Patent Publication Number: US-11041177-B2

Title: Modified lysine decarboxylase enzymes

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application is a 35 U.S.C. § 371 National Stage of International Application No. PCT/CN2016/113519, filed 30 Dec. 2016, designating the United States. Each application is incorporated herein by reference in its entirety. 
     BACKGROUND OF THE INVENTION 
     Most enzymes function optimally within a narrow pH range, because they are amphoteric molecules. The pH of the surrounding environment directly affects the charges on the acidic and basic groups of the amino acids that make up the enzyme. These changes in charge affect the net charge of the enzyme, the pKa of the active site, and the charge distribution across the surface of the enzyme. As a result, changes in pH can affect the activity, solubility, and stability of an enzyme. 
     The class of proteins known as acid decarboxylases is a group of enzymes that catalyze the decarboxylase reaction of basic amino acids (e.g., lysine, arginine, ornithine) in order to generate polyamines as part of the acid stress response in many microorganisms  Escherichia coli  has several PLP-dependent acid decarboxylases: CadA, LdcC, AdiA, SpeA, SpeC, SpeF, GadA, and GadB. All of these enzymes function within a narrow pH range, and the enzyme activity decreases significantly outside of that pH range (Kanjee et al.,  Biochemistry  50, 9388-9398, 2011). It has been previously observed that these PLP-dependent decarboxylases dimerize in order to form a complete active site. In some cases, such as CadA, the dimers form decamers that aggregate into higher molecular weight protein complexes required for optimal function. The inhibition of higher molecular weight protein complex formation (e.g., in conditions outside of the optimal pH) leads to a significant decrease in function (Kanjee et al.,  The EMBO Journal  30, 931-944, 2011). 
     The pKa values of individual amino acids in a protein are important for determining its biomolecular function, because one of the dominant reactions in a protein-water solution is the exchange of protons by certain amino acids with the environment of the protein. The pKa is a measure of the difference in free energy between the neutral and charged states, and indicates the propensity of an amino acid to donate or accept a proton. Certain amino acids have titratable groups that make them more amenable to accept and donate protons. These amino acids include aspartate, glutamate, cysteine, serine, tyrosine, threonine, histidine, lysine, and arginine. Illustrative pKa values of some amino acids are: aspartate is 4.0, glutamate is 4.4, cysteine is 8.7, tyrosine is 9.6, histidine is 6.3, lysine is 10.4, and arginine is 13.0 (Nielsen J E &amp; Vriend G,  Proteins  43, 403-412, 2001). These pKa values can vary by 0.5, depending on the literature source. 
     Whether a titratable group accepts or donates a proton will depend on its environment, such as the pH or other amino acids in its proximity. For example, when the pH is less than the pKa of the titratable group, then the group will more likely accept a proton. Conversely, when the pH is greater than the pKa of the titratable group, then the group will more likely donate a proton. When a titratable group of an amino acid either accepts or donates a proton, the amino acid can become either positively charged, negatively charged, or neutral depending on the charge it started with before the proton exchange happened. Charged groups can interact with other charged groups when the two groups are brought into proximity of one another. Like charges repel each other and opposite charges attract each other. Neutrally charged groups that are protonated can still interact with other groups through hydrogen bond interactions. 
     An understanding of the pKa values of the titratable groups of a protein is not only important for understanding how pH affects polypeptide folding and enzyme activity, but also protein-protein interactions (Jensen J E, Curr Pharm Biotechnol 9, 96-102, 2008), especially in the cases of the acid decarboxylases that undergo significant changes in their quaternary structure as a result of a change in the pH of the environment. There have been few studies in evaluating the effect of mutations at various amino acids with titratable groups on the function of the acid decarboxylases. 
     Based on prior literature (Kanjee, et al.  The EMBO Journal  30, 931-944, 2011), CadA transitions from a state that consists of decamers and high-order oligomers to a state that is composed mostly of dimers when the pH of the environment changes. The formation of decamers is a prerequisite for the formation of high-order oligomers. It has been shown that CadA functions optimally at a pH of 5.0-5.5 (Lemonnier M &amp; Lane D, Microbiology 144, 751-760, 1998). At this acidic pH, Kanjee et al. show using EM that CadA exists mainly as high-order oligomers. When the pH is increased above 6.0 or when the inhibitor ppGpp is present, the high-order oligomers do not form and decarboxylase function is significantly reduced. However, there is an absence of literature that describes how the CadA high-order oligomers form and the amino acid residues that play a role in their formation. 
     BRIEF SUMMARY OF ASPECTS OF THE DISCLOSURE 
     This invention is based, in part, on the discovery of mutations that provide the ability to stabilize the chemical interactions necessary for quaternary structure formation, and increase stability to allow a mutant acid decarboxylase protein to function across a wider pH range. The ability to function across a wider pH range is important in maintaining a high reaction rate without the need to add additional chemicals to maintain pH. The maintenance of a high reaction rate across a wide pH range would enable lysine to be converted into cadaverine faster and reduce the amount of utilities required. The tolerance of the protein for alkaline pH eliminates the need to add additional chemicals to maintain pH. These chemicals used to maintain pH oftentimes form salts (e.g., SO 4   2−  or Cl − ) that go either into the wastewater or must be removed from the process using additional purification steps. Therefore, a mutant acid decarboxylase that functions at a wider pH range than wild-type would decrease the cost and the environmental footprint of the overall process by reducing the amount of salts formed during the process. 
     In one aspect, the invention thus provides a CadA variant polypeptide comprising at least one amino acid substitution at a glutamic acid residue in a region corresponding to amino acids 276 to 509 as determined with reference to SEQ ID NO:2, where the glutamic acid residue occurs at the surface of the protein with the side chain oriented towards the external environment in a segment of the protein that lacks a defined secondary structure; and wherein the CadA variant polypeptide has at least 70% identity to any one of SEQ ID NOS:2 to 5. In some embodiments, the substitution is at a glutamic acid residue at a position selected from the group consisting of positions 291, 344, 355, 463, 482, and 499 as determined with reference to SEQ ID NO:2. In some embodiments, the amino acid substitution is E291A/C/D/H/R/V/G/K/N/S, E355C/F/H/K/L/M/N/P/Q/R/S/T/V/Y, or E482C/F/I/L/S/W/Y/A/H/K/M. In some embodiments, the amino acid substitution is E291A/C/D/H/R/V, E355C/F/H/K/L/M/N/P/Q/R/S/T/V/Y, or E482C/F/I/L/S/W/Y. In some embodiments a CadA variant polypeptide comprises substitutions of glutamic acid residues at at least two positions selected from the group consisting of positions 291, 344, 355, 463, 482, and 499. In some embodiments, a CadA variant polypeptide comprises substitutions of glutamic acid residues at at least three positions selected from the group consisting of positions 291, 344, 355, 463, 482, and 499. In some embodiments, a CadA variant polypeptide comprises substitutions of glutamic acid residues at four or five positions selected from the group consisting of positions 291, 344, 355, 463, 482, and 499; or at all six of the positions. In some embodiments, a CadA variant polypeptide as described above in this paragraph has at least 70% identity to SEQ ID NO:2. In some embodiments, the CadA variant polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:2. In some embodiments, a CadA variant polypeptide has at least 70% identity to SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. In some embodiments, the CadA variant polypeptide has at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. 
     In a further aspect, the invention provides a genetically modified host cell comprising a CadA variant polypeptide as described herein, e.g., in the preceding paragraph. In typical embodiments, the genetically modified host cell is genetically modified to over express one or more lysine biosynthesis polypeptides. In some embodiments, the host cell is a bacterium. In further embodiments, the host cell is from the genus  Escherichia, Hafnia , or  Corynebacteria . In some embodiments, the genetically modified host cell is  Escherichia coli . In some embodiments, the genetically modified host cell is  Hafnia alvei . In some embodiments, the genetically modified host cell is  Corynebacterium glutamicum.    
     In an additional aspect, the invention provides a polynucleotide comprising a nucleic acid sequence encoding a CadA variant polypeptide as described herein, e.g., in the preceding paragraphs in this section. In further aspects, the invention additionally provides an expression vector comprising a polynucleotide encoding the CadA variant, and/or a genetically modified host cell comprising the expression vector. In some embodiments, the host cell is a bacterium, e.g., from the genus  Escherichia, Hafnia , or  Corynebacteria . In some embodiments, the genetically modified host cell is  Escherichia coli . In some embodiments, the genetically modified host cell is  Hafnia alvei . In some embodiments, the genetically modified host cell is  Corynebacterium glutamicum.    
     In a further aspect, the invention provides a genetically modified host cell comprising a polynucleotide that comprises a nucleic acid sequence encoding a CadA variant polypeptide as described herein, e.g., in the preceding paragraphs in this section, wherein the nucleic acid sequence encoding the CadA variant polypeptide is integrated into the host cell chromosome. In some embodiments, the host cell is a bacterium, e.g., from the genus  Escherichia, Hafnia , or  Corynebacteria . In some embodiments, the genetically modified host cell is  Escherichia coli . In some embodiments, the genetically modified host cell is  Hafnia alvei . In some embodiments, the genetically modified host cell is  Corynebacterium glutamicum.    
     In another aspect, the invention provides a method of producing cadaverine, the method comprising culturing a genetically modified host cell as described herein, e.g., in the preceding paragraphs in this section, under conditions in which CadA variant polypeptide is expressed. 
    
    
     
       DESCRIPTION OF THE FIGURES 
         FIG. 1  shows an alignment of the  E. coli  CadA polypeptide sequence SEQ ID NO:2 with CadA homologs from  Salmonella enterica  (WP_001540636.1, SEQ ID NO:5),  Klebsiella  multispecies (WP_012968785, SEQ ID NO:3), and  Enterobacteriaeceae  multispecies (WP_002892486.1, SEQ ID NO:4).   
     
    
    
     DETAILED DESCRIPTION OF ASPECTS OF THE DISCLOSURE 
     Before the present invention is described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications and accession numbers mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. 
     Terminology 
     As used in the context of the present disclosure, a “CadA polypeptide” refers to an  Escherichia coli  CadA polypeptide having the amino acid sequence of SEQ ID NO:2, or a biologically active variant thereof that has activity, i.e., catalyzes the decarboxylation of L-lysine to produce cadaverine. Biologically active variants include alleles, mutants, fragments, and interspecies homologs of the  E. coli  CadA polypeptide. CadA has been well characterized structurally and functionally. The protein data bank ID for the structure of CadA is 3N75. Illustrative CadA polypeptides from other species include CadA from  Klebsiella  (e.g., SEQ ID NO:3),  Enterobacteriaceae  (e.g., SEQ ID NO:5), and  Salmonella enterica  (e.g., SEQ ID NO:6). Additional CadA polypeptides from other species include Serratia sp., WP 033635725.1; and  Raoultella ornithinolytica , YP 007874766.1. In some embodiments, a “CadA polypeptide” has at least 60% amino acid sequence identity, typically at least 65%, 70%, 75%, 80%, 85%, 90% identity; often at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, over a region of at least about 200, 300, 400, 500, or more, amino acids; or over the length of the CadA polypeptide of SEQ ID NO:2. In some embodiments, a “CadA polypeptide” comprises a region that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity over a region comprising amino acids residues that correspond to amino acids 261-509 of SEQ ID NO:2 where a native glutamate present in the region is substituted with another non-naturally occurring amino acid as described herein. In some embodiments, a “CadA polypeptide” has at least 60% amino acid sequence identity, often at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater, amino acid sequence identity, preferably over a region of at least about 200, 300, 400, 500, or more, amino acids, or over the length of the CadA polypeptide of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. 
     A “CadA polynucleotide” as used herein refers to a polynucleotide that encodes a CadA polypeptide. A nucleic acid or polynucleotide that encodes a CadA refers to a gene, pre-mRNA, mRNA, and the like, including nucleic acids encoding variants, alleles, fragments, mutants, and interspecies homologs of the particular amino acid sequences described herein. 
     As used herein, the term “alkaline pH” refers to a solution or surrounding environment having a pH of greater than 7.5. In one embodiment, alkaline pH refers to a solution or surrounding environment have a pH of at least 8.0, at least 8.5, or higher. 
     The term “enhanced” or “improved” in the context of the production of an amino acid derivative, e.g., cadaverine, as used herein refers to an increase in the production of the amino acid derivative produced by a host cell that expresses a CadA variant polypeptide of the invention in comparison to a control counterpart cell, such as a cell of the wildtype strain or a cell of the same strain that expresses the wildtype CadA protein. In one embodiment, activity of the CadA variant is improved by at least 10%, 15% 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater, compared to CadA activity of a counterpart cell expressing a wildtype CadA, where activity is assessed by measuring the production of an amino acid derivative, typically cadaverine, produced by the host cell and control cell under identical conditions. For example, activity of a CadA variant polypeptide of the invention can be assessed by evaluating an aliquot of a culture of host cells transformed with a polynucleotide encoding the variant CadA polypeptide compared to a corresponding aliquot from a culture of counterpart host cells of the same strain that expresses wildtype CadA. By way of further illustration, the activity of a CadA variant polypeptide of the invention compared to the counterpart widltype CadA can be determined by evaluating the production of cadaverine by cells transformed with either a vector comprising a nucleic acid sequence encoding the variant CadA polypeptide (variant host cells) or a vector comprising a nucleic acid encoding the wildtype CadA polypeptide (control host cells). Variant and control host cells that are grown under conditions to express CadA and an aliquot is incubated with lysine-HCl and PLP at a final concentration of 120 g/L and 0.1 mM, respectively at pH 8.0 for a period of time, e.g., 2 hours. Cadaverine production is measured following incubation. An exemplary assay is provided in the Examples section. 
     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 position of a variant CadA polypeptide sequence “corresponds to” a position in SEQ ID NO:2 when the variant polypeptide is aligned with SEQ ID NO:2 in a maximal alignment. 
     The terms “wild type”, “native”, and “naturally occurring” with respect to a CadA polypeptide are used herein to refer to a CadA protein that has a sequence that occurs in nature. 
     In the context of this invention, the term “mutant” with respect to a mutant polypeptide or mutant polynucleotide is used interchangeably with “variant”. A “non-naturally” occurring CadA variant refers to a variant or mutant CadA polypeptide that is not present in a cell in nature and that is produced by genetic modification, e.g., using genetic engineering technology or mutagenesis techniques, of a native CadA polynucleotide or polypeptide. A “variant” CadA polypeptide in the context of this disclosure includes any non-naturally occurring CadA polypeptide that comprises at least one amino acid substitution, where the at least one amino acid substitution is a substitution of a glutamic acid residue at positions 291, 344, 355, 463, 482, or 499, as determined with reference to SEQ ID NO:2. A variant CadA polypeptide of the invention may also have additional mutations relative to SEQ ID NO:2, including further substitutions, insertions, or deletions. 
     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. 
     The term “substantially identical,” used in the context of this disclosure for two nucleic acids or polypeptides, refers to a sequence that has at least 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. 
     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 a variant CadA polypeptide has sequence identity to SEQ ID NO:2, or another polypeptide reference sequence such as SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, is the BLAST algorithm, which is described in Altschul et al., 1990, J. Mol. Biol. 215:403-410). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the worldwide web at ncbi.nlm.nih.gov/). Illustrative software for performing protein sequence alignments include ClustalW2 and BLASTP. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expect threshold (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff &amp; Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). In the present disclosure, polypeptide sequence identity is typically determined using BLASTP Align Sequence with the default parameters. 
     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. Optimal alignments are typically conducted using BLASTP with default parameters. 
     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 refers 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. 
     The term “polypeptide” as used herein includes reference to polypeptides containing naturally occurring amino acids and amino acid backbones as well as non-naturally occurring amino acids and amino acid analogs. 
     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)). In some embodiments, conservative substitutions are employed in generating Cada variants having substitutions at sites other than a glutamate residue. 
     The term “promoter,” as used herein, refers to a polynucleotide sequence capable of driving transcription of a nucleic acid 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. 
     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” refers generally to a polynucleotide sequence or polypeptide that does not naturally occur in a wild-type cell or organism, but is typically introduced into the cell by molecular biological techniques, i.e., engineering to produce a recombinant microorganism. Examples of “exogenous”polynucleotides include vectors, plasmids, and/or man-made nucleic acid constructs encoding a desired protein. 
     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 a plasmid 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 can 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 of the invention protein 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 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, may refer to a polypeptide molecule that is free of other components of the cell, i.e., it is not associated with in vivo cellular substances. 
     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, 2009-2016). 
     Summary of Certain Aspects of the Disclosure 
     In one aspect, the invention provides a variant CadA polypeptide that comprises a mutation at a glutamic acid (glutamate) that resides at a position in one of sections of the protein in the core domain that are without a defined secondary structure (α, alpha helix; β, beta sheet; η, strand) (see Kanjee et al.,) within those domains. These sections are the amino acids 276-299 between β10 and β11 that includes η4 and α11, the amino acids 314-326 between β11 and α12 that includes β12, the amino acids 344-357 between η6 and β14, the amino acids 454-483 between β16 and β18 that includes β17, and the amino acids 494-509 between β19 and α17, wherein the positions of the amino acids are defined with reference to SEQ ID NO:2. 
     The ability of a variant CadA of the present invention to tolerate alkaline pH also allows the use of alternative nitrogen sources that have higher pH values, such as urea and ammonia (1M solution has a pH 11.6) in fermentation reactions to generate the desired product, e.g., polyamines. These alternative nitrogen sources generate less salt waste byproduct. 
     CadA Polypeptide Variants 
     CadA is a member of the subclass of Fold Type I pyridoxal 5′-phosphate (PLP)-dependent decarboxylases. This class of proteins typically contains a N-terminal wing domain, a core domain, and a C-terminal domain. The core domain has a linker region, a PLP-binding subdomain, and subdomain 4 For CadA, the N-terminal wing domain (corresponding to residues 1 to 129 as determined with reference to SEQ ID NO:2) has a flavodoxin-like fold composed of five-stranded parallel beta-sheets sandwiched between two sets of amphipathic alpha-helices. The core domain (residues 130 to 563 as determined with reference to 563 of SEQ ID NO:2) includes: a linker region, amino acid residues 130 to 183 of SEQ ID NO:2, that form a short helical bundle; the PLP-binding subdomain, amino acids 184 to 417 of SEQ ID NO:2 that form a seven-stranded beta-sheet core surrounded by three sets of alpha-helices; and subdomain 4, amino acids 418 to 563 that form a four stranded antiparallel beta-sheet core with three alpha-helices facing outward. The C-terminal domain corresponds to amino acid residues 564 to 715 as determined with referenced to SEQ ID NO:2 that form two sets of beta sheets with an alpha-helical outer surface (Kanjee et al., The EMBO Journal 30, 931-944 2011). 
     CadA protein forms a two-fold symmetric dimer that completes the active site of each monomer. Five dimers associate to form a decamer that consist of a double-ringed structure with five-fold symmetry. The decamer associates with other decamers to form higher-order oligomers. It has been shown that in acidic conditions (pH 5), CadA predominantly exists in the oligomeric state, and less oligomers and decamers are found as the environment becomes more basic. It was estimated that 25% of the enzymes exist as dimers and 75% exist as decamers at pH 6.5, while 95% of the enzymes exist as dimers at pH 8.0 (Kanjee et al.,  The EMBO Journal  30, 931-944 2011). This decrease in oligomer formation coincides with the decrease in decarboxylase activity observed as the pH of the environment of the enzyme increases above 5.0. 
     Illustrative Cad A polypeptides from  E. coli, Salmonella enterica, Klebsiella , and  Enterobacteriaeceae  are provided in SEQ ID NOS:2-5, which share greater than 90% sequence identity with one another. 
     CadA polypeptides of the present invention comprise at least one substitution of another amino acid for a glutamate at a position in one of the segments of the protein in the core domain that are without a defined secondary structure (α, alpha helix; β, beta sheet; η, strand) (see Kanjee et al.,); and where the glutamate is at the surface of the protein where the side chain is oriented toward the external environment. In the present disclosure, the amino acid that is substituted for the glutamate does not occur at that position in a native CadA sequence. A variant CadA polypeptide in accordance with the invention thus can comprise at least one amino acid substitution at position E291, E344, E355, E463, E482, and E499 as determined with reference to SEQ ID NO:2. In some embodiments a variant CadA polypeptide comprises more than one amino acid substitution, e.g., 2, 3, or more substitutions, at positions E291, E344, E355, E463, E482, and E499 as determined with reference to SEQ ID NO:2. In some embodiments, the amino acid that is substituted for a glutamate is selected from the group of amino acids consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine, where the mutant does not have the same amino acid as the wild-type sequence (SEQ ID NO: 2, 3, 4, or 5) at the same position. In some embodiments, the amino acid that is substituted for a glutamate has the ability to donate a hydrogen for hydrogen bond formation. For example, C, Y, K, and N have pKa valuates greater than 7, so their protonation state does not change when the pH increases from 5 to 8. Therefore, any hydrogen bond formed at these positions is more stable compared to when glutamate, which has an acidic pKa, is present at those positions. The sulfur of M can act as either a nucleophile or an electrophile and does not need a proton to interact with other amino acid groups. M may thus further stabilize a protein-protein interaction at that site. In some embodiments, the amino acid that is substituted for a glutamate is selected from the group consisting of C, H, K, S, A, F, L, M, N, R, V, Y, D, G, I, P, Q, T, and W. In some embodiments, the amino acid that is substituted for a glutamate is selected from the group consisting of C, H, K, S, A, F, L, M, N, R, V, and Y. In some embodiments, the amino acid that is substituted for a glutamate is selected from the group consisting of C, Y, K, S, H, R, M, and N. In some embodiments, the amino acid that is substituted for a glutamate is selected from the group consisting of C, H, K, and S. 
     In some embodiments, the variant CadA polypeptide is a variant of CadA from  E. coli  in which at least, one, two, three, four, five, or all six of the glutamates at positions E291, E344, E355, E463, E482, and E499 is substituted with another amino acid. 
     In some embodiments, a variant CadA polypeptide of the invention has at least 60% amino acid sequence identity, often at least 65%, 70%, 75%, 80%, or 85% identity; and typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, over a region of at 500 or more amino acids in length, or over the length of, the CadA polypeptide of SEQ ID NO:2; and has a substitution at a glutamic acid residue at at least one of positions E291, E344, E355, E463, E482, or E499 as determined with reference to SEQ ID NO:2. In some embodiments, the substitution is E291A/C/D/H/R/V/G/K/N/S, E355C/F/H/K/L/M/N/P/Q/R/S/T/V/Y, or E482C/F/I/L/S/W/Y/A/H/K/M. In other embodiments, the substitution is E291A/C/D/H/R/V, E355C/F/H/K/L/M/N/P/Q/R/S/T/V/Y, or E482C/F/I/L/S/W/Y. 
     In some embodiments, a variant CadA polypeptide of the invention has at least 60% amino acid sequence identity, often at least 65%, 70%, 75%, 80%, or 85% identity; and typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, over a region of at 500 or more amino acids in length, or over the length of, the CadA polypeptide of SEQ ID NO:3; and has a substitution at a glutamic acid residue at at least one of positions E291, E344, E355, E463, or E482 as determined with reference to SEQ ID NO:3. In some embodiments, the substitution is E291A/C/D/H/R/V/G/K/N/S, E355C/F/H/K/L/M/N/P/Q/R/S/T/V/Y, or E482C/F/I/L/S/W/Y/A/H/K/M. In other embodiments, the substitution is E291A/C/D/H/R/V, E355C/F/H/K/L/M/N/P/Q/R/S/T/V/Y, or E482C/F/I/L/S/W/Y. 
     In some embodiments, a variant CadA polypeptide of the invention has at least 60% amino acid sequence identity, often at least 65%, 70%, 75%, 80%, or 85% identity; and typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, over a region of at 500 or more amino acids in length, or over the length of, the CadA polypeptide of SEQ ID NO:4; and has a substitution at a glutamic acid residue at at least one of positions E291, E344, E355, E463, or E482 as determined with reference to SEQ ID NO:4. In some embodiments, the substitution is E291A/C/D/H/R/V/G/K/N/S, E355C/F/H/K/L/M/N/P/Q/R/S/T/V/Y, or E482C/F/I/L/S/W/Y/A/H/K/M. In other embodiments, the substitution is E291A/C/D/H/R/V, E355C/F/H/K/L/M/N/P/Q/R/S/T/B/Y, or E482C/F/I/L/S/W/Y. 
     In some embodiments, a variant CadA polypeptide of the invention has at least 60% amino acid sequence identity, often at least 65%, 70%, 75%, 80%, or 85% identity; and typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, over a region of at 500 or more amino acids in length, or over the length of, the CadA polypeptide of SEQ ID NO:5; and has a substitution at a glutamic acid residue at at least one of positions E291, E355, E463, or E482 as determined with reference to SEQ ID NO:5. In some embodiments, the substitution is E291A/C/D/H/R/V/G/K/N/S, E355C/F/H/K/L/M/N/P/Q/R/S/T/V/Y, or E482C/F/I/L/S/W/Y/A/H/K/M. In other embodiments, the substitution is E291A/C/D/H/R/V, E355C/F/H/K/L/M/N/P/Q/R/S/T/V/Y, or E482C/F/I/L/S/W/Y. 
     Nucleic Acids Encoding CadA Variant Polypeptides 
     Isolation or generation of CadA polynucleotide sequences can be accomplished by a number of techniques. In some embodiments, oligonucleotide probes and based on the sequences disclosed here can be used to identify the desired polynucleotide in a cDNA or genomic DNA library from a desired bacteria species. Desired substitutions may be introduced into the CadA-encoding polynucleotide sequence using appropriate primers, e.g., as illustrated in the Examples section, to incorporate the desired changes into the polynucleotide sequence. For instance, PCR may be used to amplify the sequences of the genes directly from mRNA, from cDNA, from genomic libraries or cDNA libraries and to introduce desired substitutions. 
     Appropriate primers and probes for identifying a CadA polynucleotide in bacteria can be generated from comparisons of the sequences provided herein or generated based on a CadA polynucleotide sequence from another bacteria. 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 acid decarboxylase 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., a cadA polynucleotide sequence of SEQ ID NO:1. 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 95% identity, or greater, to an acid decarboxylase polynucleotide, e.g., a cadA polynucleotide of SEQ ID NO:1, wherein the nucleic acid comprises a codon that encodes the desired amino acid to be substituted. 
     Nucleic acid sequences encoding a CadA variant protein in accordance with the invention that confers increased production of an amino acid derivative, 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 a variant CadA protein can be prepared using methods well known in the art. For example, a DNA sequence encoding a CadA variant polypeptide, 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  H. alvei, E. coli , or  C. glutamicum . In some embodiments, an expression vector that comprises an expression cassette that comprises the gene encoding the CadA variant polypeptide further comprises a promoter operably linked to the nucleic acid sequence encoding the CadA variant polypeptide. In other embodiments, a promoter and/or other regulatory elements that direct transcription of the cadA polynucleotide encoding a variant Cada polypeptide are endogenous to the host cell and an expression cassette comprising the cadA 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 polynucleotide encoding a CadA variant 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 cadA gene encoding a CadA variant polypeptide of the invention may be modified to increase expression. For example, an endogenous CadA 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 polynucleotide encoding the CadA variant 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 polynucleotide encoding a CadA variant 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 minichromosome, 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, H. alvei , or  C. glutamicum.    
     Although any suitable expression vector may be used to incorporate the desired sequences, readily available bacterial expression vectors include, without limitation: plasmids such as pSClOl, 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 M1 3 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 express a CadA variant polypeptide of the invention. 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 express a CadA variant polypeptide is performed in conjunction with modifying the host cell to overexpress one or more lysine biosynthesis polypeptides. 
     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, PflB, 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. 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                   
                   
                 GenBank  
               
               
                 Protein 
                 Gene 
                 EC Number 
                 Accession No. 
               
               
                   
               
             
            
               
                 α-ketogultarate dehydrogenase (SucA) 
                 sucA 
                 1.2.4.2 
                 YP_489005.1 
               
               
                 Phosphoenolpyruvate carboxylase (PPC) 
                 ppc 
                 4.1.1.31 
                 AAC76938.1 
               
               
                 aspartate transaminase (AspC) 
                 aspC 
                 2.6.1.1 
                 AAC74014.1 
               
               
                 aspartate kinase (LysC) 
                 lysC 
                 2.7.2.4 
                 NP_418448.1 
               
               
                 aspartate semialdehyde dehydrogenase (Asd) 
                 asd 
                 1.2.1.11 
                 AAC76458.1 
               
               
                 dihydrodipicolinate synthase (DapA) 
                 dapA 
                 4.3.3.7 
                 NP_416973.1 
               
               
                 dihydropicolinate reductase (DapB) 
                 dapB 
                 1.17.1.8 
                 AAC73142.1 
               
               
                 tetrahydrodipicoinate succinylase (DapD) 
                 dapD 
                 2.3.1.117 
                 AAC73277.1 
               
               
                 N-succinyldiaminopimelate aminotransferase (ArgD) 
                 argD 
                 2.6.1.11 
                 AAC76384.1 
               
               
                 N-succinyl-L-diaminopimelate deacylase (DapE) 
                 dapE 
                 3.5.1.18 
                 AAC75525.1 
               
               
                 diaminopimelate epimerase (DapF) 
                 dapF 
                 5.1.1.7 
                 AAC76812.2 
               
               
                 diaminopimelate decarboxylase (LysA) 
                 lysA 
                 4.1.1.20 
                 AAC75877.1 
               
               
                 meso-diaminopimelate dehydrogenase (Ddh) 
                 ddh 
                 NA 
                 P04964.1 
               
               
                 pyridine nucleotide transhydrogenase (PntAB) 
                 pntAB 
                 NA 
                 AAC74675.1,  
               
               
                   
                   
                   
                 AAC74674.1 
               
               
                 cytochrome O oxidase (CyoABE) 
                 cycABE 
                 1.10.3.10 
                 AAC73535.1,  
               
               
                   
                   
                   
                 AAC73534.1, 
               
               
                   
                   
                   
                 AAC73531.1 
               
               
                 glutamate decarboxylase (GadAB) 
                 gadAB 
                 4.1.1.15 
                 AAC76542.1,  
               
               
                   
                   
                   
                 AAC74566.1 
               
               
                 L-amino acid efflux transporter (YbjE) 
                 ybjE 
                 NA 
                 AAC73961.2 
               
               
                 glutamate dehydrogenase (GdhA) 
                 gdhA 
                 1.4.1.4 
                 AAC74831.1 
               
               
                 citrate synthase (GltA) 
                 gltA 
                 2.3.3.1/2.3.3.16 
                 AAC73814.1 
               
               
                 succinyl-coA synthase (SucC) 
                 sucC 
                 6.2.1.5 
                 AAC73822.1 
               
               
                 glutamate-GABA antiporter (GadC) 
                 gadC 
                 NA 
                 AAC74565.1 
               
               
                 aconitase B (AcnB) 
                 acnB 
                 4.2.1.99 
                 AAC73229.1 
               
               
                 pyruvate-formate lyase (PflB) 
                 pflB 
                 NA 
                 AAC73989.1 
               
               
                 aspartate kinase/homoserine dehydrogenase (ThrA) 
                 thrA 
                 2.7.2.4 
                 AAC73113.1 
               
               
                 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-D-glutamate: 
                 murE 
                 6.3.2.13 
                 AAC73196.1 
               
               
                 meso-diaminopimelate ligase (MurE) 
                   
                   
                   
               
               
                 putrescine/cadaverine 
                 speE 
                 2.5.1.16 
                 AAC73232.1 
               
               
                 aminopropyltransferase (SpeE) 
                   
                   
                   
               
               
                 spermidine acetyltransferase (SpeG) 
                 speG 
                 NA 
                 AAC74656.1 
               
               
                 glutamate-putrescine/glutamate-cadaverine 
                 puuA 
                 NA 
                 AAC74379.2 
               
               
                 ligase (PuuA) 
                   
                   
                   
               
               
                 putrescine importer (PuuP) 
                 puuP 
                 NA 
                 AAC74378.2 
               
               
                 putrescine/cadaverine aminotransferase (YgjG) 
                 ygjG 
                 2.6.1.82 
                 AAC76108.3 
               
               
                   
               
            
           
         
       
     
     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  
               
               
                   
                   
                   
                 Accession 
               
               
                 Protein 
                 Gene 
                 EC Number 
                 No. 
               
               
                   
               
             
            
               
                 PEP carboxykinase (Pck) 
                 pck 
                 4.1.1.49 
                 NP_417862 
               
               
                 Glucose-6-phosphate isomerase (Pgi) 
                 pgi 
                 5.3.1.9 
                 NP_418449 
               
               
                 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) 
                 menE 
                 6.2.1.26 
                 NP_416763 
               
               
                 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) 
                 rpoC 
                 2.7.7.6 
                 NP_418415 
               
               
                 aspartokinase I (ThrA) 
                 thrA 
                 2.7.2.4/1.1.1.3 
                 NP_414543 
               
               
                   
               
            
           
         
       
     
     Nucleic acids encoding a lysine biosynthesis polypeptide may be introduced into the host cell along with a polynucleotide encoding a CadA variant polypeptide, 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 one or more lysine biosynthesis polypeptides before or after the host cells genetically modified to express a CadA variant polypeptide. 
     A host cell engineered to express a CadA variant polypeptide is typically 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  Escherichia coli . In some embodiments, the host cell is  Hafnia alvei . In some embodiments, the host cell is  Corynebacterium glutamicum.    
     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. 
     In some embodiments, a CadA variant polypeptide of the present invention may be recovered from a host cell that expresses the variant polypeptide. In some embodiments, the recovered variant protein may be immobilized onto a solid substrate or inert material to form an immobilized enzyme. In one embodiment, the immobilized enzyme may have improved operational stability than the soluble form of the fusion protein. 
     Methods of Producing Lysine or a Lysine Derivative. 
     A host cell genetically modified to overexpress a CadA variant polypeptide of the invention can be employed to produce lysine or a derivative of lysine. In some embodiments, the host cell produces cadaverine. Thus, for example, to produce cadaverine, a host cell genetically modified to express a CadA variant 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 and/or cadaverine. A host cell modified in accordance with the invention to express a CadA variant polypeptide provides a higher yield of cadaverine relative to a counterpart host cell that expresses native CadA. 
     Host cells may be cultured using well known techniques (see, e.g., the illustrative conditions provided in the examples section. 
     In some embodiments, host cells are cultured using nitrogen sources that are not salts (e.g., ammonium sulfate or ammonium chloride), such as ammonia or urea. Host cells may be cultured at an alkaline pH during cell growth or enzyme production. 
     The lysine or lysine derivative then be separated and purified using known techniques. Lysine or lysine derivatives, e.g., cadverine, 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 
     We hypothesized that the decamer-decamer interface contains segments of the protein in the core domain, and is composed of sections of the protein without a defined secondary structure (α, alpha helix; β, beta sheet; η, strand) (see Kanjee et al.,) within those domains. These sections are the amino acids 276-299 between β10 and β11 that includes η4 and α11, the amino acids 314-326 between β11 and α12 that includes β12, the amino acids 344-357 between η6 and β14, the amino acids 454-483 between β16 and β18 that includes β17, and the amino acids 494-509 between β19 and α17. These examples show that glutamate residues at these positions can be substituted to increase production of cadaverine by host cells that are genetically modified to express the CadA variant polypeptide. 
     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 ( FIG. 1 ), digested using the restriction enzymes SacI and XbaI, and ligated into pUC18 to generate the plasmid pCIB60. The 5′ sequence upstream of the cadA gene was optimized using the PCR primers cadA-F2 and cadA-R2 to create pCIB71. 
     Example 2: Construction of Plasmid Vectors that Encode CadA with Cysteine Mutations at the Predicted Interfacial Amino Acid Residues 
     Primer pairs were designed to modify the amino acid at positions 291, 344, 355, 463, 482, or 499 to cysteine of the cadA gene in pCIB71 using Quickchange PCR. The mutations were verified using DNA sequencing, and the plasmids carrying the cysteine mutations were labeled pCIB71-E291C, pCIB71-E344C, pCIB71-E355C, pCIB71-E463C, pCIB71-E482C, or pCIB71-E499C. 
     Example 3: Lysine Decarboxylase Activity of Mutant CadA Polypeptides with Cysteine Mutations at the Predicted Interfacial Amino Acid Residues 
     [ H. alvei  was transformed with pCIB71-E291C, pCIB71-E344C, pCIB71-E355C, pCIB71-E463C, pCIB71-E482C, or pCIB71-E499C. Three single colonies from each transformation were grown overnight at 37° C. in 4 mL of LB medium with ampicillin (100 μg/mL). The following day, 0.7 mL of each overnight culture was added to 0.3 mL of lysine-HCl and PLP to a final concentration of 120 g/L and 0.1 mM, respectively. The final mixture was adjusted to pH 8.0 with 1M NaOH. Each mixture was incubated at 37° C. for 2 hours. Cadaverine production from each sample was quantified using NMR, and yield was calculated by dividing the molar amount of cadaverine produced by the molar amount of lysine added. The average yield from each sample relative to the average yield from  H. alvei  transformed with pCIB71 after 2 hours is presented in Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Relative cadaverine yield at pH 8 by  H.   alvei   
               
               
                 strains expressing plasmids encoding CadA  
               
               
                 polypeptides with mutations at the predicted 
               
               
                 interfacial amino acid residues. 
               
            
           
           
               
               
               
            
               
                   
                 Plasmid 
                 Relative Yield (%) 
               
               
                   
                   
               
               
                   
                 pCIB71 
                 100 
               
               
                   
                 pCIB71-E291C 
                 188 
               
               
                   
                 pCIB71-E344C 
                 110 
               
               
                   
                 pCIB71-E355C 
                 170 
               
               
                   
                 pCIB71-E463C 
                 120 
               
               
                   
                 pCIB71-E482C 
                 137 
               
               
                   
                 pCIB71-E499C 
                 112 
               
               
                   
                   
               
            
           
         
       
     
     As shown in Table 2, several mutations improved the activity of the CadA polypeptide at pH 8.0. The mutations E291C, E355C, and E482C significantly increased relative yield by more than 30%. The mutations E344C, E463C, and E499C increased yield by 10% to 30%. 
     Example 4: Construction of Plasmid Vectors that Encode CadA with Cysteine Mutations at Predicted Interfacial Amino Acid Residues with Deviant pKa&#39;s and Analysis of Lysine Decarboxylase of the Mutated Proteins 
     This example show that modification of predicted interfacial amino acid residues did not enhance cadaverine production. 
     Primer pairs were designed to modify the amino acid at positions 279, 288, 319, 323, 346, 353, 357, or 470 to cysteine of the cadA gene in pCIB71 using Quickchange PCR. The mutations were verified using DNA sequencing, and the plasmids carrying the cysteine mutations were labeled pCIB71-E279C, pCIB71-R288C, pCIB71-K319C, pCIB71-D323C, pCIB71-K346C, pCIB71-R353C, pCIB71-K357C, or pCIB71-D470C. 
       H. alvei  was transformed with pCIB71-E279C, pCIB71-R288C, pCIB71-K319C, pCIB71-D323C, pCIB71-K346C, pCIB71-R353C, pCIB71-K357C, and pCIB71-D470C. Three single colonies from each transformation were grown overnight at 37° C. in 4 mL of LB medium with ampicillin (100 μg/mL). The following day, 0.7 mL of each overnight culture was added to 0.3 mL of lysine-HCl and PLP to a final concentration of 120 g/L and 0.1 mM, respectively. The final mixture was adjusted to pH 8.0 with 1M NaOH. Each mixture was incubated at 37° C. for 2 hours. Cadaverine production from each sample was quantified using NMR, and yield was calculated by dividing the molar amount of cadaverine produced by the molar amount of lysine added. The average yield from each sample relative to the average yield from  H. alvei  transformed with pCIB71 after 2 hours is presented in Table 3. As shown in Table 3, all fo the mutations decreased cadaverine yield at pH 8.0. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Relative cadaverine yield at pH 8 by  H .  alvei   
               
               
                 strains expressing plasmids encoding CadA  
               
               
                 polypeptides with mutations at the predicted 
               
               
                 interfacial amino acid residues with deviant pKa&#39;s. 
               
            
           
           
               
               
               
               
            
               
                   
                 Relative 
                   
                 Relative 
               
               
                   
                 Yield 
                   
                 Yield 
               
               
                 Plasmid 
                 (%) 
                 Plasmid 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 pCIB71 
                 100 
                 pCIB71-K346C 
                 81 
               
               
                 pCIB71-E279C 
                 60 
                 pCIB71-R353C 
                 75 
               
               
                 pCIB71-R288C 
                 67 
                 pCIB71-K357C 
                 79 
               
               
                 pCIB71K319C 
                 82 
                 pCIB71-D470C 
                 60 
               
               
                 pCIB71-D323C 
                 85 
                   
                   
               
               
                   
               
            
           
         
       
     
     Example 5: Construction of Plasmid Vectors that Encode CadA with a Mutation at E291 
     Primer pairs were designed to modify the amino acid at position 291 of the cadA gene in pCIB71 using Quickchange PCR. The mutations were verified using DNA sequencing, and the plasmids carrying each mutation at amino acid position 291 were labeled pCIB71-E291X, where X is the amino acid that replaced the glutamate residue. 
     Example 6: Construction of Plasmid Vectors that Encode CadA with a Mutation at E355 
     Primer pairs were designed to modify the amino acid at position 355 of the cadA gene in pCIB71 using Quickchange PCR. The mutations were verified using DNA sequencing, and the plasmids carrying each mutation at amino acid position 355 were labeled pCIB71-E355X, where X is the amino acid that replaced the glutamate residue. 
     Example 7: Construction of Plasmid Vectors that Encode CadA with a Mutation at E482 
     Primer pairs were designed to modify the amino acid at position 468 of the cadA gene in pCIB71 using Quickchange PCR. The mutations were verified using DNA sequencing, and the plasmids carrying each mutation at amino acid position 482 were labeled pCIB71-E482X, where X is the amino acid that replaced the glutamate residue. 
     Example 8: Lysine Decarboxylase Activity of Mutant CadA Polypeptides with a E291X Mutation 
       H. alvei  was transformed with pCIB71-E291X. Three single colonies from each transformation were grown overnight at 37° C. in 4 mL of LB medium with ampicillin (100 μg/mL). The following day, 0.7 mL of each overnight culture was added to 0.3 mL of lysine-HCl and PLP to a final concentration of 120 g/L and 0.1 mM, respectively. The final mixture was adjusted to pH 8.0 with 1M NaOH. Each mixture was incubated at 37° C. for 2 hours. Cadaverine production from each sample was quantified using NMR, and yield was calculated by dividing the molar amount of cadaverine produced by the molar amount of lysine added. The yield from each sample relative to the average yield from  H. alvei  transformed with pCIB71 after 2 hours is presented in Table 4. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Relative cadaverine yield at pH 8 by  H .  alvei  strains  
               
               
                 expressing plasmids encoding CadA polypeptides 
               
               
                 with mutations at amino acid position 291. 
               
            
           
           
               
               
               
               
            
               
                   
                 Relative 
                   
                 Relative 
               
               
                   
                 Yield  
                   
                 Yield 
               
               
                 Plasmid 
                 (%) 
                 Plasmid 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 pCIB71 
                 100 
                 pCIB71-E291M 
                 97 
               
               
                 pCIB71-E291A 
                 126 
                 pCIB71-E291N 
                 120 
               
               
                 pCIB71-E291C 
                 148 
                 pCIB71-E291P 
                 4 
               
               
                 pCIB71-E291D 
                 135 
                 pCIB71-E291Q 
                 98 
               
               
                 pCIB71-E291F 
                 82 
                 pCIB71-E291R 
                 127 
               
               
                 pCIB71-E291G 
                 120 
                 pCIB71-E291S 
                 108 
               
               
                 pCIB71-E291H 
                 131 
                 pCIB71-E291T 
                 96 
               
               
                 pCIB71-E291I 
                 98 
                 pCIB71-E291V 
                 135 
               
               
                 pCIB71-E291K 
                 111 
                 pCIB71-E291W 
                 74 
               
               
                 pCIB71-E291L 
                 100 
                 pCIB71-E291Y 
                 80 
               
               
                   
               
            
           
         
       
     
     As shown in Table 4, several mutations at amino acid position 291 improved the activity of the CadA polypeptide at pH 8.0. The mutations E291A, E291C, E291D, E291H, E291R, and E291V increased relative yield by more than 25%. The mutations E291G, E291K, E291N, and E291S also increased yield. The mutations E291I, E291L, E291M, E291Q, and E291T had little effect on yield. The remaining mutations E291F, E291P, E291W, and E291Y decreased yield. 
     Example 9: Lysine Decarboxylase Activity of Mutant CadA Polypeptides with a E355X Mutation 
       H. alvei  was transformed with pCIB71-E355X. Three single colonies from each transformation were grown overnight at 37° C. in 4 mL of LB medium with ampicillin (100 μg/mL). The following day, 0.7 mL of each overnight culture was added to 0.3 mL of lysine-HCl and PLP to a final concentration of 120 g/L and 0.1 mM, respectively. The final mixture was adjusted to pH 8.0 with 1M NaOH. Each mixture was incubated at 37° C. for 2 hours. Cadaverine production from each sample was quantified using NMR, and yield was calculated by dividing the molar amount of cadaverine produced by the molar amount of lysine added. The yield from each sample relative to the average yield from  H. alvei  transformed with pCIB71 after 2 hours is presented in Table 5. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Relative cadaverine yield at pH 8 by  H .  alvei  strains 
               
               
                 expressing plasmids encoding CadA polypeptides 
               
               
                 with mutations at amino acid position 355. 
               
            
           
           
               
               
               
               
            
               
                   
                 Relative 
                   
                 Relative 
               
               
                   
                 Yield  
                   
                 Yield 
               
               
                 Plasmid 
                 (%) 
                 Plasmid 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 pCIB71 
                 100 
                 pCIB71-E355M 
                 141 
               
               
                 pCIB71-E355A 
                 0 
                 pCIB71-E355N 
                 135 
               
               
                 pCIB71-E355C 
                 157 
                 pCIB71-E355P 
                 150 
               
               
                 pCIB71-E355D 
                 97 
                 pCIB71-E355Q 
                 151 
               
               
                 pCIB71-E355F 
                 149 
                 pCIB71-E355R 
                 152 
               
               
                 pCIB71-E355G 
                 0 
                 pCIB71-E355S 
                 143 
               
               
                 pCIB71-E355H 
                 147 
                 pCIB71-E355T 
                 148 
               
               
                 pCIB71-E355I 
                 104 
                 pCIB71-E355V 
                 145 
               
               
                 pCIB71-E355K 
                 139 
                 pCIB71-E355W 
                 111 
               
               
                 pCIB71-E355L 
                 136 
                 pCIB71-E355Y 
                 141 
               
               
                   
               
            
           
         
       
     
     As shown in Table 5, the majority of mutations at amino acid position 355 improved yield at pH 8.0, with the exception of E355A, E355D, E355G, E355I, and E355W. The mutations E355D, E355I, and E355W had little effect on yield. The mutations E355A and E355G decreased yield. 
     Example 10: Lysine Decarboxylase Activity of Mutant CadA Polypeptides with a E482X Mutation 
       H. alvei  was transformed with pCIB71-E482X. Three single colonies from each transformation were grown overnight at 37° C. in 4 mL of LB medium with ampicillin (100 μg/mL). The following day, 0.7 mL of each overnight culture was added to 0.3 mL of lysine-HCl and PLP to a final concentration of 120 g/L and 0.1 mM, respectively. The final mixture was adjusted to pH 8.0 with 1M NaOH. Each mixture was incubated at 37° C. for 2 hours. Cadaverine production from each sample was quantified using NMR, and yield was calculated by dividing the molar amount of cadaverine produced by the molar amount of lysine added. The yield from each sample relative to the average yield from  H. alvei  transformed with pCIB71 after 2 hours is presented in Table 6. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Relative cadaverine yield at pH 8 by  H .  alvei  strains 
               
               
                 expressing plasmids encoding CadA polypeptides 
               
               
                 with mutations at amino acid position 482. 
               
            
           
           
               
               
               
               
            
               
                   
                 Relative 
                   
                 Relative 
               
               
                   
                 Yield  
                   
                 Yield 
               
               
                 Plasmid 
                 (%) 
                 Plasmid 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 pCIB71 
                 100 
                 pCIB71-E482M 
                 116 
               
               
                 pCIB71-E482A 
                 122 
                 pCIB71-E482N 
                 93 
               
               
                 pCIB71-E482C 
                 140 
                 pCIB71-E482P 
                 69 
               
               
                 pCIB71-E482D 
                 94 
                 pCIB71-E482Q 
                 73 
               
               
                 pCIB71-E482F 
                 135 
                 pCIB71-E482R 
                 89 
               
               
                 pCIB71-E482G 
                 86 
                 pCIB71-E482S 
                 126 
               
               
                 pCIB71-E482H 
                 119 
                 pCIB71-E482T 
                 74 
               
               
                 pCIB71-E482I 
                 147 
                 pCIB71-E482V 
                 85 
               
               
                 pCIB71-E482K 
                 112 
                 pCIB71-E482W 
                 126 
               
               
                 pCIB71-E482L 
                 128 
                 pCIB71-E482Y 
                 138 
               
               
                   
               
            
           
         
       
     
     As shown in Table 6, several mutations at amino acid position 482 improved the activity of the CadA polypeptide at pH 8.0. The mutations E482C, E482F, E482I, E482L, E482S, E482W, and E482Y increased relative yield by more than 25%. The mutations E482A, E482H, E482K, and E482M also increased yield. The mutations E482D and E482N had little effect on yield. The remaining mutations E482G, E482P, E482Q, E482R, E482T, and E482V decreased yield. 
     Example 11: In Vitro Kinetic Analysis of Mutant CadA Polypeptides at Alkaline pH Conditions 
     100 mL samples of  H. avlei  transformed with either pCIB71, pCIB71-E291C, pCIB71-E355C, and pCIB71-E482C were lysed with a french press. The lysed samples were centrifuged, and the supernatant was separated from the pellet in order to perform in vitro experiments. Each reaction was performed in Tris-HCl buffer (50 mM Tris-HCl either pH 6 or 8, 25 mM NaCl, 2 mM EDTA) with 120 g/L lysine-HCl and 0.1 mM PLP. The reaction rate of each lysed sample was measured using NMR by sampling the amount of lysine converted in the presence of PLP into cadaverine every 1.6 minutes for a total of 20 minutes, and taking the slope of the linear portion of the yield curve. The samples were diluted so that the reaction rate U (mmol/min/mL) of each sample was 4. The kinetic constants Vmax and Km for lysine of each lysed samples was measured using the same U at an initial pH of either 6 or pH 8. The results of the kinetic analysis of the two samples are shown in Table 7. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Kinetic analysis of normalized Vmax of lysed samples of  
               
               
                   H .  avlei  expressing plasmids encoding wild-type or mutant 
               
               
                 CadA polypeptides under different pH conditions. 
               
            
           
           
               
               
               
               
               
            
               
                 pH 
                 pCIB71 
                 pCIB71-E291C 
                 pCIB71-E355C 
                 pCIB71-E482C 
               
               
                   
               
               
                 6 
                 100% 
                 100% 
                 100% 
                 100% 
               
               
                 8 
                  73% 
                  88% 
                  91% 
                  90% 
               
               
                   
               
            
           
         
       
     
     As shown in Table 7, wild-type CadA (pCIB71) lost 27% of activity at pH 8 compared to pH 6. Surprisingly, the mutant CadA polypeptides (pCIB71-E291C, pCIB71-E355C, and pCIB71-E486C) showed significantly higher activity at pH 8 compared to wild-type CadA polypeptide despite there being no significant difference in activity between the wild-type and mutants at pH 6. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Table of plasmids and strains used in Examples. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Protein(s) 
                   
               
               
                   
                 Host 
                 Overexpressed 
                 Plasmid 
               
               
                   
                   
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA 
                 pCIB71 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291A 
                 pCIB71-E291A 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291C 
                 pCIB71-E291C 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291D 
                 pCIB71-E291D 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291F 
                 pCIB71-E291F 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291G 
                 pCIB71-E291G 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291H 
                 pCIB71-E291H 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291I 
                 pCIB71-E291I 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291K 
                 pCIB71-E291K 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291L 
                 pCIB71-E291L 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291M 
                 pCIB71-E291M 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291N 
                 pCIB71-E291N 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291P 
                 pCIB71-E291P 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291Q 
                 pCIB71-E291Q 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291R 
                 pCIB71-E291R 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291S 
                 pCIB71-E291S 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291T 
                 pCIB71-E291T 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291V 
                 pCIB71-E291V 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291W 
                 pCIB71-E291W 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E291Y 
                 pCIB71-E291Y 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355A 
                 pCIB71-E355A 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355C 
                 pCIB71-E355C 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355D 
                 pCIB71-E355D 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355F 
                 pCIB71-E355F 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355G 
                 pCIB71-E355G 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355H 
                 pCIB71-E355H 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355I 
                 pCIB71-E355I 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355K 
                 pCIB71-E355K 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355L 
                 pCIB71-E355L 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355M 
                 pCIB71-E355M 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355N 
                 pCIB71-E355N 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355P 
                 pCIB71-E355P 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355Q 
                 pCIB71-E355Q 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355R 
                 pCIB71-E355R 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E3555 
                 pCIB71-E3555 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355T 
                 pCIB71-E355T 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355V 
                 pCIB71-E355V 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355W 
                 pCIB71-E355W 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E355Y 
                 pCIB71-E355Y 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482A 
                 pCIB71-E482A 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482C 
                 pCIB71-E482C 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482D 
                 pCIB71-E482D 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482F 
                 pCIB71-E482F 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482G 
                 pCIB71-E482G 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482H 
                 pCIB71-E482H 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482I 
                 pCIB71-E482I 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482K 
                 pCIB71-E482K 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482L 
                 pCIB71-E482L 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482M 
                 pCIB71-E482M 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482N 
                 pCIB71-E482N 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482P 
                 pCIB71-E482P 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482Q 
                 pCIB71-E482Q 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482R 
                 pCIB71-E482R 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482S 
                 pCIB71-E482S 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482T 
                 pCIB71-E482T 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482V 
                 pCIB71-E482V 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482W 
                 pCIB71-E482W 
               
               
                   
                 
                   Hafnia 
                   alvei 
                 
                 CadA E482Y 
                 pCIB71-E482Y 
               
               
                   
                   
               
            
           
         
       
     
                     TABLE 9                  Table of primer sequences used in Examples.                     Name   Sequence (5′-3′)               cadA-F   GGCGAGCTCACACAGGAAACAGACCATGAACGTTATTGCA           ATATTGAATCAC               cadA-R   GGCTCTAGACCACTTCCCTTGTACGAGC               cadA-F2   ATTTCACACAGGAAACAGCTATGAACGTTATTGCAATATT           GAAT               cadA-R2   AGCTGTTTCCTGTGTGAAAT               E291A-F   GCGCGTGAAAAGCACACCAAACGCAACCTGGCC               E291A-R   CGTTTGGTGTGCTTTTCACGCGCTTAGCAATG               E291C-F   AAGCGCGTGAAATGTACACCAAACGCAACCTGGCCGGTAC               E291C-R   GTTGCGTTTGGTGTACATTTCACGCGCTTAGCAATGGTAG               E291D-F   GCGCGTGAAAGATACACCAAACGCAACCTGGCC               E291D-R   CGTTTGGTGTATCTTTCACGCGCTTAGCAATG               E291F-F   GCGCGTGAAATTCACACCAAACGCAACCTGGCC               E291F-R   CGTTTGGTGTGAATTTCACGCGCTTAGCAATG               E291G-F   GCGCGTGAAAGGTACACCAAACGCAACCTGGCC               E291G-R   CGTTTGGTGTACCTTTCACGCGCTTAGCAATG               E291H-F   GCGCGTGAAACATACACCAAACGCAACCTGGCC               E291H-R   CGTTTGGTGTATGTTTCACGCGCTTAGCAATG               E291I-F   GCGCGTGAAAATCACACCAAACGCAACCTGGCC               E291I-R   CGTTTGGTGTGATTTTCACGCGCTTAGCAATG               E291K-F   GCGCGTGAAAAAGACACCAAACGCAACCTGGCC               E291K-R   CGTTTGGTGTCTTTTTCACGCGCTTAGCAATG               E291L-F   GCGCGTGAAACTAACACCAAACGCAACCTGGCC               E291L-R   CGTTTGGTGTTAGTTTCACGCGCTTAGCAATG               E291M-F   GCGCGTGAAAATGACACCAAACGCAACCTGGCC               E291M-R   CGTTTGGTGTCATTTTCACGCGCTTAGCAATG               E291N-F   GCGCGTGAAAAACACACCAAACGCAACCTGGCC               E291N-R   CGTTTGGTGTGTTTTTCACGCGCTTAGCAATG               E291P-F   GCGCGTGAAACCAACACCAAACGCAACCTGGCC               E291P-R   CGTTTGGTGTTGGTTTCACGCGCTTAGCAATG               E291Q-F   GCGCGTGAAACAAACACCAAACGCAACCTGGCC               E291Q-R   CGTTTGGTGTTTGTTTCACGCGCTTAGCAATG               E291R-F   GCGCGTGAAACGTACACCAAACGCAACCTGGCC               E291R-R   CGTTTGGTGTACGTTTCACGCGCTTAGCAATG               E291S-F   GCGCGTGAAATCAACACCAAACGCAACCTGGCC               E291S-R   CGTTTGGTGTTGATTTCACGCGCTTAGCAATG               E291T-F   GCGCGTGAAAACTACACCAAACGCAACCTGGCC               E291T-R   CGTTTGGTGTAGTTTTCACGCGCTTAGCAATG               E291V-F   GCGCGTGAAAGTAACACCAAACGCAACCTGGCC               E291V-R   CGTTTGGTGTTACTTTCACGCGCTTAGCAATG               E291W-F   GCGCGTGAAATGGACACCAAACGCAACCTGGCC               E291W-R   CGTTTGGTGTCCATTTCACGCGCTTAGCAATG               E291Y-F   GCGCGTGAAATACACACCAAACGCAACCTGGCC               E291Y-R   CGTTTGGTGTGTATTTCACGCGCTTAGCAATG               E355A-F   CGGTGGCCGTGTAGCAGGGAAAGTGATTTACGAAACCCAG               E355A-R   AATCACTTTCCCTGCTACACGGCCACCGCTCATACCGCAT               E355C-F   CGGTGGCCGTGTATGTGGGAAAGTGATTTACGAAACCCAG               E355C-R   AATCACTTTCCCACATACACGGCCACCGCTCATACCGCAT               E355D-F   CGGTGGCCGTGTAGACGGGAAAGTGATTTACGAAACCCAG               E355D-R   AATCACTTTCCCGTCTACACGGCCACCGCTCATACCGCAT               E355F-F   CGGTGGCCGTGTATTCGGGAAAGTGATTTACGAAACCCAG               E355F-R   AATCACTTTCCCGAATACACGGCCACCGCTCATACCGCAT               E355G-F   CGGTGGCCGTGTAGGAGGGAAAGTGATTTACGAAACCCAG               E355G-R   AATCACTTTCCCTCCTACACGGCCACCGCTCATACCGCAT               E355H-F   CGGTGGCCGTGTACATGGGAAAGTGATTTACGAAACCCAG               E355H-R   AATCACTTTCCCATGTACACGGCCACCGCTCATACCGCAT               E355I-F   CGGTGGCCGTGTAATCGGGAAAGTGATTTACGAAACCCAG               E355I-R   AATCACTTTCCCGATTACACGGCCACCGCTCATACCGCAT               E355K-F   CGGTGGCCGTGTAAAAGGGAAAGTGATTTACGAAACCCAG               E355K-R   AATCACTTTCCCTTTTACACGGCCACCGCTCATACCGCAT               E355L-F   CGGTGGCCGTGTACTGGGGAAAGTGATTTACGAAACCCAG               E355L-R   AATCACTTTCCCCAGTACACGGCCACCGCTCATACCGCAT               E355M-F   CGGTGGCCGTGTAATGGGGAAAGTGATTTACGAAACCCAG               E355M-R   AATCACTTTCCCCATTACACGGCCACCGCTCATACCGCAT               E355N-F   CGGTGGCCGTGTAAACGGGAAAGTGATTTACGAAACCCAG               E355N-R   AATCACTTTCCCGTTTACACGGCCACCGCTCATACCGCAT               E355P-F   CGGTGGCCGTGTACCAGGGAAAGTGATTTACGAAACCCAG               E355P-R   AATCACTTTCCCTGGTACACGGCCACCGCTCATACCGCAT               E355Q-F   CGGTGGCCGTGTACAAGGGAAAGTGATTTACGAAACCCAG               E355Q-R   AATCACTTTCCCTTGTACACGGCCACCGCTCATACCGCAT               E355R-F   CGGTGGCCGTGTACGTGGGAAAGTGATTTACGAAACCCAG               E355R-R   AATCACTTTCCCACGTACACGGCCACCGCTCATACCGCAT               E355S-F   CGGTGGCCGTGTATCAGGGAAAGTGATTTACGAAACCCAG               E355S-R   AATCACTTTCCCTGATACACGGCCACCGCTCATACCGCAT               E355T-F   CGGTGGCCGTGTAACAGGGAAAGTGATTTACGAAACCCAG               E355T-R   AATCACTTTCCCTGTTACACGGCCACCGCTCATACCGCAT               E355V-F   CGGTGGCCGTGTAGTAGGGAAAGTGATTTACGAAACCCAG               E355V-R   AATCACTTTCCCTACTACACGGCCACCGCTCATACCGCAT               E355W-F   CGGTGGCCGTGTATGGGGGAAAGTGATTTACGAAACCCAG               E355W-R   AATCACTTTCCCCCATACACGGCCACCGCTCATACCGCAT               E355Y-F   CGGTGGCCGTGTATACGGGAAAGTGATTTACGAAACCCAG               E355Y-R   AATCACTTTCCCGTATACACGGCCACCGCTCATACCGCAT               E482A-F   CATCGATAACGCCCACATGTATCTTGACCCGATCAAAGTC               E482A-R   GATACATGTGGGCGTTATCGATGTTTTTGAAGCCGTGCC               E482C-F   CATCGATAACTGCCACATGTATCTTGACCCGATCAAAGTC               E482C-R   GATACATGTGGCAGTTATCGATGTTTTTGAAGCCGTGCC               E482D-F   CATCGATAACGATCACATGTATCTTGACCCGATCAAAGTC               E482D-R   GATACATGTGATCGTTATCGATGTTTTTGAAGCCGTGCC               E482F-F   CATCGATAACTTTCACATGTATCTTGACCCGATCAAAGTC               E482F-R   GATACATGTGAAAGTTATCGATGTTTTTGAAGCCGTGCC               E482G-F   CATCGATAACGGTCACATGTATCTTGACCCGATCAAAGTC               E482G-R   GATACATGTGACCGTTATCGATGTTTTTGAAGCCGTGCC               E482H-F   CATCGATAACCATCACATGTATCTTGACCCGATCAAAGTC               E482H-R   GATACATGTGATGGTTATCGATGTTTTTGAAGCCGTGCC               E482I-F   CATCGATAACATTCACATGTATCTTGACCCGATCAAAGTC               E482I-R   GATACATGTGAATGTTATCGATGTTTTTGAAGCCGTGCC               E482K-F   CATCGATAACAAACACATGTATCTTGACCCGATCAAAGTC               E482K-R   GATACATGTGTTTGTTATCGATGTTTTTGAAGCCGTGCC               E482L-F   CATCGATAACCTGCACATGTATCTTGACCCGATCAAAGTC               E482L-R   GATACATGTGCAGGTTATCGATGTTTTTGAAGCCGTGCC               E482M-F   CATCGATAACATGCACATGTATCTTGACCCGATCAAAGTC               E482M-R   GATACATGTGCATGTTATCGATGTTTTTGAAGCCGTGCC               E482N-F   CATCGATAACAACCACATGTATCTTGACCCGATCAAAGTC               E482N-R   GATACATGTGGTTGTTATCGATGTTTTTGAAGCCGTGCC               E482P-F   CATCGATAACCCGCACATGTATCTTGACCCGATCAAAGTC               E482P-R   GATACATGTGCGGGTTATCGATGTTTTTGAAGCCGTGCC               E482Q-F   CATCGATAACCAGCACATGTATCTTGACCCGATCAAAGTC               E482Q-R   GATACATGTGCTGGTTATCGATGTTTTTGAAGCCGTGCC               E482R-F   CATCGATAACCGTCACATGTATCTTGACCCGATCAAAGTC               E482R-R   GATACATGTGACGGTTATCGATGTTTTTGAAGCCGTGCC               E482S-F   CATCGATAACAGCCACATGTATCTTGACCCGATCAAAGTC               E482S-R   GATACATGTGGCTGTTATCGATGTTTTTGAAGCCGTGCC               E482T-F   CATCGATAACACCCACATGTATCTTGACCCGATCAAAGTC               E482T-R   GATACATGTGGGTGTTATCGATGTTTTTGAAGCCGTGCC               E482V-F   CATCGATAACGTGCACATGTATCTTGACCCGATCAAAGTC               E482V-R   GATACATGTGCACGTTATCGATGTTTTTGAAGCCGTGCC               E482W-F   CATCGATAACTGGCACATGTATCTTGACCCGATCAAAGTC               E482W-R   GATACATGTGCCAGTTATCGATGTTTTTGAAGCCGTGCC               E482Y-F   CATCGATAACTATCACATGTATCTTGACCCGATCAAAGTC               E482Y-R   GATACATGTGATAGTTATCGATGTTTTTGAAGCCGTGCC                    
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 
               
               
                   
               
               
                 GHMGGTAFQKSPVGSLEYDFFGPNTMKSDISISVSELGSLLDHSGPHKEA 
               
               
                   
               
               
                 EQYIARVFNADRSYMVTNGTSTANKIVGMYSAPAGSTILIDRNCHKSLTH 
               
               
                   
               
               
                 LMMMSDVTPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPVH 
               
               
                   
               
               
                 AVITNSTYDGLLYNTDFIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMS 
               
               
                   
               
               
                 GGRVEGKVIYETQSTHKLLAAFSQASMIHVKGDVNEETFNEAYMMHTTTS 
               
               
                   
               
               
                 PHYGIVASTETAAAMMKGNAGKRLINGSIERAIKFRKEIKRLRTESDGWF 
               
               
                   
               
               
                 FDVWQPDHIDTTECWPLRSDSTWHGFKNIDNEHMYLDPIKVTLLTPGMEK 
               
               
                   
               
               
                 DGTMSDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIDKTKALSLL 
               
               
                   
               
               
                 RALTDFKRAFDLNLRVKNMLPSLYREDPEFYENMRIQELAQNIHKLIVHH 
               
               
                   
               
               
                 NLPDLMYRAFEVLPTMVMTPYAAFQKELHGMTEEVYLDEMVGRINANMIL 
               
               
                   
               
               
                 PYPPGVPLVMPGEMITEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQA 
               
               
                   
               
               
                 DGRYTVKVLKEESKK 
               
               
                   
               
               
                 Polypeptide from  Klebsiella  homologous to 
               
               
                   E .  coli  CadA 
               
               
                 SEQ ID NO: 3 
               
               
                 MNVIAIMNHMGVYFKEEPIRELHRALERLDFRIVYPNDRDDLLKLIENNS 
               
               
                   
               
               
                 RLCGVIFDWDKYNLELCEEISKMNEYMPLYAFANTYSTLDVSLNDLRMQV 
               
               
                   
               
               
                 RFFEYALGAAEDIANKIKQNTDEYIDTILPPLTKALFKYVREGKYTFCTP 
               
               
                   
               
               
                 GHMGGTAFQKSPVGSIFYDFFGPNTMKSDISISVSELGSLLDHSGPHKEA 
               
               
                   
               
               
                 EEYIARVFNAERSYMVTNGTSTANKIVGMYSAPAGSTVLIDRNCHKSLTH 
               
               
                   
               
               
                 LMMMSDITPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPVH 
               
               
                   
               
               
                 AVITNSTYDGLLYNTDFIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMS 
               
               
                   
               
               
                 GGRVEGKVIYETQSTHKLLAAFSQASMIHVKGDVNEETFNEAYMMHTTTS 
               
               
                   
               
               
                 PHYGIVASTETAAAMMKGNAGKRLIDGSIERSIKFRKEIKRLKGESDGWF 
               
               
                   
               
               
                 FDVWQPEHIDGPECWPLRSDSAWHGFKNIDNEHMYLDPIKVTLLTPGMKK 
               
               
                   
               
               
                 DGTMDDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIDKTKALSLL 
               
               
                   
               
               
                 RALTDFKRAFDLNLRVKNMLPSLYREDPEFYENMRIQDLAQNIHKLIEHH 
               
               
                   
               
               
                 NLPDLMFRAFEVLPSMVMTPYAAFQKELHGQTEEVYLEEMVGRVNANMIL 
               
               
                   
               
               
                 PYPPGVPLVMPGEMITEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQA 
               
               
                   
               
               
                 DGRYTVKVLKEENNK 
               
               
                   
               
               
                 Polypeptide from  Enterobacteriaceae  homologous 
               
               
                 to  E .  coli  CadA 
               
               
                 SEQ ID NO: 4 
               
               
                 MNVIAIMNHMGVYFKEEPIRELHRALERLDFRIVYPNDRDDLLKLIENNS 
               
               
                   
               
               
                 RLCGVIFDWDKYNLELCEEISKMNEYMPLYAFANTYSTLDVSLNDLRMQV 
               
               
                   
               
               
                 RFFEYALGAAEDIANKIKQNTDEYIDTILPPLTKALFKYVREGKYTFCTP 
               
               
                   
               
               
                 GHMGGTAFQKSPVGSIFYDFFGSNTMKSDISISVSELGSLLDHSGPHKEA 
               
               
                   
               
               
                 EEYIARVFNAERSYMVTNGTSTANKIVGMYSAPAGSTVLIDRNCHKSLTH 
               
               
                   
               
               
                 LMMMSDITPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPVH 
               
               
                   
               
               
                 AVITNSTYDGLLYNTDFIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMS 
               
               
                   
               
               
                 GGRVEGKVIYETQSTHKLLAAFSQASMIHVKGDVNEETFNEAYMMHTTTS 
               
               
                   
               
               
                 PHYGIVASTETAAAMMKGNAGKRLIDGSIERSIKFRKEIKRLKGESDGWF 
               
               
                   
               
               
                 FDVWQPEHIDGPECWPLRSDSAWHGFKNIDNEHMYLDPIKVTLLTPGMKK 
               
               
                   
               
               
                 DGTMDDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIDKTKALSLL 
               
               
                   
               
               
                 RALTDFKRAFDLNLRVKNMLPSLYREDPEFYENMRIQDLAQNIHKLIEHH 
               
               
                   
               
               
                 NLPDLMFRAFEVLPSMVMTPYAAFQKELHGQTEEVYLEEMVGRVNANMIL 
               
               
                   
               
               
                 PYPPGVPLVMPGEMITEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQA 
               
               
                   
               
               
                 DGRYTVKVLKEENNK 
               
               
                   
               
               
                 Polypeptide from  Salmonella enterica  homologous 
               
               
                 to  E .  coli  CadA 
               
               
                 SEQ ID NO: 5 
               
               
                 MNVIAIMNHMGVYFKEEPIRELHRALEGLNFRIVYPNDREDLLKLIENNS 
               
               
                   
               
               
                 RLCGVIFDWDKYNLELCEEISKLNEYMPLYAFANSYSTLDVSLNDLRMQV 
               
               
                   
               
               
                 RFFEYALGAATDIAAKIRQNTDEYIDNILPPLTKALFKYVREGKYTFCTP 
               
               
                   
               
               
                 GHMGGTAFQKSPVGSIFYDFFGPNTMKSDISISVSELGSLLDHSGPHKEA 
               
               
                   
               
               
                 EEYIARVFNAERSYMVTNGTSTANKIVGMYSAPAGSTVLIDRNCHKSLTH 
               
               
                   
               
               
                 LMMMSDITPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPVH 
               
               
                   
               
               
                 AVITNSTYDGLLYNTDYIKKTLDVKSIHFDSAWVPYTNFSPIYQGKCGMS 
               
               
                   
               
               
                 GDRVEGKIIYETQSTHKLLAAFSQASMIHVKGDINEETFNEAYMMHTTTS 
               
               
                   
               
               
                 PHYGIVASTETAAAMMKGNAGKRLINGSIERAIKFRKEIKRLKSESDGWF 
               
               
                   
               
               
                 FDVWQPEHIDGAECWPLRSDSAWHGFKNIDNEHMYLDPIKVTILTPGMKK 
               
               
                   
               
               
                 DGTMDEFGIPASLVAKYLDERGIIVEKTGPYNLLFLFSIGIDKTKALSLL 
               
               
                   
               
               
                 RALTEFKRAFDLNLRVKNILPALYREAPEFYENMRIQELAQNIHKLVEHH 
               
               
                   
               
               
                 NLPDLMYRAFEVLPKMVMTPYTAFQKELHGETEEVYLEEMVGRVNANMIL 
               
               
                   
               
               
                 PYPPGVPLVMPGEMITEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQA 
               
               
                   
               
               
                 DGRYTVKVLKENTK