 
                 0.277 
                 13.4 
                 0.024 
                 1.7 
                 0.383 
                 10.2 
                 0.033 
                 0.5 
                 0.093 
                 29.8 
                 0.006 
                 1.3 
               
               
                 GLUT3-5 
                 0.242 
                 16.1 
                 0.020 
                 2.1 
                 0.280 
                 17.6 
                 0.096 
                 11.1 
                 0.106 
                 20.0 
                 0.132 
                 22.4 
               
               
                 GLUT3-7 
                 0.235 
                 15.3 
                 0.023 
                 2.5 
                 0.360 
                 11.5 
                 0.035 
                 1.1 
                 0.068 
                 6.4 
                 0.117 
                 11.0 
               
               
                 GLUT3-9 
                 0.221 
                 20.6 
                 0.011 
                 0.7 
                 0.351 
                 15.7 
                 0.023 
                 2.0 
                 0.158 
                 18.9 
                 0.062 
                 4.3 
               
               
                 GLUT4-1 
                 0.263 
                 16.3 
                 0.016 
                 0.9 
                 0.378 
                 12.8 
                 0.035 
                 0.4 
                 0.266 
                 32.9 
                 0.005 
                 3.6 
               
               
                 GLUT4-4 
                 0.242 
                 16.6 
                 0.008 
                 0.8 
                 0.388 
                 13.2 
                 0.021 
                 0.1 
                 0.265 
                 34.2 
                 0.007 
                 0.7 
               
               
                 GLUT4-10 
                 0.231 
                 15.7 
                 0.004 
                 1.3 
                 0.371 
                 11.5 
                 0.030 
                 0.7 
                 0.113 
                 20.1 
                 0.121 
                 20.5 
               
               
                 GLUT5-4 
                 0.281 
                 17.0 
                 0.038 
                 4.8 
                 0.433 
                 12.1 
                 0.026 
                 0.4 
                 0.183 
                 37.7 
                 0.014 
                 1.1 
               
               
                 GLUT5-5 
                 0.264 
                 18.4 
                 0.020 
                 0.5 
                 0.420 
                 12.8 
                 0.002 
                 0.4 
                 0.117 
                 54.9 
                 0.079 
                 4.3 
               
               
                 GLUT5-9 
                 0.244 
                 18.4 
                 0.016 
                 1.9 
                 0.428 
                 13.0 
                 0.028 
                 1.2 
                 0.143 
                 58.2 
                 0.130 
                 10.3 
               
               
                 GLUT6-4 
                 0.241 
                 14.2 
                 0.031 
                 0.5 
                 0.356 
                 11.7 
                 0.022 
                 0.6 
                 0.085 
                 26.4 
                 0.012 
                 6.6 
               
               
                 GLUT6-5 
                 0.243 
                 13.9 
                 0.010 
                 0.2 
                 0.348 
                 11.3 
                 0.003 
                 0.4 
                 0.123 
                 21.6 
                 0.030 
                 3.8 
               
               
                 GLUT6-10 
                 0.211 
                 14.4 
                 0.018 
                 1.2 
                 0.353 
                 12.1 
                 0.021 
                 0.1 
                 0.132 
                 26.2 
                 0.008 
                 2.4 
               
               
                 GLUT7-2 
                 0.223 
                 14.9 
                 0.031 
                 0.4 
                 0.349 
                 11.6 
                 0.030 
                 0.3 
                 0.173 
                 17.5 
                 0.010 
                 0.9 
               
               
                 GLUT7-6 
                 0.195 
                 17.8 
                 0.019 
                 1.0 
                 0.357 
                 14.8 
                 0.008 
                 0.5 
                 0.141 
                 18.9 
                 0.017 
                 1.2 
               
               
                 GLUT7-7 
                 0.156 
                 19.9 
                 0.015 
                 0.3 
                 0.335 
                 14.1 
                 0.024 
                 0.2 
                 0.112 
                 20.0 
                 0.033 
                 1.5 
               
               
                 GLUT8-5 
                 0.198 
                 16.4 
                 0.042 
                 1.2 
                 0.349 
                 11.8 
                 0.012 
                 0.4 
                 0.300 
                 21.4 
                 0.026 
                 0.9 
               
               
                 GLUT8-6 
                 0.236 
                 34.1 
                 0.016 
                 7.3 
                 0.184 
                 20.9 
                 0.193 
                 18.8 
                 0.464 
                 13.7 
                 0.047 
                 0.5 
               
               
                 GLUT8-9 
                 0.250 
                 38.0 
                 0.011 
                 5.5 
                 0.259 
                 11.2 
                 0.224 
                 10.1 
                 0.471 
                 14.5 
                 0.048 
                 1.3 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 29 
               
             
            
               
                   
               
               
                 Growth rates and lag times of K-12 MG1655 and adipic acid-evolved 
               
               
                 isolates in specified inhibitory concentrations of succinic, pimelic, 
               
               
                 and sebacic acid (sodium salts or neutralized with sodium hydroxide 
               
               
                 to pH 7.0), as measured in the Growth Profiler testing format. 
               
            
           
           
               
               
               
               
            
               
                   
                 50 g/L succinic acid 
                 45 g/L pimelic acid 
                 38 g/L sebacic acid 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 mean 
                 std. error 
                 mean 
                 std. error 
                 mean 
                 std. error 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
                 μ 
                 t lag   
               
               
                 strain 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
                 (h −1 ) 
                 (h) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 MG1655 
                 0.143 
                 23.8 
                 0.046 
                 5.4 
                 0.271 
                 12.4 
                 0.041 
                 1.2 
                 0.135 
                 28.2 
                 0.022 
                 5.7 
               
               
                 ADIP1-1 
                 0.299 
                 16.1 
                 0.012 
                 1.5 
                 0.432 
                 9.6 
                 0.040 
                 0.3 
                 0.244 
                 21.6 
                 0.029 
                 3.8 
               
               
                 ADIP1-9 
                 0.290 
                 14.5 
                 0.006 
                 2.3 
                 0.421 
                 8.8 
                 0.013 
                 0.9 
                 0.248 
                 17.8 
                 0.005 
                 1.1 
               
               
                 ADIP2-5 
                 0.260 
                 25.5 
                 0.028 
                 2.3 
                 0.474 
                 11.0 
                 0.011 
                 0.3 
                 0.315 
                 13.5 
                 0.054 
                 0.5 
               
               
                 ADIP2-6 
                 0.278 
                 21.7 
                 0.013 
                 1.1 
                 0.480 
                 10.6 
                 0.006 
                 0.2 
                 0.319 
                 12.9 
                 0.019 
                 0.3 
               
               
                 ADIP2-10 
                 0.262 
                 22.0 
                 0.040 
                 4.4 
                 0.479 
                 10.8 
                 0.003 
                 1.0 
                 0.290 
                 13.4 
                 0.022 
                 0.8 
               
               
                 ADIP3-2 
                 0.311 
                 13.2 
                 0.013 
                 3.2 
                 0.438 
                 8.4 
                 0.003 
                 1.3 
                 0.254 
                 17.6 
                 0.010 
                 4.6 
               
               
                 ADIP3-4 
                 0.308 
                 12.7 
                 0.006 
                 1.6 
                 0.435 
                 8.5 
                 0.010 
                 0.8 
                 0.262 
                 17.1 
                 0.007 
                 1.3 
               
               
                 ADIP3-8 
                 0.304 
                 13.8 
                 0.016 
                 2.0 
                 0.426 
                 9.1 
                 0.021 
                 1.4 
                 0.253 
                 19.8 
                 0.018 
                 4.1 
               
               
                 ADIP4-1 
                 0.206 
                 15.7 
                 0.013 
                 0.2 
                 0.348 
                 9.9 
                 0.010 
                 0.2 
                 0.308 
                 13.1 
                 0.013 
                 0.5 
               
               
                 ADIP4-8 
                 0.228 
                 15.8 
                 0.037 
                 4.0 
                 0.349 
                 10.3 
                 0.011 
                 0.8 
                 0.289 
                 12.8 
                 0.008 
                 1.3 
               
               
                 ADIP5-2 
                 0.192 
                 13.5 
                 0.028 
                 2.2 
                 0.385 
                 8.7 
                 0.114 
                 2.1 
                 0.288 
                 15.3 
                 0.107 
                 4.2 
               
               
                 ADIP5-6 
                 0.361 
                 12.1 
                 0.019 
                 1.9 
                 0.483 
                 8.5 
                 0.023 
                 1.9 
                 0.273 
                 20.7 
                 0.016 
                 0.6 
               
               
                 ADIP6-3 
                 0.246 
                 14.9 
                 0.065 
                 3.7 
                 0.346 
                 10.4 
                 0.106 
                 1.2 
                 0.268 
                 17.9 
                 0.125 
                 9.4 
               
               
                 ADIP6-9 
                 0.210 
                 18.4 
                 0.062 
                 5.2 
                 0.278 
                 11.1 
                 0.074 
                 1.9 
                 0.181 
                 23.1 
                 0.051 
                 7.6 
               
               
                 ADIP6-10 
                 0.285 
                 12.4 
                 0.033 
                 0.6 
                 0.360 
                 9.7 
                 0.014 
                 0.6 
                 0.267 
                 15.3 
                 0.066 
                 3.1 
               
               
                 ADIP7-2 
                 0.294 
                 12.5 
                 0.018 
                 0.3 
                 0.393 
                 9.0 
                 0.012 
                 0.4 
                 0.356 
                 11.1 
                 0.014 
                 1.1 
               
               
                 ADIP7-5 
                 0.274 
                 12.4 
                 0.024 
                 1.3 
                 0.376 
                 9.4 
                 0.003 
                 0.7 
                 0.353 
                 12.0 
                 0.010 
                 1.4 
               
               
                 ADIP8-3 
                 0.146 
                 22.1 
                 0.016 
                 1.6 
                 0.235 
                 12.8 
                 0.010 
                 0.2 
                 0.171 
                 28.9 
                 0.017 
                 1.4 
               
               
                 ADIP8-7 
                 0.163 
                 23.2 
                 0.014 
                 0.7 
                 0.268 
                 12.8 
                 0.060 
                 0.6 
                 0.157 
                 30.9 
                 0.012 
                 2.2 
               
               
                 ADIP8-10 
                 0.131 
                 20.0 
                 0.042 
                 0.9 
                 0.249 
                 12.8 
                 0.003 
                 0.3 
                 0.165 
                 30.0 
                 0.012 
                 2.3 
               
               
                   
               
            
           
         
       
     
     Biological Production of Diacids 
     Glutaric acid has been the target of two studies, both in engineered  E. coli . The highest reported titer of 0.82 g/L from glucose was achieved via native L-lysine production (Adkins et al., 2013) (see FIG. 1 of Adkins et al., 2013, hereby incorporated by reference). A heterologous pathway composed of genes from  Pseudomonas putida  KT2440 was expressed from plasmids and consisted of a lysine monooxygenase to convert lysine to 5-aminovaleramide, a 5-aminovaleramidase to convert 5-aminovaleramide to 5-aminovaleric acid, a 5-aminovalerate transaminase to convert 5-aminovaleric acid and α-ketoglutarate to glutarate semialdehyde and L-glutamate, and a glutarate semialdehyde dehydrogenase to convert glutarate semialdehyde to glutaric acid. To improve flux toward L-lysine, previously known feedback resistance mutations were made in DapA (4-hydroxytetrahydrodipicolinate synthase) and LysC (asparate kinase III), and these modified proteins were additionally overexpressed from plasmids. Finally, cadA and IdcC, encoding two lysine decarboxylases, were deleted to prevent side conversion of L-lysine into cadaverine. In a second study (Park et al., 2013), glutarate was not able to be produced Prom glucose, however 1.7 g/L glutarate could be produced by feeding both L-lysine and α-ketoglutarate, with only 5-aminovalerate able to be produced from glucose without supplementation. This appeared to use the same or similar heterologous genes from  P. putida  as in the previous paper, only expressed together as an artificial operon on one plasmid instead of in two operons on two plasmids. The strain additionally contained a dapA promoter replacement to allow constitutive expression of lysine biosynthesis, and deletion of speE, speG, patA, and puuPA (which would prevent production of spermidine, acetylspermidine, putrescine degradation, and putrescine import, although likely not for any targeted purpose here; use of this background strain for production of other compounds). 
     Overproduction of adipic acid, as well as other diacids that can be readily converted chemically into adipic acid, has been more heavily pursued due to the use of adipic acid in existing commercial polyamides. A wide variety of routes have been explored, with the first reported direct route in  E. coli  being a proof-of-concept demonstration, with a maximum titer of 639 μg/mL adipic acid (Yu et al., 2014). In the best-performing strain, acetyl-CoA and succinyl-CoA were condensed by a reversible 3-oxoadipyl-CoA thiolase (PaaJ from  E. coli ), 3-oxoadipyl-CoA was reduced to 3-hydroxyadipyl-CoA by a 3-hydroxyacyl-CoA dehydrogenase (PaaH1 from  Ralstonia eutropha ), 3-hydroxyadipyl-CoA was dehydrated to 2,3-dehydroadipyl-CoA by a putative enoyl-CoA hydratase (h16_AA307 gene product from  Ralstonia eutropha  H16), 2,3-dehydroadipyl-CoA was reduced to adipyl-CoA by a trans-enoyl-CoA reductase (Ter from  Euglena gracilis ), adipyl-CoA was converted to adipyl-phosphate by a phosphate butyryltransferase (Ptb from  Clostridium acetobutylicum ), and adipyl-phosphate was finally dephosphorylated to adipic acid using a butyryl kinase (Buk1 from  Clostridium acetobutylicum ) (see FIG. 1 of Yu et al., 2014, hereby incorporated by reference). The genes encoding these enzymes were heterologously expressed on two different plasmids, and additional modifications were made to the genome to improve the succinyl-CoA supply using modifications that were previously employed for succinic acid production via succinyl-CoA (Liu et al., Process Biochem. 47:1532, 2012). These were deletions of ptsG, poxB, pta, sdhA, and iciR. 
     Very recently, production of 2.5 g/L adipic acid in bioreactors, as well as smaller quantities of suberic acid (C 8 ) and sebacic acid (C 10 ), or pimelic acid (C 7 ) alone, was demonstrated in  E. coli  from glycerol using a relatively similar modular pathway (Cheong et al., 2016). It was composed of a thiolase capable of condensing a primer and extender unit (e.g. succinyl-CoA and acetyl-CoA for adipic acid), plus a hydroxyacyl-CoA hydrogenase, an enoyl-CoA hydratase, an enoyl-CoA reductase to generate the fully reduced product (for adipic acid, this would be adipyl-CoA) (see FIG. 1a of Cheong et al., 2016, hereby incorporated by reference). The major difference from previous work was the use of an acyl-CoA thioesterase to liberate the final diacid from CoA. For production of adipic acid production, a CoA transferase (Cat1 from  Clostridium kluyveri ) was expressed for activation of succinic acid to succinyl-CoA, with native sucD encoding a subunit of native  E. coli  succinyl-CoA synthetase deleted. The thiolase was  E. coli  PaaJ, the hydroxyacyl-CoA hydrogenase was  E. coli  PaaH, the enoyl-CoA hydratase was  E. coli  PaaF, the enoyl-CoA reductase was Ter from  Treponema denticola , and the acyl-CoA thioesterase was the dicarboxylic acyl-CoA thioesterase Acot8 from  Mus musculus . Additionally, fermentative pathways leading to production of acetate (pta and poxB), lactate (IdhA), and ethanol (adhE) were deleted from the background strain. To produce approximately 25 mg/L pimelic acid, glutaric acid was fed to generate glutaryl-CoA as a primer unit via the action of Cat1 in a background strain deficient in ldhA, poxB, pta, adhE, and the gene encoding fumarate reductase, frdA. To generate a mixture containing predominantly adipic acid at 95 mg/L, but also 34 mg/L suberic acid and 13 mg/L sebacic acid, an alternative thiolase (DcaF), hydroxyacyl-CoA dehydrogenase (DcaH), and enoyl-CoA hydratase (DcaE) from  Acinetobacter  sp. ADP1, with other enzymes the same as for producing adipic acid. In this case, the background strain was the same as that used for pimelic acid, with additional deletions in a number of other native  E. coli  acyl-CoA thioesterases (yciA, ybgC, ydiI, tesA, fadM, and tesB). 
     An alternative native adipate production pathway (reverse adipate degradation) has been reported in the bacterium  Thermobifida fusca  B6, where succinyl-CoA and acetyl-CoA are condensed by a p-ketothioase (EC 2.3.1.174) to form 3-oxoadipyl-CoA, followed by a series of reactions that are the same as those shown in FIG. 1 of Yu et al., 2014, to form adipyl-CoA. Adipyl-CoA is subsequently converted to adipic acid by a succinyl-CoA synthetase (Tfu_2577, Tfu_2576). A titer of over 2 g/L of adipic acid was obtained by fermentation of  T. fusca  B6 on glucose and milled corncob (Deng and Mao, 2015). The pathway has not yet been heterologously expressed in other organisms. 
     Adipic acid production from  S. cerevisiae  has been described by Verdezyne (e.g. WO 2011/003034 A2), however the starting substrates are fatty acids and the pathway for adipic acid production is therefore very different. The engineered microorganisms described have genetic modifications that add or increase the 6-oxohexanoic acid dehydrogenase, omega oxo fatty acid dehydrogenase, 6-hydroxyhexanoic acid dehydrogenase, omega hydroxyl fatty acid dehydrogenase, hexanoate synthase, monooxygenase, monooxygenase reductase, fatty alcohol oxidase, acyl-CoA ligase, acyl-CoA oxidase, enoyl-CoA hydratase, 3-L-hydroxyacyl-CoA dehydrogenase, and/or acetyl-CoA C-acetyltransferase activities. These modifications suggest a pathway where a fatty acid is broken down into multiple shorter chain diacids, derivatives are condensed with acetyl-CoA to increase the chain length where necessary, and C 6  diacid products are reduced to adipic acid following some similar steps to those shown in FIG. 1a of Cheong et al., supra). 
     In addition to directly producing the final diacid products, other groups have developed alternative pathways to cis,cis-muconic acid, which can be chemically or enzymatically reduced to adipic acid, or glucaric acid, which can be produced in few steps from glucose and can be chemically reduced to adipic acid. Basic pathway schematics are shown in FIG. 4 of Polen et al. (2013) for muconic acid, and FIG. 5 of Polen et al. (2013) for glucaric acid. FIGS. 4 and 5 of Polen et al. (2013) are hereby incorporated by reference. 
     Muconic acid has been produced at 141 mg/L from glucose in  S. cerevisiae  (Curran et al., 2013) in a strain possessing a deletion in ARO3 (a 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isoform), expression of a feedback resistant version of ARO4 (another 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isoform; the modifications included both a feedback resistant coding mutation and a constitutive promoter replacement), and a deletion in ZWF1 (glucose-6-phosphate dehydrogenase). The heterologous pathway was expressed on 3 plasmids containing codon-optimized DHS from  Podospora anserina , catechol 1,2-dioxygenase from  Candida albicans , overexpressed TKL1 (transketolase) from  S. cerevisiae , and protocatechuate decarboxylase from  Enterobacter cloacae . To obtain higher expression of protocatechuate decarboxylase, another copy of the gene was also integrated onto the chromosome. The combination of non-pathway mutations (deletion of ZWF1 and ARO3; expression of feedback-resistant ARO4; overexpression of TKL1) served to relieve feedback inhibition of the shikimate pathway that is ordinarily employed for aromatic amino acid biosynthesis, and to direct flux into the pentose phosphate pathway via transketolase instead of glucose-6-phosphate dehydrogenase, increasing the supply of the erythrose-4-phosphate precursor. 
     Muconic acid has additionally been produced (170 mg/L of muconic acid) from glucose in  E. coli  possessing deletions in ptsH, ptsI, crr, and pykF and overexpressing ubiC, a feedback resistant version of aroF, aroE, and aroL (Sengupta et al., 2015). These mutations increase the supply of the precursors erythrose-4-phosphate and phosphoenolpyruvate. The heterologous pathway was composed of pobA from  Pseudomonas putida  KT2440, aroY from  Klebsiella pneumoniae , and catA from  Acinetobacter  sp. strain ADP1.  E. coli  co-cultures consisting of strains engineered to overproduce DHS, and to convert DHS to muconic acid, have additionally been engineered to produce muconic acid from glycerol at a final titer of 2 g/L (Zhang et al., Microb. Cell Factories 14:134, 2015). 
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         US 2003/0068611 A1 (E I DuPont De Nemours and Company) 
         WO 2009/143455 A2 (Microbia Inc.) 
         WO 2011/154503 A1 (Evonik Degussa GmbH) 
         US 2014/0135526 A1 (OPX Biotechnologies Inc.) 
         WO 2014/029592 A1 (Evonik Degussa GmbH) 
         EP 2 032 711 A1 (TNO) 
         EP 2 580 315 A2 (Cobalt Technologies Inc.) 
         WO 01/05959 A1 (Ajinomoto K K) 
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         WO 2016/162442 A1 (Metabolic Explorer SA)