阅读说明：本技术 减少为了生产氨基酸或氨基酸衍生产物的亚胺/烯胺的积累 (Reduction of imine/enamine accumulation to produce amino acids or amino acid derivative products ) 是由 周豪宏 陈玲 刘修才 于 2017-11-29 设计创作，主要内容包括：提供了微生物,该微生物被遗传修饰以过表达亚胺/烯胺脱氨酶以增强该微生物的赖氨酸和赖氨酸衍生物的生产。还提供了产生这种微生物的方法,以及使用该经遗传修饰的微生物生产赖氨酸和赖氨酸衍生物的方法。(Microorganisms are provided that are genetically modified to overexpress imine/enamine deaminases to enhance the production of lysine and lysine derivatives by the microorganism. Also provided are methods of producing such microorganisms, and methods of producing lysine and lysine derivatives using the genetically modified microorganisms.)

1. A genetically modified host cell comprising an exogenous polynucleotide comprising a nucleic acid encoding an imine/enamine deaminase polypeptide, which genetically modified host cell has an increased amount of an amino acid or amino acid derivative relative to a corresponding host cell not modified to express the exogenous polynucleotide; and the genetically modified host cell has at least one further genetic modification that increases the production of the amino acid or the amino acid derivative compared to a wild-type host cell.

2. The genetically modified host cell of claim 1, wherein the amino acid is lysine and the amino acid derivative is cadaverine.

3. The genetically modified host cell of claim 1 or 2, wherein the imine/enamine deaminase polypeptide is a YoaB polypeptide.

4. The genetically modified host cell of claim 1 or 2, wherein the imine/enamine deaminase polypeptide has at least 80% identity, or at least 90% identity, to the amino acid sequence of SEQ ID No. 10.

5. The genetically modified host cell of claim 1 or 2, wherein the imine/enamine deaminase polypeptide is a YjgH polypeptide.

6. The genetically modified host cell of claim 1 or 2, wherein the imine/enamine deaminase polypeptide has at least 80% identity, or at least 90% identity, to the amino acid sequence of SEQ ID No. 12.

7. The genetically modified host cell of claim 1 or 2, wherein the imine/enamine deaminase polypeptide comprises the amino acid sequence of SEQ ID NO 10 or SEQ ID NO 12.

8. The genetically modified host cell of claim 1 or 2, wherein the imine/enamine deaminase polypeptide is heterologous to the host cell.

9. The genetically modified host cell of claim 1 or 2, wherein the imine/enamine deaminase polypeptide comprises an amino acid sequence that is native to the host cell.

10. The genetically modified host cell of any one of claims 1-9, wherein the exogenous polynucleotide is comprised in an expression vector introduced into the cell, wherein the expression vector comprises the exogenous polynucleotide operably linked to a promoter.

11. The genetically modified host cell of any one of claims 1-9, wherein the exogenous polynucleotide is integrated into the host chromosome.

12. The genetically modified host cell of any one of claims 1-11, wherein the host cell overexpresses an exogenous lysine decarboxylase polypeptide.

13. The genetically modified host cell of claim 12, wherein the exogenous lysine decarboxylase polypeptide is a CadA polypeptide.

14. The genetically modified host cell of any one of claims 1-13, wherein the host cell overexpresses one or more exogenous lysine biosynthetic polypeptides.

15. The genetically modified host cell of claim 14, wherein the exogenous lysine biosynthetic polypeptide is an aspartokinase, a dihydrodipicolinate synthase, a diaminopimelate decarboxylase, an aspartate semialdehyde dehydrogenase, a dihydropicolinate reductase, or an aspartate aminotransferase.

16. The genetically modified host cell of claim 15, wherein the aspartokinase, dihydrodipicolinate synthase, diaminopimelate decarboxylase, aspartate semialdehyde dehydrogenase, dihydropicolinate reductase, or aspartate aminotransferase is an L ysC, DapA, L ysA, Asd, DapB, or AspC polypeptide.

17. The genetically modified host cell of any one of claims 1-11, wherein the host cell overexpresses exogenous CadA, L ysC, DapA, L ysA, Asd, DapB, and AspC polypeptides.

18. The genetically modified host cell according to any one of claims 1-17, wherein the host cell belongs to the genus Escherichia (Hafnia), Hafnia (Hafnia), or Corynebacterium (Corynebacterium).

19. The genetically modified host cell according to claim 18, wherein the host cell is Escherichia coli (Escherichia coli), Hafnia alvei (Hafnia alvei), or corynebacterium glutamicum (corynebacterium glutamicum).

20. The genetically modified host cell of claim 19, wherein the host cell is e.

21. A method of engineering a host cell to increase production of an amino acid or an amino acid derivative, the method comprising introducing into the host cell a polynucleotide comprising a nucleic acid encoding an imine/enamine deaminase polypeptide, wherein the host cell has at least one additional genetic modification that increases production of the amino acid or the amino acid derivative compared to a wild-type host cell;

culturing said host cell under conditions that express said imine/enamine deaminase polypeptide, and

selecting a host cell that produces an increased amount of an amino acid or amino acid derivative relative to a corresponding host cell that has not been modified to introduce the polynucleotide encoding the imine/enamine deaminase polypeptide.

22. The method of claim 21, wherein the amino acid is lysine and the amino acid derivative is cadaverine.

23. The method of claim 21 or 22, wherein the imine/enamine deaminase polypeptide is a YoaB polypeptide.

24. The method of claim 21 or 22, wherein the imine/enamine deaminase polypeptide has at least 80% identity, or at least 90% identity, to an amino acid sequence of SEQ ID No. 10.

25. The method of claim 21 or 22, wherein the imine/enamine deaminase polypeptide is a YjgH polypeptide.

26. The method of claim 21 or 22, wherein the imine/enamine deaminase polypeptide has at least 80% identity, or at least 90% identity, to the amino acid sequence of SEQ ID NO 12.

27. The method of claim 21 or 22, wherein the imine/enamine deaminase polypeptide comprises an amino acid sequence of SEQ ID NO 10 or SEQ ID NO 12.

28. The method of any one of claims 21-27, wherein the imine/enamine deaminase polypeptide is heterologous to the host cell.

29. The method of any one of claims 21-27, wherein the imine/enamine deaminase polypeptide comprises an amino acid sequence that is native to the host cell.

30. The method of any one of claims 21-29, wherein the polynucleotide is comprised in an expression vector introduced into the cell, wherein the expression vector comprises the polynucleotide operably linked to a promoter.

31. The method of any one of claims 21-29, wherein the polynucleotide is integrated into the host chromosome.

32. The method of any one of claims 21-31, wherein the host cell is genetically modified to overexpress an exogenous lysine decarboxylase polypeptide.

33. The method of claim 32, wherein the lysine decarboxylase polypeptide is a CadA polypeptide.

34. The method of any one of claims 21-33, wherein the host cell is genetically modified to overexpress one or more exogenous lysine biosynthetic polypeptides.

35. The method of claim 34, wherein the lysine biosynthetic polypeptide is an aspartokinase, a dihydrodipicolinate synthase, a diaminopimelate decarboxylase, an aspartate semialdehyde dehydrogenase, a dihydropicolinate reductase, or an aspartate aminotransferase.

36. The method of claim 34, wherein the lysine biosynthetic polypeptide is an L ysC, DapA, L ysA, Asd, DapB, or AspC polypeptide.

37. The method of any one of claims 21-31, wherein the host cell is genetically modified to overexpress exogenous CadA, L ysC, DapA, L ysA, Asd, DapB, and AspC polypeptides.

38. The method of any one of claims 21-37, wherein the host cell is of the genus escherichia, hafnia, or corynebacterium.

39. The method of claim 38, wherein the host cell is escherichia coli, hafnia alvei, or corynebacterium glutamicum.

40. The method of claim 39, wherein the host cell is E.

41. A host cell produced by the method of any one of claims 21-40.

42. A method of producing an amino acid or amino acid derivative in increased amounts, the method comprising culturing the host cell of any one of claims 1-20 and 41 under conditions in which an imine/enamine deaminase polypeptide is expressed.

43. The method of claim 42, further comprising isolating the amino acid or amino acid derivative.

44. The method of claim 42 or 43, wherein the amino acid is lysine and the amino acid derivative is cadaverine.

Background

Overproduction of amino acids (such as lysine) and amino acid-derived products (such as cadaverine) often requires remodeling of the metabolism of the host cell to increase the flux of carbon-and nitrogen-containing compounds to the desired product. However, altering the flux of a metabolic pathway can result in the accumulation of intermediates that do not normally accumulate within the cell. Such metabolic intermediates may be the end products of an enzymatic reaction or intermediates of an enzymatic reaction that leak from the catalytic site of the enzyme. The accumulated metabolic intermediates may also be toxic to the cell, or induce activity in other pathways that convert the intermediates into compounds toxic to the cell (Danchi, microbiological Biotechnology 10:57-72,2017).

Imine/enamine intermediates are often formed during transamination, racemization, or deamination reactions that result in the formation of reactive amino acid derivatives (e.g., amino acrylates or iminopropionic acids.) imine/enamine formation sometimes involves the cofactor pyridoxal phosphate (P L P.) reactive imines/enamines are known to cause cellular damage and may accumulate intracellularly during overproduction of amino acids (such as lysine) and amino acid derivative products (such as cadaverine).

Overproduction of lysine or cadaverine involves overexpression of genes encoding one or more of dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52), diaminopimelate dehydrogenase (DAPDH, EC 1.4.1.16) and diaminopimelate decarboxylase (DAPDC, EC 4.1.1.20) (Anastasiadis, Recent Patents on Biotechnology 1:11-24,2007). DHDPS catalyzes the condensation of pyruvate and aspartate semialdehyde to form 4-hydroxy-2, 3,4, 5-tetrahydro-L-pyridinedicarboxylic acid, which involves the formation of imine intermediates (Dobson et al, protein science 17: 2080-.

The conversion of aspartate to lysine, threonine or methionine is catalysed by three different aspartate kinases, one kinase corresponding to one amino acid (L ysC, Met L, ThrA) however, increasing the flux of aspartate biosynthesis to increase lysine or cadaverine production will also increase threonine production as they share a common precursor the accumulation of threonine in the cell can trigger the activity of threonine dehydratase (EC4.3.1.19), the first enzyme involved in threonine catabolism to isoleucine threonine dehydratase, a P L P dependent enzyme that catalyses the dehydration of threonine to aminocrotonate (an enamine intermediate), aminocrotonate can tautomerize to an iminobutyrate (an imine intermediate), thus accumulation of threonine in the cell can increase the intracellular accumulation of these toxic enamine/imine intermediates.

In certain instances, flux through the threonine biosynthetic pathway is reduced or eliminated to increase the flux of carbon-containing and nitrogen-containing compounds toward lysine and cadaverine biosynthesis. However, threonine needs to be added to the medium to ensure that the intracellular concentration of threonine is sufficient for cell growth. The addition of an external threonine can result in the addition of sufficient threonine that the amino acids accumulate within the cell, in which case the accumulation of aminocrotonate and iminobutyrate can occur as described above.

The conversion of lysine to cadaverine involves the P L P dependent enzyme lysine decarboxylase therefore, overproduction of cadaverine involves increasing the intracellular concentration of P L P, which can be achieved by either addition of P L P to the culture medium or overexpression of a gene involved in P L P synthesis (e.g., pdxST). As noted above, some of the reactions leading to imine/enamine accumulation are P L P catalyzed reactions.

Salmonella enterica (Salmonella enterica) was found to produce a protein RidA (yjgf) with imine/enamine deaminase activity allowing it to catalyze the release of ammonia and the production of a more stable and less toxic intermediate from the imine/enamine compound (L ambrecht et al, j.biol.chem.287:3454-3461,2012) RidA protects Salmonella enterica from harmful imine/enamine molecules formed by the activity of P L P-dependent threonine dehydratase (IlvA) by catalyzing the removal of ammonia from the intermediate enamine/imine compound to form non-toxic 2-ketobutyric acid the activity of RidA also shows protection of cells from 2-amino acrylates (enamines formed during serine catabolism) (L ambrecht et al, mBio 4:1-8,2013) the accumulation of imine/enamines also inactivates P L catalytic enzymes in cells, thus the removal of enamines is important.

Disclosure of Invention

Provided herein are host cells that are genetically modified to enhance removal of imine and enamine compounds and thereby increase production of an amino acid or amino acid derivative of which imine/enamine is an intermediate relative to a host cell of the same strain that does not have the genetic modification to increase imine and/or enamine removal. Also provided herein are methods of producing such host cells; and methods of using the host cells to produce increased yields of amino acids or amino acid derivatives such as lysine or cadaverine.

Thus, in one aspect, provided herein is a method of engineering a host cell to increase production of an amino acid or amino acid derivative (e.g., lysine or cadaverine) comprising introducing into a host cell a polynucleotide (e.g., a heterologous polynucleotide) comprising a nucleic acid encoding an imine/enamine deaminase polypeptide, wherein the host cell has at least one additional genetic modification that increases production of an amino acid or amino acid derivative as compared to a wild-type host cell, culturing the host cell under conditions in which the imine/enamine deaminase polypeptide is expressed, and selecting a host cell that produces an increased amount of an amino acid or amino acid derivative (e.g., lysine or cadaverine) as compared to a corresponding host cell of the same strain that is not modified to express the polynucleotide encoding the imine/enamine deaminase polypeptide, wherein the imine/enamine deaminase polypeptide is a Yoqa polypeptide, in some embodiments, the imine/enamine deaminase polypeptide is a Yodoa polypeptide, wherein the imine/enamine deaminase polypeptide has at least 70% amino acid sequence identity to SEQ ID NO:10, wherein the amino acid sequence is at least one of a Corynebacterium deaminase polypeptide, wherein the amino acid sequence is introduced into a host cell, wherein the Corynebacterium deaminase polypeptide, wherein the Escherichia/deaminase polypeptide has at least one of the amino acid sequence of the Corynebacterium deaminase/deaminase polypeptide, wherein the Corynebacterium deaminase/deaminase polypeptide, wherein the Escherichia/deaminase polypeptide has at least some embodiments of the Corynebacterium deaminase/deaminase polypeptide, wherein the Corynebacterium deaminase polypeptide has at least one of the Corynebacterium deaminase/deaminase polypeptide, wherein the Corynebacterium deaminase polypeptide, wherein the Escherichia/deaminase polypeptide has at least one of the Corynebacterium strain of the Corynebacterium or Corynebacterium strain of the Corynebacterium strain of.

In another aspect, provided herein is a genetically modified host cell produced according to the method of the preceding paragraph.

In another aspect, provided herein are genetically modified host cells comprising a polynucleotide (e.g., a heterologous polynucleotide) comprising a nucleic acid encoding an imine/enamine deaminase polypeptide, which modified host cells have increased amounts of amino acids (e.g., lysine) or amino acid derivatives (e.g., cadaverine) as compared to corresponding host cells not modified to express the polynucleotide encoding the imine/enamine polypeptide, and have at least one additional genetic modification that increases production of the amino acids or amino acid derivatives as compared to wild-type host cells, in some embodiments the imine/enamine deaminase polypeptide is a YoaB polypeptide, in some embodiments the imine/enamine deaminase polypeptide has at least 70% amino acid sequence identity to SEQ ID NO:10, in some embodiments the imine/enamine deaminase polypeptide has at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO:10, in some embodiments the amino acid sequence of a deaminase polypeptide is a polynucleotide, in which DNA sequence is operably linked to a polynucleotide sequence of a deaminase polypeptide, in certain embodiments, wherein the amino acid sequence of a deaminase polypeptide has at least one of the amino acid sequence of AsgZaleneamine/enamine polypeptide, wherein the amino acid sequence of the amino acid/enamine polypeptide, or of the amino acid sequence of the amino acid/enamine polypeptide is introduced into a polypeptide.

In another aspect, provided herein is a method of producing an amino acid or amino acid derivative (e.g., lysine or cadaverine) comprising culturing a host cell as described in the preceding two paragraphs under conditions in which the imine/enamine deaminase polypeptide is expressed. In some embodiments, the method further comprises isolating the amino acid or amino acid derivative, e.g., lysine or cadaverine.

Drawings

Figure 1 provides an exemplary alignment of RidA homologous protein sequences (identified by PDB accession numbers).

FIG. 2 provides an exemplary alignment of E.coli RidA and paralog protein sequences. Positions corresponding to E120, C107, V18, K73, and E122 of RidA are underlined. The amino acids at positions D76 and K123 of YjgH (corresponding to positions K73 and E122 of RidA, respectively) are conserved in the YjgH and YoaB sequences and are shown in bold enlarged font.

Detailed Description

Term(s) for

As used in the context of the present disclosure, an "imine/enamine deaminase polypeptide" refers to an enzyme that reduces imine/enamine levels in a host cell. This polypeptide catalyzes the release of ammonia from imines/enamines. Polypeptides that reduce imine/enamine levels according to the disclosure typically reduce levels by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more when produced by a host cell genetically modified to overexpress the imine/enamine deaminase polypeptide compared to a wild-type corresponding host cell that has not been genetically modified to overexpress the imine/enamine deaminase polypeptide.

The term "imine/enamine deaminase polypeptide" encompasses biologically active variants, alleles, mutants, and interspecies homologs of the particular polypeptides described herein. Nucleic acids encoding imine/enamine deaminase polypeptides refer to genes, mRNA precursors, mrnas, and the like, including nucleic acids encoding variants, alleles, mutants, and interspecies homologs of the particular amino acid sequences described herein.

The terms "increased expression" and "overexpression" of an imine/enamine deaminase polypeptide are used interchangeably herein and refer to an increase in the amount of an imine/enamine deaminase polypeptide in a genetically modified cell (e.g., a cell into which an expression construct encoding an imine/enamine deaminase polypeptide has been introduced) as compared to the amount of an imine/enamine deaminase polypeptide in a corresponding cell that does not have the genetic modification (i.e., a cell of the same strain that does not have the modification). The increased expression level for the purposes of the present application is at least 5%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to the corresponding unmodified cell. The unmodified cells do not need to express the imine/enamine deaminase. Thus, the term "over-expression" also includes embodiments in which the imine/enamine deaminase polypeptide is expressed in a host cell that does not naturally express the imine/enamine deaminase polypeptide. Increased expression of the imine/enamine deaminase polypeptide can be assessed by any assay, including but not limited to measuring the level of RNA transcribed from the imine/enamine deaminase gene, the level of the imine/enamine deaminase polypeptide, and/or the level of imine/enamine deaminase polypeptide activity.

In the context of producing an amino acid (e.g., lysine) or an amino acid derivative (e.g., a lysine derivative, such as cadaverine), the term "increase," as used herein, refers to an increase in production of an amino acid (e.g., lysine) or derivative by a genetically modified host cell as compared to a control corresponding cell (such as a cell of a wild-type strain, or a cell of the same strain that does not have the genetic modification that increases production of the amino acid or amino acid derivative). The production of the amino acid or derivative thereof is increased by at least 5%, typically at least 0%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to control cells.

The term "reference.. numbering" or "corresponding to" or "reference.. determining," when used in the context of numbering a given amino acid or polynucleotide sequence, refers to the numbering of the residues that specify the reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. For example, a residue in a variant or homologue of a YoaB polypeptide "corresponds to" the amino acid at position in SEQ ID NO 10 when the residue is aligned with the amino acid in a comparison of SEQ ID NO 10 and the homologue or variant in the largest alignment. Similarly, a residue in a variant YjgH polypeptide "corresponds to" the amino acid at position in SEQ ID NO:12 when the residue is aligned with the amino acid in a comparison of SEQ ID NO:12 and the homologue or variant with the largest alignment.

The terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to a single-or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5 'end to the 3' end. Nucleic acids used in the invention typically contain phosphodiester linkages, although in some cases nucleic acid analogs can be used that can have alternative backbones including, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphide linkages (see Eckstein, oligonucleotides and antigens: A Practical Approach, Oxford University Press); a positive backbone; a non-ionic backbone and a non-ribose backbone. The nucleic acid or polynucleotide may also include modified nucleotides that allow for proper read-through by a polymerase. "Polynucleotide sequence" or "nucleic acid sequence" includes both the sense and antisense strands of a nucleic acid as single strands or as double strands. As will be understood by those skilled in the art, the description 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, genomic DNA and cDNA, RNA or hybrids, wherein the nucleic acid may contain a combination of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, and the like. Unless otherwise indicated, nucleic acid sequences are presented in a 5 'to 3' orientation.

The term "substantially identical" as used in the context of two nucleic acids or polypeptides refers to a sequence that has at least 40%, 45%, or 50% sequence identity to a reference sequence the 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% as compared to the reference sequence using the procedures described herein, preferably using B L AST as the standard parameter, as described below.

Two nucleic acid sequences or polypeptide sequences are considered "identical" if their sequences of nucleotides or amino acid residues, respectively, are identical when aligned for maximum correspondence as described below. The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage sequence identity is used with reference to a protein or peptide, it will be appreciated that residue positions that are not identical typically differ by conservative amino acid substitutions, wherein an amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity) and thus do not alter the functional properties of the molecule. If the sequences differ by conservative substitutions, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Means for such adjustment are known to those skilled in the art. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, if the same amino acid is scored as 1 and a non-conservative substitution is scored as 0, the score for a conservative substitution is between 0 and 1.

For sequence comparison, one sequence is typically used as a reference sequence to which it is compared to a test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters may also be used, or alternative parameters may be set. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

An algorithm that can be used to determine whether an imine/enamine deaminase polypeptide has sequence identity to SEQ ID NO:10 or 12 or another polypeptide reference sequence is the B L AST algorithm, described in Altschul et al, 1990, J.mol.biol.215: 403. multidot.410, incorporated herein by reference the software for performing the B L AST analysis is publicly available at the national center for Biotechnology information (on the world Wide Web ncbi.nlm.nih.gov.). for amino acid sequences, the B L ASTP program uses default values for the word length (W) of 3, the expected value (E) of 10, and the B L OSUM62 scoring matrix (see Henikoff & Henikoff,1989, Proc.Natl.Acad.Sci.USA 89: 10915.) other programs that can be used include the Needman-Wunsch program (J.MoI.biol.48: Acad.Sci.USA 89:10915) and the initial penalty of No. 2, the gap extension of 3975: 3402, the initial penalty of 3975: 1997, 3975. multidot.10. multidot.3, and the initial penalty of the extension of the sequence No. 10, 1997, 3973, 443, 2, respectively, the origin of the sequence No. 10, 1997, 3973, 3975, 3, the origin, 2, the origin, the.

As used herein, a "comparison window" includes reference to any one number of segments of contiguous positions selected from the Group consisting of 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein a sequence may be compared to a reference sequence of the same number of contiguous positions after optimal alignment of the two sequences.

Nucleic acid or protein sequences that are substantially identical to a reference sequence include "conservatively modified variants". Conservatively modified variants, with respect to a particular nucleic acid sequence, refers to those nucleic acids that encode identical or substantially identical amino acid sequences, or substantially identical sequences when the nucleic acid does not encode an amino acid sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where an alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are a class of conservatively modified variations. Each nucleic acid sequence herein encoding a polypeptide also describes any possible silent variation of the nucleic acid. The skilled artisan will recognize that each codon in a nucleic acid (except AUG, which is typically the only codon for methionine) can be modified to produce a functionally identical molecule. Thus, each silent variation of a nucleic acid encoding a polypeptide is encompassed by each such sequence.

With respect to amino acid sequences, the skilled artisan will recognize that when an alteration results in the substitution of an amino acid by a chemically similar amino acid, each substitution in a nucleic acid, peptide, polypeptide or protein sequence (altering individual amino acids or a small portion of amino acids in the encoded sequence) is a "conservatively modified variation". conservative substitutions for providing functionally similar amino acids are well known in the art examples of amino acid groups defined in this manner may include a "charged/polar group" comprising Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), L ys (lysine or K), Arg (arginine or R) and His (histidine or H), "aromatic or cyclic group" comprising Pro (proline or P), Phe (phenylalanine or F), Tyr (tyrosine or Y) and Trp (tryptophan or W), "and" aliphatic group "comprising Gly or G), Ala (alanine or A), Val (valine or V), L eI (leucine or L), methionine or Thr (serine or serine) and vice versa, such that substitutions may be made within the" 12 ", and optionally" serine or serine, such as a "may be maintained within a" group ", or serine, such that the amino acid sequence may be maintained as a" or serine, such that the amino acid sequence may be maintained within a "may be maintained as a" or serine, such as a "may be maintained as a" or a "which may be maintained as a", or a "which may be maintained as a" or a ", or a" which may be maintained as a "or a" or "which may be maintained as a" or "which may be maintained as a" which may include a "which may be maintained as a" or a "which may be maintained as a plurality of a", or a "which may be maintained as a plurality of a", or a.

The term "promoter" as used herein refers to a polynucleotide sequence capable of driving transcription of a DNA sequence in a cell. Thus, promoters useful in the polynucleotide constructs of the present invention include cis-and trans-acting transcriptional control elements and regulatory sequences involved in controlling or regulating the timing and/or rate of gene transcription. For example, a promoter may be a cis-acting transcriptional control element, including enhancers, repressor binding sequences, and the like. These cis-acting sequences typically interact with proteins or other biomolecules to effect (turn on/off, regulate, modulate, etc.) gene transcription. Most often, the core promoter sequence is located within 1-2kb of the translation initiation site, more often within 1kbp of the translation initiation site and often within 500bp or 200bp or less. Conventionally, a promoter sequence is typically provided as a sequence on the coding strand of the gene it controls. In the context of the present application, a promoter is generally referred to by the name of the gene whose expression is naturally regulated. The promoter used in the expression construct of the present invention is referred to by the name of the gene. Reference by name to a "promoter" includes wild-type native promoters as well as variants of promoters that retain the ability to induce expression. The mention of promoters by name is not limited to a particular species, but also encompasses promoters from corresponding genes in other species.

A "constitutive promoter" in the context of the present invention refers to a promoter capable of initiating transcription under most conditions of the cell (e.g.in the absence of an inducing molecule). An "inducible promoter" initiates transcription in the presence of an inducer molecule.

As used herein, a polynucleotide is "heterologous" to an organism or second polynucleotide sequence if it is derived from a foreign species, or if from the same species, it 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, this means that the polynucleotide coding sequence encoding the polypeptide is derived from one species, while the promoter sequence is derived from a different species; alternatively, 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, such as a different gene from the same species, or an allele from a different species). Similarly, if the native wild-type host cell does not produce the polypeptide, the polypeptide is heterologous to the host cell.

The term "exogenous" as used herein generally refers to a polynucleotide sequence or polypeptide that is introduced into a host cell by molecular biological techniques to produce a recombinant cell. Examples of "exogenous" polynucleotides include vectors, plasmids, and/or artificial nucleic acid constructs encoding the desired proteins. An "exogenous" polypeptide expressed in a host cell may occur naturally in the wild-type host cell or may be heterologous to the host cell. The term also encompasses progeny of the original host cell that have been engineered to express the exogenous polynucleotide or polypeptide sequence, i.e., the host cell expressing the "exogenous" polynucleotide may be the original genetically modified host cell or a progeny cell comprising the genetic modification.

The term "endogenous" refers to a naturally occurring polynucleotide sequence or polypeptide that can be found in a given wild-type cell or organism. In this regard, it should also be noted that even though an organism may contain endogenous copies of a given polynucleotide sequence or gene, introduction of an expression construct or vector encoding such sequence, such as to overexpress or otherwise regulate the expression of the encoded protein, represents an "exogenous" copy of the gene or polynucleotide sequence. Any pathway, gene, or enzyme described herein may utilize or rely on an "endogenous" sequence, or both, that may be provided as one or more "exogenous" polynucleotide sequences.

As used herein, "recombinant nucleic acid" or "recombinant polynucleotide" refers to a polymer of nucleic acids in which at least one of the following is authentic: (a) the nucleic acid sequence is foreign to a given host cell (i.e., not naturally found in the given host cell); (b) the sequence may be found naturally in a given host cell, but in an unnatural (e.g., greater than expected) amount; or (c) the nucleic acid sequence comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, in relation to case (c), the recombinant nucleic acid sequence will have sequences from two or more unrelated genes arranged to produce a novel functional nucleic acid.

The term "operably linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) fragments. Generally, it refers to the functional relationship of the transcriptional regulatory sequences to the sequences being transcribed. For example, a promoter or enhancer sequence is operably linked to a DNA or RNA sequence if it stimulates or regulates the transcription of the DNA or RNA sequence in an appropriate host cell or other expression system. Typically, promoter transcriptional regulatory sequences operably linked to a transcribed sequence are physically contiguous with the transcribed sequence, i.e., they are cis-acting. However, certain transcriptional regulatory sequences (such as enhancers) need not be physically contiguous or located in close proximity to the coding sequence 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 expressing a transgene, one skilled in the art will recognize that the inserted polynucleotide sequence need not be identical to the sequence of the gene from which it is derived, but may be only substantially identical to the sequence of the gene from which it is derived. As explained herein, these substantially identical variants are specifically encompassed by reference to a particular nucleic acid sequence. One example of an expression cassette is a polynucleotide construct comprising a polynucleotide sequence encoding a polypeptide for use in the present invention operably linked to a promoter (e.g., its native promoter), wherein the expression cassette is introduced into a heterologous microorganism. In some embodiments, the expression cassette comprises a polynucleotide sequence encoding a polypeptide of the invention, wherein the polynucleotide is targeted to a location in the genome of the microorganism such that expression of the polynucleotide sequence is driven by a promoter present in the microorganism.

The term "host cell" as used in the context of the present invention refers to a microorganism and includes individual cells or cell cultures which may be or have been the recipient of any recombinant vector or isolated polynucleotide of the present invention. Host cells include progeny of a single host cell, and the progeny may not necessarily be identical (in morphology or in total DNA complementarity) to the original parent cell due to natural, random, or deliberate mutation and/or variation. Host cells include cells into which a recombinant vector or polynucleotide of the invention has been introduced, including by transformation, transfection, and the like.

The term "isolated" means substantially or essentially free of components with which it normally occurs in its native state. For example, an "isolated polynucleotide" as used herein may refer to a polynucleotide that has been separated 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 DNA fragment, such as by cloning into a vector. A polynucleotide is considered isolated if, for example, it is cloned into a vector that is not part of its natural environment, or if it is artificially introduced into the genome of a cell in a manner that differs from the state in which it naturally occurs. Alternatively, "isolated peptide" or "isolated polypeptide" and the like as used herein refers to a polypeptide molecule that is free of other components of the cell, i.e., it is not associated with an in vivo substance.

Aspects of the invention

The present disclosure is based in part on the following findings: increased expression of one or more imine/enamine deaminase polypeptides in a microorganism, such as a gram-negative bacterium, increases the production of an amino acid (e.g., lysine) and/or the production of an amino acid derivative of lysine, such as cadaverine.

RidA is a member of the YjgF/YER057c/UK114 family, which is conserved in all life domains (pfam: PF 01042). members of this family are small proteins of about 15kDa and form homotrimers-trimeric barrel quaternary structures. members of this family have different phenotypes and do not have well-defined biological effects, such as most other well-defined protein families with defined substrates and products (e.g. p450 monooxygenase, DNA polymerase or lysine decarboxylase). recently, the crystal structure of RidA from arabidopsis thaliana (arapyridopsis thaliana) has been published (PBD ID:5HP7) (L u et al, Scientific Reports 6:30494,2016). the crystal structures of other members of this family have also been disclosed (PBDID: 1QD 64 Bacillus subtilis) YabJ, the first cut of sulfolobus (sulculis), the crystal structures of the first cut of sulfolobus (suis) have been shown in the sequence of several of the amino acid residues of these proteins in the codon sequence of the strain tgafotkoku @ 25, the amino acid sequences of these three amino acid residues of the heavy proteins are indicated by the amino acid sequences of the pddfa qf 1, 3, n, 3 h.

Coli also expresses the gene encoding RidA. Coli RidA has been shown to be important in thiamine synthesis (Bazurto et al, mBio 7:1-9,2016) and may also function as a chaperone protein during oxidative stress (Muller et al, Nature Communications 5:1-14,2014). Overexpression of enzymes to increase metabolic flux towards lysine production is expected to produce metabolic burden and stress on the cells; thus, it is expected that over-expression of RidA will help to remove toxic intermediates formed due to metabolic stress and increase lysine production. Surprisingly, it was found here that overexpression of E.coli RidA does not increase lysine production.

However, E.coli also contains four paralogs of RidA, which are YjgH, TdcF, RutC and YoaB. Surprisingly, it was observed that overexpression of certain paralogs did result in changes in lysine and cadaverine production. For example, overexpression of the genes encoding YjgH and YoaB increases lysine and cadaverine production.

The crystal structure of E.coli YjgH (PDB ID:1PF5) has been resolved analysis of the crystal structures of 1PF5 and 1Q9 using the Needleman-Wunsch algorithm and the B L OSUM62 matrix in UCSF Chimera showed that these two structures can be stacked on top of each other with extremely high similarity.

In FIG. 2 is shown the amino acid sequence alignment of E.coli RidA and its paralogs YjgH, YoaB, RutC and TdcF. Amino acids at the RidA positions that are important for ligand binding and trimer formation include positions E120, C107, V18, K73 and E122. E120 in RidA involved in ligand binding is conserved in all paralogs. However, E107 in RidA, which is also important for ligand binding, is not conserved in all paralogs. Furthermore, the three residues V18, K73 and E122, which are important for trimer formation, are also not conserved. Of these five amino acids, four are not conserved in all paralogs, two of which are important for trimer formation, the amino acids at positions D76 and K123 (corresponding to K73 and E122 of RidA) of YjgH, are shown to be conserved between YjgH and YoaB. These two positions are highlighted in bold in fig. 2.

Host cells engineered to overexpress an imine/enamine deaminase polypeptide (such as Yjgh or YoaB) according to the invention also overexpress at least one enzyme involved in the synthesis of an amino acid or amino acid derivative (such as a lysine decarboxylase polypeptide); and/or additional polypeptides involved in amino acid biosynthesis. Lysine decarboxylase and lysine biosynthesis polypeptides and nucleic acid sequences are available in the art.

Numerous manuals providing guidance for performing recombinant DNA manipulations are available, such as Sambrook & Russell, Molecular Cloning, A L laboratory Manual (3rd Ed,2001), and Current Protocols in Molecular Biology (Autobel, et al, John Wiley and Sons, New York, 2009-.

Polynucleotides encoding imine/enamine deaminase polypeptides

Various polynucleotides have been shown to encode polypeptides that catalyze ammonia release and reduce imine and enamine levels (e.g., yoaB or yjgH from e.

Imine/enamine deaminase nucleic acid and polypeptide sequences suitable for use in the invention include imine/enamine deaminase nucleic acid sequences encoding an imine/enamine deaminase polypeptide according to SEQ ID NO 10 or SEQ ID NO 12 or a biologically active variant having substantial identity to SEQ ID NO 10 or SEQ ID NO 12. In some embodiments, such substantially identical variants have at least 70%, or at least 75%, 80%, 85% or 90% identity to SEQ ID No. 10 or SEQ ID No. 12 or alternative imine/enamine deaminase polypeptides, such as homologues of SEQ ID No. 10 or SEQ ID No. 12. In some embodiments, a substantially identical variant (as determined with reference to the E.coli YjgH protein sequence SEQ ID NO:12) comprises an acidic amino acid residue at position 121, an acidic residue at position 76 and a basic amino acid residue at position 123. In some embodiments, a substantially identical variant (as determined with reference to the E.coli YjgH protein sequence SEQ ID NO:12) comprises a D at position 76, an E at position 121, and a K at position 123. In some embodiments, the variant has at least 90% or at least 95% identity to the amino acid sequence of SEQ ID NO 10 or SEQ ID NO 12. As used herein, the term "variant" encompasses a biologically active polypeptide having one or more substitutions, deletions or insertions relative to an imine/enamine deaminase polypeptide reference sequence (such as SEQ ID NO:10 or 12). Thus, the term "variant" includes biologically active fragments as well as substitution variants.

In some embodiments, the host is genetically modified according to the invention to express a YoaB polypeptide. An exemplary sequence is provided as SEQ ID NO 10. In some embodiments, the host cell is genetically modified to express a YoaB polypeptide having at least 90% identity, or at least 95% identity, to SEQ ID No. 10 and to increase lysine and/or cadaverine production by at least 20% or more compared to a corresponding strain that is not engineered to overexpress the YoaB polypeptide. In some embodiments, the YoaB polypeptide has at least 70% identity or at least 75% identity to SEQ ID No. 10. In some embodiments, the YoaB polypeptide has at least 80% identity or at least 85% identity to SEQ ID No. 10.

In some embodiments, the host is genetically modified according to the invention to express the YjgH polypeptide. An exemplary sequence is provided as SEQ ID NO 12. In some embodiments, the host cell is genetically modified to express a YjgH polypeptide having at least 90% identity, or at least 95% identity, to SEQ ID No. 12 and to increase lysine and/or cadaverine production by at least 20% or more compared to a corresponding strain that is not engineered to overexpress said YjgH polypeptide. In some embodiments, the YjgH polypeptide has at least 70% identity or at least 75% identity to SEQ ID No. 12. In some embodiments, the YjgH polypeptide has at least 80% identity or at least 85% identity to SEQ ID No. 12.

In some embodiments, cadaverine production is measured in an escherichia coli modified to collectively express L ysC, DapA, L ysA, Asd, DapB, AspC, and CadA, as well as variants of YoaB or YjgH to be tested, or other imine/enamine deaminase polypeptides to be tested.an exemplary assay for assessing lysine and/or cadaverine production follows, escherichia coli is modified to express L ysC, DapA, 34 ysA, Asd, DapB, AspC, and CadA variant to be tested.a gene may be introduced into escherichia coli alone or into one or more operons.e.g., 3982 ysC, DapA, 34 ysA, Asd, DapB, AspC, and CadA variant to be tested.a gene may be introduced into escherichia coli alone or into one or more operons.e.g., L ysC, DapA, 29 ysA, asysd, and capa, and a variant to be tested.5. a candidate plasmid containing a synthetic resistance marker, e.g., 0% of the antibiotic may be selected from kh56 ysC, 2. when cultured₂PO₄、0.1％MgSO₄、1.6％(NH₄)₂SO₄、0.001％FeSO₄、0.001％MnSO₄0.2% yeast extract, 0.05% L-methionine, 0.01% L-threonine, 0.005% L-isoleucine and appropriate antibiotic for selection medium culture overnightTwo days, each culture was inoculated with 30 g/L glucose, 0.7% Ca (HCO)₃)₂The lysine or cadaverine concentration can be quantified using NMR.

In some embodiments, the YoaB or YjgH polypeptide increases lysine or cadaverine production by at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or more when expressed in a host cell as compared to a corresponding host cell of the same strain comprising the same genetic modification but not a modification that overexpresses the YoaB or YjgH polypeptide. In some embodiments, the YoaB or YjgH polypeptide increases lysine or cadaverine production by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or more when expressed in a host cell modified to overexpress lysine decarboxylase, aspartokinase, dihydrodipicolinate synthase, diaminopimelate decarboxylase, aspartate semialdehyde dehydrogenase, dihydropicolinate reductase, and aspartate aminotransferase as compared to a corresponding host cell comprising the same strain modified to overexpress lysine decarboxylase, aspartokinase, dihydropicolinate synthase, diaminopimelate decarboxylase, aspartate semialdehyde dehydrogenase, and/or YjgH polypeptide.

Isolation or production of the imine/enamine deaminase polynucleotide sequence may be accomplished by a variety of techniques. This technique will be discussed in the context of an imine/enamine deaminase gene. However, one skilled in the art understands that the same techniques can be used to isolate and express other desired genes. In some embodiments, oligonucleotide probes based on the sequences disclosed herein can be used to identify a desired polynucleotide in a cDNA or genomic DNA library from a desired bacterial species. Probes can be used to hybridize to genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species.

Alternatively, the nucleic acid of interest can be amplified from a nucleic acid sample using conventional amplification techniques. For example, PCR can be used to amplify gene sequences directly from mRNA, from cDNA, from genomic libraries, or cDNA libraries. PCR and other in vitro amplification methods can also be used, for example, to clone nucleic acid sequences encoding proteins to be expressed, such that the nucleic acids serve as probes for detecting the presence of desired mRNA in a sample, for nucleic acid sequencing, or for other purposes.

Suitable primers and probes for identifying imine/enamine deaminase polynucleotides in bacteria may be generated from comparisons of sequences provided herein. For a general overview of PCR, see PCR Protocols: a guides to Methods and applications (Innis, M, Gelfand, D., Sninsky, J.and White, T., eds.) Academic Press, San Diego (1990). Exemplary primer sequences are shown in the primer tables in the examples section.

Nucleic acid sequences encoding imine/enamine deaminase polypeptides for use in the present disclosure include genes and gene products identified and characterized by techniques such as hybridization and/or sequence analysis using exemplary nucleic acid sequences (e.g., SEQ ID NO:9 or SEQ ID NO: 11). In some embodiments, the host cell is genetically modified by introducing a nucleic acid sequence having at least 60% identity, or at least 70%, 75%, 80%, 85% or 90% identity, or 100% identity to a polynucleotide comprising SEQ ID No. 9 or SEQ ID No. 11.

The nucleic acid sequence encoding the imine/enamine deaminase polypeptide, which confers increased production of an amino acid (e.g., lysine) or amino acid derivative product (e.g., cadaverine) to a host cell, may additionally be codon optimized for expression in the desired host cell. Methods and databases that may be employed are known in the art. For example, preferred codons may be determined with respect to codon usage in a single gene, a group of genes of common function or origin, a highly expressed gene, codon frequency in the agrin coding region of the whole organism, codon frequency in the agrin coding region of the relevant organism, or a combination 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 imine/enamine deaminase polypeptides may be prepared using methods well known in the art. For example, a DNA sequence encoding an imine/enamine deaminase polypeptide (described in further detail below) may be combined with transcriptional and other regulatory sequences that will direct the transcription of the gene sequence in the desired cell (e.g., a bacterial cell, such as e. In some embodiments, the expression vector comprising the expression cassette (comprising a gene encoding an imine/enamine deaminase polypeptide) further comprises a promoter operably linked to the imine/enamine deaminase gene. In other embodiments, the promoter and/or other regulatory elements that direct transcription of the imine/enamine deaminase gene are endogenous to the host cell, and an expression cassette comprising the imine/enamine deaminase gene is introduced, for example, by homologous recombination, such that the exogenous gene is operably linked to and driven by the expression of the endogenous promoter.

As described above, expression of the gene encoding the imine/enamine deaminase polypeptide may be controlled by a number of regulatory sequences including a promoter (which may be constitutive or inducible), and optionally a repressor sequence (if desired), examples of suitable promoters, particularly in bacterial host cells, are promoters obtained from the lac operon of E.coli and other promoters derived from genes involved in the metabolism of other sugars, such as galactose and maltose. further examples include promoters such as the trp promoter, bla promoter phage lambda P L and T5. furthermore, synthetic promoters such as the tac promoter (U.S. Pat. No. 4,551,433) may be used, other examples of promoters include the Streptomyces coelicolor agarolylase gene (dagA), the Bacillus subtilis sucrase gene (sacB), the Bacillus subtilis genes α -amylase gene (amy L), the Bacillus stearothermophilus gene (Bacillus licheniformis gene) 3656, the Bacillus thermophilus gene (Saybolen. et. I., Bacillus licheniformis. A. and Bacillus subtilis genes are also described in Bacillus subtilis et. Biotech.: starch 4634. A. and Bacillus amyloliquefaciens genes.

In some embodiments, promoters that affect expression of a native imine/enamine deaminase polypeptide may be modified to increase expression. For example, the endogenous YoaB or YjgH promoter may be replaced by a promoter that provides increased expression compared to the native promoter.

The expression vector may further comprise additional sequences that affect the expression of the gene encoding the imine/enamine deaminase polypeptide. Such sequences include enhancer sequences, ribosome binding sites or other sequences (such as transcription termination sequences), and the like.

The vector expressing the nucleic acid encoding the imine/enamine deaminase polypeptide of the invention may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may comprise any means for ensuring self-replication. Alternatively, the vector may be one which, when introduced into a host, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Thus, the expression vector may additionally contain elements which allow the vector to integrate into the host genome.

The expression vectors of the invention preferably contain one or more selectable markers that allow for 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 a recombinant host organism (e.g., a bacterial cell, such as e.coli).

Although any suitable expression vector can be used to incorporate the desired sequences, readily available bacterial expression vectors include, but are not limited to: plasmids such as pSClOl, pBR322, pBBRlMCS-3, pUR, pET, pEX, pMRlOO, pCR4, pBAD24, p15a, pACYC, pUC (for example pUC18 or pUC19), or plasmids derived from these plasmids; and bacteriophages, such as the Ml3 bacteriophage and λ bacteriophage. However, one of ordinary skill in the art can readily determine by routine experimentation whether any particular expression vector is suitable for any given host cell. For example, an expression vector can be introduced into a host cell, which is then tested for viability and expression of the sequences contained in the vector.

The expression vectors of the invention can 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 cell

The present invention provides genetically modified host cells engineered to overexpress exogenous imine/enamine deaminase polypeptides. Such host cells may comprise a nucleic acid encoding a heterologous imine/enamine deaminase peptide, including any non-naturally occurring imine/enamine deaminase polypeptide variant; or may be genetically modified to overexpress a native imine/enamine deaminase polypeptide relative to a wild-type host cell.

The genetically modified host strain of the invention typically comprises at least one further genetic modification to increase the production of an amino acid or amino acid derivative relative to a control strain not having the one further genetic modification (e.g. a wild-type strain or cells of the same strain without the one further genetic modification). The additional genetic modification to increase production of an amino acid or amino acid derivative may be any genetic modification. In some embodiments, the genetic modification is the introduction of a polynucleotide expressing an enzyme involved in the synthesis of an amino acid or amino acid derivative. In some embodiments, the host cell comprises a plurality of modifications to increase production of an amino acid or amino acid derivative relative to a wild-type host cell.

In some aspects, the genetic modification of a host cell to overexpress an imine/enamine deaminase polypeptide is performed in conjunction with modifying the host cell to overexpress a lysine decarboxylase polypeptide and/or one or more lysine biosynthesis polypeptides.

Lysine decarboxylase refers to an enzyme that converts L-lysine to cadaverine classified as an E.C.4.1.1.18 lysine decarboxylase polypeptide is a well characterized enzyme, the structure of which is well known in the art (see, e.g., Kanjee, et al, EMBO J.30:931-944,2011, and a review by L emonier & L ane, Microbiology 144; 751. 760, 1998; and references described therein.) the EC number of lysine decarboxylase is 4.1.1.18. exemplary lysine decarboxylase sequences are homologs of CadA from Klebsiella sp.Klebsiella sp.sp.Klebsiella sp. 012968785.1; Enterobacter aerogenes (Enterobacter aeogens), YP 004592843.1; Salmonella enterica, WP 020936842.1; Serratia sp.sp. 033635725.1; Rarra kleinia (Raylella ornula sp.), Salmonella sp.sp.sp.sp.31, Salmonella sp.sp.31, Salmonella sp.2015, and Salmonella sp.35, as well as variants of lysine decarboxylase including the naturally occurring lysine decarboxylase, as described in Escherichia sp.35, Escherichia coli, Salmonella sp.12, Salmonella sp.35, Salmonella sp.12, and Salmonella sp.11, and the variants of lysine decarboxylase, Salmonella sp.11, Salmonella sp.12, Salmonella sp.sp.sp.11, and Salmonella sp.12, the sequences of lysine decarboxylase, and the sequences of the sequences are described herein.

Examples of lysine biosynthetic polypeptides include the E.coli genes sucA, Ppc, AspC, L ysC, Asd, DapA, DapB, DapD, ArgD, DapE, DapF, L ysA, Ddh, PntAB, CyoABE, GadAB, YbjE, GdhA, GltA, SucC, GadC, AcnB, PflB, ThrA, AceA, AceB, GltB, AceE, Sd, MurE, SpeE, SpeG, PuuA, PuuP and YgjG, or corresponding genes from other organisms such genes are known in the art (see, for example, Shah et al, J.Sci.2: Anssi. 2002, Ansz. 152. and YokjG; see, for example, for lysine biosynthetic genes related to MedBiotech. Biotech. Polypeptides, see, for example, Biotech. Polypeptides, see, Biotech. 7: Polypeptides, Biotech. 12. Biotech. Polypeptides, see, Biotech. for production of lysine biosynthetic polypeptides.

In some embodiments, the host cell is genetically modified to express a lysine decarboxylase, an aspartate kinase, a dihydrodipicolinate synthase, a diaminopimelate decarboxylase, an aspartate semialdehyde dehydrogenase, a dihydropicolinate reductase, and an aspartate aminotransferase. Additional modifications may also be incorporated into the host cell.

In some embodiments, the host cell may be genetically modified to attenuate or reduce 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; Anastassiadai, S.RecentrtPatents on Biotechnol.1:11-24,2007). For reviews of genes attenuated to increase cadaverine production see also Kind, et al, appl.Microbiol.Biotechnol.91:1287-1296, 2011. Exemplary genes encoding polypeptides whose attenuation increases lysine biosynthesis are provided below.

Nucleic acids encoding lysine decarboxylase or lysine biosynthesis polypeptides can be introduced into a host cell with the imine/enamine deaminase polynucleotide, e.g., encoded on a single expression vector, or introduced simultaneously in multiple expression vectors. Alternatively, the host cell can be genetically modified to overexpress a lysine decarboxylase or one or more lysine biosynthetic polypeptides, either before or after the host cell is genetically modified to overexpress the imine/enamine deaminase polypeptide.

In alternative embodiments, host cells overexpressing a naturally occurring imine/enamine deaminase polypeptide may be obtained by other techniques, such as by mutagenizing the cells (e.g., E.coli cells), and screening the cells to identify those imine/enamine deaminase polypeptides, such as YoaB or YjhG, that are at a higher level than the cells prior to mutagenesis.

The host cell comprising the imine/enamine deaminase polypeptide described herein is a bacterial host cell. In typical embodiments, the bacterial host cell is a gram-negative bacterial host cell. In some embodiments of the invention, the bacteria are enteric bacteria. In some embodiments of the invention, the bacterium is a species of the taxonomic class Corynebacterium, Escherichia, Pseudomonas (Pseudomonas), Zymomonas (Zymomonas), Shewanella (Shewanella), Salmonella (Salmonella), Shigella (Shigella), Enterobacter (Enterobacter), Citrobacter (Citrobacter), Enterobacter sakazakii (Cronobacter), Erwinia (Erwinia), Serratia (Serratia), Yersinia (Proteus), Hafnia, Yersinia pestis (Yersinia), Morganella (Morganella), Edwardsiella (Edwardsiella), or Klebsiella (Klebsiella). In some embodiments, the host cell is a member of the genus Escherichia, Hafnia or Corynebacterium. In some embodiments, the host cell is an E.coli, Hafnia alvei or Corynebacterium glutamicum host cell.

In some embodiments, the host cell is a gram-positive bacterial host cell, such as a bacillus, e.g., bacillus subtilis or bacillus licheniformis; or another bacillus, such as bacillus alcalophilus (b.alcalophilus), bacillus aminovorans (b.aminovorans), bacillus amyloliquefaciens, bacillus caldolyticus (b.caldolyticus), bacillus circulans (b.circulans), bacillus stearothermophilus, bacillus thermoglucosaccharyase (b.thermoglucosidasius), bacillus thuringiensis (b.thuringiensis), or bacillus westernii (b.vulgatis).

Host cells modified according to the invention can be screened for increased production of lysine or lysine derivatives (such as cadaverine), as described herein.

A method for producing lysine or a lysine derivative.

Lysine or lysine derivatives can be produced using host cells that are genetically modified to overexpress imine/enamine deaminase polypeptides. In some embodiments, the host cell produces cadaverine. For the production of lysine or lysine derivatives, a host cell genetically modified to overexpress an imine/enamine deaminase polypeptide as described herein may be cultured under conditions suitable to allow expression of the polypeptide and expression of the gene encoding the enzyme for the production of lysine or lysine derivatives. Host cells modified according to the invention provide higher yields of lysine or lysine derivatives relative to unmodified corresponding host cells expressing the imine/enamine deaminase polypeptide at the native level.

Host cells can be cultured using well-known techniques (see, e.g., the exemplary conditions provided in the examples section).

Lysine or lysine derivatives can then be isolated and purified using known techniques. The lysine or lysine derivative (e.g. cadaverine) produced according to the invention can then be used in any known process, for example in the production of polyamides.

In some embodiments, lysine may be converted to caprolactam using a chemical catalyst or by using an enzyme and a chemical catalyst.

The present invention will be described in more detail by way of specific examples. The following examples are provided for illustrative purposes and are not intended to limit the invention in any way. Those skilled in the art will readily recognize that various non-critical parameters may be changed or modified to produce substantially the same result.