JP2022550084A - Method for producing L-amino acid by bacterial fermentation - Google Patents

Abstract

The present invention relates to Enterobacteriaceae modified to attenuate the expression of a gene (ipdC) encoding a protein having indole pyruvate decarboxylase activity and/or a gene (ilvA) encoding a protein having threonine deaminase activity. Provided is a method for producing L-amino acids by fermentation using bacteria belonging to

Description

The present invention relates to microbial industry, and in particular, enterobacteria modified so that the expression of a gene encoding a protein having indolepyruvate decarboxylase activity is attenuated and the production of L-amino acids is enhanced. The present invention relates to a method for producing L-amino acids by fermentation of bacteria belonging to the order Enterobacterales.

Conventionally, L-amino acids have been industrially produced by fermentation methods using microbial strains obtained from natural sources or their variants. Typically, such microorganisms have been modified to increase the production yield of L-amino acids.

Many techniques have been reported to increase the production yield of L-amino acids, including transformation of microorganisms with recombinant DNA (see, for example, US4278765 A), promoters, leader sequences and/or attenuators, or There are other modifications of the expression control region 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 enzymes involved in amino acid biosynthesis and/or releasing the feedback inhibition of the target enzyme by the produced L-amino acid (for example, WO9516042 A1 , EP0685555 A1, or US4346170A, US5661012A, and US6040160A).

Another method for increasing the production yield of an L-amino acid is to convert one or several genes involved in the degradation of the desired L-amino acid and the desired L-amino acid biosynthetic pathway into the L-amino acid. , genes involved in the redistribution of carbon, nitrogen, sulfur, and phosphate fluxes, and genes encoding toxins.

The ipdC gene encodes the IpdC protein characterized as indolepyruvate decarboxylase (Enzyme Commission (EC) number: 4.1.1.74). Indolepyruvate decarboxylase (IpdC) has been shown to be involved in the biosynthesis of indole-3-pyruvate from tryptophan and indole-3-acetic acid via indole-3-acetaldehyde in various bacteria, including Pantoea strains. (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 ).

However, no data describing the effect of attenuated expression of the ipdC gene on L-amino acid production by fermentation of L-amino acid-producing bacteria belonging to the order Enterobacteria has been reported so far.

This specification describes an improved method of producing L-amino acids by fermentation of bacteria belonging to the order Enterobacteriaceae. According to the present invention, L-amino acid production by fermentation of bacteria belonging to the order Enterobacteria can be increased. Specifically, the production of L-amino acids by fermentation of bacteria belonging to the order Enterobacteria weakens the expression of genes encoding proteins having indolepyruvate decarboxylase activity,
It can be improved by enhancing the production of L-amino acids by the bacterium modified with the above. The production of L-amino acids by modified bacteria can be further improved by further modifying the bacterium to attenuate the expression of genes encoding proteins with threonine deaminase activity.

The present invention provides the following.

One aspect of the present invention is a method for producing an L-amino acid, comprising:
(i) culturing an L-amino acid-producing bacterium belonging to the order Enterobacterales in a medium to produce and accumulate an L-amino acid in the medium or in the cells of the bacterium, or both; and (ii) recovering an L-amino acid from said medium or said bacterial cells, or both;
An object of the present invention is to provide a method, wherein the bacterium is modified so that expression of a gene encoding a protein having indole pyruvate decarboxylase activity and/or a gene encoding a protein having threonine deaminase activity is attenuated.

Another aspect of the present invention is a method for producing an L-amino acid, comprising:
(i) culturing an L-amino acid-producing bacterium belonging to the order Enterobacterales in a medium to produce and accumulate an L-amino acid in the medium or in the cells of the bacterium, or both; and (ii) recovering an L-amino acid from said medium or said bacterial cells, or both;
An object of the present invention is to provide a method, wherein the bacterium is modified so that expression of a gene encoding a protein having indole pyruvate decarboxylase activity is attenuated.

Another aspect of the present invention is to provide the above method, wherein the gene encoding the protein having indole pyruvate decarboxylase activity is the ipdC gene.

Another aspect of the present invention is to provide said method, wherein said protein having indole pyruvate 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 comprising substitutions, deletions, insertions, and/or additions of 1 to 250 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 or 55, wherein the protein exhibits indole pyruvate decarboxylase activity. and (C) a protein comprising an amino acid sequence having 50% or more identity to the entire amino acid sequence set forth in SEQ ID NO: 2 or 55, which protein has indole pyruvate decarboxylase activity.

Another aspect of the invention is to provide said method, 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 with a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or 54, which encodes a protein having indole pyruvate decarboxylase activity; gene;
(c) a gene encoding a protein comprising an amino acid sequence containing substitutions, deletions, insertions, and/or additions of 1 to 250 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 or 55, wherein the protein is 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. ,gene.

Another aspect of the present invention is to provide the above method, wherein expression of the gene encoding the protein having indole pyruvate decarboxylase activity is attenuated by inactivation of the gene.

Another aspect of the present invention is to provide the above method, wherein the gene encoding the protein having indole pyruvate decarboxylase activity is deleted.

Another aspect of the present invention is to provide the above method, wherein the bacterium is further modified to attenuate the expression of a gene encoding a protein having threonine deaminase activity.

Another aspect of the present invention is to provide the above method, wherein the gene encoding the protein having threonine deaminase activity is the ilvA gene.

Another aspect of the present invention is to provide said method, wherein said bacterium belongs to the family Enterobacteriaceae or Erwiniaceae.

Another aspect of the present invention is to provide the method, wherein the bacterium belongs to the genus Escherichia or Pantoea.

Another aspect of the present invention is to provide said method, wherein said bacterium is Escherichia coli or Pantoea ananatis.

Another aspect of the present invention is to provide the method, 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.

Another aspect of the present invention is to provide said method, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tryptophan, and L-tyrosine.

Another aspect of the present invention is that the non-aromatic L-amino acid is L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, selected from the group consisting of glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-proline, L-serine, L-threonine, and L-valine; The object is to provide said method.

Another aspect of the present invention is to provide the above method, wherein the sulfur-containing L-amino acid is selected from the group consisting of L-cysteine, L-methionine, L-homocysteine, and L-cystine.

Still other objects, features and attendant advantages of the present invention will become apparent to those skilled in the art upon reading the following detailed description of embodiments constructed in accordance therewith.

1. Bacteria The bacteria described herein contain indolepyruvate decarboxylase.
It may be an L-amino acid-producing bacterium belonging to the order Enterobacterales that has been modified to weaken the expression of a gene encoding a protein having decarboxylase activity. The bacteria described herein can be used in the methods described herein. Therefore, the following description of the same bacterium applies mutatis mutandis to any bacterium used alternatively or equivalently in the methods described herein.

Bacteria that can be used in the methods described herein can be appropriately selected depending on the type of L-amino acid of interest to be produced using the method.

Any L-amino acid-producing bacterium belonging to the order Enterobacteria can be used in the method described herein, as long as it is modified to weaken the expression of a gene encoding a protein having indolepyruvate decarboxylase activity. Any bacteria belonging to the order Enterobacteriaceae, provided that the expression of a gene encoding a protein having indolepyruvate decarboxylase activity is modified to be attenuated, so that the production of L-amino acids can be enhanced compared to unmodified bacteria. of L-amino acid-producing bacteria can be used in the methods described herein.

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

The term "L-amino acid-producing bacterium" means that when the bacterium is cultured in a medium, it produces, excretes or secretes an L-amino acid in the medium and/or the cells of the bacterium (i.e., bacterial cells), and/or bacteria belonging to the order Enterobacteriaceae having the ability to accumulate.

In addition, the term "L-amino acid-producing bacterium" includes, for example, the ability to produce, excrete or secrete, and/or accumulate L-amino acids in a medium in a larger amount than non-modified bacteria. Bacteria can also mean. The term "unmodified bacterium" may be used interchangeably or equivalently with the term "unmodified strain". The term "unmodified strain" can refer to a control strain that has not been modified to attenuate the expression of a gene encoding a protein having indolepyruvate decarboxylase activity. Examples of unmodified bacteria include wild strains or parent strains such as Pantoea ananatis (P. ananatis) AJ13355 and Escherichia coli (E. coli) K-12 strains (e.g., W3110 (ATCC 27325) and MG1655 (ATCC 47076)). mentioned. In addition, the term "L-amino acid-producing bacteria" is, for example, 0.1 g/L or more, 0.5 g/L or more, or 1.0 g/L or more, and can accumulate the desired L-amino acid in the medium. Bacteria can also mean.

Bacteria belonging to the order Enterobacteria and having L-amino acid-producing ability, which are modified so as to weaken expression of a gene encoding a protein having indolepyruvate decarboxylase activity, can also be used. Bacteria may inherently have L-amino acid-producing ability, or may be modified to have L-amino acid-producing ability. Such modifications can be achieved, for example, by mutagenesis or DNA recombination techniques. The bacterium can be obtained by attenuating the expression of a gene encoding a protein having indolepyruvate decarboxylase activity in a bacterium that inherently has an L-amino acid-producing ability. Alternatively, the bacterium can be obtained by imparting L-amino acid-producing ability to a bacterium already modified to weaken the expression of a gene encoding a protein having indolepyruvate decarboxylase activity. Alternatively, the bacterium may have acquired L-amino acid-producing ability by modifying to weaken the expression of a gene encoding a protein having indolepyruvate decarboxylase activity. Specifically, the bacterium described herein can be obtained, for example, by modifying the bacterial strain described below.

The term "L-amino acid-producing ability" means the ability of bacteria belonging to the order Enterobacteriaceae to produce, excrete or secrete, and/or accumulate L-amino acids in the medium and/or bacterial cells. obtain. Specifically, the term "L-amino acid-producing ability" refers to the extent that L-amino acids can be recovered from the medium and/or bacterial cells when the bacteria are cultured in the medium. may refer to the ability of bacteria belonging to the order Enterobacteriaceae to produce, excrete or secrete and/or accumulate L-amino acids in

The term “cultured” when referring to bacteria grown according to the methods described herein includes “cultivated,” “grown,” etc., which are well known to those skilled in the art. may be used interchangeably or equivalently with the term

Bacteria can produce L-amino acids alone or as a mixture of L-amino acids and one or more substances different from L-amino acids. For example, a bacterium can extract an L-amino acid of interest alone, or with one or more amino acids different from the L-amino acid of interest (for example, an L-amino acid (also referred to as an L-amino acid)). etc.). In other words, it is permissible that a bacterium can produce two or more L-amino acids as a mixture. Furthermore, for example, bacteria can produce the L-amino acid of interest alone or as a mixture of the L-amino acid of interest and one or more other organic acids (eg, carboxylic acids, etc.).

L-amino acids are not particularly limited, but 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 are included.

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

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 derivative forms (salts, hydrates, adducts, or combinations thereof, etc.). . An adduct can be a compound formed with an amino acid or carboxylic acid and another organic or inorganic compound. Thus, the terms "L-amino acid" and "carboxylic acid" can mean, for example, L-amino acids and carboxylic acids in free form, derivative forms, or mixtures thereof. The terms "L-amino acid" and "carboxylic acid" may, for example, denote, inter alia, L-amino acids and carboxylic acids in free form, salts thereof, or mixtures thereof. The terms "L-amino acid" and "carboxylic acid" refer to, for example, their sodium salts, potassium salts, ammonium salts, monohydrates, dihydrates, trihydrates, monohydrochlorides, dihydrochlorides. can mean any of the salts such as The terms "L-amino acid" and "carboxylic acid" do not refer to hydration state unless otherwise specified (e.g., "L-amino acid in free form", "carboxylic acid in free form", "salt of L-amino acid", and the terms "salts of carboxylic acids") can mean non-hydrates of L-amino acids and carboxylic acids, hydrates of L-amino acids and carboxylic acids, or mixtures thereof.

L-amino acids can belong to one or more L-amino acid families. By way of example, L-amino acids include the glutamic acid family, including L-arginine, L-glutamic acid, L-glutamine, and L-proline; the serine family, including L-cysteine, glycine, and L-serine; L-asparagine, L- the aspartic acid family, including aspartic acid, L-isoleucine, L-lysine, L-methionine, and L-threonine;
It can belong to the pyruvate 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" can mean L-histidine in addition to the above aromatic L-amino acids.

L-amino acids may also belong to the non-aromatic family, examples being 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. Since the biosynthetic pathway of aromatic amino acids such as L-phenylalanine, L-tryptophan, and L-tyrosine differs from the biosynthetic pathway of L-histidine, the term "non-aromatic L-amino acid" is - in addition to amino acids, can mean L-histidine.

Furthermore, L-amino acids 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 (for example, Ferra MP and Patrick WM, Bacterial methionine biosynthesis, Microbiology, 2014, 160(Pt 8): 1571 -1584). Specifically, in E. coli, L-cysteine is biochemically derived from L-serine. Specifically, L-serine is activated by serine acetyltransferase (CysE) to give O-acetylserine, which is then converted to O-acetylserine using a reduced sulfur source such as hydrogen sulfide. Serine (thiol)-lyase (O-acetylserine(thiol)-lyase; CysM
) to form L-cysteine. Next, L-cysteine is converted to O-succinylhomoserine (thiol)-lyase/O-succinylhomoserine lyase (O-succinylhomoserine (thiol)-lyase/O-succinylhomoserine lyase; MetB) and cystathionine β-lyase/cysteine desulfhydrolase. (cystathionine β-lyase/L-cysteine desulfhydrase; MetC) sequentially catalyzes transsulfation of L
- can be converted to homocysteine; L-methionine is synthesized from L-homocysteine by homocysteine transmethylase (MetE) and/or methionine synthase (MetH). L-cystine is normally produced in the medium together with L-cysteine as a result of oxidation of L-cysteine (Nakamori et al.
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).

Since some L-amino acids can be intermediate amino acids in the biosynthetic pathway of a particular L-amino acid, the amino acid family also includes other L-amino acids (e.g., non-proteinogenic L-amino acids). good. For example, L-citrulline and L-ornithine are amino acids of the arginine biosynthetic pathway. Thus, the glutamic acid family may include L-arginine, L-citrulline, L-glutamic acid, L-glutamine, L-ornithine, and L-proline.

In recent years, bacteria belonging to the Enterobacteriaceae family have undergone comprehensive comparative genomic analysis, including phylogenetic tree reconstruction based on 1548 core proteins, 53 ribosomal proteins, and 4 multilocus sequencing proteins. (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. nov., Hafniaceae fam. nov., Morgan
nov., and Budviciaceae fam. nov., Int. J. Syst. Evol. Microbiol., 2016, 66:5575-5599).

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

Species of bacteria belonging to the genus Escherichia are not particularly limited, and species classified into the genus Escherichia according to the classification known to microbiological experts can be mentioned. As Escherichia bacteria, for example, 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, ^2nd ed. ASM Press, Washington, DC, 1996). Escherichia bacteria include, in particular, Escherichia coli (E. coli). As E. coli, specifically, the prototype wild strain E. coli K-12 strain (E. coli W3110 (ATCC 27325) and E. coli
MG1655 (ATCC 47076), etc.).

Bacteria of the genus Enterobacter are not particularly limited, and include species classified into the genus Enterobacter according to classifications known to microbiological experts. Examples of bacteria belonging to the genus Enterobacter include Enterobacter agglomerans and Enterobacter aerogenes. Specific examples of Enterobacter agglomerans strains include, for example, Enterobacter agglomerans ATCC 12287. Specific examples of Enterobacter aerogenes strains include, for example, Enterobacter aerogenes ATCC 13048, 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). In addition, the strains of the genus Enterobacter include, for example, those described in European Patent Application Publication No. 0952221. Enterobacter agglomerans also includes several strains classified as Pantoea agglomerans.

The Pantoea bacterium is not particularly limited, and includes species classified into the Pantoea genus by classification known to microbiological experts. Pantoea genus bacterial species include, for example, Pantoea ananatis (P. ananatis), Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. As Pantoea ananatis strains, specifically, for example, Pantoea ananatis LMG20103, AJ13355 (FERM BP-6614), AJ13356 (FERM BP-6615), AJ13601 (FERM BP-7207), SC17 (FERM BP-11091), SC17(0) (VKPM B-9246). Some species of Enterobacter agglomerans have recently been reclassified into Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, etc., based on 16S rRNA nucleotide sequence analysis (Mergaert J. et al., Transfer of 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. nov., Int. J. Syst. Evol. Microbiol., 1993, 43:162-173). Bacteria of the genus Pantoea include bacteria reclassified into the genus Pantoea in this way.

Erwinia bacteria include Erwinia amylovora and Erwinia carotovora. Klebsiella bacteria include Klebsiella planticola.

These strains are available, for example, from the American Type Culture Collection, Address: PO Box 1549, Manassas, VA 20108, United States of America. That is, each strain has a corresponding registry number that can be used to order (see atcc.org/). Accession numbers for each strain are listed in the catalog of the American Type Culture Collection.

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

L-amino acid-producing ability can be imparted or enhanced by a method conventionally employed for breeding amino acid-producing bacteria such as Escherichia bacteria (Amino Acid Fermentation, Gakkai Shuppan Center Co., Ltd., May 30, 1986). Japanese first edition, see pages 77-100). Such methods include:
For example, the acquisition of auxotrophic mutant strains, the acquisition of L-amino acid analog-resistant strains, the acquisition of metabolic control mutant strains, and the creation of recombinant strains with enhanced activity of L-amino acid biosynthetic enzymes. In the breeding of L-amino acid-producing bacteria, properties such as auxotrophy, analog resistance, and metabolic control mutation may be imparted singly or in combination of two or more. In breeding L-amino acid-producing bacteria, the activity of L-amino acid biosynthetic enzymes may be enhanced either singly or in combination of two or more. Furthermore, imparting properties such as auxotrophy, analogue resistance, and metabolic control mutation may be combined with enhancing the activity of biosynthetic enzymes.

Auxotrophic mutants, analog-resistant strains, or metabolic regulatory mutants having L-amino acid-producing ability are obtained by subjecting the parent strain or wild strain to a typical mutation treatment, and among the resulting mutants, auxotrophic, It can be obtained by selecting those exhibiting analogue resistance or metabolic regulation mutation and having L-amino acid production ability. Typical mutagenesis treatments include irradiation with X-rays and ultraviolet rays, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and the like. treatment with a mutagen.

Also, the ability to produce L-amino acids can be imparted or enhanced by enhancing the activities of enzymes involved in the biosynthesis of the target L-amino acid. Enzyme activity can be enhanced, for example, by modifying the bacterium so that the expression of the gene encoding the same enzyme is enhanced. Methods for enhancing gene expression are described in WO00/18935, EP 1010755A, and the like.

The L-amino acid-producing ability can also be imparted or enhanced by reducing the activity of an enzyme that catalyzes a reaction that diverges from the target L-amino acid biosynthetic pathway and produces a compound other than the target L-amino acid. It can be carried out. The term "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. Enzyme activity can be reduced, for example, by modifying the bacterium so that the gene encoding the enzyme is inactivated. A method for reducing enzyme activity will be described later.

Specific examples of L-amino acid-producing bacteria and methods for imparting or enhancing L-amino acid-producing ability are given below. Any of the properties possessed by the L-amino acid-producing bacteria and modifications for conferring or enhancing L-amino acid-producing ability as exemplified below may be used alone or in combination as appropriate.

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 strain 237 (VKPM B-7925, US2002058315 A1) and mutant N- E. coli 382 strain (VKPM B-7926, EP1170358 A1), a derivative strain thereof having acetylglutamate synthase (RU2215783 C2), an arginine-producing strain (EP1170361 A1) into which the argA gene encoding N-acetylglutamate synthase was introduced (EP1170361 A1) and strains belonging to the genus Escherichia, such as the E. coli 382 ilvA+ strain, which is obtained by introducing a wild-type allele of the ilvA gene derived from the E. coli K-12 strain into the 382 strain. The mutant N-acetylglutamate synthase is, for example, a mutant N-acetylglutamate synthase in which the feedback inhibition by L-arginine is canceled by substitution of amino acid residues corresponding to positions 15 to 19 of the wild-type enzyme. (EP1170361 A1).

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

Parent strains that can be used to induce L-arginine-producing bacteria and L-arginine-producing bacteria also include strains that are resistant to amino acid analogs and the like. Such strains include, for example, α-methylmethionine, p-fluorophenylalanine, D-arginine, arginine hydroxamic acid, S-(2-aminoethyl)-cysteine, α-methylserine, β-2-thienylalanine, or E. coli mutant strains resistant to sulfaguanidine (see JP-A-56-106598) can be mentioned.

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 E. coli fumaric acid-producing strain CGMCCNO: 2301 (CN101240259 A), Aspartic acid producer ΔiclR (CCTCC NO: M 2018521) (CN109370971 A) lacking the gene iclR encoding the glyoxylate cycle regulator, its derivative strain, malate synthetase and isocitrate lyase
lacking the aceBA gene that encodes the aspartate ammonia lyase (a
AS12 with an inserted aspA gene encoding spartate ammonia-lyase (CN105296411 B)
strains belonging to the genus Escherichia such as

E. coli CM-AS-115 (CCTCC NO: M 2016457) (CN106434510 A) is directly produced by fermentation.
- is a genetically engineered strain capable of producing aspartic acid. The original strain was wild-type Escherichia coli
W1485 (ATCC12435), knockout of multiple genes (icdA (EG10489), mdh (EG10576), sfcA (EG10948), maeB (EG14193), fumAC (EG10356 and EG10358)) yielding recombinant strain CM-AS-100 . A mutant strain CM-AS-105 was obtained by subjecting the CM-AS-100 strain to metabolic evolution and acclimation. E. coli strain CM-AS-115 was induced by overexpression of two genes of mutant strain CM-AS-105 (ppc encoding phosphoenolpyruvate carboxylase and aspartase encoding aspartase). Obtained.

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

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

L-aspartic acid-producing bacteria and parent strains that can be used to induce L-aspartic acid-producing bacteria include P. ananatis strain AJ13355 (FERM BP-6614), P. ananatis strain SC17 (FERM BP-11091), and P. ananatis strain SC17 (FERM BP-11091). Ananatis SC17(0) strain (VKPM B-9246) is also included. L- of P. ananatis
The aspartic acid-producing strain 5ΔP2RM (WO2010038905 A1) is a derivative of the SC17(0) strain and contains ΔaspA, ΔsucA, ΔgltA, ΔpykA, ΔpykF, Δppc, ppc ^K620S (feedback-resistant E. coli phosphoenolpyruvate carboxylase (encoding phosphoenolpyruvate carboxylase)), ΔmdhA and other modifications have been consistently introduced.

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 harboring mutant N-acetylglutamate synthase. 237/pMADS11, 237/pMADS12, and 237/pMADS13 (RU2215783 C2, EP1170361 B1, US6790647 B2), E. coli 333 strain (VKPM) harboring carbamoyl phosphate synthetase resistant to feedback inhibition B- ^{8084) and strain 374 (VKPM B-8086) (R2264459 C2) with increased α-ketoglutarate synthase activity and ferredoxin NADP + reductase} , pyruvate synthase (pyruvate synthase) and / or α-ketoglutarate dehydrogenase (α-ketoglutarate dehydrogenase) strains belonging to the genus Escherichia such as E. coli strain (EP2133417 A1) further modified, succinate dehydrogenase (succinate dehydrogenase) and Pantoea ananantis NA1sucAsdhA strain (US2009286290 A1) having decreased α-ketoglutarate dehydrogenase activity.

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

In addition, the L-citrulline-producing bacterium is obtained by inactivating the argininosuccinate synthase encoded by the argG gene from any L-arginine-producing bacterium (eg, E. coli strain 382 (VKPM B-7926)). can be easily obtained by Methods for inactivating genes are described herein.

L-Cysteine-Producing Bacteria L-cysteine-producing bacteria and parent strains that can be used to derive L-cysteine-producing bacteria include, for example, strains with increased activity of one or more L-cysteine biosynthetic enzymes. be done. Examples of 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 into bacteria a mutant cysE gene that encodes a mutant serine acetyltransferase resistant to feedback inhibition by cysteine. Mutant serine acetyltransferases are disclosed, for example, in JP-A-11-155571 and US2005-0112731A. As such a mutant serine acetyltransferase, specifically, the Val and Asp residues at positions 95 and 96 of the wild-type serine acetyltransferase encoded by the cysE5 gene are replaced with Arg and Pro residues, respectively. (US2005-0112731A). Also, 3-phosphoglycerate dehydrogenase activity can be enhanced, for example, by introducing into bacteria a mutant serA gene encoding a mutant 3-phosphoglycerate dehydrogenase resistant to feedback inhibition by serine. Mutant 3-phosphoglycerate dehydrogenase is disclosed, for example, in US Pat. No. 6,180,373.

Examples of L-cysteine-producing bacteria and parent strains that can be used to derive L-cysteine-producing bacteria include, for example, one species that catalyzes a reaction that branches from the L-cysteine biosynthetic pathway to produce a compound other than L-cysteine. Also included are strains in which the activity of the enzyme is reduced or more. Such enzymes include, for example, enzymes involved in the degradation of L-cysteine. Enzymes involved in the degradation of L-cysteine include, but are not limited to, cystathionine-β-lyase (metC, JP-A-11-155571; Chandra et al., Biochemistry, 1982, 21:3064-3069), tryptopha nase (tnaA, JP-A-2003-169668; Austin Newton et al., J. Biol. Chem., 240 (1965) 1211-1218), O-acetylserine sulfhydrylase B (cysM, JP-A-2005-245311) , malY gene product (JP 2005-245311), d0191 of Pantoea ananatis
gene products (JP-A-2009-232844) and cysteine desulfhydrase (aecD, JP-A-2002-233384).

L-cysteine-producing bacteria and parent strains that can be used to derive L-cysteine-producing bacteria also include, for example, strains with increased activity of the L-cysteine excretion system and/or the sulfate/thiosulfate transport system. be done. Proteins of the L-cysteine excretion system include proteins encoded by the ydeD gene (JP-A-2002-233384), proteins encoded by the yfiK gene (JP-A-2004-49237), emrAB, emrKY, yojIH, acrEF, bcr, and each protein encoded by each gene of cusA (JP-A-2005-287333), and a protein encoded by yeaS gene (JP-A-2010-187552). Proteins of the sulfate/thiosulfate transport system include proteins encoded by the cysPTWA gene cluster.

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 transformed with various cysE alleles encoding mutant serine acetyltransferases resistant to feedback inhibition. JM15 (US6218168 B1, RU2279477 C2), E. coli W3110 (US5972663 A) overexpressing genes encoding proteins suitable for excreting toxic substances into cells, E. coli with reduced cysteine desulfhydrase activity. Escherichia strains (JP11155571 A2), strains belonging to the genus Escherichia such as E. coli W3110 (WO0127307 A1) in which the activity of the transcription factor of the positive cysteine regulon encoded by the cysB gene is elevated, Pantoea ananatis EYPSG8 and its derivatives. Also included is a strain overexpressing a gene involved in certain sulfur assimilation (EP2486123 B1).

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, strains belonging to the genus Escherichia, such as E. coli VL334thrC ⁺ (EP1172433 A1). . E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotroph with mutations in the thrC and ilvA genes (US4278765). A wild-type allele of the thrC gene was introduced by a general transduction method using bacteriophage P1 grown in cells of E. coli strain K-12 (VKPM B-7). As a result, an L-isoleucine-requiring strain VL334thrC ⁺ (VKPM B-8961) capable of producing L-glutamic acid was obtained.

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 with increased expression of one or more genes encoding L-glutamic acid biosynthetic enzymes. mentioned. Such genes include glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthase (gltBD), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), methyl citrate synthase (prpC), pyruvate carboxylase (pyc), phosphoenolpyruvate carboxylase (ppc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglyceromutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-
phosphate dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), glucose phosphate isomerase (pgi), 6-phosphogluconate dehydratase (edd), Genes encoding 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), transhydrogenase (pntAB).

Strains modified for increased expression of the citrate synthase, phosphoenolpyruvate carboxylase, and/or glutamate dehydrogenase genes include those disclosed in EP1078989 A2, EP955368 A2, and EP952221 A2. Strains modified to increase expression of Entner-Doudoroff pathway genes (edd, eda) include those disclosed in EP1352966B.

L-glutamic acid-producing bacteria and parent strains that can be used to derive L-glutamic acid-producing bacteria have reduced activity of enzymes that diverge from the L-glutamic acid biosynthetic pathway and catalyze the biosynthesis of compounds other than L-glutamic acid. Or the stock|strain which disappeared is also mentioned. Examples of such enzymes include, but are 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), 1-pyrroline-5-carboxylate dehydrogenase (putA).

Bacteria belonging to the genus Escherichia with deficient or reduced α-ketoglutarate dehydrogenase activity,
and methods for obtaining them are described in US5378616 and US5573945. Specific examples of these strains include the following.
E. coli W3110sucA:: ^KmR
E. coli AJ12624 (FERM BP-3853)
E. coli AJ12628 (FERM BP-3854)
E. coli AJ12949 (FERM BP-4881)

E. coli W3110sucA::Km ^R is a strain obtained by disrupting the E. coli W3110 α-ketoglutarate dehydrogenase gene (sucA). This strain is completely deficient in α-ketoglutarate dehydrogenase.

Examples of L-glutamic acid-producing bacteria and parent strains that can be used to induce L-glutamic acid-producing bacteria include P. ananatis AJ13355 strain (FERM BP-6614), P. ananatis SC17 strain (FERM BP-11091), and P. ananatis SC17. Pantoea bacteria such as (0) strain (VKPM B-9246) are also included. The AJ13355 strain was isolated from soil in Iwata City, Shizuoka Prefecture as a strain capable of growing on a medium containing L-glutamic acid and a carbon source at low pH. The SC17 strain is a strain selected from the AJ13355 strain as a mutant strain with low mucus production (US6596517). The SC17 strain was deposited on February 4, 2009 at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center (currently, National Institute of Technology and Evaluation Patent Organism Depositary Center (NITE IPOD), zip code: 292-0818. , Address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) and has been assigned the accession number FERM BP-11091. On February 19, 1998, the AJ13355 strain was acquired by the Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Institute of Biotechnology and Technology (currently NITE IPOD, Zip code: 292-0818, Address: 2-Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan). 5-8 No. 120) under the accession number FERM P-16644 and transferred to the international deposit under the Budapest Treaty on January 11, 1999, under the accession number FERM BP-6614. Share SC17(0) was granted to the Russian National Collection of Industrial Microorganisms (VKPM; FGUP GosNII Genetika, Russian Federation, 117545 Moscow, 1 ^st Dorozhny proezd, 1) on September 21, 2005 under the accession number VKPM B-9246. has been deposited with

Examples of L-glutamic acid-producing bacteria and parent strains that can be used to derive L-glutamic acid-producing bacteria include mutant strains belonging to the genus Pantoea that lack or have reduced α-ketoglutarate dehydrogenase activity, and can be obtained as described above. . Examples of such strains include P. ananatis AJ13356 (US6331419 B1), which is an E1 subunit gene (sucA)-deficient strain of α-ketoglutarate dehydrogenase of the AJ13355 strain, and Pantoea ananatis SC17sucA, which is a sucA gene-deficient strain of the SC17 strain. U.S. Pat. No. 6,596,517). P. ananatis AJ13356 was established on February 19, 1998 by the Institute of Biotechnology, Ministry of International Trade and Industry, Agency of Industrial Science and Technology (currently NITE IPOD, Zip code: 292-0818, Address: Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan). 2-5-8 Room 120) under the accession number FERM P-16645, and was transferred to the international depository under the Budapest Treaty on January 11, 1999, under the accession number FERM BP-6615. The AJ13356 strain was identified as Enterobacter agglomerans when it was isolated, and was deposited as Enterobacter agglomerans AJ13356. However, in recent years, this strain has been reclassified as P. ananatis based on analysis of the 16S rRNA nucleotide sequence and other factors. AJ13356 has been deposited with the above depository as Enterobacter agglomerans, but is referred to as P. ananatis for the purposes of this specification.

Parent strains that can be used to induce L-glutamic acid-producing bacteria and L-glutamic acid-producing bacteria include P. ananatis SC17sucA/RSFCPG+pSTVCB strain, P. ananatis AJ13601 strain, P. ananatis NP106 strain, and P. ananatis NA1 strain. and strains belonging to the genus Pantoea. The SC17sucA/RSFCPG+pSTVCB strain is composed of the SC17sucA strain, the citrate synthase gene (gltA) derived from Escherichia coli (E. coli), and the phosphoenolpyruvate carboxylase gene (ppc).
, and the plasmid RSFCPG containing the glutamate dehydrogenase gene (gdhA), and the plasmid pSTVCB containing the citrate synthase gene (gltA) derived from Brevibacterium lactofermentum. The AJ13601 strain was selected from this SC17sucA/RSFCPG+pSTVCB strain as a strain exhibiting resistance to high concentrations of L-glutamic acid at low pH. Strain NP106 was obtained by dropping plasmids RSFCPG and pSTVCB from strain AJ13601. Strain NA1 was obtained by introducing the plasmid RSFPPG into strain NP106 (EP2336347 A1). AJ13601 stock was established on August 18, 1999 by the Institute of Biotechnology, Ministry of International Trade and Industry, Agency of Industrial Science and Technology (currently NITE IPOD, Zip code: 292-0818, Address: 2-Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan). 5-8 Room 120) under the accession number FERM P-17516 and was transferred to the international deposit under the Budapest Treaty on July 6, 2000, under the accession number FERM BP-7207.

Parent strains that can be used to derive L-glutamic acid-producing bacteria and 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 auxotroph with mutations in the thrC and ilvA genes (US Pat. No. 4,278,765). E. coli VL334thrC ⁺ is an L-isoleucine auxotrophic L gene obtained by introducing a wild-type allele of the thrC gene into VL334.
- It is a glutamic acid-producing bacterium. A 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 strain K-12 (VKPM B-7).

Parent strains that can be used to derive L-glutamic acid-producing bacteria and L-glutamic acid-producing bacteria also include strains that are resistant to aspartic acid analogues. These strains may, for example, be deficient in α-ketoglutarate dehydrogenase activity. Specific examples of strains resistant to aspartic acid analogs and lacking α-ketoglutarate dehydrogenase activity include, for example, E. coli AJ13199 (FERM BP-5807; US5908768); coli FERM P-12379 (US5393671), E. coli AJ13138 (FERM BP-5565; US6110714).

L-histidine-producing bacteria 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 strain 24 (VKPM B-5945; RU2003677 C1), E. coli strain 80 (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-6675) 6676) (US6344347 B1), E.
coli H-9341 (FERM BP-6674; EP1085087 A2), E. coli AI80/pFM201 (US6258554 B1)
strains belonging to the genus Escherichia such as

L-histidine-producing bacteria and parent strains that can be used to derive L-histidine-producing bacteria also include strains with 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/phosphoribosyl-ATP pyrophosphatase (hisIE). , phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase (hisA), amidotransferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), histidinol dehydrogenase (hisD) Examples include genes encoding such as

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

As an L-histidine-producing bacterium and a parent strain that can be used to derive an L-histidine-producing bacterium, specifically, E. coli FERM P transformed with a vector carrying a DNA encoding an L-histidine biosynthetic enzyme. -5038 and FERM P-5048 (JP56-005099 A), E. coli strain (EP1016710 A2) transformed with rht, the gene for amino acid excretion, sulfaguanidine, DL-1,2,4-triazole-3 - E. coli strain 80 (VKPM B-7270; RU2119536 C1) that confers resistance to alanine and streptomycin, E. coli MG1655+hisGr hisL'_Δ
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).

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, mutant strains resistant to 6-dimethylaminopurine (JP 5-304969 A), Mutants resistant to isoleucine analogues such as thiisoleucine and isoleucine hydroxamate, and mutants resistant to DL-ethionine and/or arginine hydroxamate (JP5-130882 A) can be mentioned. Recombinant strains transformed with genes encoding proteins involved in the L-isoleucine biosynthesis system such as threonion deaminase and acetohydroxy acid synthase can also be used as L-isoleucine-producing strains or parent strains (JP2-458 A, EP0356739 A1, and US5998178).

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 such as strain 57 (VKPM B-7386; US6124121 )), β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 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 method described in WO9606926, and strains belonging to the genus Escherichia such as E. coli H-9068 (JP8-70879 A).

L-leucine-producing bacteria and parent strains that can be used to derive L-leucine-producing bacteria include strains with increased expression of one or more genes involved in L-leucine biosynthesis. Such genes include those of the leuABCD operon, which are
It can be represented by a mutant leuA gene (US6403342 B1) encoding α-isopropylmalate synthase freed from feedback inhibition by L-leucine. In addition, L-leucine-producing bacteria and parent strains that can be used to derive L-leucine-producing bacteria have increased expression of one or more genes encoding proteins that excrete L-amino acids from bacterial cells. Stocks are also included. Such genes include the b2682 and b2683 genes (ygaZH
gene) (EP1239041 A2).

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
Full or partial desensitization occurs when lysine is present in the medium. L-lysine analogs include, but are not limited to, oxalysine, lysine hydroxamate, S-(2-aminoethyl)-L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam, and the like. Mutant strains having resistance to these lysine analogues can be obtained by subjecting bacteria belonging to the genus Escherichia to conventional artificial mutagenesis treatments. Bacterial strains useful for L-lysine production specifically include, for example, E. coli AJ11442 (FERM BP-1543, NRRL B-12185; see US4346170) and E. coli VL611. In these stocks
Feedback inhibition by L-lysine on aspartokinase is released.

L-lysine-producing bacteria and parent strains that can be used to derive L-lysine-producing bacteria include strains with increased expression of one or more genes encoding L-lysine biosynthetic enzymes. Such genes include, but are not limited to, dihydrodipicolinate synthase (dapA), aspartokinase III (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate synthase. Carboxylase (diaminopimelate decarboxylase) (lysA), diaminopimelate dehydrogenase
(ddh) (US6040160), phosphoenolpyruvate carboxylase (ppc), aspartate semialdehyde dehydrogenase (asd), aspartate aminotransferase (aspartate transaminase ) (aspC), diaminopimelate epimerase (dapF), tetrahydrodipicolinate succinylase (dapD), succinyl diaminopimelate
deacylase (dapE), and aspartase (aspA) (EP1253195 A1). In the parent strain, the gene involved in energy efficiency (cyo) (EP1170376 A1), the gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (US5830716 A), the ybjE gene (WO2005073390), or The expression level of the combination may be increased. Since aspartokinase III (lysC) is subject to feedback inhibition by L-lysine, the mutant lysC gene encoding aspartokinase III freed from feedback inhibition by L-lysine is used to enhance the activity of this enzyme. may be used (US5932453). Examples of aspartokinase III freed from feedback inhibition by L-lysine include mutations that substitute a methionine residue at position 318 with an isoleucine residue, a mutation that substitutes a glycine residue at position 323 with an aspartic acid residue, and a mutation at position 352. aspartokinase III from Escherichia coli with one or more mutations selected from mutations that replace threonine residues at positions with isoleucine residues (U.S. Pat. No. 5,661,012; U.S. Pat. No. 5,661,012; Patent No. 6,040,160). In addition, dihydrodipicolinate synthase (dapA) is subject to feedback inhibition by L-lysine. A type dapA gene may be used. As the dihydrodipicolinate synthase freed from the feedback inhibition by L-lysine, dihydrodipicolinate synthase derived from Escherichia coli having a mutation in which the histidine residue at position 118 is replaced with a tyrosine residue. (U.S. Pat. No. 6,040,160).

In L-lysine-producing bacteria or parent strains that can be used to derive L-lysine-producing bacteria, the activity of enzymes that catalyze reactions that branch from the L-amino acid biosynthetic pathway to produce other compounds is reduced or eliminated. It's okay. In addition, in L-lysine-producing bacteria or parent strains that can be used to derive L-lysine-producing bacteria, the activity of enzymes that negatively affect L-lysine synthesis or accumulation may be reduced or eliminated. Examples of such enzymes include homoserine dehydrogenase, lysine decarboxylase (cadA, ldcC), malic enzyme, etc. Strains in which the activities of these enzymes are reduced or deleted , WO9523864, WO9617930, WO2005010175 and the like.

Expression of both the cadA and ldcC genes encoding lysine decarboxylase can be reduced to reduce or eliminate lysine decarboxylase activity. Expression of both genes can be reduced, for example, by the method described in WO2006078039.

L-lysine-producing bacteria or parent strains that can be used to induce L-lysine-producing bacteria include E. coli WC196 strain (FERM BP-5252; US5827698), E. coli WC196ΔcadAΔldcC strain (FERM BP-11027; “WC196LC”). also called), E. coli WC196ΔcadAΔldcC/pCABD2 strain (WO2006078039).

Strain WC196 was bred by conferring AEC resistance to strain W3110 derived from E. coli K-12 (US5827698). The WC196 strain was named E. coli AJ13069 and was transferred to the National Institute of Biotechnology and Biotechnology, Agency of Industrial Science and Technology (currently NITE IPOD, zip code: 292-0818, address: Kisarazu City, Chiba Prefecture, Japan) on December 6, 1994. Kazusa Kamatari 2-5-8 Room 120) under the accession number FERM P-14690, transferred to the international deposit under the Budapest Treaty on September 29, 1995, and was assigned the accession number FERM BP-5252. (US5827698).

The WC196ΔcadAΔldcC strain was constructed from the WC196 strain by disrupting the cadA and ldcC genes encoding lysine decarboxylase. The WC196ΔcadAΔldcC strain was named AJ110692 and was registered on October 7, 2008 at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center (currently NITE IPOD, zip code: 292-0818, address: Kisarazu City, Chiba Prefecture, Japan). Kazusa Kamatari 2-5-8 Room 120) under the accession number FERM BP-11027.

The WC196ΔcadAΔldcC/pCABD2 strain was constructed by introducing a plasmid pCABD2 (US6040160) containing a lysine biosynthetic gene into the WC196ΔcadAΔldcC strain. pCABD2 is a mutant dapA gene encoding dihydrodipicolinate synthase (DDPS) derived from Escherichia coli (E. coli) having a mutation (H118Y) that cancels the feedback inhibition by L-lysine, and A mutant lysC gene encoding aspartokinase III from E. coli with a mutation (T352I) that releases feedback inhibition and dihydrodipicolinate reductase from E. coli It contains the encoding dapB gene and the ddh gene encoding diaminopimelate dehydrogenase from Brevibacterium lactofermentum.

L-lysine-producing bacteria or parent strains that can be used to derive L-lysine-producing bacteria also include E. coli strain AJIK01 (NITE BP-01520). The AJIK01 strain, named E. coli AJ111046, was transferred to NITE IPOD (zip code: 292-0818, address: 2-5-8 120 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on January 29, 2013. It was deposited and transferred to the international deposit under the Budapest Treaty on May 15, 2014, with accession number NITE BP-01520.

L-methionine-producing bacteria L-methionine-producing bacteria and parent strains that can be used to induce L-methionine-producing bacteria are not particularly limited, but 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 the L-methionine analog norleucine (VKPM B-8125; RU2
209248 C2) and strain 73 (VKPM B-8126; RU2215782 C2); E. coli strain 218 was entrusted to the Russian National Collection of Industrial Microorganisms (VKPM; Russian Federation, 117545 Moscow, 1 ^st Dorozhny proezd, 1) on May 14, 2001 as VKPM B-8125. No. and transferred to the international deposit under the Budapest Treaty on February 1, 2002. 73 strains of E. coli were deposited on May 14, 2001 at the Lucian National Collection of Industrial Microorganisms (VKPM; Russian Federation, 117545 Moscow, 1 ^st Dorozhny proezd, 1) under accession number VKPM B-8126. and was transferred to the international deposit under the Budapest Treaty on February 1, 2002. In addition, methionine repressor-deficient strains and recombinant strains transformed with genes encoding proteins involved in L-methionine biosynthesis (homoserine transsuccinylase, cystathionine γ-synthase, etc.) (JP2000-139471 A) are also L- It can be used as a methionine-producing strain or parent strain.

Other examples of L-methionine-producing strains of the genus Escherichia and parent strains that can be used to derive L-methionine-producing strains lack the repressor (MetJ) of the L-methionine biosynthetic system and the intracellular homoserine transoxygenase. E. coli strain (US7611873 B1) with increased homoserine transsuccinylase (MetA) activity, suppressed cobalamin-independent methionine synthase (MetE) activity, and reduced cobalamin-dependent methionine synthase (MetE) activity E. coli strain (EP2861726 B1) with increased cobalamin-dependent methionine synthase (MetH) activity, capable of producing L-threonine, threonine dehydratase (tdcB, ilvA) and, at least, O - O-succinylhomoserine lyase (metB), cystathionine beta-lyase (metC), 5,10-methylenetetrahydrofolate reductase (metF), and serine hydroxymethyltransferase (glyA)
(US7790424 B2), an E. coli strain with enhanced transhydrogenase (pntAB) activity (EP2633037 B1), and the like.

L-methionine-producing bacteria may be modified to overexpress a cysteine synthase-encoding gene.

The term "cysteine synthase-encoding gene" can refer to a gene that encodes a cysteine synthase. "Cysteine synthase
)" can mean a protein with cysteine synthase activity (EC 2.5.1.47). Cysteine synthase encoding genes include cysM gene and cysK gene. The cysM gene may encode cysteine synthase B with thiosulfate as a substrate. The cysK gene may encode cysteine synthase A with a sulfide substrate. Specific examples of cysteine synthase-encoding genes include the P. ananatis-specific cysM gene. SEQ ID NO: 13 shows the nucleotide sequence of the P. ananatis-specific cysM gene.

L-methionine-producing bacteria may be modified to have a mutant metA gene.

The metA gene encodes the homoserine transsuccinylase MetA (EC 2.3.1.46). The term "mutant metA gene" can refer to a gene encoding a mutant MetA protein. The term "mutant MetA protein" has the R34C mutation, which is a mutation in which the arginine (Arg) residue at position 34 in the amino acid sequence of the wild-type MetA protein is replaced with a cysteine (Cys) residue. It can mean MetA protein. "
The term "wild-type metA gene" can refer to the gene encoding the wild-type MetA protein. The term "wild-type MetA protein" can refer to a MetA protein that does not have the R34C mutation.
Wild-type metA genes include the P. ananatis-specific metA gene and mutants thereof that do not have the mutation resulting in the R34C mutation of the encoded protein. Wild-type MetA proteins include P. ananatis-specific MetA proteins and mutants thereof that do not have the R34C mutation. In other words, the mutant metA gene may be identical to any wild-type metA gene except that it has a mutation that results in the R34C mutation of the encoded protein. A mutant MetA protein may also be identical to any wild-type MetA protein except that it has the R34C mutation. The amino acid sequence of the P. ananatis-specific MetA protein is shown in SEQ ID NO:35. Specifically, one example of the amino acid sequence of the mutant MetA protein can be the amino acid sequence shown in SEQ ID NO:37, which can be encoded by the mutant metA gene having the base sequence shown in SEQ ID NO:36. That is, the mutant metA gene may be a gene (eg, DNA) having the base sequence shown in SEQ ID NO:36, and the mutant MetA protein may be a protein having the amino acid sequence shown in SEQ ID NO:37. The mutant metA gene may be a gene (eg, DNA) having a nucleotide sequence that is the mutant nucleotide sequence of SEQ ID NO: 36 and has a mutation that results in the R34C mutation of the encoded protein. The mutant MetA protein may be a protein having a mutant amino acid sequence of SEQ ID NO: 37 with the R34C mutation. The mutant MetA protein may be a homoserine transsuccinylase that is resistant to feedback inhibition by L-methionine. In other words, the mutant MetA protein may be a protein that has homoserine transsuccinylase activity and is resistant to feedback inhibition by L-methionine. The following description of a gene encoding a protein having indolepyruvate decarboxylase activity and the indolepyruvate decarboxylase mutant encoded by the gene can be applied mutatis mutandis to metA gene mutants and MetA protein mutants. The term "position 34" does not necessarily indicate an absolute position in the wild-type MetA protein amino acid sequence, but indicates a relative position based on the amino acid sequence shown in SEQ ID NO: 35 in the wild-type MetA protein. be.

The L-methionine-producing bacterium may be modified so that expression of the metJ gene is attenuated.

The metJ gene encodes the Met repressor, which regulates the expression of the methionine regulon and the S
- can suppress the expression of enzymes involved in adenosylmethionine (SAM) synthesis. metJ genes include those unique to host bacteria such as P. ananatis. SEQ ID NO: 24 shows the nucleotide sequence of the P. ananatis-specific metJ gene.

L-methionine-producing bacteria may be modified to have a mutant thrA gene encoding a mutant aspartokinase homoserine dehydrogenase I that is resistant to feedback inhibition by threonine. Mutant thrA genes include the thrA442 gene.

L-methionine-producing bacteria may be modified to overexpress the aminomethyltransferase gene.

The term "aminomethyltransferase gene" can refer to a gene that encodes an aminomethyltransferase. The term "aminomethyltransferase" may mean a protein having aminomethyltransferase activity (EC 2.1.2.10). The aminomethyltransferase gene includes the gcvT gene.

L-methionine-producing bacteria of the genus Pantoea and parent strains that can be used to derive L-methionine-producing bacteria include, but are not limited to, P. ananatis AJ13355 strain (FERM BP-6614). The strain is also known as P. ananatis strain SC17 (FERM BP-11091).

L-ornithine-producing bacteria L-ornithine is an intermediate in the L-arginine biosynthetic pathway. strains with increased expression of one or more genes (such as those listed above) encoding systemic enzymes.

L-ornithine-producing bacteria can be easily obtained by inactivating ornithine carbamoyltransferase encoded by both argF and argI genes from any L-arginine-producing bacteria (e.g., E. coli 382 strain (VKPM B-7926), etc.). can get to Methods for inactivating genes are described herein.

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 harboring the 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 designated AJ12604 [W3110 (tyrA)/pBR-aroG4, pACMAB] (FERM BP- 3579, EP488424 B1) and other strains belonging to the genus Escherichia. Furthermore, L-phenylalanine-producing bacteria and 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 are also available as parent strains that can be used to derive L-phenylalanine-producing bacteria. (US7259003 and US7666655).

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 lacks the ilvA gene and is capable of producing L-proline.
strains belonging to the genus Escherichia such as (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 with enhanced expression of one or more genes involved in L-proline biosynthesis.
An example of such a gene that can be used in L-proline-producing bacteria is the proB gene encoding glutamate kinase freed from 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 expression of one or more genes encoding proteins responsible for L-amino acid excretion from bacterial cells. Also included are strains with enhanced . Examples of such genes include the b2682 gene and the b2683 gene (ygaZH gene) (EP1239041 A2).

Bacteria belonging to the genus Escherichia having an ability to produce L-proline include NRRL B-12403 and NRRL B-12404 (GB2075056), VKPM B-8012 (RU2207371 C2), plasmid mutants described in DE3127361 A1, Bloom FR et al. in <<The ^15th Miami winter symposium>>, 1983, p.34.

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 (US5939307), 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), E. coli VKPM B-5318 (EP0593792 A1), and other strains belonging to the genus Escherichia.

The TDH-6 strain lacks the thrC gene, is sucrose-utilizing, and has a leaky mutation in its ilvA gene. This strain also has a mutation in the rhtA gene that confers resistance to high concentrations of threonine or homoserine. The VKPM B-3996 strain contains the plasmid pVIC40
and was obtained by introducing the plasmid pVIC40 into the TDH-6 strain. Plasmid pVIC40 was obtained by inserting the thrA*BC operon containing the mutant thrA gene into the RSF1010-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I that is substantially free of feedback inhibition by threonine. The strain VKPM B-3996 was deposited on November 19, 1987 with the All-Union Scientific Center of Antibiotics, Russian Federation, 117105 Moscow, Nagatinskaya Street 3-A, under accession number RIA 1867. The VKPM B-3996 strain was also deposited with the Russian National Collection of Industrial Microorganisms (VKPM; Russian Federation, 117545 Moscow, 1 ^st Dorozhny proezd, 1) on April 7, 1987 under the accession number B-3996.

Strain B-5318 is isoleucine agnostic and has the regulatory regions of the threonine operon in plasmid pVIC40 replaced by the temperature sensitive lambda phage C1 repressor and PR promoter. VKPM B-5318 strain was deposited on May 3, 1990 with the Russian National Collection of Industrial Microorganisms (VKPM) under accession number VKPM B-5318.

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

thrA encoding E. coli aspartokinase I and homoserine dehydrogenase I
The gene has been elucidated (KEGG, Kyoto Encyclopedia of Genes and Genomes, Entry No. b0002, GenBank accession No. NC_000913.3, nucleotide positions 337-2,799, Gene ID 945803). The thrA gene is located between the thrL and thrB genes on the chromosome of E. coli K-12.

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

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.
~5,020, Gene ID 945198). The thrC gene is located between the thrB and yaaX genes on the chromosome of E. coli K-12 strain. All three of these genes function as a single threonine operon thrABC. In order to enhance the expression of the threonine operon, it is desirable to remove the attenuator region that affects transcription from the operon (WO2005049808 A1, WO2003097839 A1).

Mutant thrA genes encoding aspartokinase I and homoserine dehydrogenase I resistant to feedback inhibition by threonine, and thrB genes and thrC
The gene 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 plasmid pVIC40 are described in US5705371.

The rhtA gene, which encodes a protein of the E. coli threonine and homoserine efflux system (inner membrane transporter), has been characterized (KEGG, Entry No. b0813, GenBank accession No. NC_000913.3, nucleotide positions 849,210 to 850,097; complementary strand, Gene ID 947939). The rhtA gene is located on the chromosome of E. coli strain K-12 between the dps and ompX genes near the glnHPQ operon, which encodes elements of the glutamine transport system. The rhtA gene is identical to the ybiF gene (KEGG, entry No. B0813).

The asd gene encoding E. coli aspartate-β-semialdehyde dehydrogenase has been characterized (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 located on the same strand (the yhgN gene is on the opposite strand).

The aspartate aminotransferase-encoding aspartate aminotransferase of E. coli has also been characterized (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 E. coli K-12 chromosome between the gloC gene on the opposite strand and the ompF gene on the same strand.

L-tryptophan-producing bacteria L-tryptophan-producing bacteria and parent strains that can be used to derive L-tryptophan-producing bacteria include, but are not limited to, E. coli JP4735 deficient in tryptophanyl-tRNA synthetase encoded by the mutant trpS gene. /pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) (US5756345), a serA allele encoding phosphoglycerate dehydrogenase that is not feedback-inhibited by serine and a trpE allele that encodes anthranilate synthase that is not feedback-inhibited by tryptophan. E. coli SV164 (pGH5) (US6180373 B1) with phospho Strains belonging to the genus Escherichia such as E. coli AGX17/pGX50, pACKG4-pps (WO9708333, US6319696 B1) with enhanced enolpyruvic acid-producing ability can be mentioned. L-tryptophan-producing bacteria and parent strains that can be used to induce L-tryptophan-producing bacteria also include L-tryptophan-producing bacteria belonging to the genus Escherichia in which the activity of the protein encoded by the yedA gene or yddG gene is enhanced. (US2003148473 A1 and US2003157667 A1).

L-tryptophan-producing bacteria and parent strains that can be used to derive L-tryptophan-producing bacteria have enhanced activity of one or more enzymes selected from anthranilate synthase, phosphoglycerate dehydrogenase, and tryptophan synthase. Also included are strains that have been Since both anthranilate synthase and phosphoglycerate dehydrogenase are subject to feedback inhibition by L-tryptophan and L-serine, mutations that remove feedback inhibition may be introduced into these enzymes. Specific examples of strains having such mutations include E. coli SV164, which retains feedback-inhibited anthranilate synthase, and a mutant serA gene encoding feedback-inhibited phosphoglycerate dehydrogenase. A transformant strain obtained by introducing a plasmid pGH5 (WO9408031 A1) containing E. coli SV164 into E. coli SV164.

Examples of parent strains that can be used to induce L-tryptophan-producing bacteria and 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). Furthermore, L-tryptophan-producing ability may be imparted by enhancing the expression of the gene encoding tryptophan synthase in the tryptophan operon (trpBA). Tryptophan synthase consists of α and β subunits encoded by the trpA and trpB genes, respectively. Furthermore, L-tryptophan-producing ability may be improved by enhancing the expression of the isocitrate lyase-malate synthase operon (WO2005103275).

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 modified to overexpress the ilvGMEDA operon (US5998178). It is desirable to remove the region required for attenuation in the ilvGMEDA operon so that the produced L-valine does not attenuate the expression of the operon. Further, it is desirable that the ilvA gene in the operon is disrupted and threonine deaminase activity is reduced.

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). Examples of such strains include E. coli VL1970, which has a mutation in the ileS gene, which encodes isoleucine tRNA synthetase. E. coli VL1970 has been deposited on June 24, 1988 with the Russian National Collection of Industrial Microorganisms (VKPM; Russian Federation, 117545 Moscow, 1 ^st Dorozhny proezd, 1) under accession number VKPM B-4411.

Furthermore, mutant strains requiring lipoic acid for growth and/or lacking H ⁺ -ATPase can also be used as L-valine-producing strains or their parent strains (WO9606926 A1).

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

Genes and proteins used for breeding L-amino acid-producing bacteria may have, for example, the known nucleotide sequences and amino acid sequences of the above-exemplified genes and proteins, respectively. In addition, the genes and proteins used for breeding L-amino acid-producing bacteria may be mutants of the genes and proteins exemplified above, as long as the original functions (for example, in the case of proteins, the respective enzymatic activities) are maintained. ) (eg, variants of genes and proteins having such known base and amino acid sequences). With respect to mutants of genes and proteins, the description of genes encoding proteins having indolepyruvate decarboxylase activity described herein and mutants of indolepyruvate decarboxylase encoded by them can be applied mutatis mutandis.

Bacteria belonging to the order Enterobacteria described herein may be modified so that at least a gene encoding a protein having indolepyruvate decarboxylase activity is attenuated.

The term "gene encoding a protein having indolepyruvate decarboxylase activity" refers to the following reaction: indol-3-pyruvate + H ⁺ → indole-3-acetaldehyde + _CO2 (EC: 4.1.1.74 ) that encodes a protein having an enzymatic activity that catalyzes Methods for measuring the indolepyruvate decarboxylase activity of proteins include, for example, those described in Koga J. et al. things are mentioned. Protein concentration was determined by the Bradford protein assay using Coomassie dye with bovine serum albumin (BSA) as standard or the 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).

A specific example of a gene encoding an enzyme having indolepyruvate decarboxylase activity is the ipdC gene that encodes indolepyruvate decarboxylase. A gene encoding an enzyme having indolepyruvate decarboxylase activity can be the ipdC gene and its homologues or mutant nucleotide sequences. The ipdC gene and its homologues and mutant nucleotide sequences are described in more detail below.

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

The ipdC gene (GenBank, accession No. NC_017531.2/AP012032.2; nucleotide positions: 2461340 to 2462992, complement; Gene ID: BAK12109.1) is derived from P. ananatis AJ13355.
Located on the chromosome of the strain between the glk gene (BAK12108.1) on the same strand and the yjhZ gene (BAK12110.1) on the opposite strand. The P. ananatis AJ13355 strain-specific ipdC gene has the nucleotide sequence shown in SEQ ID NO: 1, and the amino acid sequence of the P. ananatis AJ13355 strain-specific IpdC protein encoded by the same gene is shown in SEQ ID NO: 2.

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 protein has a nucleotide sequence or amino acid sequence" can mean that the gene or protein includes the nucleotide sequence or amino acid sequence in a longer sequence. It can also mean that the protein has only said base sequence or said amino acid sequence.

As described above, protein homologues of IpdC having indolepyruvate decarboxylase activity unique to various bacteria belonging to the order Enterobacteria are known. Examples of such homologous proteins unique to bacteria belonging to the order Enterobacteria are listed in the NCBI database (National Center for Biotechnology Information, ncbi.nlm.nih.gov/protein), accession number of amino acid sequence, phylogenetic data, homology It is shown in Table 1 together with an indication of the gender value (as "identity" which is the identity of the amino acid). In particular, the E. coli-specific ipdC gene having the nucleotide sequence shown in SEQ ID NO: 54 is
It is known to encode an IpdC protein having the amino acid sequence shown in SEQ ID NO: 55 (Table 1; accession No.: WP_069192931.1).

The bacterium described herein can also be an L-amino acid-producing bacterium belonging to the order Enterobacteriaceae that has been modified so that the expression of a gene encoding a protein having threonine deaminase activity is attenuated. That is, a bacterium belonging to the order Enterobacteria that can be used in the methods described herein may be modified to attenuate the expression of a gene encoding a protein having threonine deaminase activity. The bacterium may be modified so that the expression of a gene encoding a protein having indolepyruvate decarboxylase activity and a gene encoding a protein having threonine deaminase activity is attenuated. indolepyruvate
The description of L-amino acid-producing bacteria belonging to the order Enterobacteriaceae, which has been modified so that the expression of the gene encoding the protein with decarboxylase activity is attenuated, states that the expression of the gene encoding the protein with threonine deaminase activity is attenuated. It can also be applied mutatis mutandis to L-amino acid-producing bacteria belonging to the order Enterobacteria, which have been modified to produce L-amino acids.

The term "gene encoding a protein having threonine deaminase activity" (also known as threonine dehydratase, threonine ammonia-lyase) refers to the following reaction: L-threonine → 2-oxobutanoate + ammonium (EC: 4.3.1.19 ) that encodes a protein having an enzymatic activity that catalyzes The threonine deaminase activity of a protein can be determined, for example, by measuring α-ketobutyric acid produced as a phenylhydrazone derivative, 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).

A specific example of a gene encoding an enzyme having threonine deaminase activity is the ilvA gene that encodes threonine deaminase. A gene encoding an enzyme having threonine deaminase activity can be the ilvA gene and its homologues or mutant nucleotide sequences. The nucleotide sequences of ilvA and its homologues and mutants are described in more detail below.

The ilvA gene unique to P. ananatis encodes the pyridoxal-phosphate dependent threonine dehydratase protein IlvA (BioCyc database, biocyc.org, accession ID: PAJ_RS16870; UniParc, accession No. UPI0002323460; KEGG). entry No. PAJ_3043).

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

That is, the ilvA gene may be a gene (eg, 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.

As described above, protein homologues of IlvA having threonine deaminase activity unique to various bacteria belonging to the order Enterobacteria are known. Examples of such homologous proteins unique to bacteria belonging to the order Enterobacteria are listed in the NCBI database (National Center for Biotechnology Information, ncbi.nlm.nih.gov/protein), accession number of amino acid sequence, phylogenetic data, homology It is shown in Table 2 along with an indication of the gender value (as "identity" which is the identity of the amino acid).

For example, the following descriptions of attenuation of gene expression, proteins (including mutant proteins), and genes (including mutant nucleotide sequences) refer to any of the proteins and genes described herein, including but not limited to IpdC and IlvA proteins, and genes encoding them such as the ipdC and ilvA genes, and their homologues).

There may be differences in DNA sequences between families, genera, species, or strains belonging to the order Enterobacteriaceae. Therefore, the ipdC and ilvA genes are not limited to genes having the nucleotide sequences shown in SEQ ID NOS: 1 and 3,
Genes that are variant sequences to, or homologous to, SEQ ID NOs: 1 and 3 and that encode variants of the IpdC and IlvA proteins may be included. 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 include proteins having or homologous to variant amino acid sequences of SEQ ID NOs:2 and 4. .

The term "mutant nucleotide sequence" refers to any synonymous amino acid codon according to the standard genetic code table (see, e.g., Lewin B., "Genes VIII", 2004, Pearson Education, Inc., Upper Saddle River, NJ 07458). can be used to refer to a base sequence that encodes a protein having the wild-type amino acid sequence. Therefore, a gene encoding 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.

The term "mutant nucleotide sequence" includes, but is not limited to, a nucleotide sequence complementary to the wild-type nucleotide sequence, or a nucleotide sequence thereof, as long as it encodes a protein having the desired activity, such as the above-described indolepyruvate decarboxylase activity or threonine deaminase activity. It can also mean a base sequence that can hybridize under stringent conditions with a probe that can be prepared from the base sequence. The term "stringent conditions" refers to specific hybrids, e.g. 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92%
93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more hybrids are formed, and non-specific hybrids, such as less homologous than the above can include conditions under which hybrids are not formed. Stringent conditions include, for example, 1
x SSC (standard sodium citrate or standard sodium chloride), 0.1% SDS (sodium dodecyl sulfate) at 60°C, another example is 0.1 x SSC, 0.1% SDS at 60 or 65°C. , one or more, in another example two or three washes. Wash times depend on the type of membrane used for blotting, but generally should be that recommended by the manufacturer. For example, the recommended wash time under stringent conditions for Amersham Hybond ^™ -N+ positively charged nylon membrane (GE Healthcare) is 15 minutes. Two or three washing steps can be performed. A portion of the sequence complementary to the wild-type base sequence may be used as the probe. Such probes are prepared by PCR (polymerase chain reaction; White TJ et al., The polymerase chain reaction) using oligonucleotides prepared based on the wild-type nucleotide sequence as primers and DNA fragments containing the nucleotide sequences as templates. reaction, Trends Genet., 1989, 5:185-189). The length of the probe is recommended to be over 50 bp, but can be appropriately selected depending on the hybridization 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 conditions after hybridization include 2×SSC and 0.1% SDS at 50°C, 60°C, or 65°C. obtain.

The term "mutant sequence" can also mean a sequence that encodes a variant protein.

The term "mutant protein" can refer to a protein having a mutant amino acid sequence.

The term "mutant protein" refers to the substitution, deletion, insertion and/or addition of one or several amino acid residues, whether one or several, as compared to the wild-type amino acid sequence of the protein. A protein having further mutations in its amino acid sequence, which maintains activity or function similar to that of the wild-type protein, or whose three-dimensional structure is not significantly altered relative to the unmodified protein. can mean The number of mutations in a mutant protein depends on the amino acid residue position or amino acid residue type in the three-dimensional structure of the protein. The number of alterations in the variant protein is not strictly limited, but may range from 1-300, in other instances 1-250, in other instances 1-200, in other instances 1-200, in the wild-type amino acid sequence of the protein. 1-150 in other examples 1-100 in other examples 1-90 in other examples 1-80 in other examples 1-70 in other examples 1-60 in other examples 1 in other examples ~50, another example 1-40, another example 1-30, another example 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 with each other, and such changes do not affect activity or function, or the tertiary structure of the protein is significantly different from that of the wild-type or unmodified protein. This is because it does not change. Thus, a mutant protein may be modified relative to the wild-type amino acid sequence of a protein as long as the 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. 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%
93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more,
It may be a protein having amino acid sequences with homology 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. Sequence identity between two sequences is calculated as the percentage of identical residues between the two sequences when the two sequences are aligned for maximal correspondence.

Examples of substitutions, deletions, insertions, and/or additions of one or several amino acid residues are those in which the activity or function of the mutant protein is maintained, or the three-dimensional structure of the protein is reduced to that of the unaltered protein (e.g. Conservative mutations are included such that they do not change significantly relative to the wild-type protein). A typical conservative mutation is a conservative substitution. Conservative substitutions include, but are not limited to, between Phe, Trp and Tyr when the site of substitution is an aromatic amino acid, and between Ala, Leu, Ile and Val when the site of substitution is a hydrophobic amino acid. , between Glu, Asp, Gln, Asn, Ser, His, and Thr when the substitution site is a hydrophilic amino acid, and between Gln and Asn when the substitution site is a polar amino acid, and the substitution site is basic Between Lys, Arg, and His when the substitution site is an acidic amino acid, between Asp and Glu when the substitution site is an amino acid having a hydroxyl group, and between Ser and Thr when the substitution site is an amino acid having a hydroxyl group. , are permutations that replace each other. Examples of conservative substitutions include Ala to Ser or Thr, Arg to Gln, His or Lys, Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln. , Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Asn, Gln, Lys or Asp, Gly to Pro, His to Asn, Lys, Gln, Arg or Tyr; Ile to Leu, Met, Val or Phe; Leu to Ile, Met, Val or Phe; Lys to Asn, Glu, Gln, His or substitution to Arg, Met to Ile, Leu, Val
or Phe substitution, Phe to Trp, Tyr, Met, Ile or Leu substitution, Ser to Thr or Ala substitution, Thr to Ser or Ala substitution, Trp to Phe or Tyr substitution, Tyr to Substitutions to His, Phe or Trp and substitutions of Val to Met, Ile or Leu are included. In addition, substitution, deletion, insertion, addition, etc. of amino acid residues as described above include naturally occurring mutations due to individual differences in organisms from which the amino acid sequences are derived.

Examples of substitutions, deletions, insertions and/or additions of one or several amino acid residues also include non-conservative mutations, provided that the mutations are at one or more different positions in the amino acid sequence. The second mutation above maintains the activity or function of the mutant protein, or does not significantly change the three-dimensional structure of the protein relative to the unmodified protein (e.g., wild-type protein, etc.). It is compensated.

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

The term "a bacterium modified to attenuate the expression of a gene" can mean that the bacterium has been modified such that the expression of the gene is attenuated in the modified bacterium. Gene expression can be attenuated, for example, by inactivating the gene.

The term "gene is inactivated" can mean that the modified gene encodes a completely inactive or non-functional protein compared to the wild-type or unmodified gene. Modified DNA regions include deletion of a part of a gene or deletion of the entire gene, substitution of one or more bases resulting in an amino acid substitution of a protein encoded by a gene (missense mutation), introduction of a stop codon (nonsense mutation), Deletion of one or two bases leading to a reading frame shift, insertion of drug resistance genes and/or transcription termination signals, or flanking regions of genes (which include genes such as promoters, enhancers, attenuators, ribosome binding sites, etc.) (including sequences controlling the expression of ) may not naturally express the gene. Gene inactivation can be achieved, for example, by mutagenesis using ultraviolet irradiation or nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, gene disruption using homologous recombination, and/or or "Red/ET-driven integration"
Or insertion-deletion mutagenesis based on "λRed/ET-mediated integration" (Yu D. et al.,
An efficient recombination system for chromosome engineering in Escherichia coli, Proc. Natl. Acad. Sci. USA, 2000, 97(11):5978-5983; 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).

The term "a bacterium has been modified to attenuate the expression of a gene" refers to regions to which the modified bacterium is operably linked to the gene, including promoters, enhancers, operators, attenuators and termination signals, ribosome binding sites, and other expression control elements, sequences that control the expression of genes) that are modified to attenuate the level of expression of the gene; and other examples (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).

The term "operably linked to the gene" means that the regulatory region has a base sequence expression (e.g., enhanced, increased, constitutive, basal, anti-terminating , attenuated, deregulated, reduced or repressed expression), specifically linked to a nucleic acid molecule or gene sequence so as to achieve expression of the gene product encoded by the sequence can mean that there are

The term "a bacterium has been modified such that the expression of a gene is attenuated" means that the expression level (i.e., expression amount) of a gene in a modified bacterium is It can also mean that the bacterium has been modified so as to be weakened. A decrease in the expression level of a gene can be measured, for example, as a decrease in the expression level of the gene per cell (which can be the average expression level of the gene per cell). The terms "expression level of a gene" or "expression level of a gene" can mean, for example, the amount of the expression product of a gene (eg, the amount of mRNA of the gene or the amount of protein encoded by the gene). The bacterium may be modified such that the level of gene expression per cell is reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of the unmodified bacterium.

The term "a bacterium has been modified to attenuate the expression of the metJ gene" refers to the total amount of the corresponding gene product (i.e., the encoded protein) and/or It could mean that the bacterium has been modified so that its total activity is reduced. A decrease in total protein amount or total activity can be measured, for example, as a decrease in protein amount or activity per cell (which can be the average amount or average activity of protein per cell). Bacteria have a protein activity per cell that is, for example, 50% that of unmodified bacteria
Below, 20% or less, 10% or less, 5% or less, or down to 0%.

The unmodified bacteria used for the above comparison include wild strains of bacteria belonging to the genus Escherichia such as E. coli MG1655 strain (ATCC 47076) and E. coli W3110 strain (ATCC 27325), or P. ananatis AJ13355 strain (FERM BP -6614) and other wild strains of bacteria belonging to the genus Pantoea. Examples of unmodified bacteria used for the above comparison include parent strains that have not been modified to weaken gene expression and bacteria that have not attenuated gene expression.

Gene expression can be attenuated by replacing the gene's expression control sequences, such as promoters, on the chromosomal DNA with weaker ones. Promoter strength is defined by the frequency of initiation of RNA synthesis. An example of a promoter strength evaluation method is the Goldstein MA
et al. (Goldstein MA and Doi RH, Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)). Also, WO0018935 A1
Promoters can also be modified to be weaker by introducing one or more base substitutions into the promoter region of the gene, as disclosed in . In addition, replacement of several nucleotides 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). is known to greatly affect the translation efficiency of mRNA.

Gene expression is specifically achieved, for example, by inserting transposons or insertion sequences (IS) into the coding region of the gene (US5175107) or regions controlling gene expression, or by UV irradiation or nitrosoguanidine (N-methyl -N'-Nitro-N-nitrosoguanidine; Furthermore, site-specific incorporation of mutations is based, for example, on λRed/ET-mediated recombination (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645). It can be carried out by a known chromosome editing method.

Gene copy number or the presence or absence of a gene can be determined, for example, by restriction treatment of chromosomal DNA, followed by Southern blotting using probes based on the gene sequence, or fluorescence in situ analysis.
It can be measured by performing situ hybridization (FISH) or the like. Gene expression levels can be determined by measuring the amount of mRNA transcribed from the gene using various well-known methods such as Northern blotting and quantitative RT-PCR. The amount of protein encoded by the gene was determined by SDS-PAGE followed by immunoblotting (
It can be measured by a known method such as Western blotting) or mass spectrometry of a protein sample.

Methods for recombinant molecular manipulation of DNA and molecular cloning, such as preparation of plasmid DNA, cleavage of DNA, joining of DNA, transformation of DNA, selection of oligonucleotides as primers, introduction of mutations, etc., are within the skill of the art. A well-known normal method may be used. Such methods are described, for example, in Sambrook J., Fritsch EF and Maniatis T., "Molecular Cloning: A Laboratory
Manual", ^2nd ed., Cold Spring Harbor Laboratory Press (1989) or Green MR and Sambrook JR, "Molecular Cloning: A Laboratory Manual", ^4th 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", ^4th ed., Washington, DC, ASM Press (2009).

Any method can be used for manipulation using recombinant DNA, including conventional methods such as transformation, transfection, infection, conjugation, mobilization, and the like. Transformation, transfection, infection, conjugation, or mobilization of a bacterium with DNA encoding a protein can confer on the bacterium the ability to synthesize the protein encoded by the DNA. Methods of transformation, transfection, infection, conjugation, and mobilization include any method. For example, for efficient DNA transformation and transfection, treatment of recipient cells with calcium chloride to make the E. coli K-12 cells more permeable to DNA 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). Other methods for random and/or targeted integration of DNA into host microorganisms, such as "Mu-driven integration/amplification" (Akhverdyan et al., Appl. -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) can be applied. Furthermore, for multiple insertions of desired genes, Mu-driven replicative transfer (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 chromosomal evolution based on recA-dependent homologous recombination leading to amplification of the desired gene (Tyo KEJ et al., Stabilized gene duplication enables long-term selection-free heterologous pathway expression, Nature Biotechnol.
2009, 27:760-765), as well as others utilizing various combinations of transposition, site-specific and/or homologous Red/ET-mediated recombination, and/or P1-mediated generalized transduction. (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) are available.

Since the sequences of genes encoding wild-type proteins unique to E. coli and P. ananatis species have already been sequenced (see above), mutant sequences encoding mutant proteins of the wild-type protein are: PCR (polymerase chain reaction) using DNA from the same bacterial species and primers prepared based on the base sequence of the wild-type gene; refer to White TJ et al.,
The polymerase chain reaction, Trends Genet., 1989, 5(6):185-189) or by site-directed mutagenesis in vitro treatment of DNA containing the wild-type gene with, for example, hydroxylamine or with the wild-type gene. Microorganisms, such as E. coli spp. Depending on the method of processing, it can be obtained or chemically synthesized as a full-length gene construct. Genes encoding proteins from other bacteria belonging to the order Enterobacteria or their mutant proteins can be similarly obtained.

The term "wild-type" when referring to proteins (e.g., "wild-type proteins") and genes (e.g., "wild-type genes"), which is also referred to as "native
and can be equivalent to the terms "natural"), respectively, are wild-type bacteria (e.g., E. coli strain MG1655 (ATCC 47076), E. coli strain W3110 (ATCC 27325), P. ananatis
AJ13355 strain (wild strain of bacteria belonging to the family Enterobacteriaceae or Enterobacteriaceae such as Erwiniaceae) such as strain AJ13355 (FERM BP-6614)) and/or is naturally produced , can refer to native proteins and genes. Since proteins are encoded by genes, a "wild-type protein" can be encoded by a "wild-type gene" that occurs naturally in the genome of a wild-type bacterium.

The term "native to" when referring to a protein or nucleic acid native to a particular organism (eg, bacterial species, etc.) can mean a protein or nucleic acid native to that organism. Thus, a protein or nucleic acid that is unique to a particular organism can refer to a protein or nucleic acid, respectively, that is naturally occurring in that organism and can be isolated from that organism and sequenced by methods known to those of skill in the art. . Furthermore, since the amino acid or base sequence of a protein or nucleic acid isolated from each organism in which the protein or nucleic acid resides can be readily determined, the term "unique" when referring to a protein or nucleic acid includes: As long as the resulting amino acid sequence or nucleotide sequence of the protein or nucleic acid obtained is identical to the amino acid sequence or nucleotide sequence of the protein or nucleic acid naturally occurring in the organism, genetic engineering techniques including, for example, recombinant DNA techniques or chemical synthesis It can also mean a protein or nucleic acid obtained by a method or the like. Amino acid sequences unique to a particular organism include, but are not limited to, peptides, oligopeptides, polypeptides (which include proteins, particularly enzymes), and the like. Nucleotide sequences unique to specific organisms include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are expression control sequences (including promoters, attenuators, terminators, etc.), genes, It is not limited to intergenic sequences, and base sequences encoding signal peptides, protein prosites, artificial amino acid sequences, and the like. Specific examples of amino acid and nucleotide sequences and their homologues unique to various organisms are described herein, including the proteins IpdC and IlvA ( These may be native to bacteria of the P. ananatis species and may be encoded by the corresponding genes ipdC and ilvA with the nucleotide sequences shown in SEQ ID NOS: 1 and 3, respectively).

Bacteria can have specific properties such as various auxotrophs, drug resistance, drug susceptibility, drug dependence, etc., in addition to the above properties, without departing from the scope of the present invention.

2. Methods Methods described herein include methods of producing L-amino acids using the bacteria described herein. The method for producing an L-amino acid using the bacterium described herein comprises cultivating (also referred to as culturing) the bacterium in a medium to produce an L-amino acid in the medium, bacterial cells, or both. producing, excreting or secreting, and/or accumulating; and recovering the L-amino acid from the medium and/or bacterial cells. The method may optionally further comprise the step of purifying the L-amino acid from the culture medium and/or bacterial cells. L-amino acids can be produced in the form as described above. L-amino acids can be produced in free form, or salts thereof, or mixtures thereof. For example, salts such as sodium, potassium and ammonium salts of L-amino acids or inner salts such as zwitterions can be produced by the above method. This is possible because amino acids can react under fermentation conditions with each other or with neutralizing agents such as inorganic or organic acids or alkaline substances by typical acid-base neutralization reactions to form salts. , which are chemical signatures of amino acids that are apparent to those skilled in the art. Specifically, L-cysteine monohydrochloride (L-cysteine×HCl) or L-cysteine monohydrate monohydrochloride (L-cysteine×H ₂ O×HCl) can be produced by the method. .

Cultivation of bacteria, and recovery and optional purification of L-amino acids from media and the like can be carried out in the same manner as conventional fermentation methods for producing L-amino acids using microorganisms. That is, the culture of bacteria and the recovery and purification of L-amino acids from media and the like are performed by applying conditions suitable for culturing bacteria and conditions suitable for recovery and purification of L-amino acids, which are well known to those skilled in the art. may be implemented.

The medium used is not particularly limited as long as it contains at least a carbon source and allows the bacteria described herein to grow and produce L-amino acids. The medium may be synthetic or natural, such as typical media containing carbon sources, nitrogen sources, sulfur sources, phosphorus sources, inorganic ions, and other organic and inorganic components as appropriate. Carbon sources include sugars such as glucose, sucrose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose and starch hydrolysates, alcohols such as ethanol, glycerol, mannitol and sorbitol, gluconic acid and fumaric acid. , organic acids such as 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, ammonia water, and the like can be used. In addition, peptones, yeast extracts, meat extracts, malt extracts, corn steep liquor and the like can also be used. The medium can contain one or more of these nitrogen sources. 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 the carbon, nitrogen, and sulfur sources. As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, phosphoric acid polymers such as pyrophosphoric acid, and the like can be used. 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, peptone, casamino acids, and organic nutrients such as yeast extract. , may be present in an appropriate amount, even a trace amount. In addition to these, if necessary, a small amount of calcium phosphate, iron ions, manganese ions, etc. may be added. As other organic and inorganic components, one component may be used, or two or more components may be used in combination. When using an auxotrophic strain that requires amino acids and the like for growth, it is preferable to supplement the required nutrients in the medium.

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

After culturing, the L-amino acid can be recovered from the medium. Specifically, L-amino acids present outside the cells can be recovered from the medium. In addition, after culturing, L-amino acids can be recovered from bacterial cells. Specifically, bacterial cells are crushed, solids such as bacterial cells and suspension of crushed bacterial cells (also referred to as cell debris) are removed to obtain a supernatant, and L-amino acids are recovered from the supernatant. can be done. Disruption of bacterial cells can be carried out by a well-known method such as ultrasonic disruption using high-frequency sound waves. Removal of solids can be performed, for example, by centrifugation or membrane filtration. Recovery of L-amino acids from media, supernatants, etc. can be performed, for example, by concentration, crystallization, membrane treatment, ion-exchange chromatography, flash chromatography, thin-layer chromatography, medium-pressure or high-pressure liquid chromatography, or any of these. It can be carried out by conventional techniques such as combination. These methods may be used alone or in combination as appropriate.

The invention will be explained more precisely with reference to the following non-limiting examples.

Example 1 Construction of P. ananatis Strain Deleting the ipdC Gene
A P. ananatis SC17(0)ΔipdC::λattL-kan ^R -λattR strain deleted for the ipdC gene (SEQ ID NO: 1) was constructed by λRed-dependent integration. For this purpose, P. ananatis strain SC17(0) (US8383372 B2, VKPM B-9246) carrying the RSF-Red-TER plasmid (US8383372 B2) was grown in LB liquid medium (Sambrook J. and Russell DW, Molecular Cloning: A Laboratory Manual ( ^3rd ed.), Cold Spring Harbor Laboratory Press, 2001). Then, 1 mL of the culture was added to LB containing isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM.
The cells were inoculated into 100 mL of liquid medium and cultured with shaking (250 rpm) at 32°C for 3 hours. Cells were collected and washed with 10% (v/v) glycerol three times to obtain competent cells. Using the pMW118-attL-kan ^R -attR plasmid (US7919284 B2) as a template, PCR was performed using primers P1 (SEQ ID NO: 5) and P2 (SEQ ID NO: 6), and sequences homologous to both upstream and downstream of the ipdC gene were obtained. An amplified λattL-kan ^R -λattR DNA fragment with terminal ends was obtained. The resulting DNA fragment was purified using the Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by electroporation. After culturing the cells in SOC medium (Sambrook J. and Russell DW, Molecular Cloning: A Laboratory Manual ( ^3rd ed.), Cold Spring Harbor Laboratory Press, 2001) for 2 hours, LB containing 35 mg/L kanamycin It was spread on a plate and cultured at 34°C for 16 hours. Appearing colonies were purified on the same medium. Then primer P3 (SEQ ID NO: 7)
and P4 (SEQ ID NO: 8) (TaKaRa Speed Star (R); 40 cycles of 97°C for 20 seconds, 54°C for 20 seconds, and 72°C for 100 seconds), and ipdC on the chromosome The gene is λattL- ^kanR-
It was confirmed that it was replaced with the λattR cassette. As a result, the P. ananatis SC17(0)ΔipdC::λattL-kan ^R -λattR strain was obtained.

Example 2 L-Methionine Production Using P. ananatis Strains Deleting the ipdC Gene To test the effect of deleting the ipdC gene on L-methionine production, the EdgeBio PurElute Bacterial Genomic kit was used according to the manufacturer's instructions. Chromosomal DNA was isolated from the SC17(0)ΔipdC::λattL-kan ^R -λattR strain (Example 1) using 10 μg of DNA to transform P. ananatis C2691 by electroporation. The P. ananatis L-methionine producing strain C2691 was constructed as described in Preliminary Example 1. The resulting transformant was plated on an LB plate containing 20 mg/L kanamycin and cultured overnight at 34°C until each colony was visualized. Desired transformants were identified by confirming replacement of the ipdC gene by PCR analysis using primers P3 (SEQ ID NO:7) and P4 (SEQ ID NO:8). As a result, the P. ananatis C2691ΔipdC::λattL-kan ^R -λattR strain (abbreviation: C3793) was obtained.

P. ananatis C2691 strain and C2691ΔipdC::λattL-kan ^R -λattR strain were each cultured in LB liquid medium at 32°C for 18 hours. 0.2 mL of the resulting culture was then inoculated into 2 mL of fermentation medium in a 20 x 200 mm test tube and shaken on a rotary shaker at 250 rpm for 32 minutes until the glucose was consumed.
℃ for 48 hours.

The composition (g/L) of the fermentation medium was as follows.
glucose 40.0
( _NH4 ) _{2SO4 15.0
KH2PO4 1.5
MgSO4-7H2O1.0 _}
Thiamine-HCl 0.1
_CaCO3 25.0
LB medium 4% (v/v)

The fermentation medium was sterilized at 116°C for 30 minutes. However, glucose and _CaCO3 were each sterilized separately as follows: glucose at 110°C for 30 minutes; _CaCO3 at 116°C for 30 minutes. The pH was adjusted to 7.0 with KOH solution.

After culturing, the amount of L-methionine accumulated in the medium was measured with an Agilent 1260 amino-acid analyzer. Results of three independent test tube fermentations (as mean ± 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 higher amounts of L-methionine compared to the parent strain P. ananatis C2691.

Example 3 L-Cysteine Production Using a P. ananatis Strain Deleting the ipdC Gene To test the effect of deletion of the ipdC gene on L-cysteine production, SC17(0)ΔipdC:: The P. ananatis L-cysteine-producing strain EYP197(s) (RU2458981 C2 or WO2012/137689) was transformed with the chromosomal DNA of the λattL-kan ^R -λattR strain (Example 1), and P. ananatis EYP ΔipdC::λattL Obtain the -kanR-λattR strain. The P. ananatis EYP197(s) strain was constructed from P. ananatis SC17 (FERM BP-11091) by introducing the cysE5 and yeaS genes and replacing the native promoter of the cysPTWA gene cluster with the Pnlp8 promoter.

P. ananatis EYP197(s) strain and EYP ΔipdC::λattL-kanR-λattR strain were each cultured in 3 mL of LB liquid medium at 32°C for 18 hours. and inoculate 2 mL of fermentation medium in test tubes and incubate for 24 hours at 32° C. on a rotary shaker.

After culturing, the amount of L-cysteine accumulated in the medium was measured using 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) with the following modifications: 150 μL of each sample is mixed with 150 μL of 1 MH ₂ SO ₄ and incubated at 20°C for 5 minutes, followed by 700 μL of H ₂ O was added to the mixture and the resulting mixture 150
Transfer μL 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, 13000 rp
After spinning at rpm for 10 minutes, transfer 100 μL of the mixture to a 20×200 mm test tube. then 400 μL
H ₂ O, 500 μL glacial acetic acid, and 500 μL solution B (0.63 g ninhydrin, 10 mL glacial acetic acid,
10 mL of 36% HCl) is added and the mixture is incubated in a boiling water bath for 10 minutes. Then add 4.5 mL of ethanol and measure the _OD560 . The concentration of cysteine is calculated using the formula: C (Cys, g/L) = 11.3 x _OD560 .

The composition (g/L) of the fermentation medium is as follows.
glucose 40.0
( _{NH4 ) 2S2O3 12.0
KH2PO4 1.5
MgSO4-6H2O 0.825}
Thiamine-HCl 0.1
_CaCO3 25.0
LB medium 4% (v/v)

The fermentation medium is sterilized at 116°C for 30 minutes. However, glucose, _{( NH4} ) _{2S2O3 ,} and _CaCO3 are each sterilized separately as follows: glucose at 110°C for 30 minutes; ( _NH4 ) _{2S2O3 with} 0.2 µm membrane _; Filtration; _CaCO3 at 116°C for 30 minutes. Adjust pH to 7.0 with KOH solution.

Example 4 Production of L-Glutamic Acid Using P. ananatis Strains Deleting the ipdC Gene To test the effect of deletion of the ipdC gene on L-glutamic acid production, SC17(0)ΔipdC:: In the chromosomal DNA of the λattL-kan ^R -λattR strain (Example 1), P.
An ananatis L-glutamic acid producing strain NA1 (EP2336347 A1) is transformed to obtain a P. ananatis NA1ΔipdC::λattL-kanR-λattR strain.

P. ananatis NA1 strain and NA1ΔipdC::λattL-kanR-λattR strain were each cultured in 3 mL of LB liquid medium at 32°C for 18 hours, and 0.2 mL of the resulting culture solution was placed in a 20 x 200 mm test tube. Inoculate 2 mL of filled fermentation medium and incubate for 24 hours at 32° C. on a rotary shaker.

After culturing, the amount of L-glutamic acid that accumulates in the medium is calculated as butan-1-ol: acetic acid: water = 4.
: 1:1 (v/v), stained with ninhydrin (1% solution in acetone), and L-glutamic acid treated with 50% ethanol containing 0.5% CdCl ₂ . Elute and measure the amount of L-glutamic acid at 540 nm.

The composition (g/L) of the fermentation medium 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

The fermentation medium is sterilized at 116°C for 30 minutes. However, glucose and _CaCO3 are each sterilized separately as follows: glucose at 110°C for 30 minutes; _CaCO3 at 116°C for 30 minutes. pH with KOH solution
Adjust to 7.0.

Example 5 L-Aspartic Acid Production Using a P. ananatis Strain Deleting the ipdC Gene To test the effect of deletion of the ipdC gene on L-aspartic acid production, SC17(0)ΔipdC was grown by electroporation. The chromosomal DNA of the ::λattL-kan ^R -λattR strain (Example 1) was transformed into the P. ananatis L-aspartic acid-producing strain 5ΔP2RM (WO2010038905 A1), and the P. ananatis 5ΔP2RMΔipdC::λattL-kanR-λattR strain was transformed. get

P. ananatis 5ΔP2RM strain and 5ΔP2RMΔipdC::λattL-kanR-λattR strain were each cultured in 3 mL of LB liquid medium at 32°C for 18 hours. Inoculate 2 mL of filled fermentation medium and incubate for 72 hours at 32° C. on a rotary shaker. After culturing, the amount of L-aspartic acid accumulating in the medium is measured by paper chromatography.

The composition (g/L) of the fermentation medium is as follows.
glucose 40.0
_{MgSO4-7H2O1.0 _}
( _NH4 ) _{2SO4 16.0
KH2PO4 0.3}
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
_CaCO3 30.0

The fermentation medium is sterilized at 116°C for 30 minutes. However, glucose and _CaCO3 are each sterilized separately as follows: glucose at 110°C for 30 minutes; _CaCO3 at 116°C for 30 minutes. pH with NaOH solution
to 6.5.

Example 6 Construction of L-Methionine-Producing E. coli Deleting the ipdC Gene Temperature-sensitive replication type pKD46 plasmid (Datsenko KA and Wanner BL, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products USA, 2000, 97(12):6640-6645), an L-methionine-producing strain of E. coli (examples of which are given above) in LB liquid medium overnight. culture. Then, 1 mL of the culture medium 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). 10% (v/v)
Obtain competent cells by washing three times with glycerol. PCR is performed using the pMW118-attL-kan ^R -attR plasmid (US7919284 B2) as a template to obtain an amplified λattL-kan ^R -λattR DNA fragment having sequences homologous to the upstream and downstream of the ipdC gene at both ends. The resulting DNA fragment is purified using the Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by electroporation. After culturing the cells in SOC medium for 2 hours, the cells are plated on an LB plate containing 35 mg/L kanamycin and cultured at 34°C for 16 hours. The colonies that appear are purified on the same medium. Confirm by PCR that the ipdC gene on the chromosome has been replaced with the λattL-kan ^R -λattR cassette. As a result, the E. coli L-methionine-producing strain ΔipdC::λattL-kan
^R -λattR is obtained.

Example 7 Production of L-Methionine Using an E. coli Strain Deleting the ipdC Gene
An E. coli L-methionine-producing strain ΔipdC::λattL-kan ^R -λattR and its parent strain are cultured in LB liquid medium at 37° C. for 18 hours with shaking. 0.2 mL of the resulting culture is then inoculated into 2 mL of fermentation medium in a 20×200 mm test tube and cultured for 48 hours at 32° C. on a rotary shaker at 250 rpm. After culturing, the amount of L-methionine accumulated in the medium is measured with an Agilent 1260 amino-acid analyzer.

The composition (g/L) of the fermentation medium is as follows.
glucose 40.0
( _NH4 ) _{2SO4 15.0
KH2PO4 1.5
MgSO4-7H2O1.0 _}
Thiamine-HCl 0.1
Threonine 0.5
_CaCO3 20.0
LB medium 4% (v/v)

The fermentation medium is sterilized at 116°C for 30 minutes. However, glucose and _CaCO3 are each sterilized separately as follows: glucose at 110°C for 30 minutes; _CaCO3 at 116°C for 30 minutes. Adjust pH to 7.0 with KOH solution.

Example 8 Construction of a P. ananatis Strain Deleting the ilvA Gene
A P. ananatis SC17(0)ΔilvA::λattL-kan ^R -λattR strain deleted for the ilvA gene (SEQ ID NO:3) was constructed by λRed-dependent integration. For this purpose, P. ananatis SC17(0)/RSF-Red-TER strain (US8383372 B2) was grown overnight in LB liquid medium. Thereafter, 1 mL of the culture solution 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). Cells were collected and washed with 10% (v/v) glycerol three times to obtain competent cells. Using the pMW118-attL-kan ^R -attR plasmid (US7919284 B2) as a template, PCR was performed using primers P5 (SEQ ID NO: 9) and P6 (SEQ ID NO: 10). An amplified λattL-kan ^R -λattR DNA fragment with terminal ends was obtained. The resulting DNA fragment was purified using the Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by electroporation. The cells were cultured in SOC medium for 2 hours, spread on an LB plate containing 35 mg/L kanamycin, and cultured at 34°C for 16 hours. Appearing colonies were purified on the same medium. Then, PCR analysis (TaKaRa Speed Star®) using primers P7 (SEQ ID NO: 11) and P8 (SEQ ID NO: 12); 25 cycles of 97°C for 20 seconds, 55°C for 20 seconds, and 72°C for 100 seconds ) was performed to confirm that the ilvA gene on the chromosome was replaced with the λattL-kan ^R -λattR cassette. As a result, the P. ananatis SC17(0)ΔilvA::λattL-kan ^R -λattR strain was obtained.

Example 9 L-Methionine Production Using a P. ananatis Strain Deleting the ilvA Gene To test the effect of ilvA deletion on L-methionine production, the EdgeBio PurElute Bacterial Genomic kit was used according to the manufacturer's instructions. Chromosomal DNA was isolated from the SC17(0)ΔilvA::λattL- ^kanR -λattR strain (Example 8) using 10 µg of DNA to transform P. ananatis C2792 by electroporation. The P. ananatis L-methionine producing strain C2792 was constructed as described in Preliminary Example 2. The resulting transformant was plated on an LB plate containing 20 mg/L kanamycin and cultured overnight at 34°C until each colony was visualized. Desired transformants were identified by PCR analysis as described in Example 8 to confirm replacement of the ilvA gene. As a result, P. ananatis C2792ΔilvA::λattL-kan ^R -λa
A ttR strain (abbreviation: C3653) was obtained.

P. ananatis C2792 strain and C2792ΔilvA::λattL-kan ^R -λattR strain were each cultured in LB liquid medium at 32°C for 18 hours. 0.2 mL of the resulting culture was then inoculated into 2 mL of fermentation medium in a 20 x 200 mm test tube and shaken on a rotary shaker at 250 rpm for 32 minutes until the glucose was consumed.
℃ for 48 hours.

The composition (g/L) of the fermentation medium was as follows.
glucose 40.0
( _NH4 ) _{2SO4 15.0
KH2PO4 1.5
MgSO4-7H2O1.0 _}
Thiamine-HCl 0.1
_CaCO3 25.0
LB medium 4% (v/v)

The fermentation medium was sterilized at 116°C for 30 minutes. However, glucose and _CaCO3 were each sterilized separately as follows: glucose at 110°C for 30 minutes; _CaCO3 at 116°C for 30 minutes. The pH was adjusted to 7.0 with KOH solution.

After culturing, the amount of L-methionine accumulated in the medium was measured with an Agilent 1260 amino-acid analyzer. The results of three independent test tube fermentations (as mean±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 higher amounts of L-methionine compared to the parent strain P. ananatis C2792.

Example 10 Construction of E. coli MG1655 strain lacking the ilvA gene E. coli MG1655 strain (ATCC 700926) harboring the temperature-sensitive replication type plasmid pKD46 is cultured overnight in LB liquid medium. Then, 1 mL of the culture medium 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). Cells are collected and washed three times with ice-cold 10% (v/v) glycerol to obtain competent cells. PCR is performed using the pMW118-attL-kan ^R -attR plasmid (US7919284 B2) as a template to obtain an amplified λattL-kan ^R -λattR DNA fragment having sequences homologous to the upstream and downstream of the ilvA gene at both ends. The resulting DNA fragment is purified using the Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by electroporation. After culturing the cells in SOC medium for 2 hours, the cells are plated on an LB plate containing 35 mg/L kanamycin and cultured at 34°C for 16 hours. The colonies that appear are purified on the same medium. Confirm by PCR that the ilvA gene on the chromosome has been replaced with the λattL-kan ^R -λattR cassette. As a result, E. coli MG1655ΔilvA::λattL-kan ^R -λattR strain is obtained.

Example 11 L-Methionine Production Using an E. coli Strain Deleting the ilvA Gene To test the effect of ilvA deletion on L-methionine production, strain MG1655ΔilvA::λattL-kan ^R -λattR (implementation The chromosomal DNA of Example 10) is introduced into E. coli L-methionine producing strain 218 (VKPM B-8125, RU2209248 C2) by P1 transduction. As a result, E. coli 218ΔilvA::λattL-kan ^R -λattR strain is obtained.

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

After culturing, the amount of L-methionine accumulated in the medium is measured with an Agilent 1260 amino-acid analyzer.

The composition (g/L) of the fermentation medium is as follows.
glucose 40.0
( _NH4 ) _{2SO4 15.0
KH2PO4 1.5
MgSO4-7H2O1.0 _}
Thiamine-HCl 0.1
Threonine 0.5
_CaCO3 20.0
LB medium 4% (v/v)

The fermentation medium is sterilized at 116°C for 30 minutes. However, glucose and _CaCO3 are each sterilized separately as follows: glucose at 110°C for 30 minutes; _CaCO3 at 116°C for 30 minutes. Adjust pH to 7.0 with KOH solution.

Preliminary Example 1 Construction of L-Methionine Producing Strain C2691 of P. ananatis 1.1. Construction of P. ananatis SC17(0)λattL-kan ^R -λattR-Pnlp8sd22-cysM strain
The P. ananatis strain SC17(0) λattL-kan ^R -λattR-Pnlp8sd22-cysM strain in which the promoter region of the cysM gene (SEQ ID NO: 13) was replaced with the cassette λattL-kan ^R -λattR-Pnlp8sd22 was constructed by λRed-dependent integration. did. For this purpose, the P. ananatis SC17(0)/RSF-Red-TER strain was cultured overnight in LB liquid medium. Thereafter, 1 mL of the culture solution 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). Cells were collected and washed with 10% (v/v) glycerol three times to obtain competent cells. Using primers P9 (SEQ ID NO: 14) and P10 (SEQ ID NO: 15), PCR was performed using the pMW118-attL-kan-attR-pnlp8sd22 plasmid (SEQ ID NO: 16) as a template, and homologous upstream and downstream of the promoter region of the cysM gene. An amplified DNA fragment of λattL-kan ^R -λattR-Pnlp8sd22 having a sequence at both ends was obtained. The resulting DNA fragment was purified using the Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by electroporation. SOC medium (Sambrook J. and
Russell DW, Molecular Cloning: A Laboratory Manual ( ^3rd ed.), Cold Spring Harbor Laboratory Press, 2001), plated on LB plates containing 35 mg/L kanamycin, and incubated at 34°C for 16 hours. cultured for hours. Appearing colonies were purified on the same medium. Next, primers P11 (SEQ ID NO: 17) and P12 (SEQ ID NO: 18) were used to confirm that the promoter region of the cysM gene on the chromosome was replaced with the λattL-kan ^R -λattR-Pnlp8sd22 cassette. PCR analysis (TaKaRa Speed Star (R); 40 cycles of 92°C 10 seconds, 56°C 10 seconds, 72°C 60 seconds) was performed. As a result, P. ananatis SC17(0)λattL-kan ^R -λattR-Pnlp8sd22-cysM strain (abbreviation: C2338) was obtained.

1.2. P. ananatis strain C2597 (SC17(0)ΔmdeA::λattL-kan ^R -λattR-Pnlp8sd22-cysM
) The silent gene mdeA (SEQ ID NO: 19) was inserted into the cassette λattL-kan ^R -λattR-Pnlp8sd22-cysM
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. Thereafter, 1 mL of the culture solution 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). Cells were collected and washed with 10% (v/v) glycerol three times to obtain competent cells. Using primers P13 (SEQ ID NO: 20) and P14 (SEQ ID NO: 21), PCR was performed using the chromosome isolated from the C2338 strain (preliminary example 1.1) as a template to identify sequences homologous to the upstream and downstream of the mdeA gene. An amplified DNA fragment of λattL-kan ^R -λattR-Pnlp8sd22-cysM having both ends was obtained. The resulting DNA fragment was subjected to Wizard PCR
It was purified using the Prep DNA Purification System (Promega) and introduced into competent cells by electroporation. The cells were cultured in SOC medium for 2 hours, spread on an LB plate containing 35 mg/L kanamycin, and cultured at 34°C for 16 hours. Appearing colonies were purified on the same medium. Next, PCR was performed using primers P15 (SEQ ID NO: 22) and P16 (SEQ ID NO: 23) to confirm that the mdeA gene on the chromosome was replaced with the λattL-kan ^R -λattR-Pnlp8sd22-cysM cassette. Analysis (TaKaRa Speed Star (R); 40 cycles of 92°C for 10 seconds, 56°C for 10 seconds, and 72°C for 60 seconds) was performed. As a result, P. ananatis SC17(0)ΔmdeA::λattL-kan ^R -λattR-Pnlp8sd22-cysM strain (abbreviation: C2597) was obtained.

1.3. Construction of P. ananatis strain C2603 (SC17ΔmdeA::λattL-kan ^R -λattR-Pnlp8sd22-cysM)
Chromosomal DNA was isolated from strain C2597 (SC17(0) ΔmdeA::λattL-kan ^R -λattR-Pnlp8sd22-cysM) using the EdgeBio PurElute Bacterial Genomic kit according to the manufacturer's instructions. The obtained chromosomal DNA was used for transformation of P. ananatis SC17 strain (FERM BP-11091). For this purpose, P. ananatis strain SC17 was cultured overnight in LB liquid medium. After that, 1 mL of the culture solution was inoculated into 100 mL of LB liquid medium, and the cells were cultured at 32°C for 2 hours with shaking (250 rpm). Cells were collected and washed with 10% (v/v) glycerol three times to obtain competent cells. C2597 strain (preliminary example 1
. The chromosomal DNA isolated from 2) was introduced into competent cells by electroporation. The cells were cultured in SOC medium for 2 hours, spread on an LB plate containing 35 mg/L kanamycin, and cultured at 34°C for 16 hours. Appearing colonies were purified on the same medium. PCR analysis was then 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.

1.4. Deletion of the kan gene from strain C2603 (SC17ΔmdeA::λattL-kan ^R -λattR-Pnlp8sd22-cysM)
The kanamycin resistance gene (kan) was deleted from strain C2603 using the RSF(TcR)-int-xis plasmid (US20100297716 A1). RSF(TcR)-int-xis was introduced into strain C2603 by electroporation, the cells were spread on LB medium containing tetracycline (15 mg/L) and cultured at 30°C to obtain C2603/RSF(TcR). -int-xis strain was obtained.

The resulting plasmid-bearing strains were purified in LB medium containing tetracycline (15 mg/L) and 1 mM IPTG to obtain single colonies. Next, a single colony was plated on a medium containing 50 mg/L kanamycin and cultured overnight at 37°C with shaking (250 rpm). A kanamycin-sensitive strain was plated on LB medium containing 10% sucrose (weight ratio) 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 designated C2614.

1.5. Construction of P. ananatis strain C2607 (SC17(0)ΔmetJ::λattL-cat ^R -λattR)
P. ananatis SC17(0)ΔmetJ::λattL-cat ^R -λatt lacking the metJ gene (SEQ ID NO: 24)
The R 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. 1 mL of the culture was then added to IPTG at a final concentration of 1 mM.
was inoculated into 100 mL of LB liquid medium containing, and the cells were cultured at 32°C for 3 hours with shaking (250 rpm). Cells were collected and washed with 10% (v/v) glycerol three times to obtain competent cells. PCR was performed using primers P17 (SEQ ID NO: 25) and P18 (SEQ ID NO: 26) and pMW118-attL-cat-attR plasmid (Minaeva NI et al., BMC Biotechnol., 2008, 8:63) as a template to generate metJ. An amplified DNA fragment of λattL-cat ^R -λattR having sequences homologous to the upstream and downstream of the gene at both ends was obtained. The resulting DNA fragment was purified using the Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by electroporation. After culturing the cells in SOC medium for 2 hours, they were plated on an LB plate containing 35 mg/L chloramphenicol and cultured at 34°C for 16 hours. Appearing colonies were purified on the same medium. Next, to confirm that the metJ gene on the chromosome has been replaced with the λattL-cat ^R -λattR cassette, PCR analysis (TaKaRa Speed Star(R)；92
40 cycles of 10 seconds at 56°C and 60 seconds at 72°C) were performed. As a result, P. ananatis SC17(0)ΔmetJ::λattL-cat ^R -λattR strain (abbreviation: C2607) was obtained.

1.6. Construction of P. ananatis strain C2634 (C2614ΔmetJ::λattL-cat ^R -λattR)
Chromosomal DNA was isolated from strain C2607 (SC17(0)ΔmetJ::λattL- ^catR -λattR) (preparatory example 1.5) using the EdgeBio PurElute Bacterial Genomic kit according to the manufacturer's instructions.
The resulting chromosomal DNA was used to transform strain C2614 (preliminary example 1.4). For this purpose, P. ananatis strain C2614 was cultured overnight in LB liquid medium. After that, 1 mL of the culture solution was inoculated into 100 mL of LB liquid medium, and the cells were cultured at 32°C for 2 hours with shaking (250 rpm). Cells were collected and washed with 10% (v/v) glycerol three times to obtain competent cells. Chromosomal DNA isolated from strain C2607 was introduced into competent cells by electroporation. After culturing the cells in SOC medium for 2 hours, they were plated on LB plates containing 35 mg/L chloramphenicol and incubated at 34°C for 16 hours.
cultured for hours. Appearing colonies were purified on the same medium. PCR analysis was then performed as described in Preliminary Example 1.5 to confirm the replacement of the metJ gene on the chromosome. As a result, the P. ananatis C2614ΔmetJ::λattL-cat ^R -λattR strain (abbreviation: C2634) was obtained.

1.7. Construction of P. ananatis strain C2605 (SC17(0) λattL-kan ^R -λattR-Ptac71φ10-metA)
The promoter region of the metA gene (SEQ ID NO: 29) is the cassette λattL-kan ^R -λattR-Ptac71
The φ10-substituted P. ananatis SC17(0)λattL-kan ^R -λattR-Ptac71φ10-metA strain was constructed by λRed-dependent integration. For this purpose, P. ananatis SC17(0)/RSF-Red-TER
The strain was cultured overnight in LB liquid medium. Thereafter, 1 mL of the culture solution 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). Cells were collected and washed with 10% (v/v) glycerol three times to obtain competent cells. Using primers P21 (SEQ ID NO: 30) and P22 (SEQ ID NO: 31), PCR was performed using the pMW118-attL-kan-attR-Ptac71φ10 plasmid (SEQ ID NO: 32) as a template, and homologous upstream and downstream of the promoter region of the metA gene. An amplified DNA fragment of λattL-kan ^R -λattR-Ptac71φ10 having a sequence at both ends was obtained. The resulting DNA fragment was purified using the Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by electroporation. The cells were cultured in SOC medium for 2 hours, spread on an LB plate containing 35 mg/L kanamycin, and cultured at 34°C for 16 hours.
Appearing colonies were purified on the same medium. Next, primers P23 ( ^SEQ ID NO: 33) and P24 (sequence No. 34) was used to perform PCR analysis (TaKaRa Speed Star (R); 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)λattL-kan ^R -λattR-Ptac71φ10-metA strain (abbreviation: C2605) was obtained.

1.8. Construction of P. ananatis strain C2611 (SC17λattL-kan ^R -λattR-Ptac71φ10-metA)
Chromosomal DNA was isolated from strain C2605 (SC17(0)λattL-kan ^R -λattR-Ptac71φ10-metA) (preparatory example 1.7) using the EdgeBio PurElute Bacterial Genomic kit according to the manufacturer's instructions. The resulting chromosomal DNA was used to transform strain SC17. For this purpose, P. ananatis strain SC17 was cultured overnight in LB liquid medium. After that, 1 mL of the culture solution was inoculated into 100 mL of LB liquid medium, and the cells were cultured at 32°C for 2 hours with shaking (250 rpm). Cells were collected and washed with 10% (v/v) glycerol three times to obtain competent cells. Chromosomal DNA isolated from strain C2605 was introduced into competent cells by electroporation. The cells were cultured in SOC medium for 2 hours, spread on an LB plate containing 35 mg/L kanamycin, and cultured at 34°C for 16 hours. Appearing colonies were purified on the same medium. PCR analysis was then performed as described in Preliminary Example 1.7 to confirm the replacement of the promoter region of the metA gene on the chromosome. As a result, P. ananatis SC17λattL-kan ^R -λattR-Ptac71φ10-metA strain (abbreviation: C2611) was obtained.

1.9. Construction of P. ananatis strain C2619 (C2611ΔmetJ::λattL-cat ^R -λattR)
Chromosomal DNA was isolated from strain C2607 (SC17(0)ΔmetJ::λattL- ^catR -λattR) (preparatory example 1.5) using the EdgeBio PurElute Bacterial Genomic kit according to the manufacturer's instructions.
The resulting chromosomal DNA was used to transform strain C2611 (preliminary example 1.8). For this purpose, P. ananatis strain C2611 was cultured overnight in LB liquid medium. After that, 1 mL of the culture solution was inoculated into 100 mL of LB liquid medium, and the cells were cultured at 32°C for 2 hours with shaking (250 rpm). Cells were collected and washed with 10% (v/v) glycerol three times to obtain competent cells. Chromosomal DNA isolated from strain C2607 was introduced into competent cells by electroporation. After culturing the cells in SOC medium for 2 hours, they were plated on LB plates containing 35 mg/L chloramphenicol and incubated at 34°C for 16 hours.
cultured for hours. Appearing colonies were purified on the same medium. PCR analysis was then performed as described in Preliminary Example 1.5 to confirm the replacement of the metJ gene on the chromosome. As a result, the P. ananatis C2611ΔmetJ::λattL-cat ^R -λattR strain (abbreviation: C2619) was obtained.

1.10. Selection of P. ananatis strains with mutant alleles of the metA gene encoding feedback-resistant MetA
Cells of strain C2619 (SC17λattL-kan ^R -λattR-Ptac71φ10-metA ΔmetJ::λattL-cat ^R -λattR) were inoculated into a 50 mL flask containing LB liquid medium so that the OD600 was 0.05. at 2
h, aerated (250 rpm). An exponentially growing cell culture of the same strain with an OD600 of 0.25 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 plated on M9 agar plates containing glucose (0.2%) and norleucine (600 g/L). The resulting mutants were examined for their ability to produce L-methionine. A strain with the highest L-methionine-producing ability was selected, and the base sequence of the metA gene of that strain was determined. Sequence analysis revealed a mutation in the metA gene that resulted in substitution of the arginine (Arg) residue at position 34 in the wild-type MetA amino acid sequence (SEQ ID NO: 35) with a cysteine residue (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. Thus, the P. ananatis SC17λattL-kan ^R -λattR-Ptac71φ10-metA(R34C)ΔmetJ::λattL-cat ^R -λattR strain (abbreviation: C2664) was constructed.

1.11. Construction of P. ananatis strain C2669 (C2634λattL-kan ^R -λattR-Ptac71φ10-metA(R34C))
From strain C2664 (SC17λattL-kan ^R -λattR-Ptac71φ10-metA(R34C)ΔmetJ::λattL-cat ^R -λattR) (Preliminary Example 1.10), using the EdgeBio PurElute Bacterial Genomic kit according to the manufacturer's instructions. Chromosomal DNA was isolated. The resulting chromosomal DNA was used to transform strain C2634 (preliminary example 1.6). For this purpose, P. ananatis strain C2634 was cultured overnight in LB liquid medium. After that, inoculate 1 mL of the culture solution into 100 mL of LB liquid medium and incubate the cells at 32°C for 2 hours.
Cultured with shaking (250 rpm). Cells were collected and washed with 10% (v/v) glycerol three times to obtain competent cells. Chromosomal DNA isolated from strain C2664 was introduced into competent cells by electroporation. The cells were cultured in SOC medium for 2 hours, spread on an LB plate containing 35 mg/L kanamycin, and cultured at 34°C for 16 hours. Appearing colonies were purified on the same medium. PCR analysis was then performed as described in Preliminary Example 1.7 to confirm the replacement of the promoter region of the metA gene on the chromosome. As a result, the P. ananatis C2634λattL-kan ^R -λattR-Ptac71φ10-metA(R34C) strain (abbreviation: C2669) was obtained.

1.12. Deletion of kan and cat genes from strain C2669 (C2614ΔmetJ::λattL-cat ^R -λattR λattL-kan ^R -λattR-Ptac71φ10-metA(R34C))
Kanamycin and chloramphenicol resistance genes (corresponding to kan and cat) were deleted from strain C2669 (preparatory example 1.11) using the RSF(TcR)-int-xis plasmid. RSF(TcR)-int-xis was introduced into the C2669 strain by electroporation, the cells were spread on LB medium containing tetracycline (15 mg/L) and cultured at 30°C to obtain C2669/RSF(TcR). -int-xis strain was obtained.

The resulting plasmid-bearing strains were purified in LB medium containing tetracycline (15 mg/L) and 1 mM IPTG to obtain single colonies. A 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). Strains sensitive to both kanamycin and chloramphenicol were plated on LB medium containing 10% sucrose (by weight) and 1 mM IPTG to eliminate the RSF(TcR)-int-xis plasmid from the strain. and incubated overnight at 37°C. A tetracycline-sensitive colony was selected and the corresponding strain was designated C2691.

Preliminary Example 2 Construction of L-Methionine Producing Strain C2792 of P. ananatis 2.1. Construction of P. ananatis SC17(0)ΔmetE1::λattL-kan ^R -λattR-Ptac71φ10-thrA442
P. ananatis SC17(0)ΔmetE1::λattL-kan ^R -λattR-Ptac71φ10-thrA442 in which the metE1 gene (c0742) (SEQ ID NO:38) was replaced with the cassette λattL-kan ^R -λattR-Ptac71φ10-thrA442 (SEQ ID NO:39) Strains were constructed by λRed-dependent integration. For this purpose, the P. ananatis SC17(0)/RSF-Red-TER strain was cultured overnight in LB liquid medium. Thereafter, 1 mL of the culture solution 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). Cells were collected and washed with 10% (v/v) glycerol three times to obtain competent cells. PCR was performed using primers P25 (SEQ ID NO: 40) and P26 (SEQ ID NO: 41), and λattL-kan ^R -λattR-Ptac71φ10-thrA442 having sequences homologous to the upstream and downstream of the metE1 gene at both ends.
of amplified DNA fragments were obtained. The resulting DNA fragment was subjected to Wizard PCR Prep DNA Purification System
(Promega) and introduced into competent cells by electroporation. The cells were cultured in SOC medium for 2 hours, spread on an LB plate containing 35 mg/L kanamycin, and cultured at 34°C for 16 hours. Appearing colonies were purified on the same medium. Next, PCR was performed using primers P27 (SEQ ID NO: 42) and P28 (SEQ ID NO: 43) to confirm that the metE1 gene on the chromosome was replaced with the λattL-kan ^R -λattR-Ptac71φ10-thrA442 cassette. Analysis was performed. As a result, P. ananatis SC17(0)ΔmetE1::λattL-kan ^R -λattR-Ptac71φ10-thrA442 strain was obtained.

2.2. Construction of P. ananatis strain C2707 (C2691ΔmetE1::λattL-kan ^R -λattR-Ptac71φ10-thrA442)
Chromosomal DNA was isolated from the SC17(0)ΔmetE1::λattL-kan ^R -λattR-Ptac71φ10-thrA442 strain using the EdgeBio PurElute Bacterial Genomic kit according to the manufacturer's instructions. The resulting chromosomal DNA was used to transform P. ananatis strain C2691 (preliminary example 1). For this purpose, P. ananatis strain C2691 was cultured overnight in LB liquid medium. After that, 1 mL of the culture solution was inoculated into 100 mL of LB liquid medium, and the cells were cultured at 32°C for 2 hours with shaking (250 rpm). Cells were collected and washed with 10% (v/v) glycerol three times to obtain competent cells. Chromosomal DNA isolated from the SC17(0)ΔmetE1::λattL-kan ^R -λattR-Ptac71φ10-thrA442 strain (Preliminary Example 2.1) was introduced into competent cells by electroporation. After culturing the cells in SOC medium for 2 hours,
It was plated on an LB plate containing mg/L kanamycin and cultured at 34°C for 16 hours. Appearing colonies were purified on the same medium. Next, to confirm the replacement of the metE1 gene on the chromosome,
PCR analysis was performed as described in Preparative Example 2.1. As a result, the P. ananatis C2691ΔmetE1::λattL-kan ^R -λattR-Ptac71φ10-thrA442 strain (abbreviation: C2707) was obtained.

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

The resulting plasmid-bearing strains were purified in LB medium containing tetracycline (15 mg/L) and 1 mM IPTG to obtain single colonies. Next, a single colony was plated on a medium containing 50 mg/L kanamycin and cultured overnight at 37°C with shaking (250 rpm). A kanamycin-sensitive strain was plated on LB medium containing 10% sucrose (weight ratio) 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 designated С2743.

2.4. Construction of P. ananatis SC17(0)λattL-kan ^R -λattR-Ptac71φ10-metH strain
The promoter region of the metH gene (SEQ ID NO: 44) is the cassette λattL-kan ^R -λattR-Ptac71
The φ10-substituted P. ananatis SC17(0)λattL-kan ^R -λattR-Ptac71φ10-metH strain was constructed by λRed-dependent integration. For this purpose, P. ananatis SC17(0)/RSF-Red-TER
The strain was cultured overnight in LB liquid medium. Thereafter, 1 mL of the culture solution 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). Cells were collected and washed with 10% (v/v) glycerol three times to obtain competent cells. Using primers P29 (SEQ ID NO: 45) and P30 (SEQ ID NO: 46), PCR was performed using the pMW118-attL-kan ^R -attR-Ptac71φ10 plasmid (SEQ ID NO: 32) as a template, and the upstream and downstream of the promoter region of the metH gene. An amplified DNA fragment of λattL-kan ^R -λattR-Ptac71φ10 having homologous sequences at both ends was obtained. The resulting DNA fragment was purified using the Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by electroporation. The cells were cultured in SOC medium for 2 hours, spread on an LB plate containing 35 mg/L kanamycin, and cultured at 34°C for 16 hours.
Appearing colonies were purified on the same medium. Next, to confirm that the promoter region of the metH gene on the chromosome was replaced with the λattL-kan ^R -λattR-Ptac71φ10 cassette,
PCR analysis (TaKaRa Speed Star (R); 25 cycles of 20 seconds at 97°C, 20 seconds at 54°C and 100 seconds at 72°C) 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.

2.5. Construction of P. ananatis strain C2761 (С2743 λattL-kan ^R -λattR-Ptac71φ10-metH)
Chromosomal DNA was isolated from the SC17(0)λattL-kan ^R -λattR-Ptac71φ10-metH strain (Preliminary Example 2.4) using the EdgeBio PurElute Bacterial Genomic kit according to the manufacturer's instructions. The chromosomal DNA obtained was used for transformation of P. ananatis strain С2743 (Preliminary Example 2.3).
For this purpose, strain P. ananatis С2743 was grown overnight in LB liquid medium. After that, 1 mL of the culture solution was inoculated into 100 mL of LB liquid medium, and the cells were cultured at 32°C for 2 hours with shaking (250 rpm). Cells were collected and washed with 10% (v/v) glycerol three times to obtain competent cells. Chromosomal DNA isolated from the SC17(0) λattL-kan ^R -attR-Ptac71φ10-metH strain was introduced into competent cells by electroporation. The cells were cultured in SOC medium for 2 hours, spread on an LB plate containing 35 mg/L kanamycin, and cultured at 34°C for 16 hours. Appearing colonies were purified on the same medium. Next, to confirm that the promoter region of the metH gene on the chromosome was replaced with the λattL-kan ^R -λattR-Ptac71φ10 cassette, PCR analysis was performed as described in Preliminary Example 2.4. . As a result, P. ananatis С2743λattL-kan ^R -λattR-Ptac71φ10-metH strain (abbreviation: C2761) was obtained.

2.6. Construction of P. ananatis SC17(0)λattL- ^catR -λattR-Pnlp8-gcvT strain
A P. ananatis strain SC17(0)λattL-cat ^R -λattR-Pnlp8-gcvT strain in which the promoter region of the gcvT gene (SEQ ID NO: 49) was replaced with the cassette λattL-cat ^R -λattR-Pnlp8 was constructed by λRed-dependent integration. did. For this purpose, the P. ananatis SC17(0)/RSF-Red-TER strain was cultured overnight in LB liquid medium. Thereafter, 1 mL of the culture solution 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). Cells were collected and washed with 10% (v/v) glycerol three times to obtain competent cells. Using primers P33 (SEQ ID NO: 50) and P34 (SEQ ID NO: 51), PCR was performed using the pMW118-attL-cat ^R -attR-Pnlp8 plasmid (WO2011043485) as a template to identify regions homologous to the upstream and downstream promoter regions of the gcvT gene. An amplified DNA fragment of λattL-cat ^R -λattR-Pnlp8 having sequences at both ends was obtained. The resulting DNA fragment was purified using the Wizard PCR Prep DNA Purification System (Promega) and introduced into competent cells by electroporation. The cells were cultured in SOC medium for 2 hours, spread on an LB plate containing 35 mg/L kanamycin, and cultured at 34°C for 16 hours. Appearing colonies were purified on the same medium. Next, primers P35 (SEQ ID NO: 52) and P36 (SEQ ID NO: 53) were used to confirm that the promoter region of the gcvT gene on the chromosome was replaced with the λattL-cat ^R -λattR-Pnlp8 cassette. PCR analysis (TaKaRa Speed Star (R); 40 cycles of 97°C 20 seconds, 54°C 20 seconds, 72°C 120 seconds) was performed. As a result, the SC17(0) λattL-cat ^R -λattR-Pnlp8-gcvT strain was obtained.

2.7. Construction of P. ananatis strain C2777 (C2761 λattL-cat ^R -λattR-Pnlp8-gcvT)
Chromosomal DNA was isolated from the SC17(0)λattL- ^catR -λattR-Pnlp8-gcvT strain (preparatory example 2.6) using the EdgeBio PurElute Bacterial Genomic kit according to the manufacturer's instructions. The resulting chromosomal DNA was used to transform P. ananatis strain C2761 (Preliminary Example 2.5). For this purpose, P. ananatis strain C2761 was cultured overnight in LB liquid medium. After that, 1 mL of the culture solution was inoculated into 100 mL of LB liquid medium, and the cells were cultured at 32°C for 2 hours with shaking (250 rpm). Cells were collected and washed with 10% (v/v) glycerol three times to obtain competent cells. Chromosomal DNA isolated from the SC17(0)λattL-cat ^R -λattR-Pnlp8-gcvT strain was introduced into competent cells by electroporation. After culturing the cells in SOC medium for 2 hours, LB containing 35 mg/L kanamycin
It was spread on a plate and cultured at 34°C for 16 hours. Appearing colonies were purified on the same medium. Next, to confirm that the promoter region of the gcvT gene on the chromosome was replaced with the λattL-cat ^R -λattR-Pnlp8 cassette, PCR analysis was performed as described in Preliminary Example 2.7. . As a result, the P. ananatis C2761λattL-cat ^R -λattR-Pnlp8-gcvT strain (abbreviation: C2777) was obtained.

2.8. Deletion of kan and cat genes from strain C2777 (C2761λattL-cat ^R -λattR-Pnlp8-gcvT)
Kanamycin and chloramphenicol resistance genes (corresponding to kan and cat) were deleted from strain C2777 (preliminary example 2.7) using the RSF(TcR)-int-xis plasmid. RSF(TcR)-int-xis was introduced into the C2777 strain by electroporation, and the cells were spread on LB medium containing tetracycline (15 mg/L) and cultured at 30°C to obtain C2777/RSF(TcR). -int-xis strain was obtained.

The resulting plasmid-bearing strains were purified in LB medium containing tetracycline (15 mg/L) and 1 mM IPTG to obtain single colonies. A 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). Strains sensitive to both kanamycin and chloramphenicol were plated on LB medium containing 10% sucrose (by weight) and 1 mM IPTG to eliminate the RSF(TcR)-int-xis plasmid from the strain. and incubated overnight at 37°C. A tetracycline-sensitive colony was selected and the corresponding strain designated C2792.

Although the present 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 adopted without departing from the scope of the invention.

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

Claims (15)

A method for producing an L-amino acid, comprising:
(i) culturing an L-amino acid-producing bacterium belonging to the order Enterobacterales in a medium to produce and accumulate an L-amino acid in the medium or in the cells of the bacterium, or both; and (ii) recovering an L-amino acid from said medium or said bacterial cells, or both;
The method, wherein the bacterium is modified to attenuate the expression of a gene encoding a protein having indolepyruvate decarboxylase activity. 2. The method of claim 1, wherein the gene encoding a protein having indole pyruvate decarboxylase activity is the ipdC gene. 3. The method of claim 1 or 2, wherein said protein having indole pyruvate 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 comprising substitutions, deletions, insertions, and/or additions of 1 to 250 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 or 55, wherein the protein exhibits indole pyruvate decarboxylase activity. and (C) a protein comprising an amino acid sequence having 50% or more identity to the entire amino acid sequence set forth in SEQ ID NO: 2 or 55, which protein has indole pyruvate decarboxylase activity. The method of any one of claims 1-3, 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 with a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 or 54, which encodes a protein having indole pyruvate decarboxylase activity; gene;
(c) a gene encoding a protein comprising an amino acid sequence containing substitutions, deletions, insertions, and/or additions of 1 to 250 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 or 55, wherein the protein is 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. ,gene. The method according to any one of claims 1 to 4, wherein the expression of the gene encoding the protein having indole pyruvate decarboxylase activity is attenuated by inactivation of the gene. 6. The method of claim 5, wherein the gene encoding a protein having indole pyruvate decarboxylase activity is deleted. The method according to any one of claims 1 to 6, wherein said bacterium is further modified to attenuate the expression of a gene encoding a protein having threonine deaminase activity. 8. The method according to claim 7, wherein the gene encoding a protein having threonine deaminase activity is the ilvA gene. the bacterium belongs to the family Enterobacteriaceae or Erwiniaceae
The method according to any one of claims 1 to 8, belonging to 10. The method of claim 9, wherein the bacterium belongs to the genus Escherichia or Pantoea. 11. The method of claim 10, wherein said bacterium is Escherichia coli or Pantoea ananatis. The method according to any one of claims 1 to 11, wherein said 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. 13. The method of claim 12, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tryptophan, and L-tyrosine. The non-aromatic L-amino acids are L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L- 13. The method of claim 12, wherein the isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-proline, L-serine, L-threonine, and L-valine. 13. The method of claim 12, wherein said sulfur-containing L-amino acid is selected from the group consisting of L-cysteine, L-methionine, L-homocysteine, and L-cystine.