Patent Publication Number: US-2021164003-A1

Title: Reducing the accumulation of imines/enamines for the production of amino acids or amino acid-derived products

Description:
BACKGROUND OF THE INVENTION 
     The overproduction of amino acids, such as lysine, and amino acid-derived products, such as cadaverine, often requires the remodeling of the host cell&#39;s metabolism in order to increase the flux of the carbon and nitrogen containing compounds towards the desired products. However, modifying the flux of metabolic pathways can lead to the accumulation of intermediates that do not normally accumulate inside the cell. Such metabolic intermediates may be either the final product of an enzymatic reaction or an intermediate of an enzymatic reaction that leaks out of the catalytic site of the enzyme. The metabolic intermediate that accumulates may also be toxic to the cell, or induce the activity of other pathways that transform the intermediate into a compound that is toxic to the cell (Danchin,  Microbial Biotechnology  10:57-72, 2017). 
     Imine/enamine intermediates often form during transamination, racemization, or deamination reactions that lead to the formation of reactive amino acid derivatives (e.g., aminoacrylate or iminopropionate). Imine/enamine formation sometimes involves the cofactor pyridoxal phosphate (PLP). Reactive imines/enamines are known to cause cellular damage and can accumulate inside the cell during the overproduction of amino acids, such as lysine, and amino acid-derived products, such as cadaverine. For example, the overproduction of lysine or cadaverine in  Escherichia coli  can involve the remodeling of the host metabolism in such a way that leads to the production of imine/enamine compounds. 
     The overproduction of lysine or cadaverine involves the overexpression of genes encoding one or more of the following proteins: dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52), diaminopimelate dehydrogenase (DAPDH, EC 1.4.1.16), and diaminopimelate decarboxylase (DAPDC, EC 4.1.1.20) (Anastassiadis,  Recent Patents on Biotechnology  1: 11-24, 2007). DHDPS catalyzes the condensation of pyruvate and aspartate semialdehyde to form 4-hydroxy-2,3,4,5-tetrahydro-L,L-dipicolinic acid, which involves the formation of an imine intermediate (Dobson et al.,  Protein Science  17:2080-2090, 2008). DAPDH catalyzes the reductive amination of L-2-amino-6-ketopimelate, which creates an imine intermediate that is reduced by NADPH to produce meso-DAP (Scapin et al.,  Biochemistry  37: 3278-3285, 1998). DAPDC is a PLP-dependent enzyme that catalyzes the decarboxylation of meso-DAP to lysine. Certain DAPDCs form an aldimine in the presence of PLP (Hu et al.,  J. Biol. Chem.  283: 21284-21293, 2008). 
     Lysine, threonine, and methionine share the same upstream metabolic pathway, since all three amino acids are derived from aspartate. The conversion of aspartate to the precursors of lysine, threonine, or methionine is catalyzed by three different aspartate kinases, one for each amino acid (LysC, MetL, ThrA). However, increasing the flux through aspartate biosynthesis in order to increase either lysine or cadaverine production will also increase threonine production, since they share a common precursor. The accumulation of threonine in the cell can trigger the activity of threonine dehydratase (EC 4.3.1.19), which is the first enzyme involved in the catabolism of threonine to isoleucine. Threonine dehydratase is a PLP-dependent enzyme that catalyzes the dehydration of threonine to aminocrotonoate, an enamine intermediate. Aminocrotonoate can tautomerize to iminobutyrate, an imine intermediate. Therefore, the accumulation of threonine in the cell can increase the accumulation of these toxic enamine/imine intermediates inside the cell. 
     In certain cases, the flux through the threonine biosynthesis pathway is reduced or eliminated in order to increase the flux of carbon- and nitrogen-containing compounds going towards lysine and cadaverine biosynthesis. However, threonine needs to be added to the medium in order to ensure that the intracellular concentration of threonine is sufficient for cell growth. The addition of external threonine may lead to the addition of sufficient threonine that the amino acid accumulates inside the cell, in which case, the accumulation of aminocrotonoate and iminobutyrate can result as described above. 
     The conversion of lysine to cadaverine involves the PLP-dependent enzyme lysine decarboxylase. Therefore, overproduction of cadaverine involves increasing the intracellular concentration of PLP, which can be accomplished by adding PLP to the medium or overexpressing genes involved in the synthesis of PLP (e.g., pdxST). As described above, some of the reactions that lead to the accumulation of imine/enamine are PLP-catalyzed reactions. Therefore, an increase in the intracellular concentration of PLP increases the probability for imine/enamine to form and accumulate inside the cell. 
     It was discovered that  Salmonella enterica  produces a protein RidA (YjgF) that has imine/enamine deaminase activity, allowing it to catalyze the release of ammonia and the production of a more stable and less toxic intermediate from imine/enamine compounds (Lambrecht et al.,  J. Biol. Chem.  287: 3454-3461, 2012). RidA protects  S. enterica  from the harmful imine/enamine molecules formed by the activity of the PLP-dependent threonine dehydratase (IlvA) by catalyzing the removal of ammonia from the intermediate enamine/imine compounds to form the nontoxic 2-ketobutyrate. The activity of RidA was also shown to protect cells from 2-aminoacrylate, an enamine formed during serine catabolism (Lambrecht et al.,  mBio  4: 1-8, 2013). The accumulation of imines/enamines also inactivates the PLP-catalyzed enzymes in the cell, so the removal of imines/enamines is important. 
     BRIEF SUMMARY OF ASPECTS OF THE INVENTION 
     Provided herein are host cells genetically modified to enhance removal of imine and enamine compounds and thus increase, relative to host cells of the same strain that do not have the genetic modification to enhance imine and enamine removal, the production of an amino acid or amino acid derivate for which imine and/or enamine is an intermediate. Also provided herein are methods of generating such host cells; and methods of using the host cells to produce increased yields of an amino acid or amino acid derivative, such as lysine or cadaverine. 
     Thus, in one aspect, provided herein is a method of engineering a host cell to increase production of an amino acid or an amino acid derivative, e.g., lysine or cadaverine, the method comprising introducing a polynucleotide, e.g., heterologous polynucleotide, comprising a nucleic acid that encodes an imine/enamine deaminase polypeptide into the host cell, wherein the host cell has at least one additional genetic modification that increases production of the amino acid or the amino acid derivative compared to wildtype host cell; culturing the host cell under conditions in which the imine/enamine deaminase polypeptide is expressed, and selecting a host cell that produces an increased amount of an amino acid or amino acid derivative, e.g., lysine or cadaverine, relative to a counterpart host cell of the same strain that has not been modified to express the polynucleotide encoding the imine/enamine deaminase polypeptide. In some embodiments, the imine/enamine deaminase polypeptide is a YoaB polypeptide. In some embodiments, the imine/enamine deaminase polypeptide has at least 70% amino acid sequence identity to SEQ ID NO:10. In some embodiments, the imine/enamine deaminase polypeptide has at least 80% identity to the amino acid sequence of SEQ ID NO:10. In some embodiments, the imine/enamine deaminase polypeptide has at least 90% identity to the amino acid sequence of SEQ ID NO:10. In some embodiments, the imine/enamine deaminase polypeptide has at least 95% identity to the amino acid sequence of SEQ ID NO:10. In some embodiments, the imine/enamine polypeptide comprises the amino acid sequence of SEQ ID NO:10. In some embodiments, the imine/enamine deaminase polypeptide is a YjgH polypeptide. In some embodiments, the imine/enamine deaminase polypeptide has at least 70% amino acid sequence identity to SEQ ID NO:12. In some embodiments, the imine/enamine deaminase polypeptide has at least 80% identity to the amino acid sequence of SEQ ID NO:12. In some embodiments, the imine/enamine deaminase polypeptide has at least 90% identity to the amino acid sequence of SEQ ID NO:12. In some embodiments, the imine/enamine deaminase polypeptide has at least 95% identity to the amino acid sequence of SEQ ID NO:12. In some embodiments, the imine/enamine deaminase polypeptide comprises the amino acid sequence of SEQ ID NO:12. In some embodiments, the polynucleotide is contained in an expression vector introduced into the cell, wherein the expression vector comprises the polynucleotide operably linked to a promoter. In other embodiments, the polynucleotide introduced into the host cell is integrated into the host chromosome. In some embodiments, the genetically modified host cell is additionally modified to overexpress an exogenous lysine decarboxylase; and/or one or more exogenous lysine biosynthesis polypeptides. In some embodiments, the genetically modified host cell additionally overexpresses an exogenous LysC, DapA, LysA, Asd, DapB, or AspC polypeptide. In some embodiments, the genetically modified host cell is additionally modified to overexpress exogenous CadA, LysC, DapA, LysA, Asd, DapB, and AspC polypeptides. In some embodiments, the genetically modified host cell is of the genus  Escherichia,  e.g.,  Escherichia coli; Hafnia,  e.g.,  Hafnia alvei;  or  Corynebacterium,  e.g.,  Corynebacterium glutamicum.    
     In another aspect, provided herein is a genetically modified host cell produced according to a method of the preceding paragraph. 
     In a further aspect, provided herein is a genetically modified host cell comprising a polynucleotide, e.g., a heterologous polynucleotide, comprising a nucleic acid encoding an imine/enamine deaminase polypeptide that increases the amount of an amino acid, e.g., lysine, or amino acid derivative, e.g., cadaverine, compared to a counterpart host cell that has not been modified to express the polynucleotide encoding the imine/enamine polypeptide; and has at least one additional genetic modification that increases production of the amino acid or the amino acid derivative compared to wildtype host cells. In some embodiments, the imine/enamine deaminase polypeptide is a YoaB polypeptide. In some embodiments, the imine/enamine deaminase polypeptide has at least 70% amino acid sequence identity to SEQ ID NO:10. In some embodiments, the imine/enamine deaminase polypeptide has at least 80% identity to the amino acid sequence of SEQ ID NO:10. In some embodiments, the imine/enamine deaminase polypeptide has at least 90% identity to the amino acid sequence of SEQ ID NO:10. In some embodiments, the imine/enamine deaminase polypeptide has at least 95% identity to the amino acid sequence of SEQ ID NO:10. In some embodiments, the imine/enamine deaminase polypeptide is a YjgH polypeptide. In some embodiments, the imine/enamine deaminase polypeptide has at least 70% amino acid sequence identity to SEQ ID NO:12. In some embodiments, the imine/enamine deaminase polypeptide has at least 80% identity to the amino acid sequence of SEQ ID NO:12. In some embodiments, the imine/enamine deaminase polypeptide has at least 90% identity to the amino acid sequence of SEQ ID NO:12. In some embodiments, the imine/enamine deaminase polypeptide has at least 95% identity to the amino acid sequence of SEQ ID NO:12. In some embodiments, the imine/enamine deaminase polypeptide comprises the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:12. In some embodiments, the polynucleotide is contained in an expression vector introduced into the cell, wherein the expression vector comprises the polynucleotide operably linked to a promoter. In other embodiments, the polynucleotide introduced into the host cell is integrated into the host chromosome. In some embodiments, the genetically modified host cell additionally overexpresses a lysine decarboxylase; and/or one or more lysine biosynthesis polypeptides. In some embodiments, the genetically modified host cell is of the genus  Escherichia,  e.g.,  Escherichia coli; Hafnia,  e.g.,  Hafnia alvei;  or  Corynebacterium,  e.g.,  Corynebacterium glutamicum.  In some embodiments, the genetically modified host cell additionally overexpresses a LysC, DapA, LysA, Asd, DapB, or AspC polypeptide. In some embodiments, the genetically modified host cell additionally overexpresses a CadA, LysC, DapA, LysA, Asd, DapB, and AspC polypeptide. 
     In a further aspect, provided herein is a method of producing an amino acid or an amino acid derivative, e.g., lysine or cadaverine, the method comprising culturing a host cell as set forth in the two preceding paragraphs under conditions in which the imine/enamine deaminase polypeptide is expressed. In some embodiments, the method further comprises isolating the amino acid or amino acid derivative, e.g., lysine or cadaverine. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  provides an illustrative alignment of RidA homolog protein sequences (identified by PDB accession numbers). 
         FIG. 2  provides an illustrative alignment of  E. coli  RidA and paralog protein sequences. Positions corresponding to E120, C107, V18, K73, and E122 of RidA are underlined. Amino acids at positions D76 and K123 of YjgH, corresponding to positions K73 and E122 of RidA, respectively) are conserved in the YjgH and YoaB sequences and are shown in bold, enlarged font. 
     
    
    
     DETAILED DESCRIPTION OF ASPECTS OF THE INVENTION 
     Terminology 
     As used in the context of the present disclosure, an “imine/enamine deaminase polypeptide” refers to an enzyme that decreases imine/enamine levels in a host cells. Such a polypeptide catalyzes the release of ammonia from imine/enamine. A polypeptide that decreases imine/enamine levels in accordance with the disclosure typically decreases levels by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, or greater, when produced by a host cell genetically modified to overexpress the imine/enamine deaminase polypeptide compared to a wildtype counterpart host cell that has not been genetically modified to overexpress the imine/enamine deaminase polypeptide. 
     The term “imine/enamine deaminase polypeptide” encompasses biologically active variants, alleles, mutants, and interspecies homologs to the specific polypeptides described herein. A nucleic acid that encodes an imine/enamine deaminase polypeptide refers to a gene, pre-mRNA, mRNA, and the like, including nucleic acids encoding variants, alleles, mutants, and interspecies homologs of the particular amino acid sequences described herein. 
     The terms “increased expression” and “overexpression” of an imine/enamine deaminase polypeptide are used interchangeably herein to refer to an increase in the amount of imine/enamine deaminase polypeptide in a genetically modified cell, e.g., a cell into which an expression construct encoding imine/enamine deaminase polypeptide has been introduced, compared to the amount of imine/enamine deaminase polypeptide in a counterpart cell that does not have the genetic modification, i.e., a cell of the same strain without the modification. An increased level of expression for purposes of this application is at least 5%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater, compared to the counterpart unmodified cell. The unmodified cell need not express the imine/enamine deaminase. Thus, the term “overexpression” also includes embodiments in which an imine/enamine deaminase polypeptide is expressed in a host cell that does not natively express the imine/enamine deaminase polypeptide. Increased expression of an imine/enamine deaminase polypeptide can be assessed by any number of assays, including, but not limited to, measuring the level of RNA transcribed from the imine/enamine deaminase gene, the level of imine/enamine deaminase polypeptide, and/or the level of imine/enamine deaminase polypeptide activity. 
     The term “enhanced” in the context of the production of an amino acid, e.g., lysine, or an amino acid derivative, e.g., a lysine derivative, such as cadaverine, as used herein refers to an increase in the production of amino acid, e.g., lysine, or the derivative by a genetically modified host cell in comparison to a control counterpart cell, such as a cell of the wildtype strain or a cell of the same strain that does not have the genetic modification to increase production of the amino acid or amino acid derivative. Production of the amino acid or its derivative is enhanced by at least 5%, typically at least 0%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater compared to the control cell. 
     The terms “numbered with reference to”, or “corresponding to,” or “determined with reference to” when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. For example, a residue in a YoaB polypeptide variant or homolog “corresponds to” an amino acid at a position in SEQ ID NO:10 when the residue aligns with the amino acid in a comparison of SEQ ID NO:10 and the homolog or variant in a maximal alignment. Similarly, a residue in a YjgH polypeptide variant “corresponds to” an amino acid at a position in SEQ ID NO:12 when the residue aligns with the amino acid in a comparison of SEQ ID NO:12 and the homolog or variant in a maximal alignment. 
     The terms “polynucleotide” and “nucleic acid” are used interchangeably and refer to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. A nucleic acid as used in the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs may be used that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); positive backbones; non-ionic backbones, and non-ribose backbones. Nucleic acids or polynucleotides may also include modified nucleotides that permit correct read-through by a polymerase. “Polynucleotide sequence” or “nucleic acid sequence” includes both the sense and antisense strands of a nucleic acid as either individual single strands or in a duplex. As will be appreciated by those in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus the sequences described herein also provide the complement of the sequence. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc. Nucleic acid sequences are presented in the 5′ to 3′ direction unless otherwise specified. 
     The term “substantially identical,” used in the context of two nucleic acids or polypeptides, refers to a sequence that has at least 40%, 45%, or 50% sequence identity with a reference sequence. Percent identity can be any integer from 50% to 100%. Some embodiments include at least: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. 
     Two nucleic acid sequences or polypeptide sequences are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. 
     For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. 
     An algorithm that may be used to determine whether an imine/enamine deaminase polypeptide has sequence identity to SEQ ID NO:10 or 12, or another polypeptide reference sequence, is the BLAST algorithm, which is described in Altschul et al., 1990, J. Mol. Biol. 215:403-410, which is incorporated herein by reference. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the worldwide web at ncbi.nlm.nih.gov/). For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff &amp; Henikoff, 1989, Proc. Natl. Acad. Sci. USA 89:10915). Other programs that may be used include the Needleman-Wunsch procedure, J. MoI. Biol. 48: 443-453 (1970), using BLOSUM62, a Gap start penalty of 7 and gap extend penalty of 1; and gapped BLAST 2.0 (see Altschul, et al. 1997, Nucleic Acids Res., 25:3389-3402) both 
     A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith &amp; Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman &amp; Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson &amp; Lipman, Proc. Nat&#39;l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection. 
     Nucleic acid or protein sequences that are substantially identical to a reference sequence include “conservatively modified variants.” With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence. 
     As to amino acid sequences, one of skill will recognize that individual substitutions, in a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Examples of amino acid groups defined in this manner can include: a “charged/polar group” including Glu (Glutamic acid or E), Asp (Aspartic acid or D), Asn (Asparagine or N), Gln (Glutamine or Q), Lys (Lysine or K), Arg (Arginine or R) and His (Histidine or H); an “aromatic or cyclic group” including Pro (Proline or P), Phe (Phenylalanine or F), Tyr (Tyrosine or Y) and Trp (Tryptophan or W); and an “aliphatic group” including Gly (Glycine or G), Ala (Alanine or A), Val (Valine or V), Leu (Leucine or L), Ile (Isoleucine or I), Met (Methionine or M), Ser (Serine or S), Thr (Threonine or T) and Cys (Cysteine or C). Within each group, subgroups can also be identified. For example, the group of charged/polar amino acids can be sub-divided into sub-groups including: the “positively-charged sub-group” comprising Lys, Arg and His; the “negatively-charged sub-group” comprising Glu and Asp; and the “polar sub-group” comprising Asn and Gln. In another example, the aromatic or cyclic group can be sub-divided into sub-groups including: the “nitrogen ring sub-group” comprising Pro, His and Trp; and the “phenyl sub-group” comprising Phe and Tyr. In another further example, the aliphatic group can be sub-divided into sub-groups including: the “large aliphatic non-polar sub-group” comprising Val, Leu and Ile; the “aliphatic slightly-polar sub-group” comprising Met, Ser, Thr and Cys; and the “small-residue sub-group” comprising Gly and Ala. Examples of conservative mutations include amino acid substitutions of amino acids within the sub-groups above, such as, but not limited to: Lys for Arg or vice versa, such that a positive charge can be maintained; Glu for Asp or vice versa, such that a negative charge can be maintained; Ser for Thr or vice versa, such that a free —OH can be maintained; and Gln for Asn or vice versa, such that a free —NH2 can be maintained. The following six groups each contain amino acids that further provide illustrative conservative substitutions for one another. 1) Ala, Ser, Thr; 2) Asp, Glu; 3) Asn, Gln; 4) Arg, Lys; 5) Ile, Leu, Met, Val; and 6) Phe, Try, and Trp (see, e.g., Creighton, Proteins (1984)). 
     The term “promoter,” as used herein, refers to a polynucleotide sequence capable of driving transcription of a DNA sequence in a cell. Thus, promoters used in the polynucleotide constructs of the invention include cis- and trans-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter can be a cis-acting transcriptional control element, including an enhancer, a repressor binding sequence and the like. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription. Most often the core promoter sequences lie within 1-2 kb of the translation start site, more often within 1 kbp and often within 500 bp or 200 bp or fewer, of the translation start site. By convention, promoter sequences are usually provided as the sequence on the coding strand of the gene it controls. In the context of this application, a promoter is typically referred to by the name of the gene for which it naturally regulates expression. A promoter used in an expression construct of the invention is referred to by the name of the gene. Reference to a promoter by name includes a wild type, native promoter as well as variants of the promoter that retain the ability to induce expression. Reference to a promoter by name is not restricted to a particular species, but also encompasses a promoter from a corresponding gene in other species. 
     A “constitutive promoter” in the context of this invention refers to a promoter that is capable of initiating transcription under most conditions in a cell, e.g., in the absence of an inducing molecule. An “inducible promoter” initiates transcription in the presence of an inducer molecule. 
     As used herein, a polynucleotide is “heterologous” to an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, when a polynucleotide encoding a polypeptide sequence is said to be operably linked to a heterologous promoter, it means that the polynucleotide coding sequence encoding the polypeptide is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence, e.g., from a different gene in the same species, or an allele from a different species). Similarly, a polypeptide is “heterologous” to a host cell if the native wildtype host cell does not produce the polypeptide. 
     The term “exogenous” as used herein refers generally to a polynucleotide sequence or polypeptide that is introduced into a host cell by molecular biological techniques to produce a recombinant cell. Examples of “exogenous” polynucleotides include vectors, plasmids, and/or man-made nucleic acid constructs encoding a desired protein. An “exogenous” polypeptide expressed in the host cell may occur naturally in the wildtype host cell or may be heterologous to the host cell. The term also encompasses progeny of the original host cell that has been engineered to express the exogenous polynucleotide or polypeptide sequence, i.e., a host cell that expresses an “exogenous” polynucleotide may be the original genetically modified host cell or a progeny cell that comprises the genetic modification. 
     The term “endogenous” refers to naturally-occurring polynucleotide sequences or polypeptides that may be found in a given wild-type cell or organism. In this regard, it is also noted that even though an organism may comprise an endogenous copy of a given polynucleotide sequence or gene, the introduction of an expression construct or vector encoding that sequence, such as to over-express or otherwise regulate the expression of the encoded protein, represents an “exogenous” copy of that gene or polynucleotide sequence. Any of the pathways, genes, or enzymes described herein may utilize or rely on an “endogenous” sequence, which may be provided as one or more “exogenous” polynucleotide sequences, or both. 
     “Recombinant nucleic acid” or “recombinant polynucleotide” as used herein refers to a polymer of nucleic acids wherein at least one of the following is true: (a) the sequence of nucleic acids is foreign to (i.e., not naturally found in) a given host cell; (b) the sequence may be naturally found in a given host cell, but in an unnatural (e.g., greater than expected) amount; or (c) the sequence of nucleic acids comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, regarding instance (c), a recombinant nucleic acid sequence will have two or more sequences from unrelated genes arranged to make a new functional nucleic acid. 
     The term “operably linked” refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a DNA or RNA sequence if it stimulates or modulates the transcription of the DNA or RNA sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance. 
     The term “expression cassette” or “DNA construct” or “expression construct” refers to a nucleic acid construct that, when introduced into a host cell, results in transcription and/or translation of an RNA or polypeptide, respectively. In the case of expression of transgenes, one of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only substantially identical to a sequence of the gene from which it was derived. As explained herein, these substantially identical variants are specifically covered by reference to a specific nucleic acid sequence. One example of an expression cassette is a polynucleotide construct that comprises a polynucleotide sequence encoding a polypeptide for use in the invention operably linked to a promoter, e.g., its native promoter, where the expression cassette is introduced into a heterologous microorganism. In some embodiments, an expression cassette comprises a polynucleotide sequence encoding a polypeptide of the invention where the polynucleotide that is targeted to a position in the genome of a microorganism such that expression of the polynucleotide sequence is driven by a promoter that is present in the microorganism. 
     The term “host cell” as used in the context of this invention refers to a microorganism and includes an individual cell or cell culture that can be or has been a recipient of any recombinant vector(s) or isolated polynucleotide(s) of the invention. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells into which a recombinant vector or a polynucleotide of the invention has been introduced, including by transformation, transfection, and the like. 
     The term “isolated” refers to a material that is substantially or essentially free from components that normally accompany it in its native state. For example, an “isolated polynucleotide,” as used herein, may refer to a polynucleotide that has been isolated from the sequences that flank it in its naturally-occurring or genomic state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment, such as by cloning into a vector. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment, or if it is artificially introduced in the genome of a cell in a manner that differs from its naturally-occurring state. Alternatively, an “isolated peptide” or an “isolated polypeptide” and the like, as used herein, refers to a polypeptide molecule that is free of other components of the cell, i.e., it is not associated with in vivo substances. 
     Aspects of the Disclosure 
     The present disclosure is based, in part, on the discovery that increased expression of one or more imine/enamine deaminase polypeptides in a microorganism, such as a gram-negative bacterium, enhances amino acid, e.g., lysine, production and/or production of an amino acid derivative of lysine, such as cadaverine. 
     RidA is a member of the YjgF/YER057c/UK114 family that is conserved in all domains of life (pfam: PF01042). The members of this family are small proteins of about 15 kDa, and form homotrimers—a trimeric barrel-like quaternary structure. The family members have diverse phenotypes and do not have a clearly defined biological role like most other well-defined protein families with defined substrates and products (e.g., P450 mono-oxidase, DNA polymerase, or lysine decarboxylase). Recently, the crystal structure of RidA from  Arapidopsis thaliana  was published (PBD ID: 5HP7) (Lu et al.,  Scientific Reports  6: 30494, 2016). The crystal structures of other members of this family have also been published (PBD ID: 1QD9  Bacillus subtilis  YabJ, 1X25 of  Sulfolobus tokodaii  YjgF member, 1QU9 of  E. coli  RidA, 2UYN of  E. coli  TdcF, 1ONI of human p14.5). Sequences of RidA homolog proteins (identified by PDB accession numbers) are shown in  FIG. 1 . The members of this family have several important residues involved in trimer formation and substrate binding. Five of these positions are indicated by underline in the 5HP7 amino acid sequence. 
       Escherichia coli  also expresses a gene encoding RidA.  E. coli  RidA has been shown to be important in the synthesis of thiamine (Bazurto et al.,  mBio  7: 1-9, 2016), and can also function as a chaperone protein during oxidative stress (Muller et al.,  Nature Communications  5: 1-14, 2014). The overexpression of enzymes in order to increase metabolic flux towards the production of lysine is expected to produce metabolic burden and stress on the cell; therefore, it would be expected that the overexpression of RidA would help to remove toxic intermediates formed as a result of metabolic stress and increase lysine production. Surprisingly, it was discovered here that the overexpression of  E. coli  RidA did not increase the production of lysine. 
     However,  E. coli  also contains four paralogs of RidA, which are YjgH, TdcF, RutC, and YoaB. Surprisingly, it was observed that the overexpression of certain paralogs did lead to a change in lysine and cadaverine production. For example, the overexpression of the genes encoding YjgH and YoaB increased lysine and cadaverine production. 
     The crystal structure of  E coli  YjgH has been solved (PDB ID: 1PF5). Crystal structure analysis of 1PF5 and 1QU9 using the Needleman-Wunsch algorithm and Blosum 62 matrix in UCSF Chimera shows that the two structures can be superimposed on top of each other with extremely high similarity. 
     An amino acid sequence alignment of  E. coli  RidA and its paralogs YjgH, YoaB, RutC, and TdcF are shown in  FIG. 2 . Amino acids at positions of RidA that are important for ligand binding and trimer formation include positions E120, C107, V18, K73, and E122. E120 in RidA, involved in ligand binding, is conserved across all paralogs. However, C107 in RidA, also important for ligand binding, is not conserved across all paralogs. Furthermore, the three residues V18, K73, and E122 that are important for trimer formation are also not conserved. Among these five amino acids, of the four that are not conserved across all paralogs, two of the them, amino acids at positions D76 and K123 of YjgH (corresponding to K73 and E122 of RidA), that are important for trimer formation show conservation between YjgH and YoaB. These two positions are highlighted in bold in  FIG. 2 . 
     A host cell that is engineered in accordance with the invention to overexpress an imine/enamine deaminase polypeptide, such as YjgH or YoaB, also overexpresses at least one enzyme involved in the synthesis of an amino or amino acid derivative, such as a lysine decarboxylase polypeptide; and/or an additional polypeptide that is involved in amino acid biosynthesis. Lysine decarboxylase and lysine biosynthesis polypeptides and nucleic acid sequences are available in the art. 
     The invention employs various routine recombinant nucleic acid techniques. Generally, the nomenclature and the laboratory procedures in recombinant DNA technology described below are commonly employed in the art. Many manuals that provide direction for performing recombinant DNA manipulations are available, e.g., Sambrook &amp; Russell, Molecular Cloning, A Laboratory Manual (3rd Ed, 2001); and Current Protocols in Molecular Biology (Ausubel, et al., John Wiley and Sons, New York, 2009-2014). 
     Polynucleotides Encoding Imine/Enamine Deaminase Polypeptides 
     Various polynucleotides have been shown to encode polypeptides that catalyze the release of ammonia and reduce the levels of imine and enamine (e.g., yoaB or yjgH from  E. coli ). 
     Imine/enamine deaminase nucleic acid and polypeptide sequences suitable for use in the invention include imine/enamine deaminase nucleic acid sequences that encode an imine/enamine deaminase polypeptide as illustrated by SEQ ID NO:10 or SEQ ID NO:12, or biologically active variants that share substantial identity with SEQ ID NO:10 or SEQ ID NO:12. In some embodiments, such a substantially identical variant has at least 70%, or at least 75%, 80%, 85%, or 90% identity to SEQ ID NO:10 or SEQ ID NO:12, or an alternative imine/enamine deaminase polypeptide, e.g., a homolog of SEQ ID NO:10 or SEQ ID NO:12. In some embodiments, a substantially identical variant, as determined with reference to the  E. coli  YjgH protein sequence SEQ ID NO:12, comprises an acidic amino acid residue at position 121, an acidic residue at position 76, and a basic amino acid residue at position 123. In some embodiments, a substantially identical variant, as determined with reference to the  E. coli  YjgH protein sequence SEQ ID NO:12, comprises a D at position 76, an E at position 121, and a K at position 123. In some embodiments, a variant has at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:12. As used herein, the term “variant” encompasses biologically active polypeptides having one or more substitutions, deletions, or insertions relative to an imine/enamine deaminase polypeptide reference sequence, such as SEQ ID NO:10 or 12. Thus, the term “variant” includes biologically active fragments as well as substitution variants. 
     In some embodiments, a host is genetically modified in accordance with the invention to express a YoaB polypeptide. An illustrative sequence is provided as SEQ ID NO:10. In some embodiments, the host cell is genetically modified to express a YoaB polypeptide that has at least 90% identity, or at least 95% identity to SEQ ID NO:10 and increases lysine and/or cadaverine production by at least 20%, or greater compared to a counterpart strain that is not engineered to overexpress the YoaB polypeptide. In some embodiments, the YoaB polypeptide hast at least 70% identity or at least 75% identity to SEQ ID NO:10. In some embodiments, the YoaB polypeptide hast at least 80% identity or at least 85% identity to SEQ ID NO:10. 
     In some embodiments, a host is genetically modified in accordance with the invention to express a YjgH polypeptide. An illustrative sequence is provided as SEQ ID NO:12. In some embodiments, the host cell is genetically modified to express a YjgH polypeptide that has at least 90% identity, or at least 95% identity, to SEQ ID NO:12 and increases lysine and/or cadaverine production by at least 20%, or greater compared to a counterpart strain that is not engineered to overexpress the YjgH polypeptide. In some embodiments, the YjgH polypeptide has at least 70% identity or at least 75% identity to SEQ ID NO:12. In some embodiments, the YjgH polypeptide hast at least 80% identity or at least 85% identity to SEQ ID NO:12. 
     Imine/enamine deaminase polypeptide activity can be assessed using any number of assays, including assays that evaluate the production of an amino acid or an amino acid-derived compound. In some embodiments, the production of lysine or cadaverine production is measured. Illustrative assays are provided in the examples section. In some embodiments, cadaverine production is measured in  E. coli  modified to co-express LysC, DapA, LysA, Asd, DapB, AspC, and CadA and the variant of YoaB or YjgH to be tested, or another imine/enamine deaminase polypeptide to be tested. The following is an illustrative assay that is used to assess production of lysine and/or cadaverine.  E. coli  are modified to express LysC, DapA, LysA, Asd, DapB, AspC, and CadA and the variant to be tested. The genes may be individually introduced into  E. coli,  or introduced in one or more operons. For examples, LysC, DapA, LysA, Asd, DapB, and AspC may be encoded by a synthetic operon present in one plasmid and CadA and a candidate variant may be encoded by a separate plasmid. Each plasmid has a unique antibiotic-resistance selectable marker. Antibiotic-resistant colonies are selected and cultured. For example, cultures are grown overnight at 37° C. in 3 mL of medium containing 4% glucose, 0.1% KH 2 PO 4 , 0.1% MgSO 4 , 1.6% (NH 4 ) 2 SO 4 , 0.001% FeSO 4 , 0.001% MnSO 4 , 0.2% yeast extract, 0.05% L-methionine, 0.01% L-threonine, 0.005% L-isoleucine, and appropriate antibiotics for selection. The following day, each culture is inoculated into 50 mL of fresh medium with 30 g/L of glucose, 0.7% Ca(HCO 3 ) 2 , antibiotic(s), and grown for 72 hours at 37° C., at which point the concentration of lysine is determined. Lysine or cadaverine can be quantified using NMR. Yield can be calculated by dividing the molar amount of lysine or cadaverine produced by the molar amount of glucose added. An imine/enamine deaminase polypeptide for use in the invention increases the yield of lysine or cadaverine. Alternatively, colonies are evaluated for increased production of another lysine derivative. 
     In some embodiments, a YoaB or YjgH polypeptide increases lysine or cadaverine production by at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or greater, when expressed in a host cell compared to a counterpart host cell of the same strain that comprises the same genetic modifications other than the modification to overexpress the YoaB or YjgH polypeptide. In some embodiments, YoaB or YjgH polypeptide increases lysine or cadaverine production by at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or greater, when expressed in a host cell that is modified to overexpress a lysine decarboxylase, an aspartate kinase, a dihydrodipicolinate synthase, a diaminopimelate decarboxylase, an aspartate semialdehyde dehydrogenase, a dihydropicolinate reductase, and an aspartate transaminase; compared to a counterpart host cell of the same strain that comprises the modification to overexpress the lysine decarboxylase, the aspartate kinase, the dihydrodipicolinate synthase, the diaminopimelate decarboxylase, the aspartate semialdehyde dehydrogenase, the dihydropicolinate reductase, and the aspartate transaminase, but does not overexpress the YoaB or YjgH polypeptide. 
     Isolation or generation of imine/enamine deaminase polynucleotide sequences can be accomplished by a number of techniques. Such techniques will be discussed in the context of imine/enamine deaminase genes. However, one of skill understands that the same techniques can be used to isolate and express other desired genes. In some embodiments, oligonucleotide probes based on the sequences disclosed here can be used to identify the desired polynucleotide in a cDNA or genomic DNA library from a desired bacterial species. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. 
     Alternatively, the nucleic acids of interest can be amplified from nucleic acid samples using routine amplification techniques. For instance, PCR may be used to amplify the sequences of the genes directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. 
     Appropriate primers and probes for identifying an imine/enamine deaminase polynucleotide in bacteria can be generated from comparisons of the sequences provided herein. For a general overview of PCR see PCR Protocols: A Guide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990). Illustrative primer sequences are shown in the Table of Primers in the Examples section. 
     Nucleic acid sequences encoding an imine/enamine deaminase polypeptide for use in the disclosure includes genes and gene products identified and characterized by techniques such as hybridization and/or sequence analysis using illustrative nucleic acid sequences, e.g., SEQ ID NO:9 or SEQ ID NO:11. In some embodiments, a host cell is genetically modified by introducing a nucleic acid sequence having at least 60% identity, or at least 70%, 75%, 80%, 85%, or 90% identity, or 100% identity, to a polynucleotide comprising SEQ ID NO:9 or SEQ ID NO:11. 
     Nucleic acid sequences encoding an imine/enamine deaminase polypeptide that confers increased production of an amino acid, e.g., lysine, or an amino acid-derived product, e.g., cadaverine, to a host cell, may additionally be codon-optimized for expression in a desired host cell. Methods and databases that can be employed are known in the art. For example, preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. See e.g., Henaut and Danchin in “ Escherichia coli  and  Salmonella, ” Neidhardt, et al. Eds., ASM Pres, Washington D.C. (1996), pp. 2047-2066; Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl. Acids Res. 28:292). 
     Preparation of Recombinant Vectors 
     Recombinant vectors for expression of an imine/enamine deaminase polypeptide can be prepared using methods well known in the art. For example, a DNA sequence encoding an imine/enamine deaminase polypeptide (described in further detail below), can be combined with transcriptional and other regulatory sequences which will direct the transcription of the sequence from the gene in the intended cells, e.g., bacterial cells such as  E. coli.  In some embodiments, an expression vector that comprises an expression cassette that comprises the gene encoding the imine/enamine deaminase polypeptide further comprises a promoter operably linked to the imine/enamine deaminase gene. In other embodiments, a promoter and/or other regulatory elements that direct transcription of the imine/enamine deaminase gene are endogenous to the host cell and an expression cassette comprising the imine/enamine deaminase gene is introduced, e.g., by homologous recombination, such that the exogenous gene is operably linked to an endogenous promoter and is expression driven by the endogenous promoter. 
     As noted above, expression of the gene encoding an imine/enamine deaminase polypeptide can be controlled by a number of regulatory sequences including promoters, which may be either constitutive or inducible; and, optionally, repressor sequences, if desired. Examples of suitable promoters, especially in a bacterial host cell, are the promoters obtained from the  E. coli  lac operon and other promoters derived from genes involved in the metabolism of other sugars, e.g., galactose and maltose. Additional examples include promoters such as the trp promoter, bla promoter bacteriophage lambda PL, and T5. In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No. 4,551,433), can be used. Further examples of promoters include  Streptomyces coelicolor  agarase gene (dagA),  Bacillus subtilis  levansucrase gene (sacB),  Bacillus licheniformis  alpha-amylase gene (amyL),  Bacillus stearothermophilus  maltogenic amylase gene (amyM),  Bacillus amyloliquefaciens  alpha-amylase gene (amyQ),  Bacillus licheniformis  penicillinase gene (penP),  Bacillus subtilis  xylA and xylB genes. Suitable promoters are also described in Ausubel and Sambrook &amp; Russell, both supra. Additional promoters include promoters described by Jensen &amp; Hammer,  Appl. Environ. Microbiol.  64:82, 1998; Shimada, et al.,  J. Bacteriol.  186:7112, 2004; and Miksch et al.,  Appl. Microbiol. Biotechnol.  69:312, 2005. 
     In some embodiments, a promoter that influences expression of a native imine/enamine deaminase polypeptide may be modified to increase expression. For example, an endogenous YoaB or YjgH promoter may be replaced by a promoter that provides for increased expression compared to the native promoter. 
     An expression vector may also comprise additional sequences that influence expression of a gene encoding the imine/enamine deaminase polypeptide. Such sequences include enhancer sequences, a ribosome binding site, or other sequences such as transcription termination sequences, and the like. 
     A vector expressing a nucleic acid encoding an imine/enamine deaminase polypeptide of the invention may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Thus, an expression vector may additionally contain an element(s) that permits integration of the vector into the host&#39;s genome. 
     An expression vector of the invention preferably contains one or more selectable markers which permit easy selection of transformed hosts. For example, an expression vector may comprise a gene that confers antibiotic resistance (e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance) to the recombinant host organism, e.g., a bacterial cell such as  E. coli.    
     Although any suitable expression vector may be used to incorporate the desired sequences, readily available bacterial expression vectors include, without limitation: plasmids such as pSC1O1, pBR322, pBBR1MCS-3, pUR, pET, pEX, pMR100, pCR4, pBAD24, p15a, pACYC, pUC, e.g., pUC18 or pUC19, or plasmids derived from these plasmids; and bacteriophages, such as M13 phage and λ phage. One of ordinary skill in the art, however, can readily determine through routine experimentation whether any particular expression vector is suited for any given host cell. For example, the expression vector can be introduced into the host cell, which is then monitored for viability and expression of the sequences contained in the vector. 
     Expression vectors of the invention may be introduced into the host cell using any number of well-known methods, including calcium chloride-based methods, electroporation, or any other method known in the art. 
     Host Cells 
     The present invention provides for a genetically modified host cell that is engineered to overexpress an exogenous imine/enamine deaminase polypeptide. Such a host cell may comprise a nucleic acid encoding a heterologous imine/enamine deaminase peptide, including any non-naturally occurring imine/enamine deaminase polypeptide variant; or may be genetically modified to overexpress a native imine/enamine deaminase polypeptide relative to a wildtype host cell. 
     A genetically modified host strain of the present invention typically comprises at least one additional genetic modification to enhance production of an amino acid or amino acid derivative relative to a control strain that does not have the one additional genetic modification, e.g., a wildtype strain or a cell of the same strain without the one additional genetic modification. An “additional genetic modification to enhance production of an amino acid or amino acid derivative” can be any genetic modification. In some embodiments, the genetic modification is the introduction of a polynucleotide that expresses an enzyme involved in the synthesis of the amino acid or amino acid derivative. In some embodiments, the host cell comprises multiple modifications to increase production, relative to a wildtype host cell, of an amino acid or amino acid derivative. 
     In some aspects, genetic modification of a host cell to overexpress an imine/enamine deaminase polypeptide is performed in conjunction with modifying the host cell to overexpress a lysine decarboxylase polypeptide and/or one or more lysine biosynthesis polypeptides. 
     A lysine decarboxylase refers to an enzyme that converts L-lysine into cadaverine. The enzyme is classified as E.C. 4.1.1.18. Lysine decarboxylase polypeptides are well characterized enzymes, the structures of which are well known in the art (see, e.g., Kanjee, et al., EMBO J. 30: 931-944, 2011; and a review by Lemmonier &amp; Lane, Microbiology 144; 751-760, 1998; and references described therein). The EC number for lysine decarboxylase is 4.1.1.18. Illustrative lysine decarboxylase sequences are CadA homologs from  Klebsiella  sp., WP 012968785.1;  Enterobacter aerogenes,  YP 004592843.1;  Salmonella enterica,  WP 020936842.1;  Serratia  sp., WP 033635725.1; and  Raoultella ornithinolytica,  YP 007874766.1; and LdcC homologs from  Shigella  sp., WP 001020968.1;  Citrobacter  sp., WP 016151770.1; and  Salmonella enterica,  WP 001021062.1. As used herein, a lysine decarboxylase includes variants of native lysine decarboxylase enzymes that have lysine decarboxylase enzymatic activity. Additional lysine decarboxylase enzymes are described in PCT/CN2014/080873 and PCT/CN2015/072978. 
     In some embodiments, a host cell may be genetically modified to express one or more polypeptides that affect lysine biosynthesis. Examples of lysine biosynthesis polypeptides include the  E. coli  genes SucA, Ppc, AspC, LysC, Asd, DapA, DapB, DapD, ArgD, DapE, DapF, LysA, Ddh, PntAB, CyoABE, GadAB, YbjE, GdhA, GltA, SucC, GadC, AcnB, POB, ThrA, AceA, AceB, GltB, AceE, SdhA, MurE, SpeE, SpeG, PuuA, PuuP, and YgjG, or the corresponding genes from other organisms. Such genes are known in the art (see, e.g., Shah et al.,  J. Med. Sci.  2:152-157, 2002; Anastassiadia, S.  Recent Patents on Biotechnol.  1: 11-24, 2007). See, also, Kind, et al.,  Appl. Microbiol. Biotechnol.  91: 1287-1296, 2011 for a review of genes involved in cadaverine production. Illustrative genes encoding lysine biosynthesis polypeptides are provided below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                   
                 EC 
                 GenBank 
               
               
                 Protein 
                 Gene 
                 Number 
                 Accession No. 
               
               
                   
               
             
            
               
                 α-ketogultarate dehydrogenase 
                 sucA 
                 1.2.4.2 
                 YP_489005.1 
               
               
                 (SucA) 
                   
                   
                   
               
               
                 Phosphoenolpyruvate 
                 ppc 
                 4.1.1.31 
                 AAC76938.1 
               
               
                 carboxylase (PPC) 
                   
                   
                   
               
               
                 aspartate transaminase (AspC) 
                 aspC 
                 2.6.1.1 
                 AAC74014.1 
               
               
                 aspartate kinase (LysC) 
                 lysC 
                 2.7.2.4 
                 NP_418448.1 
               
               
                 aspartate semialdehyde 
                 asd 
                 1.2.1.11 
                 AAC76458.1 
               
               
                 dehydrogenase (Asd) 
                   
                   
                   
               
               
                 dihydrodipicolinate synthase 
                 dapA 
                 4.3.3.7 
                 NP_416973.1 
               
               
                 (DapA) 
                   
                   
                   
               
               
                 dihydropicolinate reductase 
                 dapB 
                 1.17.1.8 
                 AAC73142.1 
               
               
                 (DapB) 
                   
                   
                   
               
               
                 tetrahydrodipicoinate succinylase 
                 dapD 
                 2.3.1.117 
                 AAC73277.1 
               
               
                 (DapD) 
                   
                   
                   
               
               
                 N-succinyldiaminopimelate 
                 argD 
                 2.6.1.11 
                 AAC76384.1 
               
               
                 aminotransferase (ArgD) 
                   
                   
                   
               
               
                 N-succinyl-L-diaminopimelate 
                 dapE 
                 3.5.1.18 
                 AAC75525.1 
               
               
                 deacylase (DapE) 
                   
                   
                   
               
               
                 diaminopimelate epimerase 
                 dapF 
                 5.1.1.7 
                 AAC76812.2 
               
               
                 (DapF) 
                   
                   
                   
               
               
                 diaminopimelate decarboxylase 
                 lysA 
                 4.1.1.20 
                 AAC75877.1 
               
               
                 (LysA) 
                   
                   
                   
               
               
                 meso-diaminopimelate 
                 ddh 
                 NA 
                 P04964.1 
               
               
                 dehydrogenase (Ddh) 
                   
                   
                   
               
               
                 pyridine nucleotide 
                 pntAB 
                 NA 
                 AAC74675.1, 
               
               
                 transhydrogenase (PntAB) 
                   
                   
                 AAC74674.1 
               
               
                 cytochrome O oxidase (CyoABE) 
                 cyoABE 
                 1.10.3.10 
                 AAC73535.1, 
               
               
                   
                   
                   
                 AAC73534.1, 
               
               
                   
                   
                   
                 AAC73531.1 
               
               
                 glutamate decarboxylase 
                 gadAB 
                 4.1.1.15 
                 AAC76542.1, 
               
               
                 (GadAB) 
                   
                   
                 AAC74566.1 
               
               
                 L-amino acid efflux transporter 
                 ybjE 
                 NA 
                 AAC73961.2 
               
               
                 (YbjE) 
                   
                   
                   
               
               
                 glutamate dehydrogenase (GdhA) 
                 gdhA 
                 1.4.1.4 
                 AAC74831.1 
               
               
                 citrate synthase (GltA) 
                 gltA 
                 2.3.3.1/ 
                 AAC73814.1 
               
               
                   
                   
                 2.3.3.16 
                   
               
               
                 succinyl-coA synthase (SucC) 
                 sucC 
                 6.2.1.5 
                 AAC73822.1 
               
               
                 glutamate-GABA antiporter 
                 gadC 
                 NA 
                 AAC74565.1 
               
               
                 (GadC) 
                   
                   
                   
               
               
                 aconitase B (AcnB) 
                 acnB 
                 4.2.1.99 
                 AAC73229.1 
               
               
                 pyruvate-formate lyase (PflB) 
                 pflB 
                 NA 
                 AAC73989.1 
               
               
                 aspartate kinase/homoserine 
                 thrA 
                 2.7.2.4 
                 AAC73113.1 
               
               
                 dehydrogenase (ThrA) 
                   
                   
                   
               
               
                 isocitrate lyase (AceA) 
                 aceA 
                 4.1.3.1 
                 AAC76985.1 
               
               
                 malate synthase (AceB) 
                 aceB 
                 2.3.3.9 
                 AAC76984.1 
               
               
                 glutmate synthase (GltB) 
                 gltB 
                 1.4.1.13 
                 AAC76244.2 
               
               
                 pyruvate dehydrogenase (AceE) 
                 aceE 
                 1.2.4.1 
                 AAC73225.1 
               
               
                 succinate dehydrogenase (SdhA) 
                 sdhA 
                 1.3.5.1 
                 AAC73817.1 
               
               
                 UDP-N-acetylmuramoyl-L-alanyl- 
                 murE 
                 6.3.2.13 
                 AAC73196.1 
               
               
                 D-glutamate:meso- 
                   
                   
                   
               
               
                 diaminopimelate ligase (MurE) 
                   
                   
                   
               
               
                 putrescine/cadaverine 
                 speE 
                 2.5.1.16 
                 AAC73232.1 
               
               
                 aminopropyltransferase (SpeE) 
                   
                   
                   
               
               
                 spermidine acetyltransferase 
                 speG 
                 NA 
                 AAC74656.1 
               
               
                 (SpeG) 
                   
                   
                   
               
               
                 glutamate-putrescine/glutamate- 
                 puuA 
                 NA 
                 AAC74379.2 
               
               
                 cadaverine ligase (PuuA) 
                   
                   
                   
               
               
                 putrescine importer (PuuP) 
                 puuP 
                 NA 
                 AAC74378.2 
               
               
                 putrescine/cadaverine 
                 ygjG 
                 2.6.1.82 
                 AAC76108.3 
               
               
                 aminotransferase (YgjG) 
               
               
                   
               
            
           
         
       
     
     In some embodiments, a host cell is genetically modified to express a lysine decarboxylase, an aspartate kinase, a dihydrodipicolinate synthase, a diaminopimelate decarboxylase, an aspartate semialdehyde dehydrogenase, a dihydropicolinate reductase, and an aspartate transaminase. Additional modifications may also be incorporated into the host cell. 
     In some embodiments, a host cell may be genetically modified to attenuate or reduce the expression of one or more polypeptides that affect lysine biosynthesis. Examples of such polypeptides include the  E. coli  genes Pck, Pgi, DeaD, CitE, MenE, PoxB, AceA, AceB, AceE, RpoC, and ThrA, or the corresponding genes from other organisms. Such genes are known in the art (see, e.g., Shah et al.,  J. Med. Sci.  2:152-157, 2002; Anastassiadia, S.  Recent Patents on Biotechnol.  1: 11-24, 2007). See, also, Kind, et al.,  Appl. Microbiol. Biotechnol.  91: 1287-1296, 2011 for a review of genes attenuated to increase cadaverine production. Illustrative genes encoding polypeptides whose attenuation increases lysine biosynthesis are provided below. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                   
                   
                 GenBank 
               
               
                 Protein 
                 Gene 
                 EC Number 
                 Accession No. 
               
               
                   
               
             
            
               
                 PEP carboxykinase (Pck) 
                 pck 
                 4.1.1.49 
                 NP_417862 
               
               
                 Glucose-6-phosphate isomerase 
                 pgi 
                 5.3.1.9 
                 NP_418449 
               
               
                 (Pgi) 
                   
                   
                   
               
               
                 DEAD-box RNA helicase (DeaD) 
                 deaD 
                   
                 NP_417631 
               
               
                 citrate lyase (CitE) 
                 citE 
                 4.1.3.6/4.1.3.34 
                 NP_415149 
               
               
                 o-succinylbenzoate-CoA ligase 
                 menE 
                 6.2.1.26 
                 NP_416763 
               
               
                 (MenE) 
                   
                   
                   
               
               
                 pyruvate oxidase (PoxB) 
                 poxB 
                 1.2.2.2 
                 NP_415392 
               
               
                 isocitrate lyase (AceA) 
                 aceA 
                 4.1.3.1 
                 NP_418439 
               
               
                 malate synthase A (AceB) 
                 aceB 
                 2.3.3.9 
                 NP_418438 
               
               
                 pyruvate dehydrogenase (aceE) 
                 aceE 
                 1.2.4.1 
                 NP_414656 
               
               
                 RNA polymerase b′ subunit 
                 rpoC 
                 2.7.7.6 
                 NP_418415 
               
               
                 (RpoC) 
                   
                   
                   
               
               
                 aspartokinase I (ThrA) 
                 thrA 
                 2.7.2.4/1.1.1.3 
                 NP_414543 
               
               
                   
               
            
           
         
       
     
     Nucleic acids encoding a lysine decarboxylase or a lysine biosynthesis polypeptide may be introduced into the host cell along with the imine/enamine deaminase polynucleotide, e.g., encoded on a single expression vector, or introduced in multiple expression vectors at the same time. Alternatively, the host cell may be genetically modified to overexpress lysine decarboxylase or one or more lysine biosynthesis polypeptides before or after the host cell is genetically modified to overexpress the imine/enamine deaminase polypeptide. 
     In alternative embodiments, a host cell that overexpresses a naturally occurring imine/enamine deaminase polypeptide can be obtained by other techniques, e.g., by mutagenizing cells, e.g.,  E. coli  cells, and screening cells to identify those that an imine/enamine deaminase polypeptide, e.g., YoaB or YjhG, at a higher level compared to the cell prior to mutagenesis. 
     A host cell comprising an imine/enamine deaminase polypeptide as described herein is a bacterial host cell. In typical embodiments, the bacterial host cell is a Gram-negative bacterial host cell. In some embodiments of the invention, the bacterium is an enteric bacterium. In some embodiments of the invention, the bacterium is a species of the genus  Corynebacterium, Escherichia, Pseudomonas, Zymomonas, Shewanella, Salmonella, Shigella, Enterobacter, Citrobacter, Cronobacter, Erwinia, Serratia, Proteus, Hafnia, Yersinia, Morganella, Edwardsiella,  or  Klebsiella  taxonomical classes. In some embodiments, the host cells are members of the genus  Escherichia, Hafnia,  or  Corynebacterium.  In some embodiments, the host cell is an  Escherichia coli, Hafnia alvei,  or  Corynebacterium glutamicum  host cell. 
     In some embodiments, the host cell is a gram-positive bacterial host cell, such as a  Bacillus  sp., e.g.,  Bacillus subtilis  or  Bacillus licheniformis;  or another  Bacillus  sp. such as  B. alcalophilus, B. aminovorans, B. amyloliquefaciens, B. caldolyticus, B. circulans, B. stearothermophilus, B. thermoglucosidasius, B. thuringiensis  or  B. vulgatis.    
     Host cells modified in accordance with the invention can be screened for increased production of lysine or a lysine derivative, such as cadaverine, as described herein. 
     Methods of Producing Lysine or a Lysine Derivative. 
     A host cell genetically modified to overexpress an imine/enamine deaminase polypeptide can be employed to produce lysine or a derivative of lysine. In some embodiments, the host cell produces cadaverine. To produce lysine or the lysine derivative, a host cell genetically modified to overexpress an imine/enamine deaminase polypeptide as described herein can be cultured under conditions suitable to allow expression of the polypeptide and expression of genes that encode the enzymes that are used to produce lysine or the lysine derivative. A host cell modified in accordance with the invention provides a higher yield of lysine or lysine derivatives relative to a non-modified counterpart host cell that expresses the imine/enamine deaminase polypeptide at native levels. 
     Host cells may be cultured using well known techniques (see, e.g., the illustrative conditions provided in the examples section). 
     The lysine or lysine derivative can then be separated and purified using known techniques. Lysine or lysine derivatives, e.g., cadaverine, produced in accordance with the invention may then be used in any known process, e.g., to produce a polyamide. 
     In some embodiments, lysine may be converted to caprolactam using chemical catalysts or by using enzymes and chemical catalysts. 
     The present invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters, which can be changed or modified to yield essentially the same results. 
     EXAMPLES 
     The following examples are offered to illustrate, but not to limit the claimed invention. 
     Example 1 
     Construction of Plasmid Vectors that Encode CadA 
     A plasmid vector containing wild-type  E. coli  cadA (SEQ ID NO: 1), which encodes the lysine decarboxylase CadA (SEQ ID NO: 2), was amplified from the  E. coli  MG1655 K12 genomic DNA using the PCR primers cadA-F and cadA-R, digested using the restriction enzymes SacI and BamHI, and ligated into pSTV28 to generate the plasmid pCIB39. The 5′ sequence upstream of the cadA gene was optimized using the PCR primers cadA-F2 and cadA-R2 to create pCIB40. The SacI restriction site was added back to pCIB40 using the SacI-F and SacI-R primers to create pCIB41. 
     Example 2 
     Construction of Plasmid Vectors Expressing a Gene Encoding a Imine/Enamine Deaminase 
     The  E. coli  gene, ridA (SEQ ID NO: 3), that encodes an imine/enamine deaminase, RidA (SEQ ID NO: 4), was amplified from the  E. coli  MG1655 K12 genomic DNA using the PCR primers ridA-F and ridA-R, digested with the restriction enzymes SacI and BamHI, and ligated into pCIB41 plasmid vector also digested with SacI and BamHI to create pCIB144. Similarly, rutC (SEQ ID NO: 5), which encodes RutC (SEQ ID NO: 6), was cloned into pCIB41 using the primers rutC-F and rutC-R to create the plasmid pCIB174; and tdcF (SEQ ID NO: 7), which encodes TdcF (SEQ ID NO: 8), was cloned into pCIB41 using the primers tdcF-F and tdcF-R to create the plasmid pCIB175. YoaB (SEQ ID NO: 9), which encodes YoaB (SEQ ID NO: 10), was cloned into pCIB41 using the primers yoaB-F and yoaB-R to create the plasmid pCIB177; and yjgH (SEQ ID NO: 11), that encodes YjgH (SEQ ID NO: 12), was cloned into pCIB41 using the primers yjgH-F and yjgH-R to create the plasmid pCIB194. 
     Example 3 
     Construction of Plasmid Vectors Co-Expressing Synthetic Operon I that Contains Three Proteins (LysC, DapA, LysA) from the Lysine Biosynthetic Pathway 
     Three genes from  E. coli,  lysC, dapA, and lysA, encode proteins involved in the  E. coli  lysine biosynthetic pathway: aspartate kinase (LysC or AKIII, encoded by lysC), dihydrodipicolinate synthase (DapA or DHDPS, encoded by dapA), and diaminopimelate decarboxylase (LysA, encoded by lysA). The three genes were cloned into a plasmid vector and the three proteins, LysC (SEQ ID NO: 13), DapA (SEQ ID NO: 14), and LysA (SEQ ID NO: 15) were overexpressed in  E. coli.  The gene lysC was amplified from the  E. coli  MG1655 K12 genomic DNA using the primers lysC-F and lysC-R, and the amplified fragment was digested using SacI and BamHI, and ligated into pUC18 to create pCIB7. The gene dapA was amplified from the  E. coli  MG1655 K12 genomic DNA using the primers dapA-F and dapA-R, and the amplified fragment was digested using BamHI and XbaI, and ligated into pCIB7 to create pCIB8. The gene lysA was amplified from the  E. coli  MG1655 K12 genomic DNA using the primers lysA-F and lysA-R, and the amplified fragment was digested using XbaI and SalI, and ligated into pCIB8 to create pCIB9. The three-gene operon was amplified from pCIB9 using the primers lysC-F and lysA-R. The amplified product was digested using SacI and SalI, and the digested fragment was ligated into pCIB10 to create pCIB32. 
     To construct pCIB10, the synthetic promoter sequence (SEQ ID NO:22) was synthesized using the PCR primers psyn-1 and psyn-2. Primer psyn-1 contains the promoter sequence and a sequence homologous to pUC18, and primer psyn-2 contains a sequence homologous to pUC18. These two PCR primers were used to amplify a portion of pUC18 that includes the multi-cloning site from the plasmid inserted downstream of the synthetic promoter sequence. Restriction enzymes EcoRI and SacI were used to digest the amplified DNA containing the synthetic promoter, which was further ligated into pUC18 to construct pCIB10. 
     Example 4 
     Construction of Plasmid Vectors Co-Expressing Various Aspartokinases. Various Aspartokinases were Expressed in Order to Increase Lysine Production 
     Two pairs of mutations were chosen that enabled the  E. coli  LysC to have an increased feedback resistance to lysine. The gene encoding the first mutant, LysC-1 (M318I, G323D) (SEQ. ID NO: 16) was constructed using the primers 318-F, 318-R, 323-F, 323-R. The genes encoding LysC-1 (M318I, G323D) was cloned into pCIB32 and replaced the wild-type  E. coli  aspartokinase, LysC, to create the plasmids pCIB43. The aspartokinase from  Streptomyces  strains that is capable of producing polylysine was previously suggested, but not proven, to be more feedback resistant to lysine compared to  E. coli  aspartokinase. As such, the aspartokinase gene from  Streptomyces lividans  was codon optimized, synthesized, and cloned in place of wild-type lysC in pCIB32 in order to create the plasmid pCIB55 using the primers SlysC-F and SlysC-R. The resulting aspartokinase protein that was expressed was named S-LysC (SEQ ID NO: 17). 
     Example 5 
     Construction of Plasmid Vectors Co-Expressing Synthetic Operon II that Contains Three Proteins (Asd, DapB, DapD, AspC) from the Lysine Biosynthetic Pathway 
     Next, the expression of four additional genes, asd, dapB, dapD, and aspC, which are involved in the lysine biosynthetic pathway of  E. coli,  was enhanced. These genes encode the following enzymes: aspartate semialdehyde dehydrogenase (Asd (SEQ ID NO: 18), encoded by asd), dihydrodipicolinate reductase (DapB or DHDPR (SEQ ID NO: 19), encoded by dapB), tetrahydrodipicolinate succinylase (DapD (SEQ ID NO: 20), encoded by dapD), and aspartate transaminase (AspC (SEQ ID NO: 21), encoded by aspC). The gene asd was amplified from the  E. coli  MG1655 K12 genomic DNA using the primers asd-F and asd-R, and the amplified fragment was digested using SacI and BamHI, and ligated into pUC18 to create pCIB12. The gene dapB was amplified from the  E. coli  MG1655 K12 genomic DNA using the primers dapB-F and dapB-R, and the amplified fragment was digested using BamHI and XbaI, and ligated into pCIB12 to create pCIB13. The gene dapD was amplified from the  E. coli  MG1655 K12 genomic DNA using the primers dapD-F and dapD-R, and the amplified fragment was digested using XbaI and SalI, and ligated into pCIB13 to create pCIB14. Similarly, the gene aspC was amplified from the  E. coli  MG1655 K12 genomic DNA using the primers aspC-F and aspC-R, and the amplified fragment was digested using XbaI and SalI, and ligated into pCIB13 to create pCIB31. 
     Example 6 
     Construction of Plasmid Vectors Co-Expressing Synthetic Operons I and II that Contain Proteins from the Lysine Biosynthetic Pathway 
     Synthetic Operon I was further adjusted using primers lysC-rbs2-F and lysC-rbs2-R to modify pCIB43 and create the plasmid pCIB378. Synthetic Operon II was further adjusted using the primers asd-rbs2-F and asd-rbs2-R to modify pCIB31 and create the plasmid pCIB380. pCIB380 was further modified using the primers SacI-F2, SacI-R2, ApaI-F, and ApaI-R in order to add the restriction enzyme sites for ApaI and SacI to pCIB380 in order to create the plasmid pCIB393. The two synthetic operons, Synthetic Operon I and Synthetic Operon II, consisting of the genes lysC, dapA, lysA, asd, dapB, and aspC were combined into a single vector. The operon from pCIB378 consisting of the genes lysC, dapA, and lysA was amplified using the primers LAL2-SacI-F and LAL2-ApaI-R, digested using the restriction enzymes SacI and ApaI, and ligated into pCIB393 in order to create the plasmid pCIB394. 
     Example 7 
     Production of Lysine from  E. Coli  Over-Expressing Synthetic Operons I and II and an Imine/Enamine Deaminase 
       E. coli  MG1655 K12 was transformed with one of the following combination of plasmids: pCIB394 and pSTV28, pCIB394 and pCIB144, pCIB394 and pCIB174, pCIB394 and pCIB175, pCIB394 and pCIB177, or pCIB394 and pCIB194. Three single colonies from each transformation were grown overnight at 37° C. in 3 mL of medium containing 4% glucose, 0.1% KH 2 PO 4 , 0.1% MgSO 4 , 1.6% (NH 4 ) 2 SO 4 , 0.001% FeSO 4 , 0.001% MnSO 4 , 0.2% yeast extract, 0.05% L-methionine, 0.01% L-threonine, 0.005% L-isoleucine, ampicillin (100 μg/mL), and chloramphenicol (20 μg/mL). The following day, each culture was inoculated into 100 mL of fresh medium with 30 g/L of glucose, 0.7% Ca(HCO 3 ) 2 , ampicillin (100 μg/mL), and chloramphenicol (20 μg/mL). The culture was grown for 72 hours at 37° C., at which point the concentration of lysine in each culture was determined (Table 1). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Production of lysine by  E .  coli  strains containing Synthetic  
               
               
                 Operons I and II, and an imine/enamine deaminase. 
               
            
           
           
               
               
               
            
               
                 Strain 
                 Protein(s) 
                 Lysine (g/L) 
               
               
                   
               
               
                 MG1655 
                 none 
                 n.d. 
               
               
                 pCIB394 &amp; 
                 LysC, DapA, LysA, Asd, DapB, AspC 
                 6.0 ± 0.4 
               
               
                 pSTV28 
                   
                   
               
               
                 pCIB394 &amp; 
                 LysC, DapA, LysA, Asd, DapB, AspC, RidA 
                 6.1 ± 0.3 
               
               
                 pCIB144 
                   
                   
               
               
                 pCIB394 &amp; 
                 LysC, DapA, LysA, Asd, DapB, AspC, RutC 
                 5.9 ± 0.3 
               
               
                 pCIB174 
                   
                   
               
               
                 pCIB394 &amp; 
                 LysC, DapA, LysA, Asd, DapB, AspC, TdcF 
                 6.2 ± 0.4 
               
               
                 pCIB175 
                   
                   
               
               
                 pCIB394 &amp; 
                 LysC, DapA, LysA, Asd, DapB, AspC, YoaB 
                 7.3 ± 0.2 
               
               
                 pCIB177 
                   
                   
               
               
                 pCIB394 &amp; 
                 LysC, DapA, LysA, Asd, DapB, AspC, YjgH 
                 7.1 ± 0.3 
               
               
                 pCIB 194 
               
               
                   
               
               
                 n.d.: none detected 
               
            
           
         
       
     
     As shown in Table 1, the overproduction of different imine/enamine deaminases affected lysine production differently. The overproduction of RidA did not lead to any observable change in lysine production. Similarly, the overproduction of two RidA paralogs RutC and TdcF also did not lead to any change in lysine production. Surprisingly, the overproduction of two RidA paralogs did increase lysine production from 6 g/L to 7.3 g/L for a system overproducing YoaB, and 7.1 g/L for a system overproducing YjgH. 
     Example 8 
     Construction of Plasmid Vectors Encoding a Lysine Decarboxylase and an Imine/Enamine Deaminase 
     The yoaB gene on pCIB177 was modified to remove the BamHI and SphI restriction sites using the primer pairs rmvBamHI-F and rmvBamHI-R, and rmvSphI-F and rmvSphI-R. The modified yoaB gene was amplified using the primers yoaB-F2 and yoaB-R2, the amplified fragment was digested using the restriction enzymes BamHI and SphI, and ligated into pCIB41 to form the plasmid pCIB201. Similarly, pCIB194 was modified to remove the BamHI and SphI restriction sites using the primer pairs rmvBamHI-F2 and rmvBamHI-R2, rmvSphI-F2 and rmvSphI-R2, and rmvSphI-F3 and rmvSphI-R3. The modified yjgH gene was amplified using the primers yjgH-F2 and yjgH-R2, the amplified fragment was digested using the restriction enzymes BamHI and SphI, and ligated into pCIB41 to form the plasmid pCIB208. 
     Example 9 
     Production of Lysine from  E. Coli  Co-Overexpressing Genes that Encode Imine/Enamine Deaminase Proteins and Lysine Synthetic Operons I and II 
       E. coli  MG1655 K12 was transformed with one of the following combination of plasmids: pCIB394 and pSTV28, pCIB394 and pCIB41, pCIB394 and pCIB201, or pCIB394 and pCIB208. Three single colonies from each transformation were grown overnight at 37° C. in 3 mL of medium containing 4% glucose, 0.1% KH 2 PO 4 , 0.1% MgSO 4 , 1.6% (NH 4 ) 2 SO 4 , 0.001% FeSO 4 , 0.001% MnSO 4 , 0.2% yeast extract, 0.05% L-methionine, 0.01% L-threonine, 0.005% L-isoleucine, ampicillin (100 ng/mL), and chloramphenicol (20 ng/mL). The following day, each culture was inoculated into 100 mL of fresh medium with 30 g/L of glucose, 0.7% Ca(HCO 3 ) 2 , ampicillin (100 ng/mL), and chloramphenicol (20 ng/mL). The culture was grown for 72 hours at 37° C., at which point the concentration of lysine and cadaverine in each culture was determined (Table 2). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Production of lysine and cadaverine by  E .  coli  strains that contain the 
               
               
                 lysine Synthetic Operons I and II and overproduce a lysine  
               
               
                 decarboxylase and an imine/enamine deaminase. 
               
            
           
           
               
               
               
               
            
               
                 Strain 
                 Protein(s) 
                 Lysine (g/L) 
                 Cadaverine (g/L) 
               
               
                   
               
               
                 MG1655 
                 none 
                 n.d. 
                 n.d. 
               
               
                 pCIB394 &amp; 
                 LysC, DapA, LysA, Asd, 
                 6.1 ± 0.3 
                 n.d. 
               
               
                 pSTV28 
                 DapB, AspC 
                   
                   
               
               
                 pCIB394 &amp; 
                 LysC, DapA, LysA, Asd, 
                 0.5 ± 0.2 
                 3.0 ± 0.2 
               
               
                 pCIB41 
                 DapB, AspC, CadA 
                   
                   
               
               
                 pCIB394 &amp; 
                 LysC, DapA, LysA, Asd, 
                 0.5 ± 0.2 
                 3.9 ± 0.3 
               
               
                 pCIB201 
                 DapB, AspC, CadA, YoaB 
                   
                   
               
               
                 pCIB394 &amp; 
                 LysC, DapA, LysA, Asd, 
                 0.5 ± 0.2 
                 4.1 ± 0.2 
               
               
                 pCIB208 
                 DapB, AspC, CadA, YjgH 
               
               
                   
               
               
                 n.d.: none detected 
               
            
           
         
       
     
     As shown in Table 2, overproduction of CadA led to the production of cadaverine. Furthermore, the overproduction of imine/enamine deaminase further increased cadaverine production from 3.0 g/L to 3.9 g/L for YoaB and 4.1 g/L for YjgH. 
     Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. All publications, patents, accession numbers, and patent applications cited herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate. 
     
       
         
           
               
            
               
                   
               
               
                 Table of plasmids used in Examples 
               
            
           
           
               
               
               
            
               
                   
                 Protein(s) Overexpressed 
                 Plasmid 
               
               
                   
                   
               
               
                   
                 none 
                 pSTV28 
               
               
                   
                 CadA 
                 pCIB41 
               
               
                   
                 RidA 
                 pCIB144 
               
               
                   
                 RutC 
                 pCIB174 
               
               
                   
                 TdcF 
                 pCIB175 
               
               
                   
                 YoaB 
                 pCIB177 
               
               
                   
                 YjgH 
                 pCIB194 
               
               
                   
                 LysC, DapA, LysA, Asd, DapB, AspC 
                 pCIB394 
               
               
                   
                 CadA, YoaB 
                 pCIB201 
               
               
                   
                 CadA, YjgH 
                 pCIB208 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
            
               
                   
               
               
                 Table of primer sequences used in Examples. 
               
            
           
           
               
               
               
            
               
                   
                 Name 
                 Sequence (5′-3′) 
               
               
                   
                   
               
               
                   
                 cadA-F 
                 ggcgagctcacacaggaaacagaccatgaacgt 
               
               
                   
                   
                 tattgcaatattgaatc 
               
               
                   
                   
               
               
                   
                 cadA-R 
                 ggcggatccccacttcccttgtacgagctaatt 
               
               
                   
                   
                 attttttgctttcttctttc 
               
               
                   
                   
               
               
                   
                 cadA-F2 
                 atttcacacaggaaacagctatgaacgttattg 
               
               
                   
                   
                 caatattgaatcac 
               
               
                   
                   
               
               
                   
                 cadA-R2 
                 agctgtttcctgtgtgaaat 
               
               
                   
                   
               
               
                   
                 SacI-F 
                 ggcgagctcctcctgtgtgaaattgttatccgc 
               
               
                   
                   
                 tc 
               
               
                   
                   
               
               
                   
                 SacI-R 
                 ggcgagctcatgaacgttattgcaatattgaat 
               
               
                   
                   
                 c 
               
               
                   
                   
               
               
                   
                 ridA-F 
                 ggcgagctcatgagcaaaactatcgcgacg 
               
               
                   
                   
               
               
                   
                 ridA-R 
                 ggcggatccttagcgacgaacagcgatcg 
               
               
                   
                   
               
               
                   
                 rutC-F 
                 ggcgagctcatgccaaaatccgtaattattcc 
               
               
                   
                   
               
               
                   
                 rutC-R 
                 ggcggatcctcacttggcgatatgcgcaa 
               
               
                   
                   
               
               
                   
                 tdcF-F 
                 ggcgagctcatgaaaaagattatcgaaacgcaa 
               
               
                   
                   
                 c 
               
               
                   
                   
               
               
                   
                 tdcF-R 
                 ggcggatccttacgcactacgtactgcga 
               
               
                   
                   
               
               
                   
                 yoaB-F 
                 ggcgagctcatgactatcgttcgtatcgatgct 
               
               
                   
                   
                 g 
               
               
                   
                   
               
               
                   
                 yoaB-R 
                 ggcggatccttacaccgcagccacaatct 
               
               
                   
                   
               
               
                   
                 yjgH-F 
                 ggcgagctcatggtagaaagaaccgctgttttc 
               
               
                   
                   
               
               
                   
                 yjgH-R 
                 ggcggatcctattgcttactgctcagggatg 
               
               
                   
                   
               
               
                   
                 lysC-F 
                 ggcgagctcacacaggaaacagaccatgtctga 
               
               
                   
                   
                 aattgttgtctcc 
               
               
                   
                   
               
               
                   
                 lysC-R 
                 ggcggatccttactcaaacaaattactatgcag 
               
               
                   
                   
               
               
                   
                 dapA-F 
                 ggcggatccacacaggaaacagaccatgttcac 
               
               
                   
                   
                 gggaagtattgtc 
               
               
                   
                   
               
               
                   
                 dapA-R 
                 ggctctagattacagcaaaccggcatgc 
               
               
                   
                   
               
               
                   
                 lysA-F 
                 ggctctagaacacaggaaacagaccatgccaca 
               
               
                   
                   
                 ttcactgttcagc 
               
               
                   
                   
               
               
                   
                 lysA-R 
                 ggcgtcgacttaaagcaattccagcgccag 
               
               
                   
                   
               
               
                   
                 318-F 
                 cagcctgaatatactgcattctc 
               
               
                   
                   
               
               
                   
                 318-R 
                 gagaatgcagtatattcaggctg 
               
               
                   
                   
               
               
                   
                 323-F 
                 gcattctcgcgatttcctcg 
               
               
                   
                   
               
               
                   
                 323-R 
                 cgaggaaatcgcgagaatgc 
               
               
                   
                   
               
               
                   
                 SlysC-F 
                 ggcgagctcacacaggaaacagaccatgggctt 
               
               
                   
                   
                 agttgtgcagaaa 
               
               
                   
                   
               
               
                   
                 SlysC-R 
                 ggcggatccttaacgacctgtgccgccata 
               
               
                   
                   
               
               
                   
                 asd-F 
                 ggcgagctcacacaggaaacagaccatgaaaaa 
               
               
                   
                   
                 tgttggttttatcgg 
               
               
                   
                   
               
               
                   
                 asd-R 
                 ggcggatccttacgccagttgacgaagc 
               
               
                   
                   
               
               
                   
                 dapB-F 
                 ggcacacaggaaacagaccatgcatgatgcaaa 
               
               
                   
                   
                 catccg 
               
               
                   
                   
               
               
                   
                 dapB-R 
                 ggctctagattacaaattattgagatcaagtac 
               
               
                   
                   
                 atctc 
               
               
                   
                   
               
               
                   
                 dapD-F 
                 ggctctagaacacaggaaacagaccatgcagca 
               
               
                   
                   
                 gttacagaacat 
               
               
                   
                   
               
               
                   
                 dapD-R 
                 ggcgcatgcttagtcgatggtacgcagca 
               
               
                   
                   
               
               
                   
                 aspC-F 
                 ggctctagaacacaggaaacagaccatgtttga 
               
               
                   
                   
                 gaacattaccgcc 
               
               
                   
                   
               
               
                   
                 aspC-R 
                 ggcgcatgcgacctcgaggtagtcgacttacag 
               
               
                   
                   
                 cactgccacaatcg 
               
               
                   
                   
               
               
                   
                 lysC-rbs2-F 
                 atttcacacaggaaacagctatgtctgaaattg 
               
               
                   
                   
                 ttgtctcca 
               
               
                   
                   
               
               
                   
                 lysC-rbs2-R 
                 agctgtttcctgtgtgaaat 
               
               
                   
                   
               
               
                   
                 asd-rbs2-F 
                 atttcacacaggaaacagctatgaaaaatgttg 
               
               
                   
                   
                 gttttatcggctg 
               
               
                   
                   
               
               
                   
                 asd-rbs2-R 
                 agctgtttcctgtgtgaaat 
               
               
                   
                   
               
               
                   
                 SacI-F2 
                 ggcgagctctcccctgattctgtggataa 
               
               
                   
                   
               
               
                   
                 SacI-R2 
                 ggcgagctcagcaaaaggccaggaaccgt 
               
               
                   
                   
               
               
                   
                 ApaI-F 
                 ggcgggcccgtattaccgcctttgagtgag 
               
               
                   
                   
               
               
                   
                 ApaI-R 
                 ggcgggcccacagaatcaggggagagctc 
               
               
                   
                   
               
               
                   
                 LAL2-SacI-F 
                 ggcgagctcgttggccgattcattaatgc 
               
               
                   
                   
               
               
                   
                 LAL2-ApaI-R 
                 ggcgggcccttaaagcaattccagcgccag 
               
               
                   
                   
               
               
                   
                 rmvBamHI-F 
                 tgcggtgtaaggttcctctagag 
               
               
                   
                   
               
               
                   
                 rmvBamHI-R 
                 ctctagaggaaccttacaccgca 
               
               
                   
                   
               
               
                   
                 rmvSphI-F 
                 gacctgcaggcttgcaagcttg 
               
               
                   
                   
               
               
                   
                 rmvSphI-R 
                 caagcttgcaagcctgcaggtc 
               
               
                   
                   
               
               
                   
                 yoaB-F2 
                 ggcggatcccacacaggaggagctcatgactat 
               
               
                   
                   
                 cgttcgtatcgatg 
               
               
                   
                   
               
               
                   
                 yoaB-R2 
                 ggcgcatgctcattcgccattcaggctgc 
               
               
                   
                   
               
               
                   
                 rmvSphI-F2 
                 catttgataatttacatgcgaccctg 
               
               
                   
                   
               
               
                   
                 rmvSphI-R2 
                 cagggtcgcatgtaaattatcaaatg 
               
               
                   
                   
               
               
                   
                 yjgH-F2 
                 ggcggatcccacacaggaggagctcatggtaga 
               
               
                   
                   
                 aagaaccgctgtt 
               
               
                   
                   
               
               
                   
                 yjgH-R2 
                 ggcgcatgctattgcttactgctcaggga 
               
               
                   
                   
               
               
                   
                 psyn-1 
                 ggcgaattcagtttattatgacatgtagtgagg 
               
               
                   
                   
                 gggctggtataatgagctcggtacccggggat 
               
               
                   
                   
               
               
                   
                 psyn-2 
                 ggcagtactcaaccaagtcattctgagaatagt 
               
               
                   
                   
                 g 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
            
               
                 Illustrative sequences  
               
               
                   Escherichia coli  cadA nucleic acid sequence  
               
               
                 SEQ ID NO: 1 
               
               
                 ATGAACGTTATTGCAATATTGAATCACATGGGGGTTTATTTTAAAGAAGA 
               
               
                   
               
               
                 ACCCATCCGTGAACTTCATCGCGCGCTTGAACGTCTGAACTTCCAGATTG 
               
               
                   
               
               
                 TTTACCCGAACGACCGTGACGACTTATTAAAACTGATCGAAAACAATGCG 
               
               
                   
               
               
                 CGTCTGTGCGGCGTTATTTTTGACTGGGATAAATATAATCTCGAGCTGTG 
               
               
                   
               
               
                 CGAAGAAATTAGCAAAATGAACGAGAACCTGCCGTTGTACGCGTTCGCTA 
               
               
                   
               
               
                 ATACGTATTCCACTCTCGATGTAAGCCTGAATGACCTGCGTTTACAGATT 
               
               
                   
               
               
                 AGCTTCTTTGAATATGCGCTGGGTGCTGCTGAAGATATTGCTAATAAGAT 
               
               
                   
               
               
                 CAAGCAGACCACTGACGAATATATCAACACTATTCTGCCTCCGCTGACTA 
               
               
                   
               
               
                 AAGCACTGTTTAAATATGTTCGTGAAGGTAAATATACTTTCTGTACTCCT 
               
               
                   
               
               
                 GGTCACATGGGCGGTACTGCATTCCAGAAAAGCCCGGTAGGTAGCCTGTT 
               
               
                   
               
               
                 CTATGATTTCTTTGGTCCGAATACCATGAAATCTGATATTTCCATTTCAG 
               
               
                   
               
               
                 TATCTGAACTGGGTTCTCTGCTGGATCACAGTGGTCCACACAAAGAAGCA 
               
               
                   
               
               
                 GAACAGTATATCGCTCGCGTCTTTAACGCAGACCGCAGCTACATGGTGAC 
               
               
                   
               
               
                 CAACGGTACTTCCACTGCGAACAAAATTGTTGGTATGTACTCTGCTCCAG 
               
               
                   
               
               
                 CAGGCAGCACCATTCTGATTGACCGTAACTGCCACAAATCGCTGACCCAC 
               
               
                   
               
               
                 CTGATGATGATGAGCGATGTTACGCCAATCTATTTCCGCCCGACCCGTAA 
               
               
                   
               
               
                 CGCTTACGGTATTCTTGGTGGTATCCCACAGAGTGAATTCCAGCACGCTA 
               
               
                   
               
               
                 CCATTGCTAAGCGCGTGAAAGAAACACCAAACGCAACCTGGCCGGTACAT 
               
               
                   
               
               
                 GCTGTAATTACCAACTCTACCTATGATGGTCTGCTGTACAACACCGACTT 
               
               
                   
               
               
                 CATCAAGAAAACACTGGATGTGAAATCCATCCACTTTGACTCCGCGTGGG 
               
               
                   
               
               
                 TGCCTTACACCAACTTCTCACCGATTTACGAAGGTAAATGCGGTATGAGC 
               
               
                   
               
               
                 GGTGGCCGTGTAGAAGGGAAAGTGATTTACGAAACCCAGTCCACTCACAA 
               
               
                   
               
               
                 ACTGCTGGCGGCGTTCTCTCAGGCTTCCATGATCCACGTTAAAGGTGACG 
               
               
                   
               
               
                 TAAACGAAGAAACCTTTAACGAAGCCTACATGATGCACACCACCACTTCT 
               
               
                   
               
               
                 CCGCACTACGGTATCGTGGCGTCCACTGAAACCGCTGCGGCGATGATGAA 
               
               
                   
               
               
                 AGGCAATGCAGGTAAGCGTCTGATCAACGGTTCTATTGAACGTGCGATCA 
               
               
                   
               
               
                 AATTCCGTAAAGAGATCAAACGTCTGAGAACGGAATCTGATGGCTGGTTC 
               
               
                   
               
               
                 TTTGATGTATGGCAGCCGGATCATATCGATACGACTGAATGCTGGCCGCT 
               
               
                   
               
               
                 GCGTTCTGACAGCACCTGGCACGGCTTCAAAAACATCGATAACGAGCACA 
               
               
                   
               
               
                 TGTATCTTGACCCGATCAAAGTCACCCTGCTGACTCCGGGGATGGAAAAA 
               
               
                   
               
               
                 GACGGCACCATGAGCGACTTTGGTATTCCGGCCAGCATCGTGGCGAAATA 
               
               
                   
               
               
                 CCTCGACGAACATGGCATCGTTGTTGAGAAAACCGGTCCGTATAACCTGC 
               
               
                   
               
               
                 TGTTCCTGTTCAGCATCGGTATCGATAAGACCAAAGCACTGAGCCTGCTG 
               
               
                   
               
               
                 CGTGCTCTGACTGACTTTAAACGTGCGTTCGACCTGAACCTGCGTGTGAA 
               
               
                   
               
               
                 AAACATGCTGCCGTCTCTGTATCGTGAAGATCCTGAATTCTATGAAAACA 
               
               
                   
               
               
                 TGCGTATTCAGGAACTGGCTCAGAATATCCACAAACTGATTGTTCACCAC 
               
               
                   
               
               
                 AATCTGCCGGATCTGATGTATCGCGCATTTGAAGTGCTGCCGACGATGGT 
               
               
                   
               
               
                 AATGACTCCGTATGCTGCATTCCAGAAAGAGCTGCACGGTATGACCGAAG 
               
               
                   
               
               
                 AAGTTTACCTCGACGAAATGGTAGGTCGTATTAACGCCAATATGATCCTT 
               
               
                   
               
               
                 CCGTACCCGCCGGGAGTTCCTCTGGTAATGCCGGGTGAAATGATCACCGA 
               
               
                   
               
               
                 AGAAAGCCGTCCGGTTCTGGAGTTCCTGCAGATGCTGTGTGAAATCGGCG 
               
               
                   
               
               
                 CTCACTATCCGGGCTTTGAAACCGATATTCACGGTGCATACCGTCAGGCT 
               
               
                   
               
               
                 GATGGCCGCTATACCGTTAAGGTATTGAAAGAAGAAAGCAAAAAATAA 
               
               
                   
               
               
                 CadA polypeptide sequence  
               
               
                 SEQ ID NO: 2 
               
               
                 MNVIAILNHMGVYFKEEPIRELHRALERLNFQIVYPNDRDDLLKLIENNA 
               
               
                   
               
               
                 RLCGVIFDWDKYNLELCEEISKMNENLPLYAFANTYSTLDVSLNDLRLQI 
               
               
                   
               
               
                 SFFEYALGAAEDIANKIKQTTDEYINTILPPLTKALFKYVREGKYTFCTP 
               
               
                   
               
               
                 GHMGGTAFQKSPVGSLFYDFFGPNTMKSDISISVSELGSLLDHSGPHKEA 
               
               
                   
               
               
                 EQYIARVFNADRSYMVTNGTSTANKIVGMYSAPAGSTILIDRNCHKSLTH 
               
               
                   
               
               
                 LMMMSDVTPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPVH 
               
               
                   
               
               
                 AVITNSTYDGLLYNTDFIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMS 
               
               
                   
               
               
                 GGRVEGKVIYETQSTHKLLAAFSQASMIHVKGDVNEETFNEAYMMHTTTS 
               
               
                   
               
               
                 PHYGIVASTETAAAMMKGNAGKRLINGSIERAIKFRKEIKRLRTESDGWF 
               
               
                   
               
               
                 FDVWQPDHIDTTECWPLRSDSTWHGFKNIDNEHMYLDPIKVTLLTPGMEK 
               
               
                   
               
               
                 DGTMSDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIDKTKALSLL 
               
               
                   
               
               
                 RALTDFKRAFDLNLRVKNMLPSLYREDPEFYENMRIQELAQNIHKLIVHH 
               
               
                   
               
               
                 NLPDLMYRAFEVLPTMVMTPYAAFQKELHGMTEEVYLDEMVGRINANMIL 
               
               
                   
               
               
                 PYPPGVPLVMPGEMITEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQA 
               
               
                   
               
               
                 DGRYTVKVLKEESKK 
               
               
                   
               
               
                   E. coli  ridA nucleic acid sequence  
               
               
                 SEQ ID NO: 3 
               
               
                 ATGAGCAAAACTATCGCGACGGAAAATGCACCGGCAGCTATCGGTCCTTA 
               
               
                   
               
               
                 CGTACAGGGCGTTGATCTGGGCAATATGATCATCACCTCCGGTCAGATCC 
               
               
                   
               
               
                 CGGTAAATCCGAAAACGGGCGAAGTACCGGCAGACGTCGCTGCACAGGCA 
               
               
                   
               
               
                 CGTCAGTCGCTGGATAACGTAAAAGCGATCGTCGAAGCCGCTGGCCTGAA 
               
               
                   
               
               
                 AGTGGGCGACATCGTTAAAACTACCGTGTTTGTAAAAGATCTGAACGACT 
               
               
                   
               
               
                 TCGCAACCGTAAACGCCACTTACGAAGCCTTCTTCACCGAACACAACGCC 
               
               
                   
               
               
                 ACCTTCCCGGCACGTTCTTGCGTTGAAGTTGCCCGTCTGCCGAAAGACGT 
               
               
                   
               
               
                 GAAGATTGAGATCGAAGCGATCGCTGTTCGTCGCTAA 
               
               
                   
               
               
                 RidA polypeptide sequence  
               
               
                 SEQ ID NO: 4 
               
               
                 MSKTIATENAPAAIGPYVQGVDLGNMIITSGQIPVNPKTGEVPADVAAQA 
               
               
                   
               
               
                 RQSLDNVKAIVEAAGLKVGDIVKTTVFVKDLNDFATVNATYEAFFTEHNA 
               
               
                   
               
               
                 TFPARSCVEVARLPKDVKIEIEAIAVRR 
               
               
                   
               
               
                   E. coli  rutC nucleic acid sequence  
               
               
                 SEQ ID NO: 5 
               
               
                 ATGCCAAAATCCGTAATTATTCCCGCTGGCAGCAGCGCACCGCTGGCCCC 
               
               
                   
               
               
                 CTTCGTTCCCGGCACGCTGGCTGATGGCGTGGTGTATGTCTCCGGTACGC 
               
               
                   
               
               
                 TGGCTTTTGATCAACATAATAACGTGCTGTTTGCCGATGACCCAAAGGCG 
               
               
                   
               
               
                 CAAACCCGCCACGTTCTGGAAACTATCCGCAAGGTGATCGAGACGGCGGG 
               
               
                   
               
               
                 TGGCACGATGGCGGATGTGACCTTCAACAGCATCTTTATTACCGACTGGA 
               
               
                   
               
               
                 AAAATTACGCCGCGATTAACGAAATCTACGCCGAGTTTTTTCCGGGTGAT 
               
               
                   
               
               
                 AAACCGGCGCGATTCTGCATTCAGTGCGGACTGGTAAAACCTGACGCGCT 
               
               
                   
               
               
                 GGTGGAAATCGCCACAATTGCGCATATCGCCAAGTGA 
               
               
                   
               
               
                 RutC polypeptide sequence  
               
               
                 SEQ ID NO: 6 
               
               
                 MPKSVIIPAGSSAPLAPFVPGTLADGVVYVSGTLAFDQHNNVLFADDPKA 
               
               
                   
               
               
                 QTRHVLETIRKVIETAGGTMADVTFNSIFITDWKNYAAINEIYAEFFPGD 
               
               
                   
               
               
                 KPARFCIQCGLVKPDALVEIATIAHIAK 
               
               
                   
               
               
                   E. coli  tdcF nucleic acid sequence  
               
               
                 SEQ ID NO: 7 
               
               
                 ATGAAAAAGATTATCGAAACGCAACGTGCCCCAGGCGCAATCGGCCCTTA 
               
               
                   
               
               
                 TGTTCAGGGCGTTGATTTAGGCAGCATGGTCTTCACCTCCGGGCAAATAC 
               
               
                   
               
               
                 CGGTTTGCCCACAGACCGGTGAGATCCCGGCTGATGTGCAAGATCAGGCG 
               
               
                   
               
               
                 CGTTTAAGCCTCGAAAACGTCAAAGCGATCGTGGTTGCTGCCGGGCTGAG 
               
               
                   
               
               
                 CGTGGGCGATATCATCAAGATGACCGTGTTTATCACCGATCTGAATGATT 
               
               
                   
               
               
                 TTGCCACCATCAACGAAGTCTATAAGCAGTTCTTCGATGAGCATCAGGCG 
               
               
                   
               
               
                 ACCTATCCGACCCGGAGCTGTGTGCAGGTCGCGCGTTTGCCGAAAGATGT 
               
               
                   
               
               
                 GAAGCTGGAAATTGAAGCCATCGCAGTACGTAGTGCGTAA 
               
               
                   
               
               
                 TdcF polypeptide sequence  
               
               
                 SEQ ID NO: 8 
               
               
                 MKKIIETQRAPGAIGPYVQGVDLGSMVFTSGQIPVCPQTGEIPADVQDQA 
               
               
                   
               
               
                 RLSLENVKAIVVAAGLSVGDIIKMTVFITDLNDFATINEVYKQFFDEHQA 
               
               
                   
               
               
                 TYPTRSCVQVARLPKDVKLEIEAIAVRSA 
               
               
                   
               
               
                   E. coli  yoaB nucleic acid sequence  
               
               
                 SEQ ID NO: 9 
               
               
                 ATGACTATCGTTCGTATCGATGCTGAAGCCCGCTGGTCTGATGTAGTAAT 
               
               
                   
               
               
                 CCACAACAACACGCTCTACTACACTGGTGTACCGGAAAACCTCGACGCCG 
               
               
                   
               
               
                 ATGCCTTTGAGCAAACCGCCAACACGCTGGCACAGATTGACGCCGTGCTG 
               
               
                   
               
               
                 GAAAAACAGGGCAGCAATAAATCGAGCATTCTGGATGCCACCATTTTCCT 
               
               
                   
               
               
                 GGCCGATAAAAACGACTTCGCGGCGATGAATAAAGCGTGGGATGCTTGGG 
               
               
                   
               
               
                 TTGTCGCGGGTCATGCGCCGGTGCGCTGCACGGTACAAGCGGGTTTGATG 
               
               
                   
               
               
                 AACCCGAAGTATAAAGTTGAAATTAAGATTGTGGCTGCGGTGTAA 
               
               
                   
               
               
                 YoaB polypeptide sequence  
               
               
                 SEQ ID NO: 10 
               
               
                 MTIVRIDAEARWSDVVIHNNTLYYTGVPENLDADAFEQTANTLAQIDAVL 
               
               
                   
               
               
                 EKQGSNKSSILDATIFLADKNDFAAMNKAWDAWVVAGHAPVRCTVQAGLM 
               
               
                   
               
               
                 NPKYKVEIKIVAAV 
               
               
                   
               
               
                   E. coli  yjgH nucleic acid sequence  
               
               
                 SEQ ID NO: 11 
               
               
                 ATGGTAGAAAGAACCGCTGTTTTCCCTGCTGGCCGACATTCACTATATGC 
               
               
                   
               
               
                 TGAGCATCGTTATTCTGCGGCTATTCGTTCCGGCGATTTGCTGTTTGTTT 
               
               
                   
               
               
                 CCGGGCAAGTAGGAAGTCGAGAGGACGGAACACCAGAACCCGATTTTCAG 
               
               
                   
               
               
                 CAACAAGTCAGACTGGCATTTGATAATTTGCATGCGACCCTGGCAGCTGC 
               
               
                   
               
               
                 GGGATGCACTTTTGACGATATCATTGATGTTACGAGCTTCCATACCGATC 
               
               
                   
               
               
                 CAGAAAACCAATTTGAAGACATCATGACGGTGAAAAATGAAATATTTAGC 
               
               
                   
               
               
                 GCCCCACCTTATCCAAACTGGACGGCGGTGGGTGTTACATGGCTGGCAGG 
               
               
                   
               
               
                 CTTTGATTTTGAAATTAAAGTGATAGCGCGCATCCCTGAGCAGTAA 
               
               
                   
               
               
                 YjgH polypeptide sequence  
               
               
                 SEQ ID NO: 12 
               
               
                 MVERTAVFPAGRHSLYAEHRYSAAIRSGDLLFVSGQVGSREDGTPEPDFQ 
               
               
                   
               
               
                 QQVRLAFDNLHATLAAAGCTFDDIIDVTSFHTDPENQFEDIMTVKNEIFS 
               
               
                   
               
               
                 APPYPNWTAVGVTWLAGFDFEIKVIARIPEQ 
               
               
                   
               
               
                 LysC polypeptide sequence  
               
               
                 SEQ ID NO: 13 
               
               
                 MSEIVVSKFGGTSVADFDAMNRSADIVLSDANVRLVVLSASAGITNLLVA 
               
               
                   
               
               
                 LAEGLEPGERFEKLDAIRNIQFAILERLRYPNVIREEIERLLENITVLAE 
               
               
                   
               
               
                 AAALATSPALTDELVSHGELMSTLLFVEILRERDVQAQWFDVRKVMRTND 
               
               
                   
               
               
                 RFGRAEPDIAALAELAALQLLPRLNEGLVITQGFIGSENKGRTTTLGRGG 
               
               
                   
               
               
                 SDYTAALLAEALHASRVDIWTDVPGIYTTDPRVVSAAKRIDEIAFAEAAE 
               
               
                   
               
               
                 MATFGAKVLHPATLLPAVRSDIPVFVGSSKDPRAGGTLVCNKTENPPLFR 
               
               
                   
               
               
                 ALALRRNQTLLTLHSLNMLHSRGFLAEVFGILARHNISVDLITTSEVSVA 
               
               
                   
               
               
                 LTLDTTGSTSTGDTLLTQSLLMELSALCRVEVEEGLALVALIGNDLSKAC 
               
               
                   
               
               
                 GVGKEVFGVLEPFNIRMICYGASSHNLCFLVPGEDAEQVVQKLHSNLFE 
               
               
                   
               
               
                 DapA polypeptide sequence  
               
               
                 SEQ ID NO: 14 
               
               
                 MFTGSIVAIVTPMDEKGNVCRASLKKLIDYHVASGTSAIVSVGTTGESAT 
               
               
                   
               
               
                 LNHDEHADVVMMTLDLADGRIPVIAGTGANATAEAISLTQRFNDSGIVGC 
               
               
                   
               
               
                 LTVTPYYNRPSQEGLYQHFKAIAEHTDLPQILYNVPSRTGCDLLPETVGR 
               
               
                   
               
               
                 LAKVKNIIGIKEATGNLTRVNQIKELVSDDFVLLSGDDASALDFMQLGGH 
               
               
                   
               
               
                 GVISVTANVAARDMAQMCKLAAEGHFAEARVINQRLMPLHNKLFVEPNPI 
               
               
                   
               
               
                 PVKWACKELGLVATDTLRLPMTPITDSGRETVRAALKHAGLL 
               
               
                   
               
               
                 LysA polypeptide sequence  
               
               
                 SEQ ID NO: 15 
               
               
                 MPHSLFSTDTDLTAENLLRLPAEFGCPVWVYDAQIIRRQIAALKQFDVVR 
               
               
                   
               
               
                 FAQKACSNIHILRLMREQGVKVDSVSLGEIERALAAGYNPQTHPDDIVFT 
               
               
                   
               
               
                 ADVIDQATLERVSELQIPVNAGSVDMLDQLGQVSPGHRVWLRVNPGFGHG 
               
               
                   
               
               
                 HSQKTNTGGENSKHGIWYTDLPAALDVIQRHHLQLVGIHMHIGSGVDYAH 
               
               
                   
               
               
                 LEQVCGAMVRQVIEFGQDLQAISAGGGLSVPYQQGEEAVDTEHYYGLWNA 
               
               
                   
               
               
                 AREQIARHLGHPVKLEIEPGRFLVAQSGVLITQVRSVKQMGSRHFVLVDA 
               
               
                   
               
               
                 GFNDLMRPAMYGSYHHISALAADGRSLEHAPTVETVVAGPLCESGDVFTQ 
               
               
                   
               
               
                 QEGGNVETRALPEVKAGDYLVLHDTGAYGASMSSNYNSRPLLPEVLFDNG 
               
               
                   
               
               
                 QARLIRRRQTIEELLALELL 
               
               
                   
               
               
                 LysC-1 M318I, G323D polypeptide sequence  
               
               
                 SEQ ID NO: 16 
               
               
                 MSEIVVSKFGGTSVADFDAMNRSADIVLSDANVRLVVLSASAGITNLLVA 
               
               
                   
               
               
                 LAEGLEPGERFEKLDAIRNIQFAILERLRYPNVIREEIERLLENITVLAE 
               
               
                   
               
               
                 AAALATSPALTDELVSHGELMSTLLFVEILRERDVQAQWFDVRKVMRTND 
               
               
                   
               
               
                 RFGRAEPDIAALAELAALQLLPRLNEGLVITQGFIGSENKGRTTTLGRGG 
               
               
                   
               
               
                 SDYTAALLAEALHASRVDIWTDVPGIYTTDPRVVSAAKRIDEIAFAEAAE 
               
               
                   
               
               
                 MATFGAKVLHPATLLPAVRSDIPVFVGSSKDPRAGGTLVCNKTENPPLFR 
               
               
                   
               
               
                 ALALRRNQTLLTLHSLNILHSRDFLAEVFGILARHNISVDLITTSEVSVA 
               
               
                   
               
               
                 LTLDTTGSTSTGDTLLTQSLLMELSALCRVEVEEGLALVALIGNDLSKAC 
               
               
                   
               
               
                 GVGKEVFGVLEPFNIRMICYGASSHNLCFLVPGEDAEQVVQKLHSNLFE 
               
               
                   
               
               
                 S-LysC polypeptide sequence  
               
               
                 SEQ ID NO: 17 
               
               
                 MGLVVQKYGGSSVADAEGIKRVAKRIVEAKKNGNQVVAVVSAMGDTTDEL 
               
               
                   
               
               
                 IDLAEQVSPIPAGRELDMLLTAGERISMALLAMAIKNLGHEAQSFTGSQA 
               
               
                   
               
               
                 GVITDSVHNKARIIDVTPGRIRTSVDEGNVAIVAGFQGVSQDSKDITTLG 
               
               
                   
               
               
                 RGGSDTTAVALAAALDADVCEIYTDVDGVFTADPRVVPKAKKIDWISFED 
               
               
                   
               
               
                 MLELAASGSKVLLHRCVEYARRYNIPIHVRSSFSGLQGTWVSSEPIKQGE 
               
               
                   
               
               
                 KHVEQALISGVAHDTSEAKVTVVGVPDKPGEAAAIFRAIADAQVNIDMVV 
               
               
                   
               
               
                 QNVSAASTGLTDISFTLPKSEGRKAIDALEKNRPGIGFDSLRYDDQIGKI 
               
               
                   
               
               
                 SLVGAGMKSNPGVTADFFTALSDAGVNIELISTSEIRISVVTRKDDVNEA 
               
               
                   
               
               
                 VRAVHTAFGLDSDSDEAVVYGGTGR 
               
               
                   
               
               
                 Asd polypeptide sequence  
               
               
                 SEQ ID NO: 18 
               
               
                 MKNVGFIGWRGMVGSVLMQRMVEERDFDAIRPVFFSTSQLGQAAPSFGGT 
               
               
                   
               
               
                 TGTLQDAFDLEALKALDIIVTCQGGDYTNEIYPKLRESGWQGYWIDAASS 
               
               
                   
               
               
                 LRMKDDAIIILDPVNQDVITDGLNNGIRTFVGGNCTVSLMLMSLGGLFAN 
               
               
                   
               
               
                 DLVDWVSVATYQAASGGGARHMRELLTQMGHLYGHVADELATPSSAILDI 
               
               
                   
               
               
                 ERKVTTLTRSGELPVDNFGVPLAGSLIPWIDKQLDNGQSREEWKGQAETN 
               
               
                   
               
               
                 KILNTSSVIPVDGLCVRVGALRCHSQAFTIKLKKDVSIPTVEELLAAHNP 
               
               
                   
               
               
                 WAKVVPNDREITMRELTPAAVTGTLTTPVGRLRKLNMGPEFLSAFTVGDQ 
               
               
                   
               
               
                 DapB polypeptide sequence  
               
               
                 SEQ ID NO: 19 
               
               
                 MHDANIRVAIAGAGGRMGRQLIQAALALEGVQLGAALEREGSSLLGSDAG 
               
               
                   
               
               
                 ELAGAGKTGVTVQSSLDAVKDDFDVFIDFTRPEGTLNHLAFCRQHGKGMV 
               
               
                   
               
               
                 IGTTGFDEAGKQAIRDAAADIAIVFAANFSVGVNVMLKLLEKAAKVMGDY 
               
               
                   
               
               
                 TDIEIIEAHHRHKVDAPSGTALAMGEAIAHALDKDLKDCAVYSREGHTGE 
               
               
                   
               
               
                 RVPGTIGFATVRAGDIVGEHTAMFADIGERLEITHKASSRMTFANGAVRS 
               
               
                   
               
               
                 ALWLSGKESGLFDMRDVLDLNNL 
               
               
                   
               
               
                 DapD polypeptide sequence  
               
               
                 SEQ ID NO: 20 
               
               
                 MQQLQNIIETAFERRAEITPANADTVTREAVNQVIALLDSGALRVAEKID 
               
               
                   
               
               
                 GQWVTHQWLKKAVLLSFRINDNQVIEGAESRYFDKVPMKFADYDEARFQK 
               
               
                   
               
               
                 EGFRVVPPAAVRQGAFIARNTVLMPSYVNIGAYVDEGTMVDTWATVGSCA 
               
               
                   
               
               
                 QIGKNVHLSGGVGIGGVLEPLQANPTIIEDNCFIGARSEVVEGVIVEEGS 
               
               
                   
               
               
                 VISMGVYIGQSTRIYDRETGEIHYGRVPAGSVVVSGNLPSKDGKYSLYCA 
               
               
                   
               
               
                 VIVKKVDAKTRGKVGINELLRTID 
               
               
                   
               
               
                 AspC polypeptide sequence  
               
               
                 SEQ ID NO: 21 
               
               
                 MFENITAAPADPILGLADLFRADERPGKINLGIGVYKDETGKTPVLTSVK 
               
               
                   
               
               
                 KAEQYLLENETTKNYLGIDGIPEFGRCTQELLFGKGSALINDKRARTAQT 
               
               
                   
               
               
                 PGGTGALRVAADFLAKNTSVKRVWVSNPSWPNHKSVFNSAGLEVREYAYY 
               
               
                   
               
               
                 DAENHTLDFDALINSLNEAQAGDVVLFHGCCHNPTGIDPTLEQWQTLAQL 
               
               
                   
               
               
                 SVEKGWLPLFDFAYQGFARGLEEDAEGLRAFAAMHKELIVASSYSKNFGL 
               
               
                   
               
               
                 YNERVGACTLVAADSETVDRAFSQMKAAIRANYSNPPAHGASVVATILSN 
               
               
                   
               
               
                 DALRAIWEQELTDMRQRIQRMRQLFVNTLQEKGANRDFSFIIKQNGMFSF 
               
               
                   
               
               
                 SGLTKEQVLRLREEFGVYAVASGRVNVAGMTPDNMAPLCEAIVAVL 
               
               
                   
               
               
                 synthetic promoter nucleic acid sequence  
               
               
                 SEQ ID NO: 22 
               
               
                 AGTTTATTCTTGACATGTAGTGAGGGGGCTGGTATAAT