阅读说明：本技术 调节csr系统以生产赖氨酸和赖氨酸衍生产物 (Modulation of CSR System to produce lysine and lysine derived products ) 是由 周豪宏 陈玲 雷云凤 刘修才 于 2018-01-09 设计创作，主要内容包括：本公开涉及遗传修饰的微生物,其过表达生物膜扩散相关多肽以增强微生物的赖氨酸和赖氨酸衍生物的生产,产生这种微生物的方法以及使用所述遗传修饰的微生物生产赖氨酸和赖氨酸衍生物的方法。(The present disclosure relates to genetically modified microorganisms that overexpress biofilm diffusion-associated polypeptides to enhance production of lysine and lysine derivatives by the microorganisms, 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 nucleic acid encoding a CsrB sRNA or CsrC sRNA, wherein the host cell overexpresses the CsrB sRNA or CsrC sRNA relative to a corresponding host cell that has not been modified to express the exogenous nucleic acid; and having at least one additional genetic modification to increase production of lysine or a lysine derivative compared to a wild-type host cell.

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

3. The genetically modified host cell of claim 1 or 2, wherein the CsrB sRNA comprises a nucleotide sequence having at least 85% identity to SEQ ID NO 16 and the CsrC sRNA comprises a nucleotide sequence having at least 85% identity to SEQ ID NO 17.

4. The genetically modified host cell of claim 1 or 2, wherein the CsrB sRNA comprises the nucleic acid sequence of SEQ ID NO 16 and the CsrC sRNA comprises the nucleic acid sequence of SEQ ID NO 17.

5. The genetically modified host cell of any one of claims 1 to 4, wherein said CsrB or CsrC is heterologous to said host cell.

6. The genetically modified host cell of any one of claims 1 to 5, wherein an exogenous nucleic acid encoding said CsrB or CsrC is encoded by an expression vector introduced into said cell, wherein said expression vector comprises said exogenous nucleic acid operably linked to a promoter.

7. The genetically modified host cell of any one of claims 1-5, wherein said exogenous nucleic acid is integrated into the host chromosome.

8. The genetically modified host cell of any one of claims 1-7, wherein the host cell overexpresses lysine decarboxylase.

9. The genetically modified host cell of any one of claims 1-8, wherein the host cell overexpresses one or more lysine biosynthetic polypeptides.

10. The genetically modified host cell of claim 9, wherein the one or more lysine biosynthetic polypeptides is an aspartokinase, a dihydrodipicolinate synthase, a diaminopimelate decarboxylase, an aspartate semialdehyde dehydrogenase, a dihydropicolinate reductase, or an aspartate aminotransferase.

11. The genetically modified host cell of claim 10, wherein the aspartate kinase, dihydrodipicolinate synthase, diaminopimelate decarboxylase, aspartate semialdehyde dehydrogenase, dihydropicolinate reductase, or aspartate aminotransferase is a LysC, DapA, LysA, Asd, DapB, or AspC polypeptide.

12. The genetically modified host cell of any one of claims 1-7, wherein said host cell overexpresses the CadA, LysC, DapA, LysA, Asd, DapB, and AspC polypeptides.

13. The genetically modified host cell of any one of claims 1 to 12, wherein the host cell is of the genus Escherichia (Hafnia), Hafnia (Hafnia) or Corynebacterium (Corynebacterium).

14. The genetically modified host cell of claim 13, wherein the host cell is escherichia coli (escherichia coli), Hafnia alvei (Hafnia alvei), or Corynebacterium glutamicum (Corynebacterium glutamicum).

15. The genetically modified host cell of claim 14, wherein said host cell is e.

16. A method of producing lysine or a lysine derivative, the method comprising culturing the host cell of any one of claims 1-15 under conditions in which CsrB sRNA or csrcssrna is overexpressed.

17. A method of engineering a host cell to increase production of lysine or a lysine derivative, the method comprising introducing an exogenous nucleic acid encoding CsrB or CsrC sRNA into a host cell, wherein the host cell has at least one additional genetic modification that increases production of lysine or a lysine derivative as compared to a wild-type host cell;

culturing the host cell under conditions that express the CsrB or CsrC sRNA, and

selecting a host cell that exhibits increased production of lysine or a lysine derivative relative to a corresponding control host cell that has not been modified to express the exogenous nucleic acid.

18. The method of claim 17, wherein the amino acid derivative is cadaverine.

19. The method of claim 17 or 18, wherein the CsrB or CsrC sRNA is heterologous to the host cell.

20. The method of any one of claims 17-19, wherein said exogenous nucleic acid encoding said CsrB or csrcs rna is encoded by an expression vector introduced into said cell, wherein said expression vector comprises said exogenous nucleic acid operably linked to a promoter.

21. The method of any one of claims 17-19, wherein the exogenous nucleic acid is integrated into the host chromosome.

22. The method of any one of claims 17-21, wherein the CsrB sRNA comprises the nucleic acid sequence of SEQ ID No. 16 and the CsrC sRNA comprises the nucleic acid sequence of SEQ ID No. 17.

23. The method of any one of claims 17-22, wherein the host cell overexpresses lysine decarboxylase.

24. The method of any one of claims 17-23, wherein the host cell overexpresses one or more lysine biosynthetic polypeptides.

25. The method of claim 24, wherein said lysine biosynthetic polypeptide is an aspartokinase, a dihydrodipicolinate synthase, a diaminopimelate decarboxylase, an aspartate semialdehyde dehydrogenase, a dihydropicolinate reductase, or an aspartate aminotransferase.

26. The method of claim 25, wherein the aspartokinase, dihydrodipicolinate synthase, diaminopimelate decarboxylase, aspartate semialdehyde dehydrogenase, dihydropicolinate reductase, or aspartate aminotransferase is a LysC, DapA, LysA, Asd, DapB, or AspC polypeptide.

27. The method of any one of claims 17-22, wherein the host cell overexpresses the CadA, LysC, DapA, LysA, Asd, DapB, and AspC polypeptides.

28. The method of any one of claims 17-27, wherein the host cell is of the genus escherichia, hafnia, or corynebacterium.

29. The method of claim 28, wherein the host cell is escherichia coli, hafnia alvei, or corynebacterium glutamicum.

30. The method of claim 29, wherein the host cell is e.

Background

The ability of molecules to move into and out of cells has a significant effect on the intracellular concentration of the molecule. For example, if the molecule is a nutrient, slowing the engraftment of the molecule into the cell will inhibit growth (Herbert, D & HL Kornberg, biochem. J.156(2),477-480, 1976). If the molecule is a toxin, slowing the removal of the molecule from the cell will inhibit growth. If the molecule is the substrate of the reaction, slowing the migration of the molecule into the cell will slow the reaction. If the molecule is an intermediate in a series of reactions, slowing down the removal of the molecule from the cell and allowing it to accumulate in the cell may lead to feedback inhibition (Kikuchi et al, FEMS microbiology letters 173: 211-1154, 1999; Ogawa-Miyata et al, biosci.Biotechnology.biochem.65: 1149-1154, 2001).

It was previously disclosed that a phosphodiesterase protein which increases the spread of biological membranes by decreasing the intracellular concentration of bis- (3'-5') -cyclic diguanosine-monophosphate (c-di-GMP) affects the production of amino acids such as lysine and its derivatives such as cadaverine (PCT/CN 2016/095281). Although various genes have been shown to hydrolyze c-di-GMP and to increase the biofilm diffusion activity (e.g.bdcA or yahA of E.coli; rapA, fleN, rocR or bifA of P.aeruginosa; vieA or mbaA of Vibrio cholerae; and rmdAB of S.coelicolor), it is not known to increase any effect of the biofilm diffusion activity on the production of amino acids or derivatives thereof by decreasing the intracellular c-di-GMP concentration. PCT/CN2016/095281 demonstrates that genetically modified microorganisms overexpressing a biofilm diffusing polypeptide show an increased production of lysine or lysine derived compounds such as cadaverine relative to corresponding microorganisms of the same strain not comprising said genetic modification.

Another group of genes that influence biofilm formation has been identified. Carbon storage regulator (Csr) CsrA is a global regulator protein that has been shown to inhibit biofilm formation and increase biofilm diffusion in E.coli (Jackson et al, J.Bacteriol.184: 290-. Disruption of the csrA gene has been shown to increase biofilm formation, and overexpression of csrA from plasmids inhibits biofilm formation in E.coli. CsrA has also been shown to be an mRNA binding protein that is part of a regulatory pathway affecting glycogen biosynthesis, catabolism and gluconeogenesis (Romeo et al, J.Bacteriol.175: 4744-. CsrA activity is inhibited by the binding of sRNA CsrB, a non-coding RNA consisting of 18 incomplete repeats (5 '-CAGGA (U, C, A) G-3'), forming a hairpin structure (Romeo et al, mol. Microbiol.29:1321-1330, 1998). Thus, part of the inhibition mechanism is the binding of the 18CsrA protein to one CsrB molecule of the hairpin structure.

Two other proteins/sRNA are part of the Csr system. Although CsrA regulates mRNA stability and functions at both the transcriptional and posttranscriptional levels, CsrD protein is a signaling protein that results in positive transcriptional regulation of genes affected by the Csr regulatory system (Esquerre et al, Scientific Reports 6:25057,2016). The Csr system is additionally regulated by sRNA CsrC, which also inhibits the activity of CsrA (Wellbacher et al, mol. Microbiol.48: 657-containing 670, 2003). Both CsrB and CsrC have been shown to be up-regulated during malnutrition conditions as well (Jonas et al, FEMS microbiol. lett.297: 80-86).

The Csr system has been previously manipulated to increase amino acid production. For example, it has been shown that increasing CsrA production by decreasing the amount of CsrB can increase threonine production (WO 2003/046184). It was subsequently shown (EP 2050816 and US 2009/0258399) that deletions of csrB and csrC can increase amino acid production, in particular arginine production. EP 2055771 points that attenuation of csrB can increase amino acid production, and in U.S. patent No. 8759042 shows that deletion of csrC can increase arginine production. Interestingly, it has also been shown that increasing expression of csrB can increase phenylalanine production (Yakandawala et al, appl. Microbiol. Biotechnol.78: 283-. Thus, these disclosures indicate that increasing or decreasing CsrB or CsrC can increase or decrease amino acid production. Thus, when manipulating CsrB or CsrC levels in a cell, the skilled artisan will be unable to determine whether a particular amino acid other than the previously evaluated amino acids is decreased or increased.

Brief description of the invention

The present disclosure is based, in part, on the surprising discovery that increasing CsrA production in e.coli does not increase lysine production; but rather reduced lysine production; and cells overexpressing csrB or csrC showed increased lysine production.

Thus, in one aspect, provided herein is a genetically modified host cell comprising an exogenous nucleic acid encoding a CsrB sRNA or csrcs rna, wherein the host cell overexpresses CsrB or CsrC relative to a corresponding host cell that has not been modified to express the exogenous nucleic acid; and having at least one additional genetic modification to increase production of lysine or a lysine derivative compared to a wild-type host cell. In some embodiments, the amino acid derivative is cadaverine. In some embodiments, the CsrB sRNA comprises a nucleotide sequence having at least 85% identity, or at least 90% identity, or at least 95% identity to SEQ ID No. 16. In some embodiments, the CsrC sRNA comprises a nucleotide sequence having at least 85% identity, or at least 90% identity, or at least 95% identity to SEQ ID No. 17. In a particular embodiment, the CsrBsRNA comprises the nucleic acid sequence shown in SEQ ID NO 16. In other embodiments, the CsrC sRNA comprises the nucleic acid sequence set forth in SEQ ID NO 17. In some embodiments, the CsrB or CsrC sRNA is heterologous to the host cell. In some embodiments, the exogenous nucleic acid encoding CsrB or CsrC sRNA is encoded by an expression vector introduced into the cell, wherein the expression vector comprises the exogenous nucleic acid operably linked to a promoter. In other embodiments, the exogenous nucleic acid is integrated into the host chromosome. In some embodiments, the host cell overexpresses lysine decarboxylase. In further embodiments, the host cell overexpresses one or more lysine biosynthetic polypeptides, such as aspartokinase, dihydrodipicolinate synthase, diaminopimelate decarboxylase, aspartate semialdehyde dehydrogenase, dihydropicolinate reductase, or aspartate aminotransferase. In particular embodiments, the aspartokinase, dihydrodipicolinate synthase, diaminopimelate decarboxylase, aspartate semialdehyde dehydrogenase, dihydropicolinate reductase, or aspartate aminotransferase is a LysC, DapA, LysA, Asd, DapB, or AspC polypeptide. In some embodiments, the host cell overexpresses CadA, LysC, DapA, LysA, Asd, DapB, and AspC polypeptides. In some embodiments, the host cell is an Escherichia (Escherichia), Hafnia (Hafnia), or Corynebacterium (Corynebacterium) cell. In particular embodiments, the host cell is escherichia coli, Hafnia alvei (Hafnia alvei), or corynebacterium glutamicum (corynebacterium glutamicum).

In another aspect, provided herein is a method of producing lysine or a lysine derivative, such as cadaverine, comprising culturing a host cell under conditions in which the CsrB sRNA or CsrC sRNA is overexpressed, such as described above.

In another aspect, provided herein is a method of engineering a host cell to increase production of lysine or a lysine derivative, the method comprising introducing an exogenous nucleic acid encoding CsrB sRNA or CsrC sRNA into a host cell, wherein the host cell has at least one additional genetic modification to increase production of lysine or a lysine derivative as compared to a wild-type host cell; culturing the host cell under conditions that express CsrB or CsrC sRNA, and selecting a host cell that exhibits increased production of lysine or lysine derivatives relative to a corresponding control host cell that is not modified to express the exogenous nucleic acid. In some embodiments, the amino acid derivative is cadaverine. In some embodiments, the CsrB sRNA comprises a nucleotide sequence having at least 85% identity, or at least 90% identity, or at least 95% identity to SEQ ID No. 16. In some embodiments, the CsrC sRNA comprises a nucleotide sequence having at least 85% identity, or at least 90% identity, or at least 95% identity to SEQ ID No. 17. In a further embodiment, the CsrB sRNA comprises the nucleic acid sequence shown in SEQ ID NO 16. In other embodiments, the CsrC sRNA comprises the nucleic acid sequence set forth in SEQ ID NO 17. In some embodiments, the CsrB sRNA or CsrC sRNA is heterologous to the host cell. In some embodiments, the exogenous nucleic acid encoding CsrB or CsrC sRNA is encoded by an expression vector introduced into the cell, wherein the expression vector comprises the exogenous nucleic acid operably linked to a promoter. In other embodiments, the exogenous nucleic acid is integrated into the host chromosome. In some embodiments, the host cell overexpresses lysine decarboxylase. In some embodiments, the host cell overexpresses one or more lysine biosynthetic polypeptides, such as an aspartokinase, a dihydrodipicolinate synthase, a diaminopimelate decarboxylase, an aspartate semialdehyde dehydrogenase, a dihydropicolinate reductase, or an aspartate aminotransferase. In some embodiments, the aspartokinase, dihydrodipicolinate synthase, diaminopimelate decarboxylase, aspartate semialdehyde dehydrogenase, dihydropicolinate reductase, or aspartate aminotransferase is a LysC, DapA, LysA, Asd, DapB, or AspC polypeptide. In some embodiments, the host cell overexpresses CadA, LysC, DapA, LysA, Asd, DapB, and AspC polypeptides. In some embodiments, the host cell is an escherichia, hafnia, or corynebacterium cell. In certain embodiments, the host cell is escherichia coli, hafnia alvei, or corynebacterium glutamicum.

Detailed Description

Term(s) for

As used herein, "CsrB" refers to small regulatory rnas (srnas) that contain incomplete repeats that form hairpin structures and bind CsrA. "CsrB" includes Escherichia coli CsrB and homologs of CsrB from other bacteria such as members of the Enterobacteriaceae family (Enterobacteriaceae). Coli CsrB is about 360 nucleotides in length and contains 18 incomplete repeats of 5 '-CAGGA (U, C, A) G-3' (Romeo et al, mol. Microbiol.29:1321-1330, 1998). CsrA binds to the hairpin structure CsrB, whereby one CsrA molecule binds to each hairpin structure. Thus, CsrB isolates CsrA and reduces CsrA activity. CsrA/CsrB sequences have been reported in other Enterobacteriaceae such as Salmonella (Salmonella), Shigella (Shigella) and Yersinia (Yersinia). In Salmonella, CsrB is shown to have 16 predicted stem loops, each carrying the consensus sequence GWGRHG (Altier, et al. mol. Microbiol.35:635-646,2000), where "W" is A or U; r is A or G; and H is A, C or U. An exemplary E.coli CsrB DNA sequence is provided in SEQ ID NO 16. Other CsrB sequences include those encoded by the following chromosomal regions: the chromosomal region CP015574.1 of Salmonella enterica subsp, the chromosomal region CP024470.1 of Shigella flexneri (Shigella flexneri), the chromosomal region CP023645.1 of Shigella sonnei (Shigella sonnei) and the chromosomal region LT556085.1 of Citrobacter sp.

As used herein, "CsrC" refers to sRNA that contains incomplete repeats similar to those contained in CsrB that form hairpin structures and binds CsrA, thereby isolating CsrA. The term includes escherichia coli CsrC and homologs of CsrC from other bacteria such as members of the enterobacteriaceae family. Coli CsrC is about 245 nucleotides in length and contains 9 such repeats (Weilbacher et al, mol. Microbiol.48: 657-S670, 2003). In Salmonella, CsrC is shown to have 8 predicted stem-loop structures (Fortune et al, Infect. AndImmunun.74: 1331-1339, 2006). An exemplary E.coli CsrC DNA sequence is provided in SEQ ID NO 17. Other CsrC sequences include those encoded by the following chromosomal regions: the chromosomal region CP023645.1 of Shigella sonnei, the chromosomal region CP024470.1 of Shigella flexneri, the chromosomal region CP023504.1 of Citrobacter wecker (Citrobacter werkmani) and the chromosomal region CP018661.1 of Salmonella enterica subspecies.

The terms "increased expression" and "overexpression" of CsrB or CsrC sRNA are used interchangeably herein and refer to an increase in the amount of CsrB or CsrC sRNA in a genetically modified cell, such as a cell into which an expression construct encoding CsrB or CsrC sRNA has been introduced, as compared to the amount of CsrB or CsrC sRNA 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). For the purposes of the present application, an increased expression level is typically an increase of at least 5%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to the unmodified corresponding cell. The unmodified cell need not express the CsrB or CsrC sRNA. Thus, the term "overexpressing" also includes embodiments in which the CsrB or CsrC sRNA is expressed in a host cell that does not naturally express the CsrB or CsrC sRNA. Increased expression of CsrB or CsrC sRNA can be assessed by a variety of detection methods, including but not limited to measuring RNA levels and/or the level of CsrB or CsrC sRNA activity, e.g., directly measuring CsrA binding activity or by assessing activity modulated by CsrB or CsrC sRNA.

As used herein, the term "enhanced" as used in the production of lysine or a lysine derivative, such as cadaverine, refers to an increased production of an amino acid, such as lysine or a derivative, by a genetically modified host cell as compared to a control corresponding cell (e.g., a wild-type strain cell or the same strain cell without the genetic modification to increase the production of lysine or the lysine derivative). The production of an amino acid or derivative thereof is typically enhanced by at least 5%, or at least 0%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to the control cell.

The term "reference … … numbering" or "corresponding to" or "determined with reference to … …" when used in the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a designated reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. For example, the nucleotide position in the CsrB or CsrC sRNA sequence variant "corresponding to" the nucleotide in SEQ ID NO 16 is the residue that is aligned with the nucleotide when SEQ ID NO 16 and the variant are compared in the maximum alignment.

As used herein, the terms "polynucleotide" and "nucleic acid" used interchangeably in expression vectors and sequences encoding CsrB or CsrC sRNA refer to single-or double-stranded polymers of deoxyribonucleotide or ribonucleotide bases read from the 5 'to 3' end. Nucleic acids used in the invention will typically contain phosphodiester linkages, but in some cases nucleic acid analogs with alternative backbones may be used, including, for example, phosphoramidates, phosphorothioates, phosphorodithioates, or O-methylphosphonous acid amide linkages (see Eckstein, Oligonucleotides and antibiotics: A Practical Approach, Oxford University Press); a positively charged 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 the sense and antisense strands of a nucleic acid, either as a single strand or as a duplex. As will be understood by those skilled in the art, the description of a single strand also defines the sequence of the complementary strand; the sequences described herein thus also provide the complement of the sequence. Unless otherwise indicated, a particular nucleic acid sequence, e.g., a nucleic acid sequence encoding a polypeptide, also implicitly encompasses variant degenerate codon substitutions and complementary sequences, as well as the sequences explicitly indicated. The nucleic acid can be DNA, both genomic DNA and cDNA, RNA or hybrids, wherein the nucleic acid can comprise 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 sequences having at least 60%, 65%, or 70% sequence identity to a reference sequence. The percent identity can be any integer between 60-100%. Some embodiments include BLAST programs having at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

Two nucleic acid sequences or polypeptide sequences are considered "identical" if the sequences of nucleotides or amino acid residues, respectively, in the two sequences 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 or aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm, or by manual alignment and visual inspection. When percentage sequence identity is used in relation to proteins or peptides, it will be appreciated that residue positions that are not identical will generally differ by conservative amino acid substitutions, wherein amino acid residues are substituted for other amino acid residues having similar chemical properties (e.g., charge or hydrophobicity) and therefore do not alter the functional properties of the molecule. In the case of sequences that differ by conservative substitutions, the percent sequence identity may be adjusted up to correct for the conservative nature of the substitution. Means for making such adjustments are well known to those skilled in the art. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches, thereby increasing the percent sequence identity. Thus, for example, where the same amino acid scores 1 and a non-conservative substitution scores 0, the conservative substitution scores between 0 and 1.

For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. 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 be used, or optional parameters may be specified. 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 CsrB or CsrC sRNA has sequence identity to SEQ ID NO:16 or 17 or another polynucleotide reference sequence is the BLAST algorithm, described in Altschul et al, 1990, J.mol.biol.215:403-410, which is incorporated herein by reference. Software for performing BLAST and BLAST2 analyses is publicly available through the national center for Biotechnology information (world Wide Web ncbi. nlm. nih. gov /).

As used herein, a "comparison window" includes reference to any segment selected from the group consisting of a number of contiguous positions ranging from 20 to 600, typically from about 50 to about 200, more typically from about 100 to about 150, wherein, upon optimal alignment of two sequences, the sequences can be compared to a reference sequence at the same number of contiguous positions. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of the compared sequences can be performed by algorithms such as the local homology algorithm of Smith & Waterman, adv.Appl.Math.2:482(1981), the homology alignment algorithm of Needleman & Wunsch, J.mol.biol.48:443(1970), the search similarity method of Pearson & Lipman, Proc.Nat' l.Acad.Sci.USA 85:2444(1988), Computer-implemented programs for these algorithms (GAP, BESTFIT, TA and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group,575Science Dr., Madison, Wis), or manual alignment and visual inspection.

As used herein, the term "promoter" refers to a polynucleotide sequence that is 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 as well as regulatory sequences 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 enhancers, repressor binding sequences, and the like. These cis-acting sequences typically interact with proteins or other biomolecules to effect (turn on/off, regulate, etc.) gene transcription. The core promoter sequence is most often located within 1-2kb of the translation initiation site, more often within 1kbp of the translation initiation site and typically within 500bp or 200bp or less of the translation initiation site. 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. Thus, the promoter used in the expression construct of the present invention is referred to by the gene name. The names referring to a promoter include wild-type, native promoters, and promoter variants that retain the ability to induce expression. The names referring to promoters are not limited to a particular species, but also encompass promoters from corresponding genes in other species.

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

As used herein, a polynucleotide is "heterologous" to an organism or a second polynucleotide sequence if it originates from a foreign species, or if it originates from the same species but in a modified form of its original form. For example, when a polynucleotide encoding a CsrB or CsrC sRNA sequence is said to be operably linked to a heterologous promoter, this means that the polynucleotide encoding the CsrB or CsrC sequence is from one species and the promoter sequence is 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 CsrB or CsrC sRNA, the CsrB or CsrC sRNA or the polynucleotide encoding the CsrB or CsrCsRNA is "heterologous" to the host cell; a CsrB sRNA or CsrC sRNA variant is "heterologous" to a host cell if the nucleotide sequence is different from the CsrB or CsrC polynucleotide sequence native 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 that encode a desired protein. An "exogenous" polypeptide or polynucleotide expressed in a host cell may be naturally occurring in the wild-type host cell or 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 progeny cells 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 is also noted that even though an organism may contain endogenous copies of a given polynucleotide sequence or gene, the introduction of an expression construct or vector encoding such a sequence, e.g., to overexpress or otherwise regulate the expression of the encoded protein, represents an "exogenous" copy of the gene or polynucleotide sequence. Any pathway, gene, RNA, or enzyme described herein may utilize or rely on an "endogenous" sequence, which may be provided as one or more "exogenous" polynucleotide sequences, or both.

As used herein, "recombinant nucleic acid" or "recombinant polynucleotide" refers to a genetically engineered nucleic acid polymer in which at least one of the following is true: (a) the nucleic acid sequence is foreign to (i.e., not naturally present in) a given host cell; (b) the sequence may be naturally present in a given host cell, but not in a natural (e.g., greater than expected) amount; or (c) the nucleic acid sequence comprises two or more subsequences that are not in the same relationship to each other in nature. For example, in connection with case (c), the recombinant nucleic acid sequence will have two or more sequences from unrelated genes arranged into a new functional nucleic acid. A "recombinant" nucleic acid refers to the original polynucleotide being manipulated as well as copies of the polynucleotide.

The term "operably linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Generally, it refers to the functional relationship of the transcriptional regulatory sequence to the transcribed sequence. 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 a suitable host cell or other expression system. In general, the promoter transcription regulatory sequence operably linked to the transcribed sequence is physically contiguous with the transcribed sequence, i.e., it is 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 is enhanced by it.

The term "expression cassette" or "DNA construct" or "expression construct" refers to a nucleic acid construct which 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, the skilled person will recognise that the inserted polynucleotide sequence need not be identical 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, including a single cell or cell culture, which may be or has been the recipient of any recombinant vector or isolated polynucleotide of the present invention. Host cells include progeny of a single host cell that may not necessarily be identical (morphologically or over total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or alteration. Host cells include cells into which the recombinant vector or polynucleotide of the present invention has been introduced by transformation, transfection, or the like.

The term "isolated" means that a material is substantially or essentially free of components with which it normally accompanies in its natural state. For example, as used herein, an "isolated polynucleotide" may refer to a polynucleotide that has been separated from the sequences that flank it in a naturally occurring or genomic state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent thereto, e.g., by cloning into a vector. For example, a polynucleotide is considered isolated if it is cloned into a vector that is not part of its natural environment or is artificially introduced into the genome of a cell in a manner different from that found in nature.

Aspects of the present disclosure

The present disclosure is based in part on the following findings: increased expression of CsrB or csrcssrna in a microorganism, such as a gram-negative bacterium, enhances lysine production and/or the production of lysine derivatives, such as cadaverine.

Host cells engineered to overexpress CsrB or CsrC sRNA according to the present 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 other polypeptides involved in amino acid biosynthesis. Lysine decarboxylase and lysine biosynthesis polypeptides and nucleic acid sequences are available in the art.

The present invention employs various conventional recombinant nucleic acid techniques. Generally, nomenclature and laboratory procedures used in the recombinant DNA techniques described below are those commonly employed in the art. There are many manuals that provide guidance for recombinant DNA manipulation, such as Sambrook & Russell, Molecular Cloning, A Laboratory Manual (3rd Ed, 2001); and Current protocols in Molecular Biology (Ausubel, et al, John Wiley and Sons, New York, 2009-2017).

Polynucleotides encoding CsrB or CsrC sRNAs

Various polynucleotides have been shown to encode CsrB and CsrC sRNA that bind CsrA and reduce CsrA function. Polynucleotides encoding CsrB and CsrC sRNA suitable for overexpression in a host cell to increase production of lysine or lysine derivatives include E.coli CsrB and CsrC polynucleotide sequences shown in SEQ ID NO 16 and SEQ ID NO 17, respectively.

In some embodiments, the host cell is genetically modified to overexpress a CsrB polynucleotide having at least 60% or at least 70%, 75%, 80%, 85% or at least 90% identity to SEQ ID No. 16. Unless otherwise indicated, "SEQ ID NO: 16" refers to the DNA sequences shown in the exemplified sequence Listing as well as their RNA counterparts in which uracil bases replace thymine bases. Thus, when the CsrB RNA sequence is compared to SEQ ID NO 16 to determine percent identity, it is understood that SEQ ID NO 16 will contain "U" instead of "T". The CsrB polynucleotide variant of SEQ ID NO. 16 retains the ability to bind CsrA. In some embodiments, the CsrB polynucleotide is at least 85% identical, or at least 90% or at least 95% identical to SEQ ID No. 16. In some embodiments, the polynucleotide comprises the sequence set forth in SEQ ID NO 16. Other exemplary CsrB sequences include sequences shown in SEQ ID NOs 18, 19, 20, 21, and 22 from shigella, Triticum, citrobacter, and salmonella having 99% sequence identity (shigella and Triticum), 90% sequence identity (citrobacter), and 87% sequence identity (salmonella) with SEQ ID No. 16.

In some embodiments, the host cell is genetically modified to overexpress a CsrC polynucleotide having at least 60% or at least 70%, 75%, 80%, 85% or at least 90% identity to SEQ ID No. 17. Unless otherwise indicated, "SEQ ID NO: 17" refers to the DNA sequence shown in the exemplified sequence Listing as well as its RNA counterpart in which uracil bases replace thymine bases. Thus, when the CsrC RNA sequence is compared to SEQ ID NO:17 to determine percent identity, it is understood that SEQ ID NO:17 will contain a "U" instead of a "T". The CsrC polynucleotide variant of SEQ ID NO. 17 retains the ability to bind CsrA. In some embodiments, the CsrC polynucleotide is at least 85% identical, or at least 90% or at least 95% identical to SEQ ID No. 17. In some embodiments, the polynucleotide comprises the sequence set forth in SEQ ID NO. 17. Other exemplary CsrC sequences include sequences shown in SEQ ID NOS 23, 24, 25, and 26 from Shigella sonnei, Shigella flexneri, Citrobacter westermanii, and Salmonella enterica, having 100%, 99%, 89%, and 88% sequence identity to SEQ ID NO 17, respectively.

In some embodiments, the CsrB or CsrC sRNA comprises at least 8 or at least 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; and typically less than 20 CsrA-binding hairpins. In some embodiments, the hairpin structure comprises the sequence GWGGRHG, wherein "W" is a or U; r is A or G and H is A, C or U. In some embodiments, the hairpin structure comprises the sequence CAGGA (U, C, a) G.

In some embodiments, the host cell is genetically modified to overexpress CsrB or CsrC sRNA from salmonella, citrobacter, or shigella.

The activity of wild-type or variant CsrB or CsrC sRNA can be assessed using a number of assays, including assays that assess the production of lysine or lysine-derived compounds. In some embodiments, lysine production or cadaverine production is measured. The examples section herein provides exemplary assays. In some embodiments, the measurement is in E.coli modified to co-express LysC, DapA, LysA, Asd, DapB, AspC, and CadA and CsrB or CsrC sRNAAnd (4) production of cadaverine. The following are exemplary assays for assessing lysine and/or cadaverine production. Coli was modified to express LysC, DapA, LysA, Asd, DapB, AspC and CadA and CsrB or CsrC sRNA. The genes can be introduced into E.coli or into one or more operons, respectively. For example, LysC, DapA, LysA, Asd, DapB, and AspC may be encoded by synthetic operons present in one plasmid, while CadA and candidate variants may be encoded by separate plasmids. Each plasmid has a unique antibiotic resistance selectable marker. Antibiotic resistant colonies were selected and cultured. For example, the culture is incubated at 37 ℃ with 4% glucose, 0.1% KH₂PO₄、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 antibiotics for selection in 3mL medium for overnight growth. The next day, each culture was inoculated to a medium containing 30g/L glucose, 0.7% Ca (HCO)₃)₂50mL of fresh medium of antibiotic, at 37 ℃ for 72 hours, at which time the concentration of lysine was determined. Lysine or cadaverine can be quantified using NMR. The yield can be calculated by dividing the molar amount of lysine or cadaverine produced by the molar amount of glucose added. CsrB or CsrC sRNA useful in the present invention increase the yield of lysine or cadaverine. Alternatively, colonies were evaluated for increased production of another lysine derivative.

In some embodiments, the CsrB or CsrC sRNA 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 as compared to a corresponding host cell comprising the same strain with the same genetic modification except for the modification that overexpresses the CsrB or CsrC sRNA. In some embodiments, the CsrB or CsrC sRNA, when expressed in a host cell modified to overexpress lysine decarboxylase, aspartokinase, dihydrodipicolinate synthase, diaminopimelate decarboxylase, aspartate semialdehyde dehydrogenase, dihydropicolinate reductase, and aspartate aminotransferase, increases lysine or cadaverine production by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or more, as compared to a corresponding host cell comprising the same strain modified to overexpress lysine decarboxylase, aspartate kinase, dihydropicolinate synthase, diaminopimelate decarboxylase, aspartate semialdehyde dehydrogenase, dihydropicolinate reductase, and aspartate aminotransferase, but which does not overexpress CsrB or CsrC sRNA.

In some embodiments, the activity of CsrB or CsrC sRNA can be assessed by determining the ability of RNA to bind CsrA, for example, in a quantitative migration assay.

Isolation or generation of the CsrB or CsrC sequences to incorporate the expression cassette for overexpression in a host cell can be achieved by a variety of techniques. This technique will be discussed in the context of CsrB or CsrC polynucleotide sequences. However, the skilled artisan understands that other desired genes can be isolated and expressed using the same techniques. In some embodiments, oligonucleotide probes based on the sequences disclosed herein can be used to identify a desired polynucleotide from a cDNA or genomic DNA library of a desired bacterial species. Probes can be used to hybridize to genomic DNA to isolate homologous genes in the same or different species.

In typical embodiments, the nucleic acid of interest is amplified from a nucleic acid sample using conventional amplification techniques. For example, PCR can be used to amplify sequences directly from genomic DNA. PCR and other in vitro amplification methods can also be used, for example, to clone nucleic acid sequences encoding proteins to be expressed, to use the nucleic acids as probes to detect the presence of desired mRNA in a sample, for nucleic acid sequencing, or other purposes.

Suitable primers and probes for the production of CsrB and CsrC polynucleotides in bacteria can be determined by comparing the sequences provided herein. A general overview of PCR is given in PCR Protocols A Guide 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 of the examples section.

Nucleic acid sequences encoding lysines or lysine-derived products such as cadaverine produced CsrB or csrcrna that confer increased host cell production 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 can be determined with respect to: codon usage in a single gene, a group of genes having a common function or origin, a highly expressed gene, codon frequency in the total protein coding region of the entire organism, codon frequency in the total protein coding region of a related 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 vector

Recombinant vectors expressing CsrB or CsrC sRNA can be prepared using methods well known in the art. For example, a DNA sequence encoding CsrB or CsrC sRNA (described in further detail below) can be combined with the transcribed sequence and other regulatory sequences that direct transcription of the sequence in a given cell, e.g., a bacterial cell such as an e. In some embodiments, the expression vector comprising an expression cassette comprising a polynucleotide encoding CsrB or CsrC sRNA further comprises a promoter operably linked to the CsrB or CsrC polynucleotide. In other embodiments, the promoter and/or other regulatory elements directing transcription of the CsrB or CsrC polynucleotide are endogenous to the host cell, and an expression cassette comprising the CsrB or CsrC polynucleotide is introduced, e.g., by homologous recombination, whereby the exogenous CsrB or CsrC polynucleotide is operably linked to the endogenous promoter and expression is driven by the endogenous promoter.

As noted above, expression of a CsrB or CsrC polynucleotide can be controlled by a number of regulatory sequences, including promoters, which can be constitutive or inducible; and optionally including a repressor sequence, if desired. Examples of suitable promoters, in particular in bacterial host cells, are promoters 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. Other examples include promoters such as trp promoter, bla promoter phage lambda PL and T5. In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No. 4,551,433), may be used. Other examples of promoters include Streptomyces coelicolor agar hydrolase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus raw malyase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes. Suitable promoters are also found in Ausubel and Sambrook & Russell, both supra. Other promoters include those described by Jensen & Hammer, appl.environ.Microbiol.64:82,1998; shimada, et al, j.bacteriol.186:7112,2004; and the promoter described in Miksch et al, appl.Microbiol.Biotechnol.69:312,2005.

In some embodiments, promoters that affect expression of a native CsrB or CsrC polynucleotide can be modified to increase expression. For example, the endogenous CsrB or CsrC promoter can be replaced by a promoter that provides increased expression compared to the native promoter.

The expression vector may also comprise other sequences that affect the expression of the polynucleotide encoding CsrB or CsrC sRNA. 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 CsrB or CsrC sRNA 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 ensuring autonomous 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 that allow the vector to be integrated into the host genome.

The expression vector of the invention preferably comprises one or more selectable markers which 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 may 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 such as pUC18 or pUC19, or plasmids derived from these plasmids; and bacteriophages, such as the Ml 3 bacteriophage and the λ bacteriophage. However, one of ordinary skill in the art can readily determine by routine experimentation whether any particular expression vector is appropriate for any given host cell. For example, the expression vector may be introduced into a host cell, and the viability of the host cell and the expression of the sequences contained in the vector may then be monitored.

The expression vectors of the invention can be introduced into host cells using a 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 CsrB or CsrC sRNA. Such host cells can comprise nucleic acids encoding a heterologous CsrB or CsrC sRNA (including any non-naturally occurring CsrB or CsrC sRNA variants); or can be genetically modified to overexpress native CsrB or CsrC sRNA relative to a wild-type host cell.

The genetically modified host strain of the invention typically comprises at least one further genetic modification to enhance the production of lysine or a lysine derivative relative to a cell of a control strain, e.g. a wild-type strain, which is free of said at least one further genetic modification or the same strain which is free of said at least one further genetic modification. "additional genetic modification to enhance the production of lysine or lysine derivatives" 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 lysine or a derivative, such as cadaverine. In some embodiments, the host cell comprises a plurality of modifications to increase production of lysine or lysine derivatives, such as cadaverine, relative to a wild-type host cell.

In some aspects, genetically modifying a host cell to overexpress CsrB or CsrC sRNA can be performed in conjunction with modifying a 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. The enzyme is classified as e.c.4.1.1.18. Lysine decarboxylase polypeptides are well-characterized enzymes, the structures of which are well known in the art (see, e.g., Kanjee, et al., EMBO J.30: 931. ang. 944, 2011; and for reviews, Lemmonier & Lane, Microbiology 144; 751. ang. 760, 1998; and references described therein). The EC number of lysine decarboxylase was 4.1.1.18. Exemplary lysine decarboxylase sequences are CadA homologs from: klebsiella sp, WP 012968785.1; enterobacter aerogenes (Enterobacter aeogenenes), YP 004592843.1; salmonella enterica, WP 020936842.1; serratia sp, WP 033635725.1; raoultella orinitholytica (Raoultella austria), YP 007874766.1; and LdcC homologs from: shigella (Shigellasp.), WP 001020968.1; citrobacter sp, WP 016151770.1; and Salmonella enterica, WP 001021062.1. As used herein, lysine decarboxylase includes variants of native lysine decarboxylase having lysine decarboxylase enzyme activity. Other lysine decarboxylases are described in PCT/CN2014/080873 and PCT/CN 2015/072978.

In some embodiments, the host cell may be genetically modified to express one or more polypeptides that affect lysine biosynthesis. Examples of lysine biosynthetic 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 of other organisms. These genes are known in the art (see, e.g., Shah et al, J.Med.Sci.2:152-157, 2002; Anastassiada, S.Recent Patents on Biotechnol.1:11-24,2007). See also Kind, et al, appl.Microbiol.Biotechnol.91:1287-1296,2011 for reviews on genes involved in cadaverine production. Exemplary genes encoding lysine biosynthetic polypeptides are provided below.

In some embodiments, the host cell is genetically modified to express a lysine decarboxylase, an aspartokinase, a dihydrodipicolinate synthase, a diaminopimelate decarboxylase, an aspartate semialdehyde dehydrogenase, a dihydropicolinate reductase, and an aspartate aminotransferase. Other 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. These 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). See also Kind, et al, appl. Microbiol. Biotechnol.91:1287-1296,2011 for reviews on the attenuation of genes to increase cadaverine production. 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 host cells, e.g., on one expression vector, or simultaneously in multiple expression vectors, along with polynucleotides encoding CsrB or CsrC sRNA. Alternatively, the host cell can be genetically modified to overexpress lysine decarboxylase or one or more lysine biosynthetic polypeptides either before or after the host cell is genetically modified to overexpress CsrB or CsrC sRNA.

In another embodiment, host cells that overexpress naturally occurring CsrB or csrcs rna can be obtained by other techniques, e.g., by mutagenizing cells, such as e.coli cells, and screening the cells to identify those that express CsrB or CsrB at higher levels than the cells prior to mutagenesis.

The host cell for CsrB or CsrC sRNA as described herein is a bacterial host cell. In typical embodiments, the bacterial host cell is a gram-negative bacterial host cell. In some embodiments of the invention, the bacteria are enteric bacteria. In some embodiments of the invention, the bacterium is a species of the taxonomic group corynebacterium, escherichia, Pseudomonas (Pseudomonas), Zymomonas (Zymomonas), Shewanella (Shewanella), salmonella, shigella, Enterobacter (Enterobacter), citrobacter, Enterobacter (Cronobacter), Erwinia (Erwinia), Serratia (serrrata), Proteus (Proteus), haversia, yersinia, Morganella (morgelrana), Edwardsiella (edwards) or 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 escherichia coli, hafnia alvei, or corynebacterium glutamicum host cell.

In some embodiments, the host cell is a gram-positive bacterial host cell, for example a bacillus species, such as bacillus subtilis or bacillus licheniformis; or another bacillus species, such as bacillus alcalophilus (b.alcalophilus), bacillus aminovorans (b.aminovorans), bacillus amyloliquefaciens, bacillus thermolyticus (b.caldolyticus), bacillus circulans (b.circulans), bacillus stearothermophilus, bacillus thermoglucosaccharylase (b.thermogluconasius), 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.

Method for producing lysine or lysine derivative

Host cells genetically modified to overexpress CsrB or CsrC sRNA can be used to produce lysine or derivatives of lysine. In some embodiments, the host cell produces cadaverine. To produce lysine or lysine derivatives, a host cell genetically modified to overexpress CsrB or CsrC sRNA as described herein can be cultured under conditions suitable to allow expression of CsrB or CsrC sRNA and expression of enzymes for producing 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 CsrB or CsrC sRNA at native levels.

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

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

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

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

Examples

The following embodiments are provided by way of example only and are not limiting of the claimed invention.