Method for producing L-amino acids by bacterial fermentation

Abstract

The present invention provides a method for producing L-amino acids by fermentation using a bacterium belonging to the order Enterobacteriaceae that has been modified to attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity (ipdC) and / or a gene encoding a protein having threonine deaminase activity (ilvA).

Description

[Technical field]

[0001] The present invention relates to the microbial industry, and in particular to a method for producing L-amino acids by fermentation of bacteria belonging to the order Enterobacterales that have been modified to attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity and thereby enhance production of L-amino acids. [Background technology]

[0002] Traditionally, L-amino acids have been industrially produced by fermentation using microbial strains or their mutants derived from natural sources, typically modified to increase the production yield of L-amino acids.

[0003] Many techniques have been reported to increase the production yield of L-amino acids, including recombinant DNA. Transformation of microorganisms with (see, e.g., US4278765 A), promoters, leaders Modifications of the expression control region, such as the addition of an attenuator or other sequence known to those skilled in the art (see, for example, US20060216796 A1 and WO9615246 A1). Other techniques for increasing the production yield include increasing the activity of an enzyme involved in amino acid biosynthesis and / or relieving the feedback inhibition of the target enzyme by the produced L-amino acid (see, for example, WO9516042 A1, EP0685555 A1, or US4346170 A, US5661012 A, and US6040160 A).

[0004] Another method for increasing the production yield of L-amino acids includes reducing the expression of one or more genes involved in the degradation of the desired L-amino acid, genes that divert precursors of the L-amino acid from the biosynthetic pathway of the desired L-amino acid, genes involved in the redistribution of carbon, nitrogen, sulfur, and phosphate fluxes, and genes that code for toxins.

[0005] The ipdC gene encodes the IpdC protein, which is characterized as indolepyruvate decarboxylase (Enzyme Commission (EC) number: 4.1.1.74). The indolepyruvate decarboxylase (IpdC) is expressed in various strains of Pantoea including In bacteria, it has been shown to be involved in the biosynthesis of indole-3-acetic acid via indole-3-pyruvate and indole-3-acetaldehyde from tryptophan (Brandl MT and Lindow SE Cloning and characterization of a locus encoding an indolepyruvate decarboxylase involved in indole-3-acetic acid synthesis in Erwinia herbicola, Appl. Environ. Microbiol., 1996, 62(11):4121-4128; Estenson K. et al., Characterization of indole-3-acetic acid biosynthesis and the effects of this phytohormone on the proteome of the plant-associated microbe Pantoea sp. YR343, J. Proteome Res., 2018, 17(4):1361-1374).

[0006] However, no data have been reported so far describing the effect of attenuated expression of the ipdC gene on L-amino acid production by fermentation in L-amino acid-producing bacteria belonging to the order Enterobacteriaceae. Summary of the Invention

[0007] This specification describes an improved method for producing L-amino acids by fermentation with bacteria belonging to the order Enterobacteriale. According to the present invention, the production of L-amino acids by fermentation with bacteria belonging to the order Enterobacteriale can be increased. Specifically, the production of L-amino acids by fermentation with bacteria belonging to the order Enterobacteriale is achieved by attenuating the expression of a gene encoding a protein having indolepyruvate decarboxylase activity, The production of L-amino acids by the modified bacterium can be improved by further modifying the bacterium so that expression of a gene encoding a protein having threonine deaminase activity is attenuated. Further improvements can be achieved by modification.

[0008] The present invention provides the following:

[0009] One embodiment of the present invention is a method for producing an L-amino acid, comprising the steps of: (i) Cultivate L-amino acid producing bacteria belonging to the order Enterobacterales in a medium. Producing and accumulating L-amino acids in a medium or in the bacterial cells of the bacterium, or both; and (ii) recovering the L-amino acid from the medium or from the bacterial cells, or both. Including, The bacterium has been modified to attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity and / or a gene encoding a protein having threonine deaminase activity.

[0010] Another aspect of the present invention is a method for producing an L-amino acid, comprising the steps of: (i) Cultivate L-amino acid producing bacteria belonging to the order Enterobacterales in a medium. Producing and accumulating L-amino acids in a medium or in the bacterial cells of the bacterium, or both; and (ii) recovering the L-amino acid from the medium or from the bacterial cells, or both. Including, The bacterium has been modified to attenuate expression of a gene encoding a protein having indole-pyruvate decarboxylase activity.

[0011] It is another aspect of the present invention to provide the method as described above, wherein the gene encoding a protein having indolepyruvate decarboxylase activity is an ipdC gene.

[0012] It is another aspect of the present invention to provide the method as described above, wherein the protein having indolepyruvate decarboxylase activity is selected from the group consisting of: (A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 55; (B) a protein comprising an amino acid sequence including a substitution, deletion, insertion, and / or addition of 1 to 250 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 or 55, and having indolepyruvate decarboxylase activity; and (C) an amino acid sequence having 50% or more identity to the entire amino acid sequence shown in SEQ ID NO: 2 or 55; A protein comprising an indole acid sequence and having indole-pyruvate decarboxylase activity.

[0013] It is another aspect of the present invention to provide the method as described above, wherein said gene is selected from the group consisting of: (a) a gene comprising the nucleotide sequence shown in SEQ ID NO: 1 or 54; (b) a gene comprising a nucleotide sequence capable of hybridizing under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or 54, and encoding a protein having indole-pyruvate decarboxylase activity; (c) a gene encoding a protein comprising an amino acid sequence including a substitution, deletion, insertion, and / or addition of 1 to 250 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 or 55, wherein the protein has indole-pyruvate decarboxylase activity. , genes; and (d) A gene comprising a mutant nucleotide sequence of SEQ ID NO: 1 or 54, wherein the mutant nucleotide sequence is due to the degeneracy of the genetic code.

[0014] It is another aspect of the present invention to provide the method as described above, wherein expression of a gene encoding a protein having indole-pyruvate decarboxylase activity is attenuated by inactivating said gene.

[0015] It is another aspect of the present invention to provide the method as described above, wherein the gene encoding the protein having indole-pyruvate decarboxylase activity is deleted.

[0016] It is another aspect of the present invention to provide the method as described above, wherein the bacterium has been further modified so that expression of a gene encoding a protein having threonine deaminase activity is attenuated.

[0017] It is another aspect of the present invention to provide the method as described above, wherein the gene encoding a protein having threonine deaminase activity is an ilvA gene.

[0018] It is another aspect of the present invention to provide the method as described above, wherein the bacterium belongs to the family Enterobacteriaceae or Erwiniaceae.

[0019] Another aspect of the present invention is that the bacterium is Escherichia or Pantoea The present invention provides a method for producing a microbial organism that belongs to the genus Pantoea.

[0020] It is another aspect of the present invention to provide the method as described above, wherein said bacterium is Escherichia coli or Pantoea ananatis.

[0021] It is another aspect of the present invention to provide the method as described above, wherein the L-amino acid is selected from the group consisting of aromatic L-amino acids, non-aromatic L-amino acids, and sulfur-containing L-amino acids.

[0022] It is another aspect of the present invention to provide the method as described above, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tryptophan, and L-tyrosine.

[0023] It is another aspect of the present invention to provide the method as described above, wherein the non-aromatic L-amino acid is selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-proline, L-serine, L-threonine, and L-valine.

[0024] It is another aspect of the present invention to provide the method as described above, wherein the sulfur-containing L-amino acid is selected from the group consisting of L-cysteine, L-methionine, L-homocysteine, and L-cystine.

[0025] Still other objects, features and attendant advantages of the present invention will become apparent to those skilled in the art from the following detailed description of embodiments constructed in accordance therewith. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] 1. Bacteria The bacteria described herein are capable of expressing indolepyruvate decarboxylase. The bacterium may be an L-amino acid producing bacterium belonging to the order Enterobacteriale that has been modified to attenuate expression of a gene encoding a protein having L-amino acid decarboxylase activity. The bacterium described herein can be used in the methods described herein. Thus, the following description of the bacterium applies mutatis mutandis to any bacterium that can be used alternatively or equivalently in the methods described herein.

[0027] The bacteria that can be used in the methods described herein may be appropriately selected depending on the type of L-amino acid of interest to be produced using the method.

[0028] Any L-amino acid producing bacterium belonging to the order Enterobacteriaceae can be used in the methods described herein, provided that it has been modified to attenuate the expression of a gene encoding a protein having indolepyruvate decarboxylase activity. It is also acceptable to use any L-amino acid producing bacterium belonging to the order Enterobacteriaceae in the methods described herein, provided that it has been modified to attenuate the expression of a gene encoding a protein having indolepyruvate decarboxylase activity, thereby enhancing the production of L-amino acids compared to unmodified bacteria.

[0029] The term "L-amino acid producing bacteria" may be used interchangeably or equivalently with the terms "bacteria capable of producing L-amino acids", "bacteria having the ability to produce L-amino acids", or "bacteria having the ability to produce L-amino acids".

[0030] The term "L-amino acid producing bacteria" may refer to a bacterium belonging to the order Enterobacteriaceae that has the ability to produce, excrete or secrete, and / or accumulate L-amino acids in a medium and / or in the bacterial body (i.e., the bacterial cell) when the bacterium is cultured in a medium.

[0031] The term "L-amino acid producing bacteria" may also refer to bacteria that have the ability to produce, excrete or secrete, and / or accumulate L-amino acids in a medium in greater amounts than, for example, unmodified bacteria. The term "unmodified bacteria" may be used interchangeably or equivalently with the term "unmodified strain". The term "unmodified strain" may refer to a control strain that has not been modified to attenuate the expression of a gene encoding a protein having indolepyruvate decarboxylase activity. Unmodified bacteria include wild-type or parent strains such as, for example, Pantoea ananatis (P. ananatis) AJ13355 and Escherichia coli (E. coli) K-12 strains (e.g., W3110 (ATCC 27325) and MG1655 (ATCC 47076)). The term "L-amino acid producing bacteria" may also refer to, for example, For example, the desired L-amino acid is cultured at 0.1 g / L or more, 0.5 g / L or more, or 1.0 g / L or more. It may also refer to bacteria that can accumulate in the soil.

[0032] Also usable is a bacterium belonging to the order Enterobacteriaceae, which has been modified to attenuate the expression of a gene encoding a protein having indolepyruvate decarboxylase activity and has an ability to produce L-amino acids. The bacterium may inherently have an ability to produce L-amino acids, or may be modified to have an ability to produce L-amino acids. Such modification can be achieved, for example, by a mutation method or a DNA recombination technique. The bacterium may be a bacterium inherently having an ability to produce L-amino acids. The bacterium can be obtained by attenuating the expression of a gene encoding a protein having indolepyruvate decarboxylase activity in a bacterium. Alternatively, the bacterium can be obtained by imparting L-amino acid producing ability to a bacterium that has already been modified to attenuate the expression of a gene encoding a protein having indolepyruvate decarboxylase activity. Alternatively, the bacterium may have acquired L-amino acid producing ability by being modified to attenuate the expression of a gene encoding a protein having indolepyruvate decarboxylase activity. Specifically, the bacteria described herein can be obtained, for example, by modifying the bacterial strains described below.

[0033] The term "L-amino acid producing ability" may refer to the ability of a bacterium belonging to the order Enterobacteriale to produce, excrete or secrete, and / or accumulate an L-amino acid in a medium and / or bacterial cells. Specifically, the term "L-amino acid producing ability" may refer to the ability of a bacterium belonging to the order Enterobacteriale to produce, excrete or secrete, and / or accumulate an L-amino acid in a medium and / or bacterial cells to an extent that the L-amino acid can be recovered from the medium and / or bacterial cells when the bacterium is cultured in the medium.

[0034] The term "cultured" when referring to bacteria grown according to the methods described herein may be used interchangeably or equivalently with terms such as "cultivated" and "grown", which are well known to those of skill in the art.

[0035] Bacteria can produce L-amino acids alone or as a mixture of the L-amino acid and one or more substances different from the L-amino acid. For example, bacteria can produce a target L-amino acid alone or as a mixture of the target L-amino acid and one or more amino acids different from the target L-amino acid (e.g., L-amino acids (also called L-amino acids), etc.). In other words, it is acceptable that bacteria can produce two or more L-amino acids as a mixture. Furthermore, for example, bacteria can produce a target L-amino acid alone or as a mixture of the target L-amino acid and one or more other organic acids (e.g., carboxylic acids, etc.).

[0036] L-amino acids include, but are not limited to, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, and derivatives thereof.

[0037] Carboxylic acids include, but are not limited to, formic acid, acetic acid, citric acid, butyric acid, lactic acid, propionic acid, and derivatives thereof.

[0038] The terms "L-amino acid" and "carboxylic acid" are not limited to amino acids and carboxylic acids in free form, but may also refer to their derived forms (such as salts, hydrates, adducts, or combinations thereof). Adducts may be compounds formed between amino acids or carboxylic acids and other organic or inorganic compounds. That is, the terms "L-amino acid" and "carboxylic acid" may refer to, for example, L-amino acids and carboxylic acids in free form, derived form, or mixtures thereof. The terms "L-amino acid" and "carboxylic acid" may refer, for example, to L-amino acids and carboxylic acids in free form, salts thereof, or mixtures thereof, among others. The terms "L-amino acid" and "carboxylic acid" may refer to, for example, any of their salts, such as sodium salts, potassium salts, ammonium salts, monohydrates, dihydrates, trihydrates, monohydrochlorides, dihydrochlorides, etc. Unless otherwise specified, the terms "L-amino acid" and "carboxylic acid" without reference to the hydration state (e.g., the terms "L-amino acid in free form", "carboxylic acid in free form", "salt of an L-amino acid", and "salt of a carboxylic acid") can refer to non-hydrated L-amino acids and carboxylic acids, hydrated L-amino acids and carboxylic acids, or mixtures thereof.

[0039] L-amino acids may belong to one or more L-amino acid families. By way of example, L-amino acids include the glutamic acid family, which includes L-arginine, L-glutamic acid, L-glutamine, and L-proline; the serine family, which includes L-cysteine, glycine, and L-serine; the aspartic acid family, which includes L-asparagine, L-aspartic acid, L-isoleucine, L-lysine, L-methionine, and L-threonine; It may belong to the pyruvic acid family, which includes L-alanine, L-isoleucine, L-valine, and L-leucine; and the aromatic family, which includes L-phenylalanine, L-tryptophan, and L-tyrosine. Since L-histidine has an aromatic moiety such as an imidazole ring, the term "aromatic L-amino acid" may refer to L-histidine in addition to the aromatic L-amino acids listed above.

[0040] L-amino acids may also belong to the non-aromatic family, examples of which include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-proline, L-serine, L-threonine, and L-valine. The biosynthetic pathways of aromatic amino acids such as L-phenylalanine, L-tryptophan, and L-tyrosine are different from that of L-histidine, so the term "non-aromatic L-amino acids" may refer to L-histidine in addition to the above non-aromatic L-amino acids.

[0041] Furthermore, the L-amino acid may also belong to the sulfur-containing L-amino acid family, examples of which include L-cysteine, L-methionine, L-homocysteine, and L-cystine. It is known that the biosynthetic pathways of sulfur-containing L-amino acids in bacteria are closely related to each other (see, for example, Ferra MP and Patrick WM, Bacterial methionine biosynthesis, Microbiology, 2014, 160(Pt 8):1571-1584). In particular, in E. coli, L-cysteine ​​is biochemically derived from L-serine. Specifically, L-serine is activated by serine acetyltransferase (CysE) to obtain O-acetylserine, which is then converted to O-acetylserine(thiol)-lyase (CysM) using a reduced sulfur source, such as hydrogen sulfide. ) to produce L-cysteine. L-Cysteine ​​is then converted to L-cysteine ​​via L-cystathionine in a sulfation pathway catalyzed in sequence by O-succinylhomoserine(thiol)-lyase / O-succinylhomoserine lyase (MetB) and cystathionine β-lyase / L-cysteine ​​desulfhydrase (MetC). -homocysteine. L-Methionine is synthesized from L-homocysteine ​​by homocysteine ​​transmethylase (MetE) and / or methionine synthase (MetH). L-Cystine is usually produced in the medium together with L-cysteine ​​as a result of the oxidation of L-cysteine ​​(Nakamori S. et al., Overproduction of L-cysteine ​​and L-cystine by Escherichia coli strains with a genetically altered serine acetyltransferase, Appl. Environ. Microbiol., 1998, 64(5):1607-1611).

[0042] Some L-amino acids may be intermediate amino acids in the biosynthetic pathway of certain L-amino acids, so the above amino acid family may also include other L-amino acids (for example, non-proteinogenic L-amino acids).For example, L-citrulline and L-ornithine are amino acids in the arginine biosynthetic pathway.Therefore, the glutamic acid family may include L-arginine, L-citrulline, L-glutamic acid, L-glutamine, L-ornithine, and L-proline.

[0043] In addition, bacteria belonging to the Enterobacteriaceae family have recently been classified into a phylogenetic tree including 1548 core proteins, 53 ribosomal proteins, and 4 multilocus sequenced proteins. It has been reclassified based on comprehensive comparative genomic analysis (Adelou M. et al., Genome-based phylogeny and taxonomy of the 'Enterobacteriales': proposal for Enterobacterales ord. nov. divided into the families Enterobacteriaceae, Erwiniaceae fam. nov., Pectobacteriaceae fam. nov., Yersiniaceae fam. nov., Hafniaceae fam. nov., Morgan ellaceae fam. nov., and Budviciaceae fam. nov., Int. J. Syst. Evol. Microbiol., 2016, 66:5575-5599).

[0044] Based on the reclassification, bacteria previously classified in the family Enterobacteriaceae are now classified in different families within the order Enterobacteriaceae, such as the family Enterobacteriaceae and the family Erwiniaceae. Based on the above analysis, the bacteria belonging to the order Enterobacteriaceae used in the method described herein are those of the family Enterobacteriaceae and Erwiniaceae, including Enterobacter, Escherichia, Klebsiella, Salmonella, and Elsevier. Erwinia, Pantoea, Morganella, Photorhabdus, Providencia, Yersinia and the like, and may have the ability to produce L-amino acids. Specifically, bacteria classified into the order Enterobacteriales according to the classification used in the NCBI (National Center for Biotechnology Information) database (ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgi?id=91347) can be used. Strains belonging to the order Enterobacteriales that can be modified include bacteria of the family Enterobacteriaceae or Erwiniaceae, specifically bacteria of the genus Escherichia, Enterobacter, or Pantoea.

[0045] The species of bacteria belonging to the genus Escherichia are not particularly limited, and examples thereof include species classified into the genus Escherichia according to classifications known to experts in microbiology. Examples of bacteria belonging to the genus Escherichia include those described in the book by Neidhardt et al. (Bachmann, BJ, Derivations and genotypes of some mutant derivatives of E. coli K-12, p. 2460-2488. In FC Neidhardt et al. (ed.), E. coli and Salmonella: cellular and molecular biology, 2 nd ed. ASM Press, Washington, DC, 1996). Examples of Escherichia bacteria include Escherichia coli (E. coli). The prototype wild-type strain E. coli K-12 (E. coli W3110 (ATCC 27325) and E. coli MG1655 (ATCC 47076), etc.

[0046] The bacteria of the genus Enterobacter are not particularly limited, and include species classified into the genus Enterobacter according to classification known to experts in microbiology. Examples of bacteria of the genus Enterobacter include Enterobacter agglomerans and Enterobacter aerogenes. Specific examples of Enterobacter agglomerans strains include Enterobacter agglomerans ATCC 12287. Specific examples of Enterobacter aerogenes strains include Enterobacter aerogenes ATCC 13048 and NBRC 12010 (Sakai S. and Yaqishita T., Microbial production of hydrogen and ethanol from glycerol-containing wastes discharged from a biodiesel fuel production plant in a bioelectrochemical reactor with thionine, Biotechnol. Bioeng., 2007, 98(2):340-348), AJ110637 (FERM BP-10955). Examples of Enterobacter bacteria strains include those described in European Patent Application Publication No. 0952221. The species of Lomerans is classified as Pantoea agglomerans. This also includes several strains.

[0047] The Pantoea bacteria is not particularly limited, and examples thereof include species classified into the genus Pantoea according to classification known to experts in microbiology. Examples of Pantoea bacteria include Pantoea ananatis (P. ananatis), Pantoea stuartii, ... Examples of the Pantoea ananatis strain include Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Examples of the Pantoea ananatis strain include Pantoea ananatis LMG20103, AJ13355 (FERM BP-6614), AJ13356 (FERM BP-6615), AJ13601 (FERM BP-7207), SC17 (FERM BP-11091), and SC17(0) (VKPM B-9246). Some species of Enterobacter agglomerans Recently, Erwinia ananas (synonym, Erwinia uredovora) and Erwinia stewartii to the Genus Pantoea emend. as Pantoea ananas (Serrano 1928) comb. nov. and Pantoea stewartii (Smith 1898) comb. nov., respectively, and description of Pantoea stewartii subsp. indologenes subsp. nov., Int. J. Syst. Evol. Microbiol., 1993, 43:162-173). The genus Pantoea includes bacteria that have been reclassified into the genus Pantoea.

[0048] Erwinia bacteria include Erwinia amylovora, Erwinia Examples of bacteria belonging to the genus Klebsiella include Erwinia carotovora and Klebsiella planticola.

[0049] These strains can be obtained, for example, from the American Type Culture Collection (Address: PO Box 1549, Manassas, VA 20108, United States of America); each strain has a corresponding accession number, which can be used to order (see atcc.org / ). The accession numbers for each strain are listed in the American Type Culture Collection (Address: PO Box 1549, Manassas, VA 20108, United States of America). Listed in the catalogue of cultural collections.

[0050] The bacterium may be one that inherently has an L-amino acid producing ability, or may be one that has been modified to have an L-amino acid producing ability. Bacteria having an L-amino acid producing ability can be obtained, for example, by imparting an L-amino acid producing ability to the above-mentioned bacteria, or by enhancing the L-amino acid producing ability of the above-mentioned bacteria.

[0051] The L-amino acid producing ability can be imparted or enhanced by a method that has been conventionally employed in breeding amino acid producing bacteria such as Escherichia bacteria (see Amino Acid Fermentation, Academic Press, first edition published May 30, 1986, pp. 77-100). Such a method includes the following: For example, these include obtaining auxotrophic mutants, obtaining strains resistant to L-amino acid analogs, obtaining metabolically controlled mutants, and creating recombinant strains with enhanced activity of L-amino acid biosynthetic enzymes. In breeding L-amino acid producing bacteria, the properties such as auxotrophy, analog resistance, metabolic control mutation, etc., that are imparted may be one, or two or more. In addition, in breeding L-amino acid producing bacteria, the activity of the L-amino acid biosynthetic enzymes that are enhanced may be one, or two or more. Furthermore, imparting properties such as auxotrophy, analog resistance, metabolic control mutation, etc. may be combined with enhancing the activity of the biosynthetic enzymes.

[0052] Auxotrophic mutants, analogue-resistant mutants, or metabolically regulated mutants having L-amino acid production ability can be obtained by subjecting a parent strain or a wild-type strain to a typical mutation treatment and selecting from the obtained mutants those that exhibit auxotrophy, analogue-resistant, or metabolically regulated mutation and have L-amino acid production ability. Typical mutation treatments include irradiation with X-rays or ultraviolet rays, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0053] Alternatively, L-amino acid producing ability can be imparted or enhanced by enhancing the activity of an enzyme involved in the biosynthesis of the target L-amino acid. Enzyme activity can be enhanced, for example, by modifying a bacterium so that expression of a gene encoding the enzyme is enhanced. Methods for enhancing gene expression are described in WO00 / 18935, EP 1010755 A, etc.

[0054] In addition, imparting or enhancing L-amino acid producing ability reduces the activity of an enzyme that catalyzes a reaction that branches off from the biosynthetic pathway of the target L-amino acid to produce a compound other than the target L-amino acid. It can also be achieved by reducing the activity of the enzyme. The "enzyme that catalyzes a reaction that branches off from the biosynthetic pathway of the target L-amino acid to produce a compound other than the target L-amino acid" also includes enzymes involved in the decomposition of the target amino acid. The enzyme activity can be reduced, for example, by modifying the bacterium so that the gene encoding the enzyme is inactivated. Methods for reducing the enzyme activity will be described later.

[0055] Specific examples of L-amino acid producing bacteria and methods for imparting or enhancing L-amino acid producing ability are given below. The properties of L-amino acid producing bacteria and modifications for imparting or enhancing L-amino acid producing ability as exemplified below may be used alone or in appropriate combination.

[0056] L-arginine producing bacteria L-arginine producing bacteria and parent strains that can be used to derive L-arginine producing bacteria include, but are not limited to, E. coli 237 strain (VKPM B-7925, US2002058315 A1) and mutants. and its derivative strain (RU2215783 C2) having N-acetylglutamate synthase type 1, the E. coli 382 strain (VKPM B-7926, EP1170358 A1), an arginine-producing strain (EP1170361 A1) into which the argA gene encoding N-acetylglutamate synthase has been introduced, and the E. coli 382 ilvA+ strain, which is a strain obtained by introducing a wild-type allele of the ilvA gene derived from the E. coli K-12 strain into the 382 strain. Examples of mutant N-acetylglutamate synthase include strains belonging to the genus Escherichia. An example of such an N-acetylglutamate synthase is a mutant N-acetylglutamate synthase in which feedback inhibition by L-arginine is released by substituting amino acid residues corresponding to positions 15 to 19 of the wild-type enzyme (EP1170361 A1).

[0057] L-arginine producing bacteria and parent strains that can be used to derive L-arginine producing bacteria also include strains that have increased expression of one or more genes encoding L-arginine biosynthetic enzymes. Examples of such genes include those encoding N-acetylglutamate synthase (argA), as well as N-acetyl-γ-glutamylphosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), N-acetylornithine aminotransferase (argD), ornithine carbamoyltransferase (argF), argininosuccinate synthase (argG), argininosuccinate lyase (argH), and carbamoyl phosphate synthetase (carAB).

[0058] L-arginine producing bacteria and parent strains that can be used to derive L-arginine producing bacteria also include strains that have resistance to amino acid analogs, etc. Examples of such strains include E. coli mutants (particularly those that have resistance to α-methylmethionine, p-fluorophenylalanine, D-arginine, arginine hydroxamic acid, S-(2-aminoethyl)-cysteine, α-methylserine, β-2-thienylalanine, or sulfaguanidine. (See Patent Publication No. 56-106598).

[0059] L-Aspartic acid producing bacteria L-aspartic acid producing bacteria and parent strains that can be used to derive L-aspartic acid producing bacteria include, but are not limited to, the fumaric acid producing strain CGMCCNO: 2301 (CN101240259 A) of E. coli and its derivatives that contain the gene iclR encoding the glyoxylate cycle regulator. Aspartate-producing strain ΔiclR (CCTCC NO: M 2018521) (CN109370971 A) defective in malate synthetase and isocitrate lyase The aceBA gene, which encodes the aspartate ammonia-lyase (a AS12 (CN105296411 B) with the aspA gene encoding spartate ammonia-lyase inserted Examples of such strains include those belonging to the genus Escherichia.

[0060] E. coli CM-AS-115 (CCTCC NO: M 2016457) (CN106434510 A) was used to directly produce L -It is a genetically engineered strain that can produce aspartic acid. The original strain is wild-type Escherichia coli W1485 (ATCC12435), and by knocking out multiple genes (icdA (EG10489), mdh (EG10576), sfcA (EG10948), maeB (EG14193), and fumAC (EG10356 and EG10358)). The recombinant strain CM-AS-100 was obtained. The mutant strain CM-AS-105 was obtained by subjecting the CM-AS-100 strain to metabolic evolution and acclimation. The E. coli strain CM-AS-115 was obtained by overexpressing two genes (ppc encoding phosphoenolpyruvate carboxylase and aspA encoding aspartase) of the mutant strain CM-AS-105.

[0061] L-aspartic acid producing bacteria and parent strains that can be used to derive L-aspartic acid producing bacteria also include strains that are resistant to aspartic acid analogues. These strains may, for example, lack α-ketoglutarate dehydrogenase activity. Specific examples of strains that are resistant to aspartic acid analogues and lack α-ketoglutarate dehydrogenase activity include E. coli AJ13199 (FERM BP-5807, US5908768) and E. coli AJ13138 (FERM BP-5565, US6110714).

[0062] L-aspartic acid producing bacteria and parent strains that can be used to derive L-aspartic acid producing bacteria include bacteria modified to reduce α-ketoglutarate dehydrogenase (encoded by sucA, sucB, and lpdA) activity, bacteria modified to reduce citrate synthase (gltA) activity, bacteria modified to increase phosphoenolpyruvate carboxylase (ppc) activity, and bacteria modified to increase glutamate dehydrogenase (glutamate dehydrogenase) activity. Increased activity of glutamate synthase (gltBD) or glutamate synthase (gltBD) The bacterium may be further modified to attenuate expression of a gene encoding aspartate ammonia-lyase (aspartase; aspA).

[0063] L-aspartic acid producing bacteria and parent strains that can be used to derive L-aspartic acid producing bacteria also include P. ananatis strain AJ13355 (FERM BP-6614), P. ananatis strain SC17 (FERM BP-11091), and P. ananatis strain SC17(0) (VKPM B-9246). The aspartic acid producing strain 5ΔP2RM (WO2010038905 A1) is a derivative of the SC17(0) strain and contains the following genes: ΔaspA, ΔsucA, ΔgltA, ΔpykA, ΔpykF, Δppc, ppc K620S This strain has been consistently modified with modifications such as ΔmdhA (encoding a feedback-resistant form of E. coli phosphoenolpyruvate carboxylase).

[0064] L-citrulline producing bacteria L-citrulline producing bacteria and parent strains that can be used to derive L-citrulline producing bacteria include, but are not limited to, E. coli 237 / pMADS11, 237 / pMADS12, and 237 / pMADS13 strains carrying mutant N-acetylglutamate synthase. (RU2215783 C2, EP1170361 B1, US6790647 B2), E. coli 333 strain carrying carbamoyl phosphate synthetase resistant to feedback inhibition (VKPM B-8084) and 374 (VKPM B-8086) (R2264459 C2), the activity of α-ketoglutarate synthase was increased and the ferredoxin-NADP + Reductase (ferredoxin NADP +strains belonging to the genus Escherichia, such as E. coli strains (EP2133417 A1) further modified for the activities of succinate dehydrogenase and α-ketoglutarate dehydrogenase; Examples include Pantoea ananantis NA1sucAsdhA strain (US2009286290 A1), which has reduced α-ketoglutarate dehydrogenase activity.

[0065] Since L-citrulline is an intermediate in the L-arginine biosynthetic pathway, L-citrulline-producing bacteria and parent strains that can be used to derive L-citrulline-producing bacteria include strains with increased expression of one or more genes encoding L-arginine biosynthetic enzymes, including, but not limited to, N-acetylglutamate synthase (argA), N-acetylglutamate kinase (argB), N-acetylglutamyl phosphate reductase (argC), acetylornithine transaminase (argD), acetylornithine deacetylase (argE), ornithine carbamoyltransferase (ornithine carbamyltransferase), and / or N-acetylglutamate synthase (argG). Examples of genes encoding carbamoyltransferase (argFI), carbamoyl phosphate synthetase (carAB), and combinations thereof include genes encoding

[0066] In addition, the L-citrulline producing bacteria may be any L-arginine producing bacteria (e.g., E. coli 382 strain). It can be easily obtained by inactivating the argininosuccinate synthase encoded by the argG gene from the recombinant argininosuccinate synthase gene (VKPM B-7926). Methods for inactivation are described herein.

[0067] L-cysteine ​​producing bacteria Examples of L-cysteine ​​producing bacteria and parent strains that can be used to derive L-cysteine ​​producing bacteria include strains in which the activity of one or more L-cysteine ​​biosynthetic enzymes is increased. Such enzymes include, but are not limited to, serine acetyltransferase (cysE) and 3-phosphoglycerate dehydrogenase (serA). Serine acetyltransferase activity can be enhanced, for example, by introducing a mutant cysE gene encoding a mutant serine acetyltransferase that is resistant to feedback inhibition by cysteine ​​into bacteria. Mutant serine acetyltransferases are disclosed, for example, in JP-A-11-155571 and US2005-0112731A. Specific examples of such mutant serine acetyltransferases include wild-type serine acetyltransferases encoded by the cysE5 gene. The Val and Asp residues at positions 95 and 96 of acetyltransferase were replaced with Arg residues, respectively. and Pro residues are substituted (US2005-0112731A). In addition, 3-phosphoglycerate dehydrogenase activity can be enhanced by, for example, introducing a mutant serA gene encoding a mutant 3-phosphoglycerate dehydrogenase resistant to feedback inhibition by serine into a bacterium. Mutant 3-phosphoglycerate dehydrogenases are disclosed, for example, in U.S. Pat. No. 6,180,373.

[0068] L-cysteine ​​producing bacteria and parent strains that can be used to derive L-cysteine ​​producing bacteria also include strains in which the activity of one or more enzymes that catalyze reactions that branch off from the L-cysteine ​​biosynthetic pathway to produce compounds other than L-cysteine ​​is reduced, for example, enzymes involved in the decomposition of L-cysteine. Enzymes involved in the decomposition of L-cysteine ​​include, but are not limited to, cystathionine-β-lyase (metC, JP 11-155571 A; Chandra et al., Biochemistry, 1982, 21:3064-3069), tryptophanase (tnaA, JP 2003-169668 A; Austin Newton et al., J. Biol. Chem., 240 (1965) 1211-1218), O-acetylserine sulfhydrylase B (cysM, JP 2005-245311 A), the malY gene product (JP 2005-245311 A), d0191 of Pantoea ananatis, and the like. Examples of the gene product include cysteine ​​desulfhydrase (aecD, JP 2002-233384).

[0069] It can also be used to induce L-cysteine ​​producing bacteria and L-cysteine ​​producing bacteria. Examples of parent strains include strains in which the activity of the L-cysteine ​​export system and / or the sulfate / thiosulfate transport system is increased. Examples of proteins in the L-cysteine ​​export system include the protein encoded by the ydeD gene (JP Patent Publication 2002-233384), the protein encoded by the yfiK gene, and the protein encoded by the yfiK gene. The genes emrAB, emrKY, yojIH, acrEF, bcr, and cusA Each protein encoded by the yeaS gene (JP Patent Publication 2005-287333) (JP 2010-187552 A). Proteins of the sulfate / thiosulfate transport system include proteins encoded by the cysPTWA gene cluster.

[0070] L-cysteine ​​producing bacteria and parent strains that can be used to derive L-cysteine ​​producing bacteria include, but are not limited to, E. coli JM15 transformed with various cysE alleles encoding mutant serine acetyltransferases resistant to feedback inhibition (US6218168 B1, RU2279477 C2), E. coli W3110 overexpressing a gene encoding a protein suitable for excreting substances toxic to cells (US5972663 A), E. coli strains with reduced cysteine ​​desulfhydrase activity (JP11155571 A2), and E. coli strains with positive cysteine ​​reductase activity encoded by the cysB gene (US6218168 B1, RU2279477 C2). The activity of transcription factors involved in sulfur assimilation was increased in strains belonging to the Escherichia genus, such as E. coli W3110 (WO0127307 A1), and in Pantoea ananatis EYPSG8 and its derivatives. and a strain overexpressing the same (EP2486123 B1).

[0071] L-Glutamic acid producing bacteria L-glutamic acid producing bacteria and parent strains that can be used to derive L-glutamic acid producing bacteria include, but are not limited to, E. coli VL334thrC + (EP1172433 A1) and other Escherichia spp. E. coli VL334 (VKPM B-1641) is an auxotroph for L-isoleucine and L-threonine with mutations in the thrC and ilvA genes (US4278765). The wild-type allele of the thrC gene was introduced by transduction. As a result, the L-isoleucine auxotrophic strain VL334thrC, which can produce L-glutamic acid, was obtained. + (VKPM B-8961) was obtained.

[0072] L-glutamic acid producing bacteria and parent strains that can be used to derive L-glutamic acid producing bacteria include, but are not limited to, strains that have increased expression of one or more genes encoding L-glutamic acid biosynthetic enzymes. Such genes include glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthase (gltBD), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), methylcitrate synthase (prpC), pyruvate synthase (prpD), pyruvate synthase (prpE), pyruvate synthase (prpF ... pyruvate carboxylase (pyc), phosphoenolpyruvate carboxylase (ppc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), Phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglyceryl esterase (EGSA), pgmA, pgmI, phosphoglycerate kinase (pgk), glyceraldehyde-3- phosphate dehydrogenase (gapA), triosephosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), glucose Glucosyl phosphate isomerase (pgi), 6-phosphogluconate dehydratase (edd), 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), transhydrogenase Examples of genes encoding pntAB include

[0073] Examples of strains modified to increase expression of the citrate synthase gene, the phosphoenolpyruvate carboxylase gene, and / or the glutamate dehydrogenase gene include those disclosed in EP1078989 A2, EP955368 A2, and EP952221 A2. Examples of strains modified to increase expression of the Entner-Doudoroff pathway genes (edd, eda) include those disclosed in EP1352966B.

[0074] L-glutamic acid producing bacteria and parents that can be used to derive L-glutamic acid producing bacteria The strain may also have reduced or eliminated activity of an enzyme that catalyzes the biosynthesis of a compound other than L-glutamic acid by branching off from the L-glutamic acid biosynthesis pathway, including, but not limited to, isocitrate lyase (aceA), α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxyacid synthase (ilvG), acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), glutamate decarboxylase (gadAB), succinate dehydrogenase (sdhABCD), and 1-pyrroline-5-carboxylate dehydrogenase (putA).

[0075] Escherichia bacteria lacking or with reduced α-ketoglutarate dehydrogenase activity, The methods for obtaining them are described in US 5,378,616 and US 5,573,945. Specific examples of these strains include the following: E. coli W3110sucA::Km R E. coli AJ12624 (FERM BP-3853) E. coli AJ12628 (FERM BP-3854) E. coli AJ12949 (FERM BP-4881)

[0076] E. coli W3110sucA::Km R The α-ketoglutarate dehydrogenase gene of E. coli W3110 was This strain was obtained by disrupting the sucA gene. This strain is completely deficient in α-ketoglutarate dehydrogenase.

[0077] Examples of L-glutamic acid producing bacteria and parent strains that can be used to derive L-glutamic acid producing bacteria include Pantoea bacteria such as P. ananatis AJ13355 strain (FERM BP-6614), P. ananatis SC17 strain (FERM BP-11091), and P. ananatis SC17(0) strain (VKPM B-9246). The AJ13355 strain was isolated from soil in Iwata City, Shizuoka Prefecture, as a strain capable of growing in a medium containing L-glutamic acid and a carbon source at low pH. The SC17 strain was isolated from the AJ13355 strain as a low-mucus producing mutant. The SC17 strain was selected on February 4, 2009, by the Independent Administrative Agency of Japan. National Institute of Technology, Patent Organism Depository Center (currently National Institute of Technology and Evaluation, Patent Organism Depository Center (NITE IPOD), Postal Code: 292-0818, Address: Kisarazu, Chiba Prefecture, Japan The AJ13355 strain was deposited on February 19, 1998 at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently NITE IPOD, Postal Code: 292-0818, Address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number FERM P-16644, and was deposited under the international depository system under the Budapest Treaty on January 11, 1999. The SC17(0) strain was transferred to the Russian National Collection of Industrial Microorganisms (VKPM; FGUP GosNII Genetika, Russian Federation, 117545 Moscow, Russia) on September 21, 2005 and assigned the accession number FERM BP-6614. st It has been deposited in the Dorozhny proezd, 1) under the accession number VKPM B-9246.

[0078] L-glutamic acid producing bacteria and parent strains that can be used to derive L-glutamic acid producing bacteria include Pantoea spp. with deficient or reduced α-ketoglutarate dehydrogenase activity. The mutant strains belonging to the group A and B can be obtained as described above. Such a strain is a mutant strain lacking the E1 subunit gene (sucA) of α-ketoglutarate dehydrogenase of the AJ13355 strain. Examples of such strains include P. ananatis AJ13356 (US6331419 B1), which is a strain of P. ananatis, and Pantoea ananatis SC17sucA (US6,596,517), which is a sucA gene-deficient strain of the SC17 strain. P. ananatis AJ13356 was deposited on February 19, 1998 at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently NITE IPOD, postal code: 292-0818, address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number FERM P-16645, and was transferred to an international deposit based on the Budapest Treaty on January 11, 1999, and was assigned the accession number FERM BP-6615. At the time of isolation, the AJ13356 strain was identified as Enterobacter agglomerans and deposited as Enterobacter agglomerans AJ13356. However, in recent years, this strain has been reclassified as P. ananatis based on the analysis of the 16S rRNA base sequence. AJ13356 has been deposited at the above depository institution as Enterobacter agglomerans. However, for the purposes of this specification it is referred to as P. ananatis.

[0079] Examples of L-glutamic acid producing bacteria and parent strains that can be used to derive L-glutamic acid producing bacteria include strains belonging to the genus Pantoea, such as P. ananatis SC17sucA / RSFCPG+pSTVCB, P. ananatis AJ13601, P. ananatis NP106, and P. ananatis NA1. The SC17sucA / RSFCPG+pSTVCB strain is a strain that has been modified by adding the citrate synthase gene (gltA) and phosphoenolpyruvate carboxylase gene (ppc) derived from Escherichia coli (E. coli) to the SC17sucA strain. , and the plasmid RSFCPG containing the glutamate dehydrogenase gene (gdhA), and the Brevibacterium lactofermentum-derived The AJ13601 strain was obtained by introducing the plasmid pSTVCB containing the citrate synthase gene (gltA) of the SC17sucA / RSFCPG+pSTVCB strain. The NP106 strain was obtained by removing the plasmids RSFCPG and pSTVCB from the AJ13601 strain. The NA1 strain was obtained by introducing the plasmid RSFPPG into the NP106 strain (EP2336347 A1). The AJ13601 strain was obtained on August 18, 1999, at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently NITE IPOD, Postal Code: 292-0818, Address: Kazusa, Kisarazu City, Chiba Prefecture, Japan). The material was deposited at the University of Tokyo (Room 120, 2-5-8 Kamatari) under the accession number FERM P-17516 and transferred to the Budapest University Hospital on July 6, 2000. The material has been transferred to international deposit under the Treaty on Biological Digestion and has been assigned the accession number FERM BP-7207.

[0080] L-glutamic acid producing bacteria and parent strains that can be used to derive L-glutamic acid producing bacteria also include auxotrophic mutants. Specific examples of auxotrophic mutants include E. coli VL334thrC. + (VKPM B-8961; EP1172433). E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotrophic strain having mutations in the thrC gene and the ilvA gene (U.S. Patent No. 4,278,765). + The thrC gene The L-isoleucine-requiring L -glutamic acid producing bacteria. The wild-type allele of the thrC gene was introduced by a general transduction method using bacteriophage P1 grown in cells of the wild-type E. coli K-12 strain (VKPM B-7).

[0081] L-glutamic acid producing bacteria and parent strains that can be used to derive L-glutamic acid producing bacteria also include strains that are resistant to aspartic acid analogues. These strains may, for example, lack α-ketoglutarate dehydrogenase activity. Specific examples of strains that are resistant to aspartic acid analogues and lack α-ketoglutarate dehydrogenase activity include E. coli AJ13199 (FERM BP-5807; US5908768), and E. coli FERM P-12379 (US5393671) and E. coli AJ13138 (FERM BP-5565; US6110714) that have reduced L-glutamic acid decomposition ability.

[0082] L-histidine producing bacteria Specific examples of L-histidine producing bacteria and parent strains that can be used to derive L-histidine producing bacteria include, but are not limited to, E. coli 24 strain (VKPM B-5945; RU2003677 C1), E. coli 80 strain (VKPM B-7270, RU2119536 C1), E. coli NRRL B-12116 to B-12121 (US4388405), E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (US6344347 B1), E. coli H-9341 (FERM BP-6674; EP1085087 A2), E. coli AI80 / pFM201 (US6258554 B1) Examples of such strains include those belonging to the genus Escherichia.

[0083] L-histidine producing bacteria and parent strains that can be used to derive L-histidine producing bacteria also include strains that have increased expression of one or more genes encoding L-histidine biosynthetic enzymes. Such genes include ATP phosphoribosyltransferase (hisG), phosphoribosyl-ATP pyrophosphatase (hisE), phosphoribosyl-AMP cyclohydrolase (hisI), bifunctional phosphoribosyl-AMP cyclohydrolase (hisI), and phospholipase A (phospholipase B) enzymes. Dorolase / phosphoribosyl-ATP pyrophosphatase (hisIE), phosphoribosyl Examples of such genes include genes encoding formimino-5-aminoimidazolecarboxamide ribotide isomerase (hisA), amidotransferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), histidinol dehydrogenase (hisD), and the like.

[0084] It is known that the L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are inhibited by L-histidine, and therefore the L-histidine-producing ability can be efficiently enhanced by, for example, introducing a mutation that confers resistance to feedback inhibition to ATP phosphoribosyltransferase (RU2003677 C1 and RU2119536 C1).

[0085] Specifically, L-histidine-producing bacteria and parent strains that can be used to derive L-histidine-producing bacteria include vectors carrying DNA encoding L-histidine biosynthetic enzymes. Transformed E. coli FERM P-5038 and FERM P-5048 (JP56-005099 A), for amino acid excretion The E. coli strain 80 (VKPM B-7270; RU2119536 C1) was transformed with the rht gene, which is a nucleotide sequence of the E. coli MG1655+hisGr hisL'_Δ gene, and was conferred resistance to sulfaguanidine, DL-1,2,4-triazole-3-alanine, and streptomycin. ΔpurR (RU2119536 and Doroshenko VG et al., The directed modification of Escherichia coli MG1655 to obtain histidine-producing mutants, Prikl. Biochim. Mikrobiol. (Russian), 2013, 49(2):149-154).

[0086] L-isoleucine producing bacteria L-isoleucine producing bacteria and parent strains that can be used to derive L-isoleucine producing bacteria include, but are not limited to, mutants resistant to 6-dimethylaminopurine (JP 5-304969 A), isoleucine analogs such as thiaisoleucine and isoleucine hydroxamate, Examples of such mutants include mutants resistant to arginine, and mutants resistant to DL-ethionine and / or arginine hydroxamate (JP5-130882 A). Recombinant strains transformed with genes encoding proteins involved in the L-isoleucine biosynthesis system, such as threonion deaminase and acetohydroxyacid synthase, can also be used as L-isoleucine producing bacteria or parent strains (JP2-458 A, EP0356739 A1, and US5998178).

[0087] L-leucine producing bacteria L-leucine producing bacteria and parent strains that can be used to derive L-leucine producing bacteria include, but are not limited to, leucine resistant E. coli strains (e.g., strain 57 (VKPM B-7386; US6124121)), β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucin, 5, E. coli strains resistant to leucine analogues such as 5,5-trifluoroleucine (JP62-34397 B and JP8-70879 A), E. coli strains obtained by the genetic engineering methods described in WO9606926, and strains belonging to the genus Escherichia such as E. coli H-9068 (JP8-70879 A).

[0088] L-leucine producers and parent strains that can be used to derive L-leucine producers include strains that have increased expression of one or more genes involved in L-leucine biosynthesis. Such genes include the genes of the leuABCD operon, which are A representative example is a mutant leuA gene (US6403342 B1) encoding α-isopropylmalate synthase that is desensitized to feedback inhibition by L-leucine. L-leucine producing bacteria and parent strains that can be used to derive L-leucine producing bacteria also include strains in which the expression of one or more genes encoding proteins that excrete L-amino acids from bacterial cells is increased. Examples of such genes include the b2682 and b2683 genes (ygaZH Genes) are included (EP1239041 A2).

[0089] L-lysine producing bacteria L-lysine producing bacteria and parent strains that can be used to derive L-lysine producing bacteria include mutant strains belonging to the genus Escherichia and having resistance to L-lysine analogues. L-lysine analogues inhibit the growth of bacteria belonging to the genus Escherichia, and this inhibition is When lysine is present in the medium, it is completely or partially desensitized. L-lysine analogs include, but are not limited to, oxalysine, lysine hydroxamate, S-(2-aminoethyl)-L-cysteine ​​(AEC), γ-methyllysine, α-chlorocaprolactam, etc. Mutants resistant to these lysine analogues include Escherichia sp. L-lysine can be obtained by subjecting bacteria belonging to the species E. coli to a conventional artificial mutation treatment. Specific examples of bacterial strains useful for producing L-lysine include E. coli AJ11442 (FERM BP-1543, NRRL B-12185; see US4346170) and E. coli VL611. In these strains, The feedback inhibition of aspartokinase by L-lysine is relieved.

[0090] L-lysine producing bacteria and parent strains that can be used to derive L-lysine producing bacteria include strains that have increased expression of one or more genes encoding L-lysine biosynthetic enzymes, including, but not limited to, dihydrodipicolinate synthase (dapA), aspartokinase III (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (diaminopimelate dehydrogenase), and the like. (ddh) (US6040160), phosphoenolpyruvate carboxylase (ppc), aspartate semialdehyde dehydrogenase (asd), aspartate aminotransferase (aspartate transaminase) (aspC), diaminopimelate epimerase (dapF), tetrahydrodipicolinate succinylase (dapD), succinyl diaminopimelate deacylase (succin ... deacylase (dapE), and aspartase (aspA) (EP1253195 A1) In addition, the parent strain may have an increased expression level of a gene involved in energy efficiency (cyo) (EP1170376 A1), a gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (US5830716 A), a ybjE gene (WO2005073390), or a combination thereof. Aspartokinase III (lysC) is subject to feedback inhibition by L-lysine. To enhance the activity of this enzyme, a mutant lysC gene encoding aspartokinase III that is desensitized to feedback inhibition by L-lysine may be used (US Pat. No. 5,932,453). Examples of aspartokinase III that is desensitized to feedback inhibition by L-lysine include a mutation in which the methionine residue at position 318 is replaced with an isoleucine residue, and a mutation in which the glycine residue at position 323 is replaced with an asparagine residue. and a mutation that replaces the threonine residue at position 352 with an isoleucine residue. Examples of the aspartokinase III from Escherichia coli include those having one or more mutations selected from the following mutations (U.S. Patent Nos. 5,661,012 and 6,040,160). Dihydrodipicolinate synthase (dapA) is an enzyme that synthesizes dihydrodipicolinate from L-lysine. Since the enzyme is subject to feedback inhibition, a mutant dapA gene encoding a dihydrodipicolinate synthase that is desensitized to feedback inhibition by L-lysine may be used to enhance the activity of the enzyme. The dihydrodipicolinate synthase that is desensitized to feedback inhibition by L-lysine has a mutation in which the histidine residue at position 118 is replaced with a tyrosine residue. An example of such an enzyme is dihydrodipicolinate synthase from Escherichia coli (U.S. Patent No. 6,040,160).

[0091] In the L-lysine producing bacteria or parent strain that can be used to derive the L-lysine producing bacteria, the activity of an enzyme that catalyzes a reaction that branches off from the L-amino acid biosynthetic pathway to produce other compounds may be reduced or eliminated. In the parent strain that can be used for this purpose, the activity of an enzyme that has a negative effect on the synthesis or accumulation of L-lysine may be reduced or eliminated. Examples of such enzymes include homoserine dehydrogenase, lysine decarboxylase (cadA, ldcC), malic enzyme, etc., and strains in which the activities of these enzymes are reduced or deleted are disclosed in WO9523864, WO9617930, WO2005010175, etc.

[0092] In order to reduce or eliminate the lysine decarboxylase activity, the expression of both the cadA and ldcC genes encoding lysine decarboxylase can be reduced. The expression of both genes can be reduced, for example, by the method described in WO2006078039.

[0093] L-lysine-producing bacteria or parent strains that can be used to derive L-lysine-producing bacteria also include E. coli WC196 strain (FERM BP-5252; US5827698), E. coli WC196ΔcadAΔldcC strain (FERM BP-11027; also referred to as "WC196LC"), and E. coli WC196ΔcadAΔldcC / pCABD2 strain (WO2006078039).

[0094] The WC196 strain was developed by conferring AEC resistance to the W3110 strain derived from E. coli K-12. The WC196 strain was named E. coli AJ13069 and was transferred to the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently NITE IPOD, Postal Code: 292-0818, Address: Chiba, Japan) on December 6, 1994. The material was originally deposited at the National Institute of Biological Sciences (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Tokyo) under the accession number FERM P-14690, and was transferred to an international deposit under the Budapest Treaty on September 29, 1995, and assigned the accession number FERM BP-5252 (US5827698).

[0095] The WC196ΔcadAΔldcC strain was constructed by disrupting the cadA and ldcC genes encoding lysine decarboxylase from the WC196 strain. The WC196ΔcadAΔldcC strain was designated AJ110692. The name was changed to National Institute of Advanced Industrial Science and Technology (AIST) Patent Biological Deposit Center on October 7, 2008. It was deposited internationally at the NITE IPOD (currently NITE IPOD, postal code: 292-0818, address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number FERM BP-11027.

[0096] The WC196ΔcadAΔldcC / pCABD2 strain contains lysine biosynthesis genes in the WC196ΔcadAΔldcC strain. The plasmid pCABD2 (US6040160) was constructed by introducing the L-lysine Escherichia coli carrying a mutation (H118Y) that relieves feedback inhibition by gin A mutant dapA gene encoding dihydrodipicolinate synthase (DDPS) from E. coli and Escherichia coli with a mutation (T352I) that relieves feedback inhibition by L-lysine. A mutant lysC gene encoding aspartokinase III from E. coli and a mutant lysC gene encoding dihydrodipicolinate reductase from E. coli were The gene contains the dapB gene encoding the α-aminopimelate dehydrogenase from Brevibacterium lactofermentum and the ddh gene encoding the diaminopimelate dehydrogenase from Brevibacterium lactofermentum.

[0097] Another example of an L-lysine producing bacterium or a parent strain that can be used to derive an L-lysine producing bacterium is the E. coli AJIK01 strain (NITE BP-01520). The AJIK01 strain is a clone of E. coli AJ111046. The DNA was named after the author and deposited at NITE IPOD (Address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, Postal Code: 292-0818) on January 29, 2013, and transferred to international deposit under the Budapest Treaty on May 15, 2014, and assigned the accession number NITE BP-01520.

[0098] L-Methionine producing bacteria L-methionine producing bacteria and parent strains that can be used to derive L-methionine producing bacteria include, but are not limited to, E. coli AJ11539 strain (NRRL B-12399), AJ11540 strain (NRRL B-12400), AJ11541 strain (NRRL B-12401), AJ 11542 strain (NRRL B-12402) (Patent GB2075055); E. coli 218 strain resistant to norleucine, an L-methionine analog (VKPM B-8125; RU2 209248 C2) and 73 strains belonging to the genus Escherichia (VKPM B-8126; RU2215782 C2); The E. coli 218 strain was transferred to the Russian National Collection of Industrial Microorganisms (VKPM; Russian Federation, 117545 Moscow, Russia) on May 14, 2001. st The E. coli strain 73 was deposited at the Russian National Collection of Industrial Microorganisms (VKPM; Russian Federation, 117545 Moscow, Russia) under the accession number VKPM B-8125 and transferred to the international depository under the Budapest Treaty on February 1, 2002. The E. coli strain 73 was deposited at the Russian National Collection of Industrial Microorganisms (VKPM; Russian Federation, 117545 Moscow, Russia) under the accession number VKPM B-8125 and transferred to the international depository under the Budapest Treaty on February 1, 2002. st The strain was deposited in the Dorozhny proezd, 1) under the accession number VKPM B-8126, and transferred to the international depository under the Budapest Treaty on February 1, 2002. In addition, a methionine repressor-deficient strain and a recombinant strain (JP2000-139471 A) transformed with genes encoding proteins involved in L-methionine biosynthesis (homoserine transsuccinylase, cystathionine γ-synthase, etc.) are also available as L-methionine-producing bacteria or parent strains. It can be used as:

[0099] To induce L-methionine producing bacteria and L-methionine producing bacteria of Escherichia spp. Another example of a parent strain that can be used is a strain that lacks the repressor of L-methionine biosynthesis (MetJ) and has intracellular homoserine transsuccinylase (MetA) activity. An E. coli strain (US7611873 B1) with increased cytotoxicity, suppressed cobalamin-independent methionine synthase (MetE) activity and increased cobalamin-dependent methionine synthase (MetH) activity. The strain of E. coli (EP2861726 B1) has the ability to produce L-threonine and is capable of converting threonine derivatives into L-threonine. threonine dehydratase (tdcB, ilvA) and at least O-succinimidyl O-succinylhomoserine lyase (metB), cystathionine beta-lyase (metC), 5,10-methylenetetrahydrofolate reductase (metF), and serine hydroxymethyltransferase (glyA). An E. coli strain (US7790424 B2) transformed with a vector expressing Transhydrogenase The strain may be an E. coli strain (EP2633037 B1) having enhanced transhydrogenase (pntAB) activity.

[0100] L-methionine producing bacteria lack the gene encoding cysteine ​​synthase. The gene may be modified to overexpress the gene.

[0101] The term "cysteine ​​synthase-encoding gene" may refer to a gene that encodes cysteine ​​synthase. The term "cysteine ​​synthase-encoding gene" may refer to a protein having cysteine ​​synthase activity (EC 2.5.1.47). Examples of cysteine ​​synthase-encoding genes include the cysM gene and the cysK gene. The cysM gene may encode cysteine ​​synthase B using thiosulfate as a substrate. The cysK gene may encode cysteine ​​synthase A using sulfide as a substrate. A specific example of a cysteine ​​synthase-encoding gene is the cysM gene specific to P. ananatis. The nucleotide sequence of the cysM gene specific to P. ananatis is shown in SEQ ID NO:13.

[0102] The L-methionine producing bacterium may be modified to have a mutant metA gene.

[0103] The metA gene encodes homoserine transsuccinylase MetA (EC 2.3.1.46). The term "mutated metA gene" may refer to a gene encoding a mutant MetA protein. The term "mutated MetA protein" may refer to a gene encoding a mutant MetA protein having an R34C mutation (wherein the arginine (Arg) residue at position 34 in the amino acid sequence of the wild-type MetA protein is an arginine (Arg) residue at position 34 in the amino acid sequence of the wild-type MetA protein). "The term "MetA" may refer to a MetA protein having a mutation in which a cysteine ​​(Cys) residue is substituted with a cysteine ​​(Cys) residue." The term "wild-type metA gene" may refer to a gene encoding a wild-type MetA protein. The term "wild-type MetA protein" may refer to a MetA protein that does not have the R34C mutation. The wild-type metA gene includes the metA gene specific to P. ananatis and its mutants, Examples of wild-type MetA proteins include those that do not have a mutation that results in the R34C mutation in the protein that is regulated by the enzyme. Examples of wild-type MetA proteins include the MetA protein specific to P. ananatis and its mutants that have the R34C mutation. In other words, the mutant metA gene may be identical to any wild-type metA gene except for the mutation that results in the R34C mutation in the encoded protein. The mutant MetA protein may be identical to any wild-type MetA protein except for the R34C mutation. The amino acid sequence of the mutant MetA protein is shown in SEQ ID NO: 35. Specifically, an example of the amino acid sequence of the mutant MetA protein may be the amino acid sequence shown in SEQ ID NO: 37, which may be encoded by a mutant metA gene having the nucleotide sequence shown in SEQ ID NO: 36. That is, the mutant metA gene may be a gene (e.g., DNA) having the nucleotide sequence shown in SEQ ID NO: 36, and the mutant MetA protein may be The mutant metA gene may be a protein having the amino acid sequence shown in SEQ ID NO: 37. The mutant metA gene may be a gene (e.g., DNA) having a mutant nucleotide sequence of SEQ ID NO: 36, which has a mutation that results in the R34C mutation in the encoded protein. The mutant MetA protein may be a protein having a mutant amino acid sequence of SEQ ID NO: 37, which has an R34C mutation. The mutant MetA protein may be a homosine transsuccinylase resistant to feedback inhibition by L-methionine. In other words, the mutant MetA protein may have homosine transsuccinylase activity and be resistant to feedback inhibition by L-methionine. The description of the gene encoding a protein having indolepyruvate decarboxylase activity and the mutant indolepyruvate decarboxylase encoded thereby described below can also be applied mutatis mutandis to mutants of metA gene and mutants of MetA protein. The term "position 34" does not necessarily indicate an absolute position in the amino acid sequence of the wild-type MetA protein, but indicates a relative position based on the amino acid sequence shown in SEQ ID NO: 35 in the wild-type MetA protein.

[0104] The L-methionine producing bacterium may be modified so that expression of the metJ gene is weakened.

[0105] The metJ gene encodes the Met repressor, which regulates expression of the methionine regulon and S -The expression of the enzyme involved in adenosylmethionine (SAM) synthesis can be suppressed. Examples of such genes include those specific to host bacteria such as P. ananatis. The nucleotide sequence of the metJ gene specific to P. ananatis is shown in SEQ ID NO:24.

[0106] The L-methionine producing bacterium may be modified to have a mutant thrA gene encoding a mutant aspartokinase homoserine dehydrogenase I that is resistant to feedback inhibition by threonine. An example of the gene is the thrA442 gene.

[0107] The L-methionine producing bacteria may be modified to overexpress the aminomethyltransferase gene.

[0108] The term "aminomethyltransferase gene" may refer to a gene encoding an aminomethyltransferase. The term "aminomethyltransferase" may refer to a protein having aminomethyltransferase activity (EC 2.1.2.10). Examples of aminomethyltransferase genes include the gcvT gene. Examples include genes.

[0109] L-methionine producing bacteria of the genus Pantoea and used to induce L-methionine producing bacteria The parent strain from which the fermentation can be performed is not particularly limited, but the P. ananatis AJ13355 strain (FERM BP-6614) is preferred. This strain is also known as P. ananatis SC17 strain (FERM BP-11091).

[0110] L-ornithine producing bacteria Since L-ornithine is an intermediate in the L-arginine biosynthetic pathway, L-ornithine producing bacteria and parent strains that can be used to derive L-ornithine producing bacteria include strains with increased expression of one or more genes (such as those listed above) encoding L-arginine biosynthetic enzymes.

[0111] L-ornithine producing bacteria can be easily obtained from any L-arginine producing bacteria (such as E. coli 382 strain (VKPM B-7926)) by inactivating ornithine carbamoyltransferase encoded by both argF and argI genes. Methods for inactivating the genes are described herein.

[0112] L-Phenylalanine producing bacteria L-phenylalanine producing bacteria and parent strains that can be used to derive L-phenylalanine producing bacteria include, but are not limited to, E. coli AJ12739 (tyrA::Tn10, tyrR) (VKPM B-8197), E. coli HW1089 carrying a mutant pheA34 gene (ATCC 55371; US5354672), E. coli MWEC101-b (KR8903681), E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146, and NRRL B-12147 (US4407952), E. coli K-12 [W3110 (tyrA) / pPHAB] (FERM BP-3566), E. coli K-12 [W3110 (tyrA) / pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA) / pPHATerm] (FERM BP-12662), and E. coli K-12 [W3110 (tyrA) / pBR-aroG4, pACMAB] (FERM BP-3579, EP488424 B1) designated as AJ12604. Furthermore, L-phenylalanine producing bacteria and parent strains that can be used to derive L-phenylalanine producing bacteria include L-phenylalanine producing bacteria belonging to the genus Escherichia in which the activity of the protein encoded by the yedA gene or the yddG gene is enhanced. (US7259003 and US7666655).

[0113] L-Proline producing bacteria L-proline producing bacteria and parent strains that can be used to derive L-proline producing bacteria include, but are not limited to, E. coli 702ilvA, which is deficient in the ilvA gene and capable of producing L-proline. (VKPM B-8012, EP1172433 A1). L-proline producing bacteria and parent strains that can be used to derive L-proline producing bacteria also include strains in which the expression of one or more genes involved in L-proline biosynthesis is enhanced. An example of such a gene that can be used in L-proline producing bacteria is the proB gene encoding glutamate kinase desensitized to feedback inhibition by L-proline (DE3127361 A1). Furthermore, L-proline producing bacteria and parent strains that can be used to derive L-proline producing bacteria include strains in which expression of one or more genes encoding proteins responsible for the export of L-amino acids from bacterial cells is enhanced. Examples of such genes include the b2682 gene and the b2683 gene (the ygaZH gene). Examples include the ribosome-specific endothelial cell line (EP1239041 A2).

[0114] Examples of bacteria belonging to the genus Escherichia having L-proline producing ability include the bacteria described in NRRL B-12403 and NRRL B-12404 (GB2075056), VKPM B-8012 (RU2207371 C2), and DE3127361 A1. Smid mutants, Bloom FR et al. in < <The 15 th Examples of such E. coli strains include the plasmid mutants described in Miami winter symposium>>, 1983, p. 34.

[0115] L-Threonine producing bacteria L-threonine producing bacteria and parent strains that can be used to derive L-threonine producing bacteria include, but are not limited to, E. coli TDH-6 / pVIC40 (VKPM B-3996; US5175107 and US5705371), E. coli 472T23 / pYN7 (ATCC 98081; US5631157), E. coli NRRL-21593 (US 5939307), E. coli FERM BP-3756 (US5474918), E. coli FERM BP-3519 and FERM BP-3520 (US5376538), E. coli MG442 (Gusyatiner MM et al., Study of relA gene function II. Effect of the relA gene allelic state on threonine over-production by Escherichia coli K-12 mutants resistant to β-hydroxynorvaline acid, Genetika (Russian), 1978, 14(6):957-968), E. coli VL643 and VL2055 (EP1149911 A2), and E. coli VKPM B-5318 (EP0593792 A1).

[0116] The TDH-6 strain lacks the thrC gene and is sucrose-utilizing, and its ilvA gene is a leak This strain also has a leaky mutation in the rhtA gene that inhibits the synthesis of threonine at high concentrations or The VKPM B-3996 strain contains a mutation that confers resistance to homoserine. This strain was obtained by introducing the plasmid pVIC40 into the TDH-6 strain. The plasmid pVIC40 was obtained by inserting the thrA*BC operon containing the mutant thrA gene into a vector derived from RSF1010. The mutant thrA gene encodes aspartokinase homoserine dehydrogenase I, which is substantially desensitized to feedback inhibition by threonine. The VKPM B-3996 strain was received at the All-Union Scientific Center of Antibiotics (Russian Federation, 117105 Moscow, Nagatinskaya Street 3-A) on November 19, 1987. The VKPM B-3996 strain was deposited on April 7, 1987 at the Russian National Collection of Industrial Microorganisms (VKPM; Russian Federation, 117545 Moscow, Russia) under the accession number RIA 1867. st It has also been deposited in the US Pat. No. 6,399,433 (Dorozhny proezd, 1) under accession number B-3996.

[0117] The VKPM B-5318 strain is non-requiring for isoleucine, and the control region of the threonine operon in the plasmid pVIC40 has been replaced by a temperature-sensitive lambda phage C1 repressor and PR promoter. The VKPM B-5318 strain was deposited at the Russian National Collection of Industrial Microorganisms (VKPM) on May 3, 1990 under the accession number VKPM B-5318.

[0118] The L-threonine producing bacteria or parent strains that can be used to derive the L-threonine producing bacteria may be further modified to enhance the expression of one or more of the following genes: good: - a mutant thrA gene encoding aspartokinase homoserine dehydrogenase I that is resistant to feedback inhibition by threonine; - the thrB gene encoding homoserine kinase, - the thrC gene encoding threonine synthase; - the rhtA gene, which encodes a putative transmembrane protein of the threonine and homoserine export system; - the asd gene, which codes for aspartate-β-semialdehyde dehydrogenase, - the aspC gene, which codes for aspartate aminotransferase (aspartate transaminase).

[0119] thrA, which encodes aspartokinase I and homoserine dehydrogenase I in E. coli The genes have been identified (KEGG, Kyoto Encyclopedia of Genes and Genomes, En (Tree No. b0002, GenBank accession No. NC_000913.3, nucleotide positions 337 to 2,799, Gene ID 945803). The thrA gene is located between the thrL and thrB genes on the chromosome of E. coli K-12.

[0120] The thrB gene, which encodes homoserine kinase in E. coli, has been elucidated (KEGG, Entry No. b0003, GenBank accession No. NC_000913.3, nucleotide positions 2,801 to 3,733, Gene ID 947498). The thrB gene is located between the thrA and thrC genes on the chromosome of E. coli K-12.

[0121] The thrC gene encoding threonine synthase in E. coli has been elucidated (KEGG , entry no. b0004, GenBank accession no. NC_000913.3, nucleotide position 3,734 The thrC gene is located on the chromosome of E. coli K-12, and is identical to the thrB gene. The threonine operon thrABC is located between the threonine and yaaX genes. All three of these genes function as a single threonine operon, thrABC. To enhance expression of the threonine operon, it is desirable to remove the attenuator region from the operon, which affects transcription (WO2005049808 A1, WO2003097839 A1).

[0122] Aspartokinase I and homokinase are resistant to feedback inhibition by threonine. A mutant thrA gene encoding serine dehydrogenase I, as well as the thrB and thrC genes The genes can be obtained as a single operon from the well-known plasmid pVIC40 present in the E. coli threonine-producing strain VKPM B-3996. Details of the plasmid pVIC40 are described in US5705371. There are.

[0123] The rhtA gene, which encodes a protein of the threonine and homoserine export system (inner membrane transporter) of E. coli, has been elucidated (KEGG, entry no. b0813, GenBank accession no. NC_000913.3, nucleotide positions 849,210 to 850,097, complementary strand, Gene ID 947939). The gene is located on the chromosome of E. coli K-12 between the dps and ompX genes near the glnHPQ operon that encodes components of the glutamine transport system. The rhtA gene is linked to the ybiF gene (KEGG, entry - Same as No. B0813).

[0124] The asd gene encoding aspartate-β-semialdehyde dehydrogenase of E. coli has been elucidated (KEGG, entry no. b3433, GenBank accession no. NC_000913.3, nucleotide positions 3,573,775 to 3,574,878, complementary strand, Gene ID 947939). The asd gene is located on the chromosome of E. coli K-12 strain between the glgB and gntU genes, which are located on the same strand (the yhgN gene is on the opposite strand).

[0125] In addition, the aspC gene encoding aspartate aminotransferase in E. coli (KEGG, entry no. b0928, GenBank accession no. NC_000913.3, nucleotide positions 984,519 to 985,709, complementary strand, Gene ID 945553). The aspC gene is located on the chromosome of E. coli K-12 between the gloC gene on the opposite strand and the ompF gene on the same strand.

[0126] L-tryptophan producing bacteria L-tryptophan producers and parent strains that can be used to derive L-tryptophan producers include, but are not limited to, E. coli JP4735 / pMU3028 (DSM10122) and JP6015 / pMU91 (DSM10123) (US5756345) lacking tryptophanyl-tRNA synthetase encoded by a mutant trpS gene, E. coli SV164 (pGH5) (US6180373 B1) with a serA allele encoding a phosphoglycerate dehydrogenase that is not subject to feedback inhibition by serine and a trpE allele encoding anthranilate synthase that is not subject to feedback inhibition by tryptophan, E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) (US4371614), phosphoenol Examples of strains that can be used to derive L-tryptophan-producing bacteria include strains belonging to the genus Escherichia, such as E. coli AGX17 / pGX50, pACKG4-pps (WO9708333, US6319696 B1), which have enhanced ability to produce tryptophan. Examples of L-tryptophan-producing bacteria and parent strains that can be used to derive L-tryptophan-producing bacteria include strains belonging to the genus Escherichia, which have enhanced activity of the protein encoded by the yedA gene or yddG gene. Also included are putophane-producing bacteria (US2003148473 A1 and US2003157667 A1).

[0127] L-tryptophan producing bacteria and parent strains that can be used to derive L-tryptophan producing bacteria also include strains that have enhanced activity of one or more of the enzymes selected from anthranilate synthase, phosphoglycerate dehydrogenase, and tryptophan synthase. Since both enzymes are subject to feedback inhibition by L-tryptophan and L-serine, mutations that relieve the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such mutations include E. coli SV164 carrying an anthranilate synthase that is desensitized to feedback inhibition, and E. coli SV164 carrying a phosphoglycerate synthase that is desensitized to feedback inhibition. Examples of such transformants include those obtained by introducing the plasmid pGH5 (WO9408031 A1) containing a mutant serA gene encoding isopropyl dehydrogenase into E. coli SV164.

[0128] L-tryptophan producing bacteria and parent strains that can be used to derive L-tryptophan producing bacteria include strains into which a tryptophan operon containing a gene encoding deinhibited anthranilate synthase has been introduced (JP57-71397 A, JP62-244382 A, US4371614). In addition, tryptophan synthase in the tryptophan operon (trpBA) can be expressed by the codon L-tryptophan producing ability may be imparted to the strain by enhancing the expression of the gene encoding the tryptophan synthase. Tryptophan synthase is composed of α and β subunits encoded by the trpA and trpB genes, respectively. In addition, the isocitrate lyase-malate synthase operon may be regulated by the isocitrate lyase-malate synthase operon. The L-tryptophan-producing ability may be improved by enhancing the expression of the gene (WO2005103275).

[0129] L-valine producing bacteria L-valine producing bacteria and parent strains that can be used to derive L-valine producing bacteria include, but are not limited to, strains engineered to overexpress the ilvGMEDA operon (US5998178). It is preferable to remove the region necessary for attenuation in the ilvGMEDA operon so that the expression of the operon is not attenuated by the produced L-valine. Furthermore, it is preferable to disrupt the ilvA gene in the operon to reduce threonine deaminase activity.

[0130] L-valine producing bacteria and parent strains that can be used to derive L-valine producing bacteria also include mutant strains with mutations in aminoacyl t-RNA synthetases (US5658766). An example of such a strain is E. coli VL1970, which has a mutation in the ileS gene encoding isoleucine tRNA synthetase. E. coli VL1970 was obtained on June 24, 1988 from the Russian National Collection of Industrial Microorganisms (VKPM; Russian Federation, 117545 Moscow, Russia). st It has been deposited in the Dorozhny proezd, 1) under the accession number VKPM B-4411.

[0131] In addition, they require lipoic acid for growth and / or H + A mutant strain lacking -ATPase can also be used as an L-valine producing bacterium or its parent strain (WO9606926 A1).

[0132] L-valine producing bacteria and parent strains that can be used to derive L-valine producing bacteria also include E. coli H81 strain (VKPM B-8066, see e.g. EP1942183 B1), E. coli NRRL B-12287 and NRRL B-12288 (US4391907), E. coli VKPM B-4411 (US5658766), E. coli VKPM B-7707 (EP1016710 A2), and the like.

[0133] The genes and proteins used in breeding L-amino acid producing bacteria may have, for example, the known base sequences and amino acid sequences of the above-exemplified genes and proteins, respectively. Furthermore, the genes and proteins used in breeding L-amino acid producing bacteria may be variants of the above-exemplified genes and proteins (for example, variants of such known base sequences) as long as their original functions (for example, in the case of proteins, their respective enzymatic activities) are maintained. and mutants of genes and proteins having the amino acid sequences thereof). The descriptions of genes encoding proteins having indolepyruvate decarboxylase activity and mutants of indolepyruvate decarboxylase encoded thereby described herein can be applied mutatis mutandis to the mutants of genes and proteins encoding indolepyruvate decarboxylase.

[0134] The bacteria belonging to the order Enterobacteriaceae described herein may be modified so that at least a gene encoding a protein having indolepyruvate decarboxylase activity is weakened.

[0135] The term "gene encoding a protein having indolepyruvate decarboxylase activity" refers to a gene encoding a protein having the following reaction: indol-3-pyruvate + H +It may mean a gene encoding a protein having an enzyme activity that catalyzes → indole-3-acetaldehyde + CO2 (EC: 4.1.1.74). Methods for measuring the indolepyruvate decarboxylase activity of a protein include, for example, those described in Koga J. et al. (Purification and characterization of indolepyruvate decarboxylase, J. Biol. Chem., 1992, 267(22):15823-15828). Protein concentration was measured using bovine serum albumin (BSA) as a standard. It can be determined by Bradford protein assay using Coomassie dye or Lowry method (Bradford MM, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 1976, 72:248-254; Lowry OH et al., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 1951, 193:265-275) or Western blot analysis (Hirano S., Western blot analysis, Methods Mol. Biol., 2012, 926:87-97).

[0136] A specific example of a gene encoding an enzyme having indolepyruvate decarboxylase activity is the ipdC gene encoding indolepyruvate decarboxylase. The gene encoding an enzyme having indolepyruvate decarboxylase activity may be the ipdC gene and its homologs or mutant base sequences. The ipdC gene and its homologs and mutant base sequences will be described in more detail below.

[0137] The P. ananatis specific ipdC gene encodes the indolepyruvate decarboxylase protein IpdC ( Name: indole-3-pyruvate decarboxylase, 3-(indol-3-yl)pyruvate carboxy-lyase (BioCyc database, biocyc.org, accession ID: PAJ_RS11190; UniProtKB / Swiss-Prot, accession No. A0A0H3L5I5; UniParc, accession No. UPI0002040446; KEGG entry No. PAJ_2029).

[0138] The ipdC gene (GenBank, accession no. NC_017531.2 / AP012032.2; nucleotide positions: 2461340 to 2462992, complement; Gene ID: BAK12109.1) was cloned from P. ananatis AJ13355. In the chromosome of the strain, it is located between the glk gene (BAK12108.1) on the same strand and the yjhZ gene (BAK12110.1) on the opposite strand. The ipdC gene specific to the P. ananatis AJ13355 strain is shown in SEQ ID NO: 1. The IpdC protein specific to the P. ananatis AJ13355 strain is encoded by the gene having the nucleotide sequence. The amino acid sequence of this is shown in SEQ ID NO:2.

[0139] That is, the ipdC gene can be a gene (eg, DNA) having the nucleotide sequence shown in SEQ ID NO:1, and the IpdC protein can be a protein having the amino acid sequence shown in SEQ ID NO:2. Unless otherwise specified, the expression "a gene or a protein has a nucleotide sequence or an amino acid sequence" may mean that the gene or protein contains the nucleotide sequence or the amino acid sequence in a longer sequence, or may mean that the gene or protein has only the nucleotide sequence or the amino acid sequence.

[0140] As described above, protein homologs of IpdC having indolepyruvate decarboxylase activity specific to various bacteria belonging to the order Enterobacteria are known. Examples of such homolog proteins specific to bacteria belonging to the order Enterobacteria are shown in Table 1 together with the accession numbers of the amino acid sequences in the NCBI database (National Center for Biotechnology Information, ncbi.nlm.nih.gov / protein), phylogenetic classification data, and homology values ​​(as "identity", which is the identity of amino acids). In particular, the ipdC gene specific to E. coli having the base sequence shown in SEQ ID NO:54 is It is known to encode the IpdC protein having the amino acid sequence shown in SEQ ID NO:55. (Table 1; accession No.: WP_069192931.1).

[0141] [Table 1]

[0142] The bacteria described herein have threonine deaminase activity. In other words, the bacteria belonging to the order Enterobacteriaceae that can be used in the method described herein can be L-amino acid producing bacteria that have been modified to attenuate the expression of a gene encoding a protein having threonine deaminase activity. The bacterium may be modified to attenuate expression of a gene encoding a protein having indolepyruvate decarboxylase activity and a gene encoding a protein having threonine deaminase activity. The gene encoding the protein may be modified to attenuate expression. A description of an L-amino acid producing bacterium belonging to the order Enterobacteriaceae, which has been modified to attenuate the expression of a gene encoding a protein having decarboxylase activity, is given in the following: The same can be applied to L-amino acid producing bacteria belonging to the order Bacteria.

[0143] "Encodes a protein with threonine deaminase activity. The term "gene encoding a threonine dehydratase" (synonym: threonine ammonia-lyase) can refer to a gene encoding a protein with the enzymatic activity of catalyzing the reaction: L-threonine → 2-oxobutanoate + ammonium (EC: 4.3.1.19). The threonine deaminase activity of a protein can be measured, for example, by measuring the production of α-ketobutyric acid as its phenylhydrazone derivative. The amount of threonine deaminase in Escherichia coli can be determined by the addition of threonine to Escherichia coli or by other known techniques (Calhoun DH et al., Threonine deaminase from Escherichia coli. I. Purification and properties, J. Biol. Chem., 1973, 248(10):3511-3516; Eisenstein E., Cloning, expression, purification, and characterization of biosynthetic threonine deaminase from Escherichia coli, J. Biol. Chem., 1991, 266(9):5801-5807).

[0144] A specific example of a gene encoding an enzyme having threonine deaminase activity is the ilvA gene, which encodes threonine deaminase. The gene encoding the enzyme may be the ilvA gene and its homologs or mutant nucleotide sequences. ilvA and its homologs and mutant nucleotide sequences will be described in more detail below.

[0145] The P. ananatis-specific ilvA gene is a pyridoxal phosphate-dependent threonine dehydratase The enzyme IlvA encodes the pyridoxal-phosphate dependent threonine dehydratase protein. (BioCyc database, biocyc.org, accession ID: PAJ_RS16870; UniParc, accession No. UPI0002323460; KEGG entry No. PAJ_3043).

[0146] The ilvA gene (GenBank, accession No. NC_017531.2 / AP012032.2; nucleotide positions: 3640258 to 3641871, complement; Gene ID: BAK13123.1) is derived from P. ananatis AJ13355 In the chromosome of the strain, it is located between the ilvY gene (BAK13122.1) on the opposite strand and the ilvD gene (BAK13124.1) on the same strand. The ilvA gene specific to the P. ananatis AJ13355 strain has the nucleotide sequence shown in SEQ ID NO: 3, and the IlvA protein specific to the P. ananatis AJ13355 strain encoded by the gene The amino acid sequence of this is shown in SEQ ID NO:4.

[0147] That is, the ilvA gene may be a gene (e.g., DNA) having the nucleotide sequence shown in SEQ ID NO:3, and the IlvA protein may be a protein having the amino acid sequence shown in SEQ ID NO:4. good.

[0148] As mentioned above, various bacteria belonging to the order Enterobacteriaceae have threonine deaminase activity specific to them. Protein homologues of IlvA, which are specific to bacteria belonging to the order Enterobacteriaceae, are known. Examples of such homologue proteins are listed in Table 2, along with the accession numbers of the amino acid sequences in the NCBI database (National Center for Biotechnology Information, ncbi.nlm.nih.gov / protein), phylogenetic data, and homology values ​​(as "identity", which is the identity of amino acids). show.

[0149] [Table 2]

[0150] For example, the following descriptions of attenuated gene expression, proteins (including mutant proteins), and genes (including mutant nucleotide sequences) can be applied mutatis mutandis to any of the proteins and genes described herein, including but not limited to the IpdC and IlvA proteins and the genes encoding them, such as the ipdC and ilvA genes, and their homologs.

[0151] There may be differences in DNA sequences between families, genera, species, or strains belonging to the order Enterobacteriaceae. Thus, the ipdC and ilvA genes are not limited to genes having the nucleotide sequences shown in SEQ ID NOs: 1 and 3, It may also include genes that are variant nucleotide sequences relative to, or homologous to, SEQ ID NOs: 1 and 3, and that encode variants of IpdC and IlvA proteins. Similarly, IpdC and IlvA proteins are not limited to proteins having the amino acid sequences shown in SEQ ID NOs: 2 and 4, but may also include proteins having variant amino acid sequences of SEQ ID NOs: 2 and 4, or homologous thereto.

[0152] The term "variant sequence" refers to any variant of the standard genetic code (see, e.g., Lewin B., "Genes VIII", 2004, Pearson Education, Inc., Upper Saddle River, NJ 07458). It can mean a nucleotide sequence that encodes a protein having a wild-type amino acid sequence using synonymous amino acid codons. Thus, a gene that encodes a protein having a wild-type amino acid sequence can be a gene having a mutant nucleotide sequence due to the degeneracy of the genetic code.

[0153] The term "mutant nucleotide sequence" is not limited to, but may be used to encode a protein having a desired activity, such as the above-mentioned indolepyruvate decarboxylase activity or threonine deaminase activity. The term "stringent conditions" may also refer to a base sequence that can hybridize under stringent conditions with a base sequence complementary to the wild-type base sequence or a probe that can be prepared from the base sequence, as long as the base sequence is a specific hybrid, for example, a specific hybrid having a homology defined as the parameter "identity" when using the computer program blastn of 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, or a specific hybrid having a homology defined as the parameter "identity" when using the computer program blastn. High: 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more Stringent conditions may include conditions under which specific hybrids, such as hybrids with less homology than those described above, are formed and non-specific hybrids, such as hybrids with less homology than those described above, are not formed. × SSC (standard sodium citrate or standard sodium chloride), 0.1% SDS (dodecyl sulfate For example, 0.1x SSC, 0.1% SDS at 60°C or 65°C, and one or more times, or two or three times, may be used. The washing time depends on the type of membrane used for blotting, but should generally be as recommended by the manufacturer. For example, Amersham Hybond TM The recommended washing time for -N+ positively charged nylon membrane (GE Healthcare) under stringent conditions is 15 minutes. The washing step can be performed two or three times. A part of the sequence complementary to the wild-type base sequence may be used as the probe. Such a probe is prepared by using an oligonucleotide prepared based on the wild-type base sequence as a primer and a DNA fragment containing the base sequence as a template. The probe can be prepared by PCR (polymerase chain reaction; see White TJ et al., The polymerase chain reaction, Trends Genet., 1989, 5:185-189) using the nucleotide sequence as a probe for the hybridization. It is recommended that the probe length be more than 50 bp, but it may be necessary to use a probe with a length of at least 100 bp. The length can be appropriately selected depending on the conditions, and is usually 100 bp to 1 kbp. For example, when a DNA fragment having a length of about 300 bp is used as a probe, washing after hybridization Examples of conditions include 2×SSC, 0.1% SDS at 50° C., 60° C., or 65° C. .

[0154] The term "mutant nucleotide sequence" may also refer to a nucleotide sequence that encodes a mutant protein.

[0155] The term "mutant protein" may refer to a protein having a variant amino acid sequence.

[0156] The term "mutant protein" refers to a protein that has one or more amino acids that differ from the wild-type amino acid sequence of the protein. may be a substitution, deletion, insertion, and / or addition of several amino acid residues. The term may refer to a protein having 1 to 300 or more mutations in its amino acid sequence, which maintains the same activity or function as the wild-type protein, or whose three-dimensional structure is not significantly altered compared to the unmodified protein. The number of mutations in a mutant protein depends on the position of the amino acid residue in the three-dimensional structure of the protein or the type of amino acid residue. The number of changes in a mutant protein is not strictly limited, but may be 1 to 300, in another example, 1 to 250, in another example, 1 to 200, in another example, 1 to 150, in another example, 1 to 100, in another example, 1 to 90, in another example, 1 to 80, in another example, 1 to 70, in another example, 1 to 80, in another example, 1 to 9 ... In another example, it is 1-60, in another example, it is 1-50, in another example, it is 1-40, in another example, it is 1-30, in another example, it is 1-20, In another example, it may be 1-15, in another example, 1-10, or in another example, 1-5. This is because some amino acids have a high degree of homology to one another, and such changes do not affect activity or function or do not significantly change the three-dimensional structure of the protein relative to the wild-type or unmodified protein. Thus, a mutant protein may have 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more of the entire wild-type amino acid sequence of the protein, so long as activity or function is maintained or the three-dimensional structure of the protein is not significantly altered relative to the wild-type or unmodified protein. or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, The homology may be a protein having an amino acid sequence with which the homology is defined as the parameter "identity" when using the computer program blastp. As used herein, the term "homology" may mean "identity" (which is the identity between amino acid residues). The sequence identity between two sequences is calculated as the ratio of residues that match between the two sequences when the two sequences are aligned for maximum correspondence.

[0157] Examples of the substitution, deletion, insertion, and / or addition of one or several amino acid residues include conservative mutations that maintain the activity or function of the mutant protein or do not significantly change the three-dimensional structure of the protein compared to the unmodified protein (e.g., wild-type protein, etc.). A representative conservative mutation is conservative substitution. Conservative substitution is not limited, but when the substitution site is an aromatic amino acid, the substitution site is preferably between Phe, Trp, and Tyr. If the amino acid is a hydrophobic amino acid, it is between Ala, Leu, Ile, and Val. If the amino acid is a hydrophilic amino acid, it is between Glu, Asp, Gln, Asn, Ser, His, and Thr. If the amino acid is a polar amino acid, it is between Ala, Leu, Ile, and Val. between Gln and Asn in the case of a substitution site of a basic amino acid, between Lys, Arg, and His Conservative substitutions are between Asp and Glu when the substitution site is an acidic amino acid, and between Ser and Thr when the substitution site is an amino acid having a hydroxyl group. Examples of conservative substitutions include substitution of Ala with Ser or Thr, substitution of Arg with Gln, His or Lys, substitution of Asn with Glu, Gln, Lys, His or Asp, substitution of Asp with Asn, Glu or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp or Arg, substitution of Glu with Asn, Gln, Lys or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg or Asp. or Tyr; Ile to Leu, Met, Val, or Phe; Leu to Ile, Met, Val, or Phe; Lys to Asn, Glu, Gln, His, or Arg; Met to Ile, Leu, Val or Phe, substitution of Phe with Trp, Tyr, Met, Ile, or Leu, substitution of Ser with Thr or Ala, substitution of Thr with Ser or Ala, substitution of Trp with Phe or Tyr, substitution of Tyr with His, Phe, or Trp, and substitution of Val with Met, Ile, or Leu. The above-mentioned substitutions, deletions, insertions, additions, and the like of amino acid residues also include naturally occurring mutations due to individual differences in the organism from which the amino acid sequence is derived.

[0158] Examples of substitutions, deletions, insertions, and / or additions of one or several amino acid residues include non-conservative mutations, provided that the mutations are at one or more different positions in the amino acid sequence. Further, secondary mutations may maintain the activity or function of the mutant protein or may result in The modification is such that the three-dimensional structure of the protein is not significantly altered relative to the unmodified protein (eg, the wild-type protein, etc.).

[0159] The percentage of identity of the amino acid sequence can be calculated by the blastp algorithm. More specifically, the percentage of identity of the amino acid sequence can be calculated by the blastp algorithm provided by the National Center for Biotechnology Information (NCBI) using the default settings. The percentage of identity of the base sequence can be calculated using the scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment) of the BLOSUM62 Gap Costs Matrix. The percentage of identity of the base sequence can be calculated using the blastn algorithm. More specifically, the percentage of identity of the base sequence can be calculated using the default scoring parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear) of the blastn algorithm provided by NCBI.

[0160] The term "a bacterium modified to attenuate expression of a gene" can mean that the bacterium has been modified such that expression of a gene is attenuated in the modified bacterium. Expression of a gene can be attenuated, for example, by inactivation of the gene.

[0161] The term "gene is inactivated" may mean that the modified gene encodes a protein that is completely inactive or non-functional compared to the wild-type or non-modified gene. The modified DNA region may be a deletion of a part of a gene or a deletion of the entire gene, a deletion of the gene coding region, or a deletion of the entire gene coding region. Substitution of one or more bases that results in an amino acid substitution in the protein that is encoded by the gene (missense mutation), Introduction of a codon (nonsense mutation), resulting in a shift in the reading frame of a gene deletion of one or two bases, insertion of a drug resistance gene and / or a transcription termination signal, The gene may not be able to be expressed naturally by modifying the adjacent region of the gene (which includes sequences that control the expression of the gene, such as promoters, enhancers, attenuators, ribosome binding sites, etc.). Gene inactivation can be achieved, for example, by UV irradiation or mutagenesis using nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, etc. insertion, gene disruption using homologous recombination, and / or "Red / ET-driven integration" or insertion-deletion mutagenesis based on "λRed / ET-mediated integration" (Yu D. et al., This can be carried out by conventional methods such as those described in An efficient recombination system for chromosome engineering in Escherichia coli, Proc. Natl. Acad. Sci. USA, 2000, 97(11):5978-5983; Datsenko KA and Wanner BL, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645; Zhang Y. et al., A new logic for DNA engineering using recombination in Escherichia coli, Nature Genet., 1998, 20:123-128).

[0162] The term "a bacterium modified to attenuate expression of a gene" refers to a bacterium that has a region operably linked to a gene (which includes sequences that control expression of the gene, such as promoters, enhancers, operators, attenuators and termination signals, ribosome binding sites, and other expression control elements) that is modified to attenuate the expression level of the gene; and other examples (see, e.g., WO9534672 A1; Carrier TA and Keasling JD, Library of synthetic 5' secondary structures to manipulate mRNA stability in Escherichia coli, Biotechnol. Prog., 1999, 15:58-64). (see below).

[0163] The term "operably linked to the gene" refers to a regulatory It may mean that the region is linked to the base sequence of a nucleic acid molecule or gene in such a way as to achieve expression of the base sequence (e.g., enhanced, increased, constitutive, basal, terminated, attenuated, deregulated, reduced or suppressed expression), specifically expression of a gene product encoded by the base sequence.

[0164] The term "bacteria modified to attenuate gene expression" may also mean that the bacterium has been modified such that the expression level (i.e., amount of expression) of the gene in the modified bacterium is attenuated compared to an unmodified strain (e.g., a wild-type or parent strain). A reduction in the expression level of a gene may be measured, for example, as a reduction in the expression level of the gene per cell (which may be the average expression level of the gene per cell). The term "expression level of a gene" or "expression amount of a gene" may mean, for example, the amount of an expression product of the gene (e.g., the amount of mRNA of the gene or the amount of a protein encoded by the gene). The bacterium may have an expression level of the gene per cell that is, for example, 50% or less, 20% or less, 10% or less, 5% or less, or less than that of an unmodified bacterium. or may be modified to reduce it to 0%.

[0165] The term "bacteria modified to attenuate expression of the metJ gene" may mean that the bacterium has been modified such that the total amount and / or activity of the corresponding gene product (i.e., the encoded protein) is reduced in the modified bacterium compared to the unmodified bacterium. A reduction in the total amount or activity of the protein may be measured, for example, as a reduction in the amount or activity of the protein per cell (which may be the average amount or activity of the protein per cell). The bacterium may have an activity of the protein per cell that is, for example, 50% of that of the unmodified bacterium. It may be modified to reduce to below 20%, below 10%, below 5%, or to 0%.

[0166] Examples of unmodified bacteria used in the comparison include wild-type strains of bacteria belonging to the genus Escherichia, such as E. coli MG1655 strain (ATCC 47076) and E. coli W3110 strain (ATCC 27325), and wild-type strains of bacteria belonging to the genus Pantoea, such as P. ananatis AJ13355 strain (FERM BP-6614). The unmodified bacteria subjected to the method also include parent strains that have not been modified to attenuate gene expression or bacteria in which gene expression has not been attenuated.

[0167] Gene expression is controlled by weaker promoters and other gene expression control sequences on chromosomal DNA. The strength of the promoter can be determined by the ratio of the promoter It is defined by the frequency of initiation. An example of a method for assessing promoter strength is given in Goldstein MA et al. (Goldstein MA and Doi RH, Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)). As disclosed in, the promoter can also be modified to be weaker by introducing one or more base substitutions into the promoter region of the gene. In addition, the number of base substitutions in the Shine-Dalgarno (SD) sequence, and / or the spacer between the SD sequence and the start codon, and / or the sequence immediately upstream or downstream of the start codon in the ribosome binding site (RBS) can be reduced. It is known that the substitution of a single nucleotide greatly affects the translation efficiency of mRNA.

[0168] The expression of a gene can be specifically, for example, the coding region of a gene (US5175107) or By inserting a transposon or insertion sequence (IS) into the region that controls gene expression, or by UV irradiation or nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), It can also be weakened by standard methods such as mutagenesis using NTG. The incorporation of specific mutations can be achieved, for example, by known dye transfer methods based on λRed / ET-mediated recombination (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645). This can be done using color body editing methods.

[0169] The copy number of a gene or the presence or absence of a gene can be determined, for example, by restriction digestion of chromosomal DNA. Then, Southern blotting using probes based on the gene sequence or fluorescent in This can be measured by performing fluorescence in situ hybridization (FISH) or other techniques. The level of gene expression can be determined by measuring the amount of mRNA transcribed from the gene using a variety of well-known methods, such as Northern blotting and quantitative RT-PCR. The amount of protein encoded by the gene can be determined by SDS-PAGE followed by immunoblotting ( This can be measured by known methods such as Western blotting or mass spectrometry of protein samples.

[0170] Preparation of plasmid DNA, DNA cleavage, DNA ligation, DNA transformation, selection of oligonucleotides as primers, introduction of mutations, and other molecular manipulations of recombinant DNA molecules and molecular cloning. Methods for cloning may be conventional methods well known to those skilled in the art. Such methods are described, for example, in Sambrook J., Fritsch EF and Maniatis T., "Molecular Cloning: A Laboratory Manual”, 2 nded., Cold Spring Harbor Laboratory Press (1989) or Green MR and Sambrook JR, “Molecular Cloning: A Laboratory Manual”, 4 th ed., Cold Spring Harbor Laboratory Press (2012); Bernard R. Glick, Jack J. Pasternak and Cheryl L. Patten, “Molecular Biotechnology: principles and applications of recombinant DNA”, 4 th ed., Washington, DC, ASM Press (2009).

[0171] Any method can be used for engineering with recombinant DNA, including conventional methods such as transformation, transfection, infection, conjugation, mobilization, etc. Transformation, transfection, infection, conjugation, or mobilization of bacteria with DNA encoding a protein can confer the bacteria the ability to synthesize the protein encoded by the DNA. Methods of transformation, transfection, infection, conjugation, and mobilization can include any method. For example, for efficient DNA transformation and transfection, the recipient organism of E. coli K-12 can be modified to increase the cell's permeability to DNA. A method of treating cells with calcium chloride has been reported (Mandel M. and Higa A., Calcium-dependent bacteriophage DNA infection, J. Mol. Biol., 1970, 53:159-162). Specialized and / or generalized transformation methods have been described (Morse ML et al., Transduction in Escherichia coli K-12, Genetics, 1956, 41(1):142-156; Miller JH, Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor La. Press, 1972). Random and / or targeted transduction of DNA into a host microorganism. Other methods for integration can be applied, such as "Mu-driven integration / amplification" (Akhverdyan et al., Appl. Microbiol. Biotechnol., 2011, 91:857-871), "Red / ET-driven integration" or "λRed / ET-mediated integration" (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA 2000, 97(12):6640-45; Zhang Y., et al., Nature Genet., 1998, 20:123-128). In addition, for multiple insertions of desired genes, , Mu-driven replicative transposition (Akhverdyan et al., Application of the bacteriophage Mu-driven system for the integration / amplification of target genes in the chromosomes of engineered Gram-negative bacteria-mini review, Appl. Microbiol. Biotechnol., 2011, 91:857-871) and chemically inducible chromosome evolution based on recA-dependent homologous recombination leading to amplification of desired genes (Tyo KEJ et al., Stabilized gene duplication enables long-term selection-free heterologous pathway expression, Nature Biotechnol., 2009, 27:760-765), as well as translocation, site-specific and / or homologous Red / ET-mediated Other methods utilizing various combinations of plasmid-based recombination, P1-mediated generalized transduction, and / or P1-mediated generalized transduction (see, e.g., Minaeva N. et al., Dual-In / Out strategy for genes integration into bacterial chromosome: a novel approach to step-by-step construction of plasmid-less marker-less recombinant E. coli strains with predesigned genome structure, BMC Biotechnology, 2008, 8:63; Koma D. et al., A convenient method for multiple insertions of desired genes into target loci on the Escherichia coli chromosome, Appl. Microbiol. Biotechnol., 2012, 93(2):815-829) can be utilized.

[0172] The sequences of the genes encoding the wild-type proteins specific to E. coli and P. ananatis have already been elucidated (see above). Therefore, mutant sequences encoding mutant proteins of the wild-type proteins were prepared based on the DNA of the same bacterial species and the sequences of the wild-type genes. PCR (polymerase chain reaction; refer to White TJ et al., The polymerase chain reaction, Trends Genet., 1989, 5(6):185-189), or by treating DNA containing the wild-type gene in vitro with, for example, hydroxylamine. Microorganisms carrying mutant or wild-type genes, such as E. coli or P. ananatis species, are treated with ultraviolet (UV) irradiation or N-methyl-N'-nitro-nitrosamine, a method commonly used for such treatments. It can be obtained by treating with mutagens such as guanidine (NTG) or nitrous acid, or by extracting the full-length gene. The gene constructs can be chemically synthesized. Genes encoding the protein or mutant proteins from other bacteria belonging to the order Enterobacteriaceae can be obtained as well.

[0173] The term "wild-type" (which means "native") when referring to proteins (e.g., a "wild-type protein") and genes (e.g., a "wild-type gene") " and "natural" refer to wild-type bacteria (e.g., For example, E. coli MG1655 strain (ATCC 47076), E. coli W3110 strain (ATCC 27325), P. ananatis A "wild-type protein" may refer to native proteins and genes that are naturally present and / or expressed and / or naturally produced in a wild-type strain of bacteria belonging to the order Enterobacteriaceae, such as Enterobacteriaceae or the family Erwiniaceae, such as strain AJ13355 (FERM BP-6614). Since proteins are encoded by genes, a "wild-type protein" may be encoded by a "wild-type gene" that occurs naturally in the genome of a wild-type bacterium.

[0174] The term "native to" when referring to a protein or nucleic acid that is native to a particular organism (e.g., a bacterial species), can mean a protein or nucleic acid that is native to that organism. That is, a protein or nucleic acid unique to a particular organism may refer to a protein or nucleic acid, respectively, that naturally occurs in that organism and can be isolated and sequenced from that organism by methods known to those skilled in the art. Furthermore, since the amino acid or base sequence of a protein or nucleic acid, respectively, isolated from an organism in which the protein or nucleic acid occurs can be easily determined, the term "unique" when referring to a protein or nucleic acid may also refer to a protein or nucleic acid obtained, for example, by genetic engineering techniques, including recombinant DNA technology, or chemical synthesis, etc., so long as the resulting amino acid or base sequence of the protein or nucleic acid is identical to the amino acid or base sequence of the protein or nucleic acid naturally occurring in that organism. Amino acid sequences unique to a particular organism include, but are not limited to, peptides, oligopeptides, polypeptides (which include proteins, specifically enzymes), and the like. Examples of sequences specific to a particular organism include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), including, but not limited to, sequences encoding expression regulatory sequences (including promoters, attenuators, terminators, etc.), genes, intergenic sequences, and sequences encoding signal peptides, protein pro-sites, and artificial amino acid sequences. Specific examples of amino acid and nucleotide sequences and their homologs specific to various organisms are described herein, including, for example, the proteins IpdC and IlvA, which have the amino acid sequences shown in SEQ ID NOs: 2 and 4, respectively, and which may be specific to bacteria of the species P. ananatis, and which have the nucleotide sequences shown in SEQ ID NOs: 1 and 3, respectively. The corresponding genes ipdC and ilvA are encoded by the corresponding ipdC and ilvA genes.

[0175] In addition to the above-mentioned properties, the bacteria may have specific properties such as various nutritional requirements, drug resistance, drug sensitivity, drug dependency, etc., without departing from the scope of the present invention.

[0176] 2. Method The methods described herein include methods for producing L-amino acids using the bacteria described herein. The methods for producing L-amino acids using the bacteria described herein may include cultivating the bacteria in a medium to produce, excrete or secrete, and / or accumulate the L-amino acid in the medium or in the bacterial cells, or both, and recovering the L-amino acid from the medium and / or the bacterial cells. The methods may further optionally include purifying the L-amino acid from the medium and / or the bacterial cells. The L-amino acids may be produced in the forms described above. The L-amino acids may be produced in the free form, or as a salt thereof, or a mixture thereof. For example, salts such as sodium salts, potassium salts, ammonium salts, etc., or internal salts such as zwitterions of L-amino acids may be produced by the above-mentioned method. This is possible because amino acids may react with each other or with neutralizing agents such as inorganic or organic acids or alkaline substances under fermentation conditions to produce salts by typical acid-base neutralization reactions, which is a chemical characteristic of amino acids that is obvious to those skilled in the art. Specifically, monohydrochloride of L-cysteine ​​(L-cysteine ​​x HCl) or L-cysteine ​​x HCl may be produced. The monohydrochloride salt of L-cysteine ​​monohydrate (L-cysteine ​​x H2O x HCl) can be prepared by the above method.

[0177] Cultivation of bacteria and recovery and optional purification of L-amino acids from the medium etc. can be carried out in the same manner as in conventional fermentation methods for producing L-amino acids using microorganisms. That is, cultivation of bacteria and recovery and purification of L-amino acids from the medium etc. may be carried out by applying conditions suitable for culturing bacteria and conditions suitable for recovery and purification of L-amino acids that are well known to those skilled in the art.

[0178] The medium used is not particularly limited as long as it contains at least a carbon source and the bacteria described herein can grow and produce L-amino acids. The medium may be a synthetic medium or a natural medium, such as a typical medium containing a carbon source, a nitrogen source, a sulfur source, a phosphorus source, inorganic ions, and other organic and inorganic components as necessary. As the carbon source, sugars such as glucose, sucrose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose, starch hydrolysate, alcohols such as ethanol, glycerol, mannitol, sorbitol, organic acids such as gluconic acid, fumaric acid, citric acid, malic acid, succinic acid, and fatty acids can be used. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, and ammonia water can be used. In addition, peptone, yeast extract, meat extract, malt extract, corn steep liquor, and the like can also be used. The medium may contain one or more of these nitrogen sources. Examples of sulfur sources include ammonium sulfate, magnesium sulfate, iron sulfate, manganese sulfate, sodium thiosulfate, ammonium thiosulfate, sodium sulfide, and ammonium sulfide. The medium may contain a phosphorus source in addition to a carbon source, a nitrogen source, and a sulfur source. Examples of phosphorus sources that can be used include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. Vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12, and other necessary substances, such as adenine, nucleic acids such as RNA, amino acids, peptones, casamino acids, and organic nutrients such as yeast extract, can be present in appropriate amounts (even trace amounts). In addition to these, small amounts of calcium phosphate, iron ions, manganese ions, and the like may be added if necessary. As other organic and inorganic components, one type of component may be used, or two or more types of components may be used in combination. In addition, when using an auxotrophic strain that requires amino acids for growth, it is preferable to supplement the medium with the required nutrients.

[0179] The culture can be carried out under conditions suitable for culturing the bacteria used in the method for producing an L-amino acid. For example, the culture can be carried out under aerobic conditions for 16 to 72 hours, or 16 to 24 hours. The culture temperature during the culture can be controlled within the range of 30 to 45°C, or 30 to 37°C. The pH can be adjusted between 5 and 8, or between 6.0 and 7.5. The pH can be adjusted by using an inorganic or organic acidic or alkaline substance, such as urea, calcium carbonate, an inorganic acid, an inorganic alkali, or ammonia gas.

[0180] After the cultivation, the L-amino acid can be collected from the medium. Specifically, the L-amino acid present outside the bacterial cells can be collected from the medium. The L-amino acid can be recovered from the culture medium or the supernatant by disrupting the cells, removing solids such as the cells and cell disruption suspension (also called cell debris) to obtain a supernatant, and recovering the L-amino acid from the supernatant. The disruption of the cells can be carried out by a well-known method such as ultrasonic disruption using high-frequency sound waves. The removal of solids can be carried out by centrifugation or membrane filtration. The recovery of the L-amino acid from the medium or the supernatant can be carried out by a conventional technique such as concentration, crystallization, membrane treatment, ion exchange chromatography, flash chromatography, thin layer chromatography, medium or high pressure liquid chromatography, or a combination thereof. These methods may be used alone or in appropriate combination. EXAMPLES

[0181] The invention will now be more precisely described with reference to the following non-limiting examples.

[0182] Example 1 Construction of a P. ananatis strain with a deleted ipdC gene P. ananatis SC17(0)ΔipdC::λattL-kan lacking the ipdC gene (SEQ ID NO:1) R-λattR strains were constructed by λRed-dependent integration. For this purpose, P. ananatis SC17(0) strain (US8383372 B2, VKPM B-9246) carrying the RSF-Red-TER plasmid (US8383372 B2) was cultured in LB liquid medium (Sambrook J. and Russell DW, Molecular Cloning: A Laboratory Manual (3 rd ed.), Cold Spring Harbor Laboratory Press, 2001) overnight. Then, 1 mL of the culture was transferred to LB medium containing 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The cells were inoculated into 100 mL of liquid medium and cultured at 32°C for 3 hours with shaking (250 rpm). The cells were harvested and washed three times with 10% (v / v) glycerol to obtain competent cells. R PCR was performed using the -attR plasmid (US7919284 B2) as a template and primers P1 (SEQ ID NO: 5) and P2 (SEQ ID NO: 6) to generate λattL-kan 1A-K1A ... R The resulting amplified λattR DNA fragment was purified using the Wizard PCR Prep DNA Purification System (Promega) and transformed into a competent The cells were then cultured in SOC medium (Sambrook J. and Russell DW, Molecular Cloning: A Laboratory Manual (3 rd ed.), Cold Spring Harbor Laboratory Press, 2001) After culturing for 1 h, the mixture was spread on an LB plate containing 35 mg / L kanamycin and cultured at 34°C for 16 h. The colonies that emerged were purified on the same medium. Then, primer P3 (SEQ ID NO: 7) PCR analysis (TaKaRa Speed ​​Star®; 40 cycles of 97°C for 20 seconds, 54°C for 20 seconds, and 72°C for 100 seconds) was performed using λattL-kan and P4 (SEQ ID NO: 8). R - It was confirmed that the λattR cassette was replaced by P. ananatis SC17(0)ΔipdC::λattL-kan R -λattR strain was obtained.

[0183] Example 2: Production of L-methionine using P. ananatis strain lacking the ipdC gene To test the effect of the deletion of the ipdC gene on L-methionine production, SC17(0)ΔipdC::λattL-kan was transformed with the EdgeBio PurElute Bacterial Genomic kit according to the manufacturer's instructions. R Chromosomal DNA was isolated from the λattR strain (Example 1) and 10 μg of DNA was electroporated. The L-methionine producing strain C2691 of P. ananatis was constructed as described in Preliminary Example 1. The resulting transformants were plated on LB plates containing 20 mg / L kanamycin and cultured overnight at 34°C until individual colonies were visible. PCR analysis using primers P3 (SEQ ID NO: 7) and P4 (SEQ ID NO: 8) identified the ipdC gene. The desired transformant was identified by confirming the gene replacement. R -λattR strain (abbreviation: C3793) was obtained.

[0184] P. ananatis strain C2691 and C2691ΔipdC::λattL-kan REach of the -λattR strains was cultured in LB liquid medium at 32°C for 18 hours. Then, 0.2 mL of the resulting culture was inoculated into 2 mL of fermentation medium in a 20 × 200 mm test tube, and the mixture was fermented on a rotary shaker at 250 rpm for 32 hours until glucose was consumed. The plates were incubated at 4°C for 48 hours.

[0185] The composition of the fermentation medium (g / L) was as follows: Glucose 40.0 (NH4)2SO4 15.0 KH2PO41.5 MgSO4-7H2O 1.0 Thiamine-HCl 0.1 CaCO325.0 LB medium 4% (v / v)

[0186] The fermentation medium was sterilized at 116° C. for 30 min, except for glucose and CaCO3, which were sterilized separately as follows: glucose at 110° C. for 30 min; CaCO3 at 116° C. for 30 min. The pH was adjusted to 7.0 with KOH solution.

[0187] After cultivation, the amount of L-methionine accumulated in the medium was measured by Agilent 1260 amino-acid analyzer. The results of three independent test tube fermentations (as the mean value ± standard deviation) are shown in Table 3. As can be seen from Table 3, the modified strain P. ananatis C2691ΔipdC::λattL-kanR-λattR was able to accumulate a higher amount of L-methionine compared to the parent strain P. ananatis C2691.

[0188] [Table 3]

[0189] Example 3: Production of L-cysteine ​​using P. ananatis strain lacking the ipdC gene To test the effect of deletion of the ipdC gene on L-cysteine ​​production, SC17(0)ΔipdC::λattL-kan was transformed by electroporation. R - Transformation of the L-cysteine ​​producing strain EYP197(s) of P. ananatis with chromosomal DNA of the λattR strain (Example 1) (RU2458981 C2 or WO2012 / 137689) The P. ananatis EYP ΔipdC::λattL-kanR-λattR strain was obtained by introducing the cysE5 and yeaS genes from P. ananatis SC17 (FERM BP-11091) and replacing the native promoter of the cysPTWA gene cluster with the Pnlp8 promoter. Constructed.

[0190] P. ananatis EYP197(s) and EYP ΔipdC::λattL-kanR-λattR strains were cultured in 3 mL of LB liquid medium at 32°C for 18 hours, and 0.2 mL of the resulting culture was placed in a 20 × 200 mm test tube. Inoculate 2 mL of the fermentation medium and cultivate for 24 hours at 32° C. on a rotary shaker.

[0191] After the cultivation, the amount of L-cysteine ​​accumulated in the medium was measured by the method described by Gaitonde MK (A spectrophotometric method for the direct determination of cysteine ​​in the presence of other naturally occurring amino acids, Biochem. J., 1967, 104(2):627-633). The measurements are carried out with the following modifications: 150 μL of each sample is mixed with 150 μL of 1 M H2SO4. After incubating at 20°C for 5 min, 700 μL of HO was added to the mixture, and the resulting mixture was diluted with 150 Transfer 1 μL of the solution to a new vial and add 800 μL of solution A (1 M Tris-HCl pH 8.0, 5 mM dithiothreitol (DTT)). Incubate the resulting mixture at 20 °C for 5 min and centrifuge at 13,000 rp After spinning at 3500 nm for 10 min, transfer 100 μL of the mixture to a 20 × 200 mm test tube. Then, transfer 400 μL of HO, 500 μL of glacial acetic acid, and 500 μL of solution B (0.63 g of ninhydrin, 10 mL of glacial acetic acid, 10 mL of 36% HCl) is added and the mixture is incubated in a boiling water bath for 10 minutes. Add 4.5 mL of ethanol and measure the OD 560 The concentration of cysteine ​​is calculated using the formula: C (Cys, g / L) = 11.3 × OD 560 It is calculated using:

[0192] The composition of the fermentation medium (g / L) is as follows: Glucose 40.0 (NH4)2S2O312.0 KH2PO41.5 MgSO4-6H2O 0.825 Thiamine-HCl 0.1 CaCO325.0 LB medium 4% (v / v)

[0193] The fermentation medium is sterilized at 116°C for 30 min, except for glucose, (NH4)2S2O3, and CaCO3, which are each sterilized separately as follows: glucose at 110°C for 30 min, (NH4)2S2O3 filtered through a 0.2 μm membrane, and CaCO3 at 116°C for 30 min. The pH is adjusted to 7.0 with KOH solution.

[0194] Example 4: Production of L-glutamic acid using P. ananatis strain lacking the ipdC gene To test the effect of deletion of the ipdC gene on L-glutamic acid production, SC17(0)ΔipdC::λattL-kan was transformed by electroporation. R- Chromosomal DNA of the λattR strain (Example 1) P. The L-glutamic acid producing strain P. ananatis NA1 (EP2336347 A1) was transformed to obtain the P. ananatis NA1ΔipdC::λattL-kanR-λattR strain.

[0195] The P. ananatis NA1 strain and the NA1ΔipdC::λattL-kanR-λattR strain were each cultured in 3 mL of LB liquid medium at 32°C for 18 hours. 0.2 mL of the resulting culture was inoculated into 2 mL of fermentation medium in a 20 x 200 mm test tube and cultured on a rotary shaker at 32°C for 24 hours.

[0196] After cultivation, the amount of L-glutamic acid accumulated in the medium was determined by the ratio of butan-1-ol:acetic acid:water = 4 The nin activity was measured by paper chromatography using a mobile phase consisting of 1:1 (v / v) The cells were stained with CdCl2 (1% solution in acetone) and then dehydrated with L-glutamate in 50% ethanol containing 0.5% CdCl2. L-glutamic acid is eluted and the amount of L-glutamic acid is measured at 540 nm.

[0197] The composition of the fermentation medium (g / L) is as follows: Glucose 30.0 MgSO4-6H2O 0.5 (NH4)2SO4 20.0 KH2PO4 2.0 Yeast Extract 2.0 FeSO4-7H2O 0.02 MnSO4-4H2O 0.02 Thiamine-HCl 0.01 L-Lysine hydrochloride 0.2 L-Methionine 0.2 DL-α,ε-diaminopimelic acid 0.2 CaCO3 20.0

[0198] The fermentation medium is sterilized at 116°C for 30 minutes, except for glucose and CaCO3, which are sterilized separately as follows: glucose at 110°C for 30 minutes; CaCO3 at 116°C for 30 minutes. The pH is adjusted with KOH solution. Adjust to 7.0.

[0199] Example 5: Production of L-aspartic acid using P. ananatis strain lacking the ipdC gene To test the effect of deletion of the ipdC gene on L-aspartic acid production, SC17(0)ΔipdC::λattL-kan was transformed by electroporation. R The chromosomal DNA of the -λattR strain (Example 1) is transformed into the L-aspartic acid producing strain 5ΔP2RM of P. ananatis (WO2010038905 A1) to obtain the P. ananatis 5ΔP2RMΔipdC::λattL-kanR-λattR strain.

[0200] P. ananatis 5ΔP2RM strain and 5ΔP2RMΔipdC::λattL-kanR-λattR strain are each cultured in 3 mL of LB liquid medium at 32°C for 18 hours, and 0.2 mL of the resulting culture is inoculated into 2 mL of fermentation medium in a 20 x 200 mm test tube and cultured on a rotary shaker at 32°C for 72 hours. After cultivation, the amount of L-aspartic acid accumulated in the medium is measured by paper chromatography.

[0201] The composition of the fermentation medium (g / L) is as follows: Glucose 40.0 MgSO4-7H2O 1.0 (NH4)2SO4 16.0 KH2PO40.3 KCl 1.0 MES 10.0 Calcium pantothenate 0.01 Betaine 1.0 FeSO4-7H2O 0.01 MnSO4-5H2O 0.01 Thiamine-HCl 0.01 L-Lysine hydrochloride 0.1 L-Methionine 0.1 DL-α,ε-diaminopimelic acid 0.1 L-Glutamic Acid 3.0 CaCO330.0

[0202] The fermentation medium is sterilized at 116°C for 30 minutes, except for glucose and CaCO3, which are sterilized separately as follows: glucose at 110°C for 30 minutes; CaCO3 at 116°C for 30 minutes. The pH is adjusted with NaOH solution. Adjust to 6.5.

[0203] Example 6 Construction of an L-methionine producing strain of E. coli lacking the ipdC gene L-methionine synthesis in E. coli carrying the temperature-sensitive replicating pKD46 plasmid (Datsenko KA and Wanner BL, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645) The production strain (an example of which is described above) is cultured overnight in LB liquid medium. Then, 1 mL of the culture is inoculated into 100 mL of LB liquid medium containing arabinose at a final concentration of 50 mM and ampicillin at 50 mg / L, and the cells are cultured at 37°C for 2 hours with shaking (250 rpm). The cells are harvested and transferred to ice-cooled 10% (v / v) Wash with glycerol three times to obtain competent cells. R PCR was performed using the -attR plasmid (US7919284 B2) as a template to identify sequences homologous to the upstream and downstream of the ipdC gene. λattL-kan at both ends R The amplified λattR DNA fragment was obtained. The resulting DNA fragment was purified using the Wizard PCR Prep DNA Purification System (Promega) and subjected to electroporation. The cells were cultured in SOC medium for 2 hours, and then incubated with 35 mg / L kanamycin. The strain is then spread onto an LB plate containing λattL-kan and cultured at 34°C for 16 hours. The colonies that appear are purified on the same medium. R The L-methionine producing strain ΔipdC::λattL-kan was confirmed by PCR. R −λattR is obtained.

[0204] Example 7: Production of L-methionine using an E. coli strain lacking the ipdC gene L-methionine producing strain of E. coli ΔipdC::λattL-kan R -λattR and its parent strain, respectively. The resulting culture was then cultured at 37°C for 18 hours with shaking in LB liquid medium. Next, 0.2 mL of the culture was inoculated into 2 mL of fermentation medium in a 20 x 200 mm test tube, and cultured on a rotary shaker at 250 rpm at 32°C for 48 hours. After cultivation, the amount of L-methionine accumulated in the medium is measured using an Agilent 1260 amino-acid analyzer.

[0205] The composition of the fermentation medium (g / L) is as follows: Glucose 40.0 (NH4)2SO4 15.0 KH2PO41.5 MgSO4-7H2O 1.0 Thiamine-HCl 0.1 Threonine 0.5 CaCO3 20.0 LB medium 4% (v / v)

[0206] The fermentation medium is sterilized at 116° C. for 30 min, except for glucose and CaCO3, which are sterilized separately as follows: glucose at 110° C. for 30 min; CaCO3 at 116° C. for 30 min. The pH is adjusted to 7.0 with KOH solution.

[0207] Example 8 Construction of a P. ananatis strain with a deleted ilvA gene P. ananatis SC17(0)ΔilvA::λattL-kan lacking the ilvA gene (SEQ ID NO:3) R The pMW118-attL-kan-λattR strain was constructed by λRed-dependent integration. For this purpose, the P. ananatis SC17(0) / RSF-Red-TER strain (US8383372 B2) was cultured overnight in LB liquid medium. Then, 1 mL of the culture was inoculated into 100 mL of LB liquid medium containing IPTG at a final concentration of 1 mM, and the cells were cultured at 32°C for 3 h with shaking (250 rpm). The cells were harvested and washed three times with 10% (v / v) glycerol to obtain competent cells. R The -attR plasmid (US7919284 B2) was used as a template, and primer P5 (sequence PCR was performed using P6 (SEQ ID NO: 10) and P7 (SEQ ID NO: 11) to obtain λattL-kan 1A, which has sequences at both ends that are homologous to the upstream and downstream of the ilvA gene. R The resulting DNA fragment was purified using the Wizard PCR Prep DNA Purification System (Promega) and subjected to electroporation. The bacteria were cultured in SOC medium for 2 hours and then incubated with 35 mg / L of The strain was spread on an LB plate containing kanamycin and cultured at 34°C for 16 hours. The colonies that emerged were purified in the same medium. PCR analysis (TaKaRa Speed ​​Star®; 25 cycles of 97°C for 20 seconds, 55°C for 20 seconds, and 72°C for 100 seconds) was then performed using primers P7 (SEQ ID NO: 11) and P8 (SEQ ID NO: 12). The ilvA gene on the chromosome was identified as λattL-kan R The results confirmed that the P. ananatis SC17(0)ΔilvA::λattL-kan R -λattR strain was obtained.

[0208] Example 9: Production of L-methionine using P. ananatis strain lacking the ilvA gene To test the effect of the deletion of ilvA on L-methionine production, SC17(0)ΔilvA::λattL-kan was transformed with the EdgeBio PurElute Bacterial Genomic kit according to the manufacturer's instructions. R Chromosomal DNA was isolated from the -λattR strain (Example 8) and 10 μg of DNA was electroporated. The L-methionine producing strain C2792 of P. ananatis was transformed with The transformants were grown in 20 mg / L of Kana. The resulting mixture was spread onto an LB plate containing .mycin and cultured at 34° C. overnight until individual colonies were visible. The replacement of the ilvA gene was confirmed by PCR analysis as described in Example 8. The desired transformant was identified. R -λa The ttR strain (abbreviation: C3653) was obtained.

[0209] P. ananatis strains C2792 and C2792ΔilvA::λattL-kan R Each of the -λattR strains was cultured in LB liquid medium at 32°C for 18 hours. Then, 0.2 mL of the resulting culture was inoculated into 2 mL of fermentation medium in a 20 × 200 mm test tube, and the mixture was fermented on a rotary shaker at 250 rpm for 32 hours until glucose was consumed. The plates were incubated at 4°C for 48 hours.

[0210] The composition of the fermentation medium (g / L) was as follows: Glucose 40.0 (NH4)2SO4 15.0 KH2PO41.5 MgSO4-7H2O 1.0 Thiamine-HCl 0.1 CaCO325.0 LB medium 4% (v / v)

[0211] The fermentation medium was sterilized at 116° C. for 30 min, except for glucose and CaCO3, which were sterilized separately as follows: glucose at 110° C. for 30 min; CaCO3 at 116° C. for 30 min. The pH was adjusted to 7.0 with KOH solution.

[0212] After cultivation, the amount of L-methionine accumulated in the medium was measured by Agilent 1260 amino-acid analyzer. The results of three independent test tube fermentations (as the mean value ± standard deviation) are shown in Table 4. As can be seen from Table 4, the modified strain P. ananatis C2792ΔilvA::λattL-kanR-λattR was able to accumulate a higher amount of L-methionine compared to the parent strain P. ananatis C2792.

[0213] [Table 4]

[0214] Example 10 Construction of E. coli MG1655 strain lacking ilvA gene E. coli MG1655 strain (ATCC 700926) carrying the temperature-sensitive replicative plasmid pKD46 is cultured overnight in LB liquid medium. Then, 1 mL of the culture is inoculated into 100 mL of LB liquid medium containing 50 mM arabinose and 50 mg / L ampicillin at a final concentration, and the cells are cultured at 37°C for 2 hours with shaking (250 rpm). The cells are harvested and washed three times with ice-cold 10% (v / v) glycerol to obtain competent cells. pMW118-attL-kan R PCR was performed using the -attR plasmid (US7919284 B2) as a template to obtain λattL-kan 1A, which has sequences at both ends that are homologous to the upstream and downstream of the ilvA gene. R The resulting DNA fragment was amplified using the Wizard PCR Prep DNA Purification System (Promega). The bacteria are then purified using the ELISA kit and introduced into competent cells by electroporation. After culturing for 2 hours on LB medium, the strain was spread onto an LB plate containing 35 mg / L kanamycin and cultured at 34°C for 16 hours. The colonies that appeared were purified on the same medium. R The replacement of the ΔilvA::λattL-kan with the ΔilvA-λattR cassette was confirmed by PCR. R -λattR strains are obtained.

[0215] Example 11: Production of L-methionine using an E. coli strain lacking the ilvA gene To test the effect of the deletion of ilvA on L-methionine production, MG1655ΔilvA::λattL-kan R The chromosomal DNA of the -λattR strain (Example 10) was introduced into the L-methionine producing strain 218 of E. coli (VKPM B-8125, RU2209248 C2) by P1 transduction. As a result, E. coli 218ΔilvA::λattL-kan R -λattR strains are obtained.

[0216] E. coli strain 218 and 218ΔilvA::λattL-kan R Each of the -λattR strains is cultured in LB liquid medium with shaking at 37°C for 18 hours. Next, 0.2 mL of the resulting culture is inoculated into 2 mL of fermentation medium in a 20 x 200 mm test tube, and cultured at 32°C for 48 hours on a rotary shaker at 250 rpm.

[0217] After cultivation, the amount of L-methionine accumulated in the medium is measured using an Agilent 1260 amino-acid analyzer.

[0218] The composition of the fermentation medium (g / L) is as follows: Glucose 40.0 (NH4)2SO4 15.0 KH2PO41.5 MgSO4-7H2O 1.0 Thiamine-HCl 0.1 Threonine 0.5 CaCO3 20.0 LB medium 4% (v / v)

[0219] The fermentation medium is sterilized at 116° C. for 30 min, except for glucose and CaCO3, which are sterilized separately as follows: glucose at 110° C. for 30 min; CaCO3 at 116° C. for 30 min. The pH is adjusted to 7.0 with KOH solution.

[0220] Preparatory Example 1 Construction of the L-methionine producing strain C2691 of P. ananatis 1.1. P. ananatis SC17(0)λattL-kan R -Construction of λattR-Pnlp8sd22-cysM strain The promoter region of the cysM gene (SEQ ID NO: 13) is inserted into the cassette λattL-kan R P. ananatis SC17(0)λattL-kan replaced with -λattR-Pnlp8sd22 R The -λattR-Pnlp8sd22-cysM strain was transformed with λRed-dependent For this purpose, the P. ananatis SC17(0) / RSF-Red-TER strain was constructed by selective integration. was cultured overnight in LB liquid medium. Then, 1 mL of the culture was inoculated into 100 mL of LB liquid medium containing IPTG at a final concentration of 1 mM, and the cells were cultured at 32°C for 3 hours with shaking (250 rpm). The cells were collected and washed three times with 10% (v / v) glycerol to obtain competent cells. The pMW118-attL-kan-attR-pnlp8sd22 plasmid (SEQ ID NO: 15) was cloned using primers P9 (SEQ ID NO: 14) and P10 (SEQ ID NO: 15). PCR was performed using the cysM gene promoter region as a template, and the upstream and downstream regions of the promoter region were identified as homologous. λattL-kan RThe amplified DNA fragment of -λattR-Pnlp8sd22 was obtained. The obtained DNA fragment was purified using Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by electroporation. The cells were cultured in SOC medium (Sambrook J. and Russell DW, Molecular Cloning: A Laboratory Manual (3 rd After culturing for 2 hours on LB plates containing 35 mg / L kanamycin, the plates were cultured at 34°C for 16 hours. The colonies that appeared were purified on the same medium. Next, the promoter region of the cysM gene on the chromosome was transformed into λattL-kan R To confirm that the -λattR-Pnlp8sd22 cassette had been substituted, PCR analysis was performed using primers P11 (SEQ ID NO: 17) and P12 (SEQ ID NO: 18) (TaKaRa Speed ​​Star®; 92°C for 10 seconds, 56°C for 10 seconds, and 72°C for 60 seconds for 40 cycles). As a result, P. ananatis SC17(0)λattL-kan R -λattR-Pnlp8sd22-cysM strain (abbreviation: C2338) was obtained.

[0221] 1.2. P. ananatis strain C2597 (SC17(0)ΔmdeA::λattL-kan R -λattR-Pnlp8sd22-cysM ) Construction The silent gene mdeA (SEQ ID NO: 19) was inserted into the cassette λattL-kan R -λattR-Pnlp8sd22-cysM P. ananatis SC17(0)ΔmdeA::λattL-kan RThe -λattR-Pnlp8sd22-cysM strain was constructed by λRed-dependent integration. For this purpose, the P. ananatis SC17(0) / RSF-Red-TER strain was cultivated overnight in LB liquid medium. Then, 1 mL of the culture was inoculated into 100 mL of LB liquid medium containing IPTG at a final concentration of 1 mM, and the cells were cultivated at 32°C for 3 hours with shaking (250 rpm). The cells were harvested and washed three times with 10% (v / v) glycerol to obtain competent cells. The chromosome fragment isolated from the C2338 strain (preparatory example 1.1) was amplified using primers P13 (SEQ ID NO: 20) and P14 (SEQ ID NO: 21). PCR was performed using the above as a template to obtain λattL-kan, which has sequences at both ends that are homologous to the upstream and downstream of the mdeA gene. R The amplified DNA fragment of -λattR-Pnlp8sd22-cysM was obtained. The obtained DNA fragment was subjected to Wizard PCR. The DNA was purified using the Prep DNA Purification System (Promega) and then introduced into competent cells by electroporation. The cells were cultured in SOC medium for 2 hours, and then incubated with 35 mg / L kanamycin. The strain was then spread onto an LB plate containing λattL-kan and cultured at 34°C for 16 hours. The colonies that appeared were purified using the same medium. R -λattR-Pnlp8sd22-cysM cassette To confirm that the gene had been substituted with ΔmdeA, primers P15 (SEQ ID NO: 22) and P16 (SEQ ID NO: 23) were used to perform PCR analysis (TaKaRa Speed ​​Star®; 40 cycles of 92°C for 10 seconds, 56°C for 10 seconds, and 72°C for 60 seconds). As a result, P. ananatis SC17(0)ΔmdeA::λattL-kan R -λattR-Pnlp8sd22-cysM strain (abbreviation: C2597) was obtained.

[0222] 1.3. P. ananatis C2603 strain (SC17ΔmdeA::λattL-kan R -λattR-Pnlp8sd22-cysM) C2597 strain (SC17(0) ΔmdeA::λattL-kan R Chromosomal DNA was isolated from the resulting plasmid (λattR-Pnlp8sd22-cysM) using the EdgeBio PurElute Bacterial Genomic kit according to the manufacturer's instructions. The isolated chromosomal DNA was used to transform P. ananatis strain SC17 (FERM BP-11091). For this purpose, P. ananatis strain SC17 was cultured overnight in LB liquid medium. Then, 1 mL of the culture was inoculated into 100 mL of LB liquid medium, and the cells were cultured at 32°C for 2 hours with shaking (250 rpm). The cells were harvested and washed three times with 10% (v / v) glycerol to obtain competent cells. C2597 strain (Preliminary Example 1 2) Chromosomal DNA isolated from the strain was introduced into competent cells by electroporation. The cells were cultured in SOC medium for 2 hours and then spread onto an LB plate containing 35 mg / L kanamycin. The resulting colonies were purified on the same medium. Next, PCR analysis was performed as described in Preliminary Example 1.2 to confirm the replacement of the mdeA gene on the chromosome. As a result, P. ananatis SC17ΔmdeA::λattL-kan R -λattR-Pnlp8sd22-cysM A strain (abbreviation: C2603) was obtained.

[0223] 1.4. C2603 strain (SC17ΔmdeA::λattL-kan R -λattR-Pnlp8sd22-cysM) Loss of a child The RSF(TcR)-int-xis plasmid (US20100297716 A1) was used to transfect C2603 with kanamycin. The resistance gene (kan) was deleted. RSF(TcR)-int-xis was introduced into the C2603 strain by electroporation, and the cells were plated on LB medium containing tetracycline (15 mg / L) and cultured at 30°C. The C2603 / RSF(TcR)-int-xis strain was obtained.

[0224] The resulting plasmid-containing strain was purified in LB medium containing tetracycline (15 mg / L) and 1 mM IPTG to obtain a single colony. It was then transferred to a medium containing 50 mg / L kanamycin. A single colony was plated and cultured overnight at 37°C with shaking (250 rpm). Kanamycin sensitivity The strain was plated on LB medium containing 10% sucrose (by weight) and 1 mM IPTG and cultured overnight at 37°C to remove the RSF(TcR)-int-xis plasmid from the strain. A viable colony was selected and the corresponding strain was designated C2614.

[0225] 1.5. P. ananatis C2607 strain (SC17(0)ΔmetJ::λattL-cat R - λattR) P. ananatis SC17(0)ΔmetJ::λattL-cat lacking the metJ gene (SEQ ID NO:24) R -λatt The R strain was constructed by λRed-dependent integration. For this purpose, P. ananatis SC17(0) / RSF-Red-TER strain was cultivated overnight in LB liquid medium. Then, 1 mL of the culture was diluted with IPTG at a final concentration of 1 mM. The cells were inoculated into 100 mL of LB liquid medium containing 10% (v / v) glycerol and cultured at 32°C for 3 hours with shaking (250 rpm). The cells were harvested and washed three times with 10% (v / v) glycerol to obtain competent cells. PCR was performed using primers P17 (SEQ ID NO: 25) and P18 (SEQ ID NO: 26) and the pMW118-attL-cat-attR plasmid (Minaeva NI et al., BMC Biotechnol., 2008, 8:63) as a template to clone the metJ gene. λattL-cat has upstream and downstream homologous sequences at both ends R - λattR amplified DNA fragment The obtained DNA fragment was purified using the Wizard PCR Prep DNA Purification System (Promega). The bacteria were then purified and introduced into competent cells by electroporation. After culturing for 2 hours at 34°C, the mixture was spread on an LB plate containing 35 mg / L chloramphenicol and cultured at 34°C for 16 hours. The colonies that appeared were purified on the same medium. Next, the metJ gene on the chromosome was transformed into λattL-cat R To confirm that the -λattR cassette had been replaced, PCR analysis was performed using primers P19 (SEQ ID NO: 27) and P20 (SEQ ID NO: 28) (TaKaRa Speed ​​Star®; 92 The results showed that P. ananatis SC17(0)ΔmetJ::λattL-cat R -λattR strain (abbreviation: C2607) was obtained.

[0226] 1.6. P. ananatis C2634 strain (C2614ΔmetJ::λattL-cat R - λattR) C2607 strain (SC17(0)ΔmetJ::λattL-cat R -λattR) (Preparatory Example 1.5) using the EdgeBio PurElute Bacterial Genomic kit according to the manufacturer's instructions. The obtained chromosomal DNA was used to transform the strain C2614 (Preparatory Example 1.4). For this purpose, the P. ananatis strain C2614 was cultivated overnight in LB liquid medium. Then, 1 mL of the culture was added to LB liquid The cells were inoculated into 100 mL of medium and cultured at 32°C for 2 hours with shaking (250 rpm). The cells were harvested and washed three times with 10% (v / v) glycerol to obtain competent cells. The DNA was introduced into competent cells by electroporation. The cells were incubated in SOC medium for 2 h. After culturing for 1 h, the mixture was spread on an LB plate containing 35 mg / L chloramphenicol and incubated at 34°C for 16 h. The resulting colonies were purified on the same medium. Then, to confirm the replacement of the metJ gene on the chromosome, PCR analysis was carried out as described in Preliminary Example 1.5. As a result, P. ananatis C2614ΔmetJ::λattL-cat R -λattR strain (abbreviation: C2634) was obtained.

[0227] 1.7. P. ananatis C2605 strain (SC17(0) λattL-kan R -λattR-Ptac71φ10-metA) Construction The promoter region of the metA gene (SEQ ID NO: 29) is R -λattR-Ptac71 P. ananatis SC17(0)λattL-kan substituted with φ10 R -λattR-Ptac71φ10-metA strain was constructed by λRed-dependent integration. The strain was cultured overnight in LB liquid medium. Then, 1 mL of the culture was inoculated into 100 mL of LB liquid medium containing IPTG at a final concentration of 1 mM, and the cells were cultured at 32°C for 3 hours with shaking (250 rpm). The cells were collected and washed three times with 10% (v / v) glycerol to obtain competent cells. The pMW118-attL-kan-attR-Ptac71φ10 plasmid (sequence number 30) was cloned using primers P21 (sequence number 31) and P22 (sequence number 31). PCR was performed using the (column 32) as a template, and the upstream and downstream of the promoter region of the metA gene were cloned. λattL-kan with homologous sequences at both ends R The amplified DNA fragment of -λattR-Ptac71φ10 was obtained. The obtained DNA fragment was purified using Wizard PCR Prep DNA Purification System (Promega). The plasmid was then introduced into competent cells by electroporation. The cells were cultured in SOC medium for 2 hours, then spread onto an LB plate containing 35 mg / L kanamycin and cultured at 34°C for 16 hours. The colonies that emerged were purified on the same medium. Next, the metA gene on the chromosome of the SC17(0) strain was cloned. The promoter region is λattL-kan R - Confirm that it has been replaced with λattR-Ptac71φ10 cassette To detect the nucleotide sequence of the nucleotide sequence, PCR analysis (TaKaRa Speed ​​Star®; 40 cycles of 92°C for 10 seconds, 56°C for 10 seconds, and 72°C for 60 seconds) was performed using primers P23 (SEQ ID NO: 33) and P24 (SEQ ID NO: 34). Result, P. ananatis SC17(0)λattL-kan R -λattR-Ptac71φ10-metA strain (abbreviation: C2605) was obtained.

[0228] 1.8. P. ananatis strain C2611 (SC17λattL-kan R -λattR-Ptac71φ10-metA) C2605 strain (SC17(0)λattL-kan R Chromosomal DNA was isolated from the 1.1-λattR-Ptac71φ10-metA (Preliminary Example 1.7) using the EdgeBio PurElute Bacterial Genomic kit according to the manufacturer's instructions. The chromosomal DNA obtained was used to transform the SC17 strain. For this purpose, the P. ananatis SC17 strain was cultured overnight in LB liquid medium. Then, 1 mL of the culture was inoculated into 100 mL of LB liquid medium. The cells were seeded and cultured at 32°C for 2 hours with shaking (250 rpm). The cells were harvested and diluted with 10% (v / v) glycerol. The competent cells were obtained by washing three times with a roll. The chromosomal DNA isolated from the C2605 strain was The bacteria were introduced into competent cells by troporation. The bacteria were cultured in SOC medium for 2 hours, then spread onto an LB plate containing 35 mg / L kanamycin and cultured at 34°C for 16 hours. Colonies were purified on the same medium. Then, to confirm the replacement of the promoter region of the metA gene on the chromosome, PCR analysis was carried out as described in Preliminary Example 1.7. Fruit, P. ananatis SC17λattL-kan R -λattR-Ptac71φ10-metA strain (abbreviation: C2611) was obtained. Ta.

[0229] 1.9. P. ananatis C2619 strain (C2611ΔmetJ::λattL-cat R - λattR) C2607 strain (SC17(0)ΔmetJ::λattL-cat R -λattR) (Preparatory Example 1.5) using the EdgeBio PurElute Bacterial Genomic kit according to the manufacturer's instructions. The obtained chromosomal DNA was used to transform the strain C2611 (Preparatory Example 1.8). For this purpose, the P. ananatis strain C2611 was cultivated overnight in LB liquid medium. Then, 1 mL of the culture was added to LB liquid The cells were inoculated into 100 mL of medium and cultured at 32°C for 2 hours with shaking (250 rpm). The cells were harvested and washed three times with 10% (v / v) glycerol to obtain competent cells. The DNA was introduced into competent cells by electroporation. The cells were incubated in SOC medium for 2 h. After culturing for 1 h, the mixture was spread on an LB plate containing 35 mg / L chloramphenicol and incubated at 34°C for 16 h. The resulting colonies were purified on the same medium. Then, to confirm the replacement of the metJ gene on the chromosome, PCR analysis was carried out as described in Preliminary Example 1.5. As a result, P. ananatis C2611ΔmetJ::λattL-cat R -λattR strain (abbreviation: C2619) was obtained.

[0230] 1.10. Selection of P. ananatis strains carrying mutant alleles of the metA gene encoding feedback-resistant MetA C2619 strain (SC17λattL-kan R -λattR-Ptac71φ10-metA ΔmetJ::λattL-cat R The cells of the strain were inoculated into a 50 mL flask containing LB liquid medium to an OD600 of 0.05 and incubated at 34°C for 2 h. The cells were cultured for 2 hours under aeration (250 rpm). When the OD600 reached 0.25, the logarithmic growth phase cell culture of the same strain was treated with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) (final concentration 25 mg / L) for 20 minutes. The resulting culture was centrifuged, washed twice with fresh LB liquid medium, and then plated on M9 agar plates containing glucose (0.2%) and norleucine (600 g / L). The L-methionine producing ability of each of the strains was examined. The strain with the highest L-methionine producing ability was selected, and the base sequence of the metA gene of the strain was determined. The sequence analysis revealed that the metA gene has a cytosine residue of arginine (Arg) at position 34 in the amino acid sequence of wild-type MetA (SEQ ID NO: 35). A mutation was found that results in a substitution of an isoform residue with an isoform (R34C mutation). The amino acid sequence of the mutant MetA protein having the R34C mutation is shown in SEQ ID NO: 37, and the nucleotide sequence of the mutant metA gene encoding the mutant MetA protein is shown in SEQ ID NO: 36. R -λattR-Ptac71φ10-metA(R34C)ΔmetJ::λattL-cat R - λattR strain (abbreviation: C2664) was constructed. Built it.

[0231] 1.11. P. ananatis C2669 strain (C2634λattL-kan R -λattR-Ptac71φ10-metA(R34C)) C2664 strain (SC17λattL-kan R -λattR-Ptac71φ10-metA(R34C)ΔmetJ::λattL-cat R -λattR) (Preparatory Example 1.10) was purchased from the manufacturer. Chromosomal DNA was isolated using the method described in the instructions. The obtained chromosomal DNA was transformed into C2634 strain (preliminary example 1). 1.6) was used for the transformation of P. ananatis C2634 in LB liquid medium. After overnight culture, 1 mL of the culture was inoculated into 100 mL of LB liquid medium, and the cells were incubated at 32°C for 2 hours. The cells were harvested and washed three times with 10% (v / v) glycerol to isolate the competent cells. The chromosomal DNA isolated from the C2664 strain was introduced into the competent cells by electroporation. The cells were cultured in SOC medium for 2 hours and then incubated in a 35 mg / L kanamycin-containing medium. The resulting clone was spread on an LB plate containing 100 kb of P. ananatis C2634 λattL-kan and cultured at 34°C for 16 hours. The colonies that appeared were purified using the same medium. Next, to confirm the replacement of the promoter region of the metA gene on the chromosome, PCR analysis was performed as described in Preliminary Example 1.7. As a result, P. ananatis C2634 λattL-kan R -λattR-Ptac71φ10-metA(R34C) strain (abbreviation: C2669) was obtained.

[0232] 1.12. C2669 strain (C2614ΔmetJ::λattL-cat R -λattR λattL-kan R -λattR-Ptac71φ10-metA(R34C) Kanamycin was isolated from strain C2669 (Preparatory Example 1.11) using the RSF(TcR)-int-xis plasmid. The RSF(TcR)-int-xis gene was introduced into the C2669 strain by electroporation, and the cells were plated on LB medium containing tetracycline (15 mg / L) and cultured at 30°C to obtain the C2669 / RSF(TcR)-int-xis strain.

[0233] The resulting plasmid-carrying strain was purified in LB medium containing tetracycline (15 mg / L) and 1 mM IPTG to obtain a single colony. The single colony was then plated on a medium containing 50 mg / L kanamycin and 35 mg / L chloramphenicol and cultured overnight at 37°C with shaking (250 rpm). The strain sensitive to both kanamycin and chloramphenicol was plated on LB medium containing 10% sucrose (by weight) and 1 mM IPTG and cultured overnight at 37°C to remove the RSF(TcR)-int-xis plasmid from the strain. A tetracycline-sensitive colony was selected, and the corresponding strain was designated C2691.

[0234] Preparatory Example 2 Construction of L-methionine producing strain C2792 of P. ananatis 2.1. P. ananatis SC17(0)ΔmetE1::λattL-kan R -λattR-Ptac71φ10-thrA442 structure Construction The metE1 gene (c0742) (SEQ ID NO: 38) was inserted into the cassette λattL-kan R - P. ananatis SC17(0)ΔmetE1::λattL-kan replaced with λattR-Ptac71φ10-thrA442 (SEQ ID NO: 39) RThe -λattR-Ptac71φ10-thrA442 strain was constructed by λRed-dependent integration. For this purpose, the P. ananatis SC17(0) / RSF-Red-TER strain was cultured overnight in LB liquid medium. Then, 1 mL of the culture was inoculated into 100 mL of LB liquid medium containing IPTG at a final concentration of 1 mM, and the cells were cultured at 32°C for 3 h with shaking (250 rpm). The cells were harvested and washed three times with 10% (v / v) glycerol to obtain competent cells. PCR was performed using primers P25 (SEQ ID NO: 40) and P26 (SEQ ID NO: 41) to obtain λattL-kan 10-kDa ... R -λattR-Ptac71φ10-thrA442 The amplified DNA fragment was purified using the Wizard PCR Prep DNA Purification System. (Promega) and then introduced into competent cells by electroporation. The cells were cultured in SOC medium for 2 hours and then spread onto an LB plate containing 35 mg / L kanamycin. The resulting colonies were purified on the same medium. R To confirm that the -λattR-Ptac71φ10-thrA442 cassette had been replaced, PCR was performed using primers P27 (SEQ ID NO: 42) and P28 (SEQ ID NO: 43). As a result, P. ananatis SC17(0)ΔmetE1::λattL-kan R The -λattR-Ptac71φ10-thrA442 strain was obtained.

[0235] 2.2. P. ananatis C2707 strain (C2691ΔmetE1::λattL-kan R -λattR-Ptac71φ10-thrA442) SC17(0)ΔmetE1::λattL-kan RChromosomal DNA was isolated from the -λattR-Ptac71φ10-thrA442 strain using the EdgeBio PurElute Bacterial Genomic kit according to the manufacturer's instructions. The obtained chromosomal DNA was used to transform the P. ananatis strain C2691 (Preliminary Example 1). For this purpose, the P. ananatis strain C2691 was cultivated overnight in LB liquid medium. Then, 1 mL of the culture was added to the LB liquid medium. The cells were inoculated into 100 mL of medium and cultured at 32°C for 2 hours with shaking (250 rpm). The cells were harvested and washed three times with 10% (v / v) glycerol to obtain competent cells. R Chromosomal DNA isolated from the -λattR-Ptac71φ10-thrA442 strain (Preliminary Example 2.1) was introduced into competent cells by electroporation. The cells were cultured in SOC medium for 2 hours and then incubated for 35 The bacteria were spread onto an LB plate containing 100 mg / L kanamycin and cultured at 34°C for 16 hours. Ronnie was purified on the same medium. Next, to confirm the replacement of the metE1 gene on the chromosome, PCR analysis was performed as described in Preliminary Example 2.1. As a result, P. ananatis C2691ΔmetE1::λattL-kan R -λattR-Ptac71φ10-thrA442 strain (abbreviation: C2707) was obtained.

[0236] 2.3. C2707 strain (C2691ΔmetE1::λattL-kan R -λattR-Ptac71φ10-thrA442) The kanamycin resistance gene (kan) was deleted from the C2707 strain using the RSF(TcR)-int-xis plasmid. RSF(TcR)-int-xis was introduced into the C2707 strain by electroporation, and the tet The cells were plated on LB medium containing lacycline (15 mg / L) and cultured at 30°C to obtain the C2707 / RSF(TcR)-int-xis strain.

[0237] The resulting plasmid-containing strain was purified in LB medium containing tetracycline (15 mg / L) and 1 mM IPTG to obtain a single colony. It was then transferred to a medium containing 50 mg / L kanamycin. A single colony was plated and cultured overnight at 37°C with shaking (250 rpm). Kanamycin sensitivity The strain was plated on LB medium containing 10% sucrose (by weight) and 1 mM IPTG and cultured overnight at 37°C to remove the RSF(TcR)-int-xis plasmid from the strain. A viable colony was selected and the corresponding strain was designated С2743.

[0238] 2.4. P. ananatis SC17(0)λattL-kan R -Construction of λattR-Ptac71φ10-metH strain The promoter region of the metH gene (SEQ ID NO: 44) is R -λattR-Ptac71 P. ananatis SC17(0)λattL-kan substituted with φ10 R -λattR-Ptac71φ10-metH strain was constructed by λRed-dependent integration. The strain was cultured overnight in LB liquid medium. Then, 1 mL of the culture was inoculated into 100 mL of LB liquid medium containing IPTG at a final concentration of 1 mM, and the cells were cultured at 32°C for 3 hours with shaking (250 rpm). The cells were harvested and washed three times with 10% (v / v) glycerol to obtain competent cells. The pMW118-attL-kan was cloned using primers P29 (SEQ ID NO: 45) and P30 (SEQ ID NO: 46). R PCR was performed using the -attR-Ptac71φ10 plasmid (SEQ ID NO: 32) as a template to clone the upstream and downstream of the promoter region of the metH gene. λattL-kan with homologous sequences at both ends RThe amplified DNA fragment of -λattR-Ptac71φ10 was obtained. The obtained DNA fragment was purified using Wizard PCR Prep DNA Purification System (Promega). The plasmid was then introduced into competent cells by electroporation. The cells were cultured in SOC medium for 2 hours, then spread onto an LB plate containing 35 mg / L kanamycin and cultured at 34°C for 16 hours. The colonies that emerged were purified on the same medium. Next, the promoter region of the metH gene on the chromosome was transformed into λattL-kan R -To confirm that the λattR-Ptac71φ10 cassette has been replaced, PCR analysis (TaKaRa Speed ​​Star®; 25 cycles of 97°C for 20 seconds, 54°C for 20 seconds, and 72°C for 100 seconds) was performed using primers P31 (SEQ ID NO: 47) and P32 (SEQ ID NO: 48). As a result, P. ananatis SC17(0)λattL-kan R -λattR-Ptac71φ10-metH strain was obtained.

[0239] 2.5. P. ananatis C2761 strain (С2743 λattL-kan R -λattR-Ptac71φ10-metH) SC17(0)λattL-kan R Chromosomal DNA was isolated from the -λattR-Ptac71φ10-metH strain (Preparatory Example 2.4) using the EdgeBio PurElute Bacterial Genomic kit according to the manufacturer's instructions. The obtained chromosomal DNA was used to transform the P. ananatis strain C2743 (Preparatory Example 2.3). For this purpose, P. ananatis С2743 strain was cultured overnight in LB liquid medium. Then, 1 mL of the culture was inoculated into 100 mL of LB liquid medium, and the cells were cultured at 32°C for 2 h with shaking (250 rpm). The cells were harvested and washed three times with 10% (v / v) glycerol to obtain competent cells. SC17(0) λattL-kan RChromosomal DNA isolated from the -attR-Ptac71φ10-metH strain was electroporated. The cells were then cultured in SOC medium for 2 hours, and then treated with 35 mg / L kanamycin. The resulting colonies were purified on the same medium. R -λattR-Ptac71φ10 cassette was replaced with the As a result, P. ananatis С2743λattL-kan R -λattR-Ptac71φ10-metH strain (abbreviation: C2761) was obtained.

[0240] 2.6. P. ananatis SC17(0)λattL-cat R -Construction of λattR-Pnlp8-gcvT strain The promoter region of the gcvT gene (SEQ ID NO: 49) is inserted into the cassette λattL-cat R - P. ananatis SC17(0)λattL-cat replaced with λattR-Pnlp8 R -λattR-Pnlp8-gcvT strain was used as a λRed-dependent recombinant For this purpose, the P. ananatis SC17(0) / RSF-Red-TER strain was transformed into LB liquid. The bacteria were cultured overnight in LB medium. Then, 1 mL of the culture was inoculated into 100 mL of LB liquid medium containing IPTG at a final concentration of 1 mM, and the bacteria were cultured at 32°C for 3 hours with shaking (250 rpm). The bacteria were harvested and then diluted with 10% (v / v) glucose. The competent cells were obtained by washing three times with glycerol. R PCR was performed using the -attR-Pnlp8 plasmid (WO2011043485) as a template to obtain sequences homologous to the upstream and downstream of the promoter region of the gcvT gene. λattL-cat at both ends R The amplified DNA fragment of -λattR-Pnlp8 was obtained. The obtained DNA fragment was purified using Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by electroporation. The cells were cultured in SOC medium for 2 hours and then incubated with 35 mg / L of The strain was spread on an LB plate containing kanamycin and cultured at 34°C for 16 hours. The colonies that appeared were purified in the same medium. Next, the promoter region of the gcvT gene on the chromosome was transformed into λattL-cat R To confirm that the -λattR-Pnlp8 cassette had been substituted, PCR analysis (TaKaRa Speed ​​Star®; 40 cycles of 97°C for 20 seconds, 54°C for 20 seconds, and 72°C for 120 seconds) was performed using primers P35 (SEQ ID NO: 52) and P36 (SEQ ID NO: 53). R -λattR-Pnlp8-gcvT strain was obtained.

[0241] 2.7. P. ananatis strain C2777 (C2761 λattL-cat R -λattR-Pnlp8-gcvT) SC17(0)λattL-cat R Chromosomal DNA was isolated from the -λattR-Pnlp8-gcvT strain (Preparatory Example 2.6) using the EdgeBio PurElute Bacterial Genomic kit according to the manufacturer's instructions. The chromosomal DNA was used to transform the P. ananatis strain C2761 (Preparatory Example 2.5). For this purpose, the P. ananatis strain C2761 was cultured overnight in LB liquid medium. Then, 1 mL of the culture was inoculated into 100 mL of LB liquid medium, and the cells were cultured at 32° C. for 2 hours with shaking (250 rpm). The cells were harvested. The cells were then washed three times with 10% (v / v) glycerol to obtain competent cells. RChromosomal DNA isolated from the -λattR-Pnlp8-gcvT strain was transformed into competent The cells were cultured in SOC medium for 2 hours and then transferred to LB medium containing 35 mg / L kanamycin. The resulting colonies were purified using the same medium. R - PCR analysis was performed as described in preliminary example 2.7 to confirm the replacement with the λattR-Pnlp8 cassette. As a result, P. ananatis C2761λattL-cat R -λattR-Pnlp8-gcvT strain (abbreviation: C2777) was obtained.

[0242] 2.8. C2777 strain (C2761λattL-cat R -λattR-Pnlp8-gcvT) Deletion of From strain C2777 (Preparatory Example 2.7) using the RSF(TcR)-int-xis plasmid to kanamycin The RSF(TcR)-int-xis gene was introduced into the C2777 strain by electroporation, and the cells were plated on LB medium containing tetracycline (15 mg / L) and cultured at 30°C to obtain the C2777 / RSF(TcR)-int-xis strain.

[0243] The resulting plasmid-containing strain was purified in LB medium containing tetracycline (15 mg / L) and 1 mM IPTG to obtain single colonies. A single colony was plated on a medium containing kanamycin and chloramphenicol and cultured overnight at 37°C with shaking (250 rpm). A strain sensitive to both kanamycin and chloramphenicol was plated on LB medium containing 10% sucrose (by weight) and 1 mM IPTG to remove the RSF(TcR)-int-xis plasmid from the strain, and cultured overnight at 37°C. A tetracycline-sensitive colony was selected, and the corresponding strain was designated C2792.

[0244] Although the invention has been described in detail with reference to preferred embodiments, it will be apparent to those skilled in the art that various modifications and equivalents can be made without departing from the scope of the invention. [Industrial Applicability]

[0245] The method of the present invention is useful for producing L-amino acids by bacterial fermentation.

Claims

1. 1. A method for producing an L-amino acid, comprising the steps of: (i) Cultivating L-amino acid producing bacteria belonging to the order Enterobacterales in a medium producing and accumulating an L-amino acid in a medium or in the bacterial cells of the bacterium, or both; and (ii) recovering an L-amino acid from the medium or from the bacterial cells, or both. Including, The bacterium belongs to the genus Pantoea, the L-amino acid is L-methionine; the bacterium has a modified expression of a gene encoding a protein having indole-pyruvate decarboxylase activity; The modification reduces the amount of the expression product of the gene per cell to 50% or less of that of an unmodified bacterium. The activity of the protein, which is the expression product of the gene, per cell is 50% of that of the unmodified bacterium. The method of claim 1, wherein the modification reduces the expression of the gene and / or the gene is deleted.

2. The method according to claim 1 , wherein the gene encoding a protein having indolepyruvate decarboxylase activity is the ipdC gene.

3. 3. The method of claim 1 or 2, wherein the protein having indolepyruvate decarboxylase activity is selected from the group consisting of: (A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 55; (B) Substitution or deletion of 1 to 50 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 or 55 , insertions, and / or additions, wherein the protein has indole-pyruvate decarboxylase activity; and (C) an amino acid sequence having 90% or more identity to the entire amino acid sequence shown in SEQ ID NO: 2 or 55; A protein comprising an indole acid sequence and having indole-pyruvate decarboxylase activity.

4. The method according to any one of claims 1 to 3, wherein the gene is selected from the group consisting of: (a) a gene comprising the nucleotide sequence shown in SEQ ID NO: 1 or 54; (b) a gene comprising a nucleotide sequence capable of hybridizing under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or 54, the gene encoding a protein having indole-pyruvate decarboxylase activity, the stringent conditions being such that a hybrid having an identity of 90% or more is formed, but a hybrid having an identity of less than 90% is not formed; (c) Substitution or deletion of 1 to 50 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 or 55. a gene encoding a protein comprising an amino acid sequence containing an amino acid sequence including an insertion and / or addition, the protein having indole-pyruvate decarboxylase activity; and (d) A gene comprising a mutant nucleotide sequence of SEQ ID NO: 1 or 54, wherein the mutant nucleotide sequence is due to the degeneracy of the genetic code.

5. The method according to any one of claims 1 to 4, wherein expression of a gene encoding a protein having indole-pyruvate decarboxylase activity is attenuated by inactivation of the gene.

6. The method of claim 5 , wherein the gene encoding the protein having indolepyruvate decarboxylase activity is deleted.

7. The method according to any one of claims 1 to 6, wherein the bacterium has been further modified so that expression of a gene encoding a protein having threonine deaminase activity is attenuated.

8. The method according to claim 7, wherein the gene encoding a protein having threonine deaminase activity is the ilvA gene.

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