ПРОДУЦИРУЮЩАЯ L-ГЛУТАМИНОВУЮ КИСЛОТУ БАКТЕРИЯ И СПОСОБ ПОЛУЧЕНИЯ L-ГЛУТАМИНОВОЙ КИСЛОТЫ

Abstract

Provided are a method for producing L-glutamic acid, and bacterium used therein. L-glutamic acid is produced by culturing, in a culture medium, a Corynebacterium having L-glutamic acid-producing ability, the Corynebacterium being modified so as to retain a mutant acetyl-CoA hydrolase gene that codes for a mutant acetyl-CoA hydrolase and having a substitution of a serine residue at position 383 in an amino acid sequence of a wild-type acetyl-CoA hydrolase to another amino acid residue, and collecting L-glutamic acid from the culture medium and / or a bacterial cell.

Description

L-glutamic acid producing bacteria and method for producing L-glutamic acid

[0001] The present invention relates to the fermentation industry, and more particularly to a method for producing L-glutamic acid and a bacterium used therein. L-glutamic acid is industrially useful as a raw material for seasonings, etc.

[0002] L-amino acids are industrially produced by fermentation using microorganisms, such as bacteria, capable of producing L-amino acids (Non-Patent Document 1). Examples of such microorganisms include strains isolated from nature and their mutants. Furthermore, the L-amino acid-producing ability of microorganisms can be improved by recombinant DNA technology.

[0003] Acetyl-CoA hydrolase is an enzyme that catalyzes the hydrolysis of acetyl-CoA to produce coenzyme A and acetic acid, and / or the reverse reaction (Non-Patent Document 2), and it is known that reducing the enzyme activity enhances the ability to produce L-glutamic acid, L-valine, and L-alanine (Patent Document 1). However, it was not known that specific mutations in the amino acid sequence of this enzyme contribute to the enhancement of L-glutamic acid production.

[0004] Japanese Patent Application Laid-Open No. 2006-149214

[0005] Kunihiko Akashi et al., Amino Acid Fermentation, Academic Press, pp. 195-215, 1986. KEGG (Kyoto Encyclopedia of Genes and Genomes) website: https: / / www.genome.jp / entry / 3.1.2.1

[0006] An objective of the present invention is to develop a novel technique for improving the L-glutamic acid-producing ability of bacteria, and to provide an efficient method for producing L-glutamic acid and bacteria for use therein.

[0007] As a result of intensive research conducted by the present inventors to solve the above-mentioned problems, they discovered that the L-glutamic acid-producing ability of coryneform bacteria can be improved by modifying the bacteria so that they have a specific mutation in acetyl-CoA hydrolase, and thus completed the present invention.

[0008] That is, the present invention can be exemplified as follows: [1] A coryneform bacterium having the ability to produce L-glutamic acid, which has been modified to harbor a mutant acetyl-CoA hydrolase gene encoding a mutant acetyl-CoA hydrolase having a substitution of the serine residue at position 383 in the amino acid sequence of wild-type acetyl-CoA hydrolase with another amino acid residue. [2] The coryneform bacterium according to [1], wherein the other amino acid is lysine, glutamic acid, threonine, aspartic acid, asparagine, glutamine, arginine, cysteine, histidine, or methionine. [3] The coryneform bacterium according to [1], wherein the other amino acid is cysteine. [4] The coryneform bacterium according to any of [1] to [3], wherein the wild-type acetyl-CoA hydrolase is a protein set forth in the following (a), (b), or (c): (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2; (b) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 with substitution, deletion, insertion and / or addition of 1 to 10 amino acid residues, and having acetyl-CoA hydrolase activity; (c) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2, and having acetyl-CoA hydrolase activity. [5] The coryneform bacterium according to any of [1] to [4], wherein the coryneform bacterium belongs to the genus Corynebacterium. [6] The coryneform bacterium according to any of [1] to [4], wherein the coryneform bacterium is Corynebacterium glutamicum. [7] The coryneform bacterium according to any of [1] to [6], which has been modified to retain a mutant yggB gene, and wherein the mutant yggB gene is a gene encoding a protein having an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 8. [8] The coryneform bacterium according to [7], wherein the mutant yggB gene is a gene encoding a protein having the amino acid sequence of SEQ ID NO: 10.[9] A method for producing L-glutamic acid, comprising culturing the coryneform bacterium according to any one of [1] to [8] in a medium, accumulating L-glutamic acid in the medium and / or within the cells of the bacterium, and collecting L-glutamic acid from the medium and / or the cells.

[10] A mutant acetyl-CoA hydrolase having a substitution of the serine residue at position 383 in the amino acid sequence of wild-type acetyl-CoA hydrolase with another amino acid residue.

[11] The mutant acetyl-CoA hydrolase according to

[10] , wherein the other amino acid is lysine, glutamic acid, threonine, aspartic acid, asparagine, glutamine, arginine, cysteine, histidine, or methionine.

[12] The mutant acetyl-CoA hydrolase according to

[10] , wherein the other amino acid is cysteine.

[13] The mutant acetyl-CoA hydrolase according to any of

[10] to

[12] , wherein the wild-type acetyl-CoA hydrolase is a protein described in the following (a), (b), or (c): (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (b) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, but with substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues, and having acetyl-CoA hydrolase activity; (c) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 2, and having acetyl-CoA hydrolase activity.

[14] A mutant acetyl-CoA hydrolase gene encoding the mutant acetyl-CoA hydrolase according to any of

[10] to

[13] .

[0009] 1 is a diagram showing the amount of L-glutamic acid accumulated in a wild-type acetyl-CoA hydrolase gene-introduced strain and a mutant acetyl-CoA hydrolase gene-introduced strain, and a diagram showing the sugar-based L-glutamic acid yield in a wild-type acetyl-CoA hydrolase gene-introduced strain and a mutant acetyl-CoA hydrolase gene-introduced strain.

[0010] The present invention will be described in detail below.

[0011] The method of the present invention is a method for producing L-glutamic acid, comprising culturing a coryneform bacterium capable of producing L-glutamic acid in a medium, accumulating L-glutamic acid in the medium and / or within the bacterial cells, and collecting L-glutamic acid from the medium and / or the bacterial cells, wherein the coryneform bacterium has been modified to carry a specific gene mutation. The bacterium used in this method is also referred to as the "bacterium of the present invention."

[0012] <1> Bacteria of the Present Invention The bacterium of the present invention is a coryneform bacterium having the ability to produce L-glutamic acid, which has been modified to have a specific gene mutation.

[0013] <1-1> Coryneform Bacteria Capable of Producing L-Glutamic Acid In the present invention, "Coryneform Bacteria Capable of Producing L-Glutamic Acid" refers to coryneform bacteria that, when cultured in a medium, produce L-glutamic acid and accumulate it in the medium and / or intracellularly to an extent that it can be recovered. Coryneform bacteria capable of producing L-glutamic acid may be coryneform bacteria that are capable of accumulating a greater amount of L-glutamic acid in the medium and / or intracellularly than unmodified strains. An "unmodified strain" refers to a control strain that has not been modified to retain a specific genetic mutation. Examples of unmodified strains include wild-type strains and parent strains. Furthermore, coryneform bacteria capable of producing L-glutamic acid may be coryneform bacteria that are capable of accumulating a target L-amino acid in a medium at a concentration of preferably 0.5 g / L or more, more preferably 1.0 g / L or more.

[0014] The bacterium of the present invention can produce L-glutamic acid alone or as a mixture of L-glutamic acid and one or more amino acids other than L-glutamic acid, such as L-amino acids (also referred to as L-amino acids). L-amino acids include, but are not limited to, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

[0015] In the present invention, the terms "glutamic acid" and "amino acid" mean L-glutamic acid and L-amino acid, respectively, unless otherwise specified. Furthermore, in the present invention, the terms "L-glutamic acid" and "L-amino acid" mean free L-glutamic acid, free L-amino acid, salts thereof, or mixtures thereof, unless otherwise specified. Salts will be described later.

[0016] Examples of coryneform bacteria include bacteria belonging to genera such as Corynebacterium, Brevibacterium, and Microbacterium.

[0017] Specific examples of coryneform bacteria include the following species: Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Corynebacterium crenatum Corynebacterium glutamicum Corynebacterium lilium Corynebacterium melassecola Corynebacterium thermoaminogenes (Corynebacterium efficiens) Corynebacterium herculis Brevibacterium divaricatum (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium glutamicum) Brevibacterium immariophilum Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalisCorynebacterium ammoniagenes (Corynebacterium stationis) Brevibacterium album Brevibacterium cerinum Microbacterium ammoniaphilum

[0018] Corynebacterium glutamicum (formerly known as Brevibacterium lactofermentum) is particularly an example of a coryneform bacterium.

[0019] Specific examples of coryneform bacteria include the following strains: Corynebacterium acetoacidophilum ATCC 13870, Corynebacterium acetoglutamicum ATCC 15806, Corynebacterium alkanolyticum ATCC 21511, Corynebacterium callunae ATCC 15991, and Corynebacterium crenatum AS1.542 Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734 Corynebacterium lilium ATCC 15990 Corynebacterium melassecola ATCC 17965 Corynebacterium efficiens (Corynebacterium thermoaminogenes) AJ12340 (FERM BP-1539) Corynebacterium herculis ATCC 13868 Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020 Brevibacterium flavum (Corynebacterium glutamicum) ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205) Brevibacterium immariophilum ATCC 14068 Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC 13869 Brevibacterium roseum ATCC 13825 Brevibacterium saccharolyticum ATCC 14066 Brevibacterium thiogenitalis ATCC 19240 Corynebacterium ammoniagenes (Corynebacterium stationis) ATCC 6871, ATCC 6872 Brevibacterium album ATCC 15111 Brevibacterium cerinum ATCC 15112 Microbacterium ammoniaphilum ATCC 15354.

[0020] A more particular example of a coryneform bacterium is Brevibacterium lactofermentum (new name: Corynebacterium glutamicum) ATCC 13869. Another example of a coryneform bacterium is the C. glutamicum 2256ΔsucAΔldhA yggB* strain, which is deficient in the ldhA and sucA genes of Corynebacterium glutamicum ATCC 13869 and has an IS mutation (V419::IS) in the yggB gene (WO2014 / 185430).

[0021] The genus Corynebacterium also includes bacteria that were previously classified as Brevibacterium but have now been integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255(1991)). Corynebacterium stationis also includes bacteria that were previously classified as Corynebacterium ammoniagenes but have been reclassified as Corynebacterium stationis based on 16S rRNA sequence analysis and other factors (Int. J. Syst. Evol. Microbiol., 60, 874-879(2010)).

[0022] These strains can be obtained, for example, from the American Type Culture Collection (address: 12301 Parklawn Drive, Rockville, Maryland 20852, PO Box 1549, Manassas, VA 20108, United States of America). Each strain is assigned a corresponding accession number, and can be obtained using this accession number (see http: / / www.atcc.org / ). The accession numbers corresponding to each strain are listed in the catalog of the American Type Culture Collection. These strains can also be obtained, for example, from the depository institution where they were deposited.

[0023] The bacterium of the present invention may be one that inherently has the ability to produce L-glutamic acid, or may be one that has been modified to retain the ability to produce L-glutamic acid. Bacteria capable of producing L-glutamic acid can be obtained, for example, by imparting the ability to produce L-glutamic acid to the above-mentioned bacteria or by enhancing the L-glutamic acid-producing ability of the above-mentioned bacteria.

[0024] L-glutamic acid-producing ability can be imparted or enhanced by methods conventionally employed in breeding amino acid-producing bacteria, such as Corynebacterium or Escherichia bacteria (see Amino Acid Fermentation, Academic Press, first published May 30, 1986, pp. 77-100). Examples of such methods include obtaining auxotrophic mutants, obtaining L-glutamic acid analog-resistant strains, obtaining metabolically controlled mutants, and creating recombinant strains with enhanced activity of L-glutamic acid biosynthetic enzymes. When breeding L-glutamic acid-producing bacteria, the properties imparted, such as auxotrophy, analog resistance, and metabolically controlled mutations, may be one, two, or three or more. Furthermore, when breeding L-glutamic acid-producing bacteria, the activity of the L-glutamic acid biosynthetic enzymes may be enhanced, either one, or two, or three or more. Furthermore, the conferring of properties such as auxotrophy, analogue resistance, and metabolic control mutation may be combined with the enhancement of biosynthetic enzyme activity.

[0025] Auxotrophic mutants, analog-resistant mutants, or metabolically regulated mutants capable of producing L-glutamic acid can be obtained by subjecting a parent strain or a wild-type strain to a conventional mutagen treatment and selecting from the resulting mutants those that exhibit auxotrophy, analog-resistant, or metabolically regulated mutations and also have the ability to produce L-glutamic acid. Conventional mutagen treatments include irradiation with X-rays or ultraviolet light, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0026] Alternatively, L-glutamic acid-producing ability can be imparted or enhanced by enhancing the activity of an enzyme involved in L-glutamic acid biosynthesis. Enzyme activity can be enhanced, for example, by modifying the bacterium so that expression of the gene encoding the enzyme is enhanced. Methods for enhancing gene expression are described in WO 00 / 18935, EP 1010755A, etc.

[0027] Alternatively, L-glutamic acid-producing ability can be imparted or enhanced by reducing the activity of an enzyme that catalyzes a reaction that branches off from the L-glutamic acid biosynthetic pathway to produce a compound other than L-glutamic acid. Note that the term "enzyme that catalyzes a reaction that branches off from the L-glutamic acid biosynthetic pathway to produce a compound other than L-glutamic acid" as used herein also includes enzymes involved in the degradation of L-glutamic acid.

[0028] Specific examples of L-glutamic acid-producing bacteria and methods for imparting or enhancing L-glutamic acid-producing ability are described below. The properties of L-glutamic acid-producing bacteria and the modifications for imparting or enhancing L-glutamic acid-producing ability as exemplified below may be used alone or in appropriate combination.

[0029] Methods for imparting or enhancing L-glutamic acid-producing ability include, for example, modifying bacteria to enhance the activity of one or more enzymes selected from L-glutamic acid biosynthesis enzymes, including, but not limited to, glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthase (gltBD), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), methylcitrate synthase (prpC), pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglycerin (pgmA, Examples of such enzymes include glutamate dehydrogenase (pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triosephosphate isomerase (tpiA), fructose-bisphosphate aldolase (fbp), glucosephosphate isomerase (pgi), 6-phosphogluconate dehydratase (edd), 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), and transhydrogenase (pntAB). The genes encoding these enzymes are shown in parentheses (the same applies to the following descriptions). Among these enzymes, it is preferable to enhance the activity of one or more enzymes selected from glutamate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, and methylcitrate synthase.

[0030] Examples of coryneform bacteria modified to increase expression of the glutamate synthase gene (gltBD) include those disclosed in WO99 / 07853.

[0031] Another method for imparting or enhancing L-glutamic acid-producing ability is to modify a bacterium so that the activity of one or more enzymes selected from enzymes that catalyze reactions that branch off from the L-glutamic acid biosynthetic pathway to produce compounds other than L-glutamic acid is reduced. Such enzymes include, but are not limited to, isocitrate lyase (aceA), α-ketoglutarate dehydrogenase (sucA, odhA), acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), alcohol dehydrogenase (adh), glutamate decarboxylase (gadAB), and succinate dehydrogenase (sdhABCD). Among these enzymes, reducing or eliminating α-ketoglutarate dehydrogenase activity is preferred.

[0032] Coryneform bacteria with reduced or no α-ketoglutarate dehydrogenase activity and methods for obtaining them are described in WO 2008 / 075483. Specific examples of coryneform bacteria with reduced or no α-ketoglutarate dehydrogenase activity include the following strains: Corynebacterium glutamicum (Brevibacterium lactofermentum) L30-2 strain (JP 2006-340603 A) Corynebacterium glutamicum (Brevibacterium lactofermentum) ΔS strain (WO95 / 34672) Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ12821 (FERM BP-4172; French Patent No. 9401748) Corynebacterium glutamicum (Brevibacterium flavum) AJ12822 (FERM BP-4173; French Patent No. 9401748) Corynebacterium glutamicum AJ12823 (FERM BP-4174; French Patent No. 9401748)

[0033] L-glutamic acid-producing bacteria or parent strains for deriving them also include strains in which both α-ketoglutarate dehydrogenase (sucA) activity and succinate dehydrogenase (sdh) activity are reduced or deleted (JP 2010-041920 A). Specific examples of such strains include the odhAsdhA double-deficient strain of Corynebacterium glutamicum ATCC14067 (Corynebacterium glutamicum 8L3GΔSDH strain) (JP 2010-041920 A).

[0034] Furthermore, methods for imparting or enhancing L-glutamic acid-producing ability to coryneform bacteria include methods for imparting resistance to organic acid analogs or respiratory inhibitors, and methods for imparting sensitivity to cell wall synthesis inhibitors. Specific examples of such methods include a method of imparting monofluoroacetic acid resistance (Japanese Patent Laid-Open No. 50-113209), a method of imparting adenine resistance or thymine resistance (Japanese Patent Laid-Open No. 57-065198), a method of weakening urease (Japanese Patent Laid-Open No. 52-038088), a method of imparting malonic acid resistance (Japanese Patent Laid-Open No. 52-038088), a method of imparting resistance to benzopyrones or naphthoquinones (Japanese Patent Laid-Open No. 56-1889), a method of imparting HOQNO resistance (Japanese Patent Laid-Open No. 56-140895), a method of imparting α-ketomalonic acid resistance (Japanese Patent Laid-Open No. 57-2689), a method of imparting guanidine resistance (Japanese Patent Laid-Open No. 56-35981), and a method of imparting sensitivity to penicillin (Japanese Patent Laid-Open No. 4-88994).

[0035] Specific examples of such resistant or sensitive bacteria include the following strains: Corynebacterium glutamicum (Brevibacterium flavum) AJ3949 (FERM BP-2632; JP-A-50-113209) Corynebacterium glutamicum AJ11628 (FERM P-5736; JP-A-57-065198) Corynebacterium glutamicum (Brevibacterium flavum) AJ11355 (FERM P-5007; Japanese Patent Application Publication No. 1889-1989) Corynebacterium glutamicum AJ11368 (FERM P-5020; Japanese Patent Application Publication No. 1889-1989) Corynebacterium glutamicum (Brevibacterium flavum) AJ11217 (FERM P-4318; JP-A-57-2689) Corynebacterium glutamicum AJ11218 (FERM P-4319; JP 57-2689 A) Corynebacterium glutamicum (Brevibacterium flavum) AJ11564 (FERM P-5472; JP 56-140895 A) Corynebacterium glutamicum (Brevibacterium flavum) AJ11439 (FERM P-5136; Japanese Patent Application Laid-Open No. 56-35981) Corynebacterium glutamicum H7684 (FERM BP-3004; Japanese Patent Application Publication No. 04-88994) Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ11426 (FERM P-5123; Japanese Patent Application Publication No. 56-048890) Corynebacterium glutamicum AJ11440 (FERM P-5137; Japanese Patent Application Publication No. 56-048890) Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ11796 (FERM P-6402; Japanese Patent Application Publication No. 58-158192)

[0036] Methods for imparting or enhancing L-glutamic acid-producing ability include, for example, modifying bacteria so that they have increased activity to secrete L-glutamic acid from bacterial cells. The activity to secrete L-glutamic acid can be increased, for example, by increasing the expression of a gene encoding a protein that secretes L-glutamic acid. Examples of genes encoding proteins that secrete various amino acids include the b2682 gene (ygaZ), the b2683 gene (ygaH), the b1242 gene (ychE), and the b3434 gene (yhgN) (Japanese Patent Laid-Open Publication No. 2002-300874).

[0037] Methods for imparting or enhancing L-glutamic acid-producing ability include, for example, modifying bacteria so that the activity of proteins involved in sugar metabolism or energy metabolism is increased.

[0038] Proteins involved in carbohydrate metabolism include proteins involved in carbohydrate uptake and glycolytic enzymes. Genes encoding proteins involved in carbohydrate metabolism include the glucose 6-phosphate isomerase gene (pgi; WO01 / 02542), pyruvate carboxylase gene (pyc; WO99 / 18228, EP1092776A), phosphoglucomutase gene (pgm; WO03 / 04598), fructose bisphosphate aldolase genes (pfkB, fbp; WO03 / 04664), transaldolase gene (talB; WO03 / 008611), fumarase gene (fum; WO01 / 02545), non-PTS sucrose uptake gene (csc; EP1149911A), and sucrose utilization genes (scrAB operon; U.S. Patent No. 7,179,623).

[0039] Examples of genes encoding proteins involved in energy metabolism include the transhydrogenase gene (pntAB; US Pat. No. 5,830,716) and the cytochrome bo type oxidase gene (cyoB; EP1,070,376A).

[0040] Furthermore, methods for imparting or enhancing L-glutamic acid-producing ability to coryneform bacteria include methods for enhancing expression of the yggB gene and methods for introducing a mutant yggB gene with a mutation introduced into the coding region (WO 2006 / 070944). That is, the bacterium of the present invention may be modified to enhance expression of the yggB gene, or may be modified to retain (have) a mutant yggB gene.

[0041] The yggB gene encodes a mechanosensitive channel. Examples of the yggB gene include those of coryneform bacteria. Specific examples of the yggB gene of coryneform bacteria include the yggB genes of Corynebacterium glutamicum ATCC13869, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14967, and Corynebacterium melassecola ATCC17965 (WO2006 / 070944). The yggB genes of Corynebacterium glutamicum ATCC13032 and Corynebacterium callunae ATCC 15991 correspond to the complementary sequence of positions 1,336,091 to 1,337,692 in the genome sequence registered in the NCBI database under GenBank Accession No. NC_003450, and are also referred to as NCgl1221. The YggB protein encoded by the yggB gene of Corynebacterium glutamicum ATCC13032 is registered under GenBank Accession No. NP_600492. The nucleotide sequence of the yggB gene of Corynebacterium glutamicum 2256 (ATCC 13869) and the amino acid sequence of the YggB protein encoded by this gene are shown in SEQ ID NOs: 7 and 8, respectively.

[0042] In the present invention, a yggB gene having a "specific mutation" described below is also referred to as a mutant yggB gene, and the protein encoded thereby is also referred to as a mutant YggB protein. In the present invention, a yggB gene not having a "specific mutation" described below is also referred to as a wild-type yggB gene, and the protein encoded thereby is also referred to as a wild-type YggB protein. With regard to the YggB protein, a change in amino acid sequence caused by a "specific mutation" in the yggB gene is also referred to as a "specific mutation." The term "wild-type" used here is a convenient description to distinguish it from a "mutant" and is not limited to naturally occurring proteins as long as they do not have the "specific mutation." Examples of wild-type YggB proteins include the YggB proteins exemplified above, such as a protein having the amino acid sequence shown in SEQ ID NO: 8. Examples of wild-type YggB proteins also include conservative variants (variants that maintain the original function) of the above-exemplified YggB proteins that do not have the "specific mutation." The "original function" of the YggB protein may be, for example, its function as a mechanosensitive channel, or its property of improving the L-glutamic acid-producing ability of coryneform bacteria when its expression is increased in the coryneform bacterium.

[0043] The "specific mutation" is not particularly limited, as long as it is a mutation that alters the amino acid sequence of the wild-type YggB protein as described above and improves the L-glutamic acid-producing ability of the coryneform bacterium. Examples of the "specific mutation" include a C-terminal mutation and a mutation in the transmembrane domain (WO2006 / 070944). The "specific mutation" may also be a combination of these mutations.

[0044] (1) C-Terminal Mutation A C-terminal mutation is a mutation in the region of the wild-type yggB gene that encodes amino acid residues 419 to 533 of the wild-type YggB protein. The C-terminal mutation may be introduced at one or more sites in this region. The type of change in the amino acid sequence caused by the C-terminal mutation is not particularly limited. The C-terminal mutation may be, for example, a substitution of an amino acid residue (missense mutation), an insertion of an amino acid residue, a deletion of an amino acid residue, the appearance of a stop codon (nonsense mutation), a frameshift mutation, or a combination thereof. Preferred C-terminal mutations include, for example, insertion of a base sequence such as an insertion sequence (hereinafter also referred to as "IS") or a transposon.

[0045] (1-1) Insertion of Nucleotide Sequence. Examples of C-terminal mutations include a mutation (2A-1 type mutation) in which a nucleotide sequence is inserted into the region encoding the valine residue at position 419 of the wild-type YggB protein. The 2A-1 type mutation may result in the partial or complete deletion or substitution of amino acid residues 419 to 533 of the wild-type YggB protein. A specific example of a mutant yggB gene having a 2A-1 type mutation is a yggB gene in which an IS is inserted after the "G" at position 1255 of SEQ ID NO: 7, thereby encoding a mutant YggB protein with a total length of 423 amino acid residues, shorter than the original wild-type YggB protein (SEQ ID NO: 8). The nucleotide sequence of this mutant yggB gene (V419::IS) and the amino acid sequence of the mutant YggB protein (V419::IS) encoded by this gene are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. In SEQ ID NO: 9, positions 1 to 1269 represent the CDS of the mutant YggB protein (V419::IS). Specific examples of L-glutamic acid-producing bacteria having the mutant yggB gene (V419::IS) include C. glutamicum 2256ΔsucAΔldhA yggB * strain (WO2014 / 185430).

[0046] (1-2) Substitution of Proline Residues C-terminal mutations include, for example, substitution of the proline residues at positions 419 to 533 of the wild-type YggB protein with other amino acids. Examples of such proline residues include those at positions 424, 437, 453, 457, 462, 469, 484, 489, 497, 515, 529, and 533 of the wild-type YggB protein. Substitution of the proline residues at positions 424 and / or 437 with other amino acids is particularly preferred. The "other amino acids" are not particularly limited as long as they are naturally occurring amino acids other than proline. Examples of "other amino acids" include Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, and His. For example, the proline residue at position 424 may be substituted with a hydrophobic amino acid (Ala, Gly, Val, Leu, or Ile), more preferably with a branched-chain amino acid (Leu, Val, or Ile).Also, for example, the proline residue at position 437 may be substituted with an amino acid having a hydroxyl group in the side chain (Thr, Ser, or Tyr), more preferably with Ser.

[0047] (2) Mutations in the Transmembrane Region The YggB protein is presumed to have five transmembrane regions. These transmembrane regions correspond to amino acid residues at positions 1 to 23 (first transmembrane region), 25 to 47 (second transmembrane region), 62 to 84 (third transmembrane region), 86 to 108 (fourth transmembrane region), and 110 to 132 (fifth transmembrane region) of the wild-type YggB protein. The transmembrane region mutations are mutations in the region encoding these transmembrane regions in the wild-type yggB gene. The transmembrane region mutations may be introduced at one or more sites within the region. The transmembrane region mutations preferably result in the substitution, deletion, addition, insertion, or inversion of one or several amino acids, and are free of frameshift and nonsense mutations. "One or several" preferably refers to 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3. Mutations in the transmembrane region include mutations that insert one or several amino acids (e.g., Cys-Ser-Leu) between the leucine residue at position 14 and the tryptophan residue at position 15 of the wild-type YggB protein, mutations that substitute the alanine residue at position 100 with another amino acid residue (e.g., an amino acid having a hydroxyl group in its side chain (Thr, Ser, or Tyr), preferably Thr), and mutations that substitute the alanine residue at position 111 with another amino acid residue (e.g., Val or an amino acid having a hydroxyl group in its side chain (Thr, Ser, or Tyr), preferably Val or Thr).

[0048] In the present invention, unless otherwise specified, the "amino acid residue at position X of the wild-type YggB protein" refers to the amino acid residue corresponding to the amino acid residue at position X in SEQ ID NO: 8. "Position X" in an amino acid sequence refers to the Xth position counting from the N-terminus of the amino acid sequence, with the N-terminal amino acid residue being the first amino acid residue. Note that the position of an amino acid residue indicates a relative position, and its absolute position may change due to amino acid deletion, insertion, addition, or the like. For example, "amino acid residue at position 419 of the wild-type YggB protein" refers to the amino acid residue corresponding to the amino acid residue at position 419 in SEQ ID NO: 8. If one amino acid residue N-terminal to position 419 has been deleted, the amino acid residue 418 from the N-terminus is considered to be the "amino acid residue at position 419 of the wild-type YggB protein." Furthermore, if one amino acid residue N-terminal to position 419 has been inserted, the amino acid residue 420 from the N-terminus is considered to be the "amino acid residue at position 419 of the wild-type YggB protein." Specifically, for example, in the YggB protein of Corynebacterium glutamicum ATCC14967 strain, the amino acid residues at positions 419 to 529 correspond to the amino acid residues at positions 419 to 533 in the wild-type YggB protein. Also, for example, the alanine residue at position 98 in the YggB protein of Corynebacterium callunae corresponds to the alanine residue at position 100 in the wild-type YggB protein.

[0049] In the amino acid sequence of any YggB protein, the amino acid residue corresponding to the amino acid residue at position X in SEQ ID NO: 8 can be determined by aligning the amino acid sequence of the YggB protein with the amino acid sequence of SEQ ID NO: 8. Alignment can be performed using, for example, known genetic analysis software. Specific examples of such software include DNASIS manufactured by Hitachi Solutions and GENETYX manufactured by Genetyx (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24(1), 72-96, 1991; Barton GJ et al., Journal of molecular biology, 198(2), 327-37, 1987).

[0050] A mutant yggB gene can be obtained by modifying a wild-type yggB gene to have the above-mentioned "specific mutation." DNA modification can be performed by known techniques. Specific examples of site-specific mutagenesis, which introduces a desired mutation into a target site in DNA, include PCR-based methods (Higuchi, R., 61, in PCR Technology, Erlich, H.A. Eds., Stockton Press (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)) and phage-based methods (Kramer, W. and Frits, H.J., Meth. In Enzymol., 154, 350 (1987); Kunkel, T.A. et al., Meth. In Enzymol., 154, 367 (1987)). A mutant yggB gene can also be obtained by chemical synthesis.

[0051] Modification of coryneform bacteria to have a mutant yggB gene can be achieved by introducing the mutant yggB gene into the coryneform bacterium, or by introducing a mutation into the yggB gene of the bacterium by natural mutation or mutagen treatment.

[0052] <1-2> Modification of the Mutant Acetyl-CoA Hydrolase Gene The coryneform bacterium of the present invention has been modified to harbor a mutant acetyl-CoA hydrolase gene encoding a mutant acetyl-CoA hydrolase in which the serine residue at position 383 in the amino acid sequence of wild-type acetyl-CoA hydrolase has been substituted with another amino acid residue. The coryneform bacterium of the present invention may be a coryneform bacterium having L-glutamic acid-producing ability that has been modified to harbor the mutant acetyl-CoA hydrolase gene, as described above, or a coryneform bacterium obtained by imparting L-glutamic acid-producing ability to a coryneform bacterium that has been modified to harbor a mutant acetyl-CoA hydrolase gene. Coryneform bacteria that have been modified to harbor a mutant acetyl-CoA hydrolase gene and thereby become capable of producing L-glutamic acid are also included.

[0053] The acetyl-CoA hydrolase gene is a gene that encodes acetyl-CoA hydrolase. Examples of acetyl-CoA hydrolase genes include those of coryneform bacteria. Specific examples of acetyl-CoA hydrolase genes of coryneform bacteria include the acetyl-CoA hydrolase genes of Corynebacterium glutamicum ATCC13869, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14967, and Corynebacterium melassecola ATCC17965. The acetyl-CoA hydrolase gene of Corynebacterium glutamicum ATCC13032 corresponds to the complementary sequence of positions 2,729,376 to 2,730,884 in the genome sequence registered in the NCBI database under GenBank Accession No. NC_003450, and is also referred to as NCgl2480. The acetyl-CoA hydrolase encoded by the acetyl-CoA hydrolase gene of Corynebacterium glutamicum ATCC 13032 has been registered as GenBank accession No. WP_003858947.1. The nucleotide sequence of the wild-type acetyl-CoA hydrolase gene of Corynebacterium glutamicum 2256 (ATCC 13869) and the amino acid sequence of the acetyl-CoA hydrolase encoded by the gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0054] "Acetyl-CoA hydrolase" may refer to a protein having the activity of catalyzing the reaction of hydrolyzing acetyl-CoA to produce coenzyme A and acetic acid and / or the reverse reaction (e.g., EC 3.1.2.1). This activity is also referred to as "acetyl-CoA hydrolase activity." Specifically, acetyl-CoA hydrolase activity may be the activity of catalyzing the reaction of converting acetyl-CoA and HO to coenzyme A and acetic acid and / or the reverse reaction. Acetyl-CoA hydrolase is also referred to as "acetyl-CoA deacylase." An example of a gene encoding acetyl-CoA hydrolase is the NCgl2480 gene. The nucleotide sequences of acetyl-CoA hydrolase genes, such as the NCgl2480 gene, contained in the bacterium to be modified and the amino acid sequences of the acetyl-CoA hydrolases encoded thereby can be obtained from public databases such as NCBI.

[0055] In the present invention, an acetyl-CoA hydrolase gene having a "specific genetic mutation" described below is also referred to as a mutant acetyl-CoA hydrolase gene, and a protein encoded thereby is also referred to as a mutant acetyl-CoA hydrolase. In the present invention, an acetyl-CoA hydrolase gene not having a "specific genetic mutation" described below is also referred to as a wild-type acetyl-CoA hydrolase gene, and a protein encoded thereby is also referred to as a wild-type acetyl-CoA hydrolase. In the case of acetyl-CoA hydrolase, a change in amino acid sequence caused by a "specific genetic mutation" in the acetyl-CoA hydrolase gene is also referred to as a "specific genetic mutation." The term "wild-type" used here is a convenient term to distinguish it from a "mutant" and is not limited to naturally occurring acetyl-CoA hydrolases as long as they do not have the "specific genetic mutation." Examples of wild-type acetyl-CoA hydrolases include the acetyl-CoA hydrolases exemplified above, such as a protein having the amino acid sequence shown in SEQ ID NO: 2. Examples of wild-type acetyl-CoA hydrolases also include conservative variants (variants that maintain the original function) of the acetyl-CoA hydrolases exemplified above that do not have the "specific genetic mutation."

[0056] The "specific gene mutation" is a mutation that alters the amino acid sequence of the wild-type acetyl-CoA hydrolase described above and improves the L-glutamic acid-producing ability of the coryneform bacterium. Specifically, the mutation is at amino acid residue 383 in the amino acid sequence of the wild-type acetyl-CoA hydrolase in the wild-type acetyl-CoA hydrolase gene.

[0057] Examples of mutations at the amino acid residue at position 383 of the amino acid sequence of wild-type acetyl-CoA hydrolase include substitutions of the amino acid residue (missense mutations).

[0058] Mutations at the amino acid residue at position 383 of wild-type acetyl-CoA hydrolase include, for example, substitution of the serine residue at position 383 of wild-type acetyl-CoA hydrolase with another amino acid residue. The "other amino acid" is not particularly limited as long as it is a naturally occurring amino acid other than serine. Examples of the "other amino acid" include Lys, Glu, Thr, Gly, Val, Leu, Ile, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Pro, Ala, and His. For example, the serine residue at position 383 may be substituted with a hydrophilic amino acid (Lys, Glu, Thr, Asp, Asn, Gln, Arg, Cys, or His), preferably with Cys. Furthermore, for example, the serine residue at position 383 may be substituted with a sulfur-containing amino acid (Cys or Met), preferably with Cys.

[0059] In the present invention, unless otherwise specified, the "amino acid residue at position X in wild-type acetyl-CoA hydrolase" refers to the amino acid residue corresponding to the amino acid residue at position X in SEQ ID NO: 2. The "position X" in an amino acid sequence refers to the Xth position counting from the N-terminus of the amino acid sequence, with the N-terminal amino acid residue being the first amino acid residue. The position of an amino acid residue indicates a relative position, and its absolute position may change due to amino acid deletion, insertion, addition, or the like. For example, the "amino acid residue at position 383 in wild-type acetyl-CoA hydrolase" refers to the amino acid residue corresponding to the amino acid residue at position 383 in SEQ ID NO: 2. When one amino acid residue N-terminal to position 383 is deleted, the amino acid residue at position 382 from the N-terminus is considered to be the "amino acid residue at position 383 in wild-type acetyl-CoA hydrolase." Furthermore, when one amino acid residue N-terminal to position 383 is inserted, the amino acid residue at position 384 from the N-terminus is considered to be the "amino acid residue at position 383 in wild-type acetyl-CoA hydrolase."

[0060] In the amino acid sequence of any acetyl-CoA hydrolase, which amino acid residue corresponds to the "amino acid residue at position X in SEQ ID NO: 2" can be determined by aligning the amino acid sequence of the acetyl-CoA hydrolase with the amino acid sequence of SEQ ID NO: 2. Alignment can be performed using, for example, known genetic analysis software. Specific examples of such software include DNASIS manufactured by Hitachi Solutions and GENETYX manufactured by Genetyx (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24(1), 72-96, 1991; Barton GJ et al., Journal of molecular biology, 198(2), 327-37, 1987).

[0061] A mutant acetyl-CoA hydrolase gene can be obtained by modifying a wild-type acetyl-CoA hydrolase gene to have the above-mentioned "specific gene mutation." DNA modification can be performed by known techniques. Specific examples of site-specific mutagenesis, which introduces a desired mutation into a target site in DNA, include PCR-based methods (Higuchi, R., 61, in PCR technology, Erlich, H.A. Eds., Stockton Press (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)) and phage-based methods (Kramer, W. and Frits, H.J., Meth. In Enzymol., 154, 350 (1987); Kunkel, T.A. et al., Meth. In Enzymol., 154, 367 (1987)). Alternatively, a mutant acetyl-CoA hydrolase gene can be obtained by chemical synthesis.

[0062] The wild-type acetyl-CoA hydrolase gene may be, for example, a gene having the nucleotide sequence of the acetyl-CoA hydrolase gene exemplified above (e.g., the nucleotide sequence shown in SEQ ID NO: 1). Furthermore, the acetyl-CoA hydrolase may be, for example, a protein having the amino acid sequence of the acetyl-CoA hydrolase exemplified above (e.g., the amino acid sequence shown in SEQ ID NO: 2). Unless otherwise specified, the expression "having an (amino acid or nucleotide) sequence" means "including the (amino acid or nucleotide) sequence" and also encompasses the case where the protein "consists of the (amino acid or nucleotide) sequence."

[0063] The wild-type acetyl-CoA hydrolase gene may also be a variant of the above-exemplified acetyl-CoA hydrolase gene (e.g., a gene having the nucleotide sequence shown in SEQ ID NO: 1), so long as the original function is maintained. Similarly, the acetyl-CoA hydrolase may be a variant of the above-exemplified acetyl-CoA hydrolase (e.g., a protein having the amino acid sequence shown in SEQ ID NO: 2), so long as the original function is maintained. Such variants that maintain the original function may also be referred to as "conservative variants." The term "acetyl-CoA hydrolase gene" encompasses the above-exemplified acetyl-CoA hydrolase genes as well as their conservative variants. Similarly, the term "acetyl-CoA hydrolase" encompasses the above-exemplified acetyl-CoA hydrolases as well as their conservative variants. Examples of conservative variants include homologs and artificially modified forms of the above-exemplified acetyl-CoA hydrolase genes and acetyl-CoA hydrolases.

[0064] "Maintaining the original function" means that a variant of a gene or protein has a function (e.g., activity or property) corresponding to the function (e.g., activity or property) of the original gene or protein. "Maintaining the original function" with respect to a gene means that a variant of the gene encodes a protein that maintains the original function. In other words, "maintaining the original function" with respect to an acetyl-CoA hydrolase gene may mean that a variant of the acetyl-CoA hydrolase gene encodes a protein that has acetyl-CoA hydrolase activity. Furthermore, "maintaining the original function" with respect to an acetyl-CoA hydrolase may mean that a variant of the acetyl-CoA hydrolase has acetyl-CoA hydrolase activity.

[0065] Acetyl-CoA hydrolase activity can be measured, for example, by incubating the enzyme with the corresponding substrate (e.g., acetyl-CoA and HO) and measuring the enzyme- and substrate-dependent production of the corresponding product (e.g., coenzyme A and acetate).

[0066] Examples of conservative variants are shown below.

[0067] Homologues of the acetyl-CoA hydrolase gene or acetyl-CoA hydrolase can be easily obtained from public databases, for example, by BLAST or FASTA search using the nucleotide sequences of the acetyl-CoA hydrolase genes exemplified above or the amino acid sequences of the acetyl-CoA hydrolases exemplified above as query sequences. Homologues of the acetyl-CoA hydrolase gene can also be obtained, for example, by PCR using the chromosomes of various organisms as templates and oligonucleotides prepared based on the nucleotide sequences of these known acetyl-CoA hydrolase genes as primers.

[0068] The acetyl-CoA hydrolase gene may be a gene encoding an acetyl-CoA hydrolase having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added at one or several positions in the above amino acid sequence (e.g., the amino acid sequence shown in SEQ ID NO: 2), so long as the original function is maintained. For example, the encoded protein may be extended or shortened at its N-terminus and / or C-terminus. Note that the term "one or several" varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but specifically means, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0069] The above-mentioned substitution, deletion, insertion, and / or addition of one or several amino acids is a conservative mutation that maintains normal protein function. A typical conservative mutation is a conservative substitution. A conservative substitution is a mutation in which Phe, Trp, and Tyr are substituted with each other when the substitution site is an aromatic amino acid; Leu, Ile, and Val are substituted with each other when the substitution site is a hydrophobic amino acid; Gln and Asn are substituted with each other when the substitution site is a polar amino acid; Lys, Arg, and His are substituted with each other when the substitution site is a basic amino acid; Asp and Glu are substituted with each other when the substitution site is an acidic amino acid; and Ser and Thr are substituted with each other when the substitution site is an amino acid having a hydroxyl group. Specific examples of substitutions that are considered to be conservative substitutions include substitutions of Ala with Ser or Thr, substitutions of Arg with Gln, His, or Lys, substitutions of Asn with Glu, Gln, Lys, His, or Asp, substitutions of Asp with Asn, Glu, or Gln, substitutions of Cys with Ser or Ala, substitutions of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitutions of Glu with Gly, Asn, Gln, Lys, or Asp, substitutions of Gly with Pro, substitutions of His with Asn, Lys, Gln, Arg, or Tyr, substitutions of Ile substitution of Lys with Leu, Met, Val, or Phe, substitution of Leu with Ile, Met, Val, or Phe, substitution of Lys with Asn, Glu, Gln, His, or Arg, substitution of Met with Ile, Leu, Val, or Phe, substitution of Phe with Trp, Tyr, Met, Ile, or Leu, substitution of Ser with Thr or Ala, substitution of Thr with Ser or Ala, substitution of Trp with Phe or Tyr, substitution of Tyr with His, Phe, or Trp, and substitution of Val with Met, Ile, or Leu. The above-mentioned amino acid substitutions, deletions, insertions, or additions also include those resulting from naturally occurring mutations (mutants or variants) based on individual differences or differences in species of the organism from which the gene is derived.

[0070] Furthermore, the acetyl-CoA hydrolase gene may be a gene encoding a protein having an amino acid sequence that is, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identical to the entire amino acid sequence described above, so long as the original function is maintained.

[0071] Furthermore, the acetyl-CoA hydrolase gene may be a gene (e.g., DNA) that hybridizes under stringent conditions with a probe that can be prepared from the above-mentioned nucleotide sequence (e.g., the nucleotide sequence shown in SEQ ID NO: 1), such as a sequence complementary to all or part of the above-mentioned nucleotide sequence, so long as the original function is maintained. "Stringent conditions" refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. One example of such conditions is a condition under which DNAs with high identity, for example, DNAs with an identity of 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more, hybridize with each other, while DNAs with lower identity do not hybridize with each other; or a condition in which washing is performed once, preferably two to three times, at a salt concentration and temperature equivalent to the washing conditions for conventional Southern hybridization, namely, 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0072] As mentioned above, the probe used in the hybridization may be a portion of the complementary sequence of the gene. Such a probe can be prepared by PCR using oligonucleotides prepared based on a known gene sequence as primers and a DNA fragment containing the gene as a template. For example, a DNA fragment of about 300 bp in length can be used as the probe. When a DNA fragment of about 300 bp in length is used as the probe, washing conditions for the hybridization include 50°C, 2×SSC, and 0.1% SDS.

[0073] Furthermore, since codon degeneracy differs depending on the host, the acetyl-CoA hydrolase gene may be one in which any codon is substituted with an equivalent codon. That is, the acetyl-CoA hydrolase gene may be a variant of the acetyl-CoA hydrolase gene exemplified above due to the degeneracy of the genetic code. For example, the acetyl-CoA hydrolase gene may be modified to have an optimal codon depending on the codon usage frequency of the host used.

[0074] The "identity" between amino acid sequences refers to the identity between amino acid sequences calculated by blastp using default scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). The "identity" between nucleotide sequences refers to the identity between nucleotide sequences calculated by blastn using default scoring parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear).

[0075] The above descriptions regarding conservative variants of genes and proteins can also be applied mutatis mutandis to any proteins and the genes encoding them. That is, the genes and proteins used to breed L-glutamic acid-producing bacteria may have the nucleotide sequence and amino acid sequence of known genes and proteins, such as the genes and proteins exemplified above. Furthermore, the genes and proteins used to breed L-glutamic acid-producing bacteria may be conservative variants of known genes and proteins, such as the genes and proteins exemplified above. Specifically, for example, the genes used to breed L-glutamic acid-producing bacteria may be genes encoding proteins having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added at one or several positions in the amino acid sequence of a known protein, as long as the original function is maintained.

[0076] Methods for modifying a coryneform bacterium to retain the mutant acetyl-CoA hydrolase gene include a method for introducing a mutant acetyl-CoA hydrolase gene into the coryneform bacterium, in which a mutation that substitutes the serine residue at position 383 has been introduced into the coding region of the wild-type acetyl-CoA hydrolase gene, and a method for introducing the same mutation into the coding region of the wild-type acetyl-CoA hydrolase gene possessed by the coryneform bacterium.

[0077] Modification of a coryneform bacterium to have a mutant acetyl-CoA hydrolase gene can be achieved by introducing the mutant acetyl-CoA hydrolase gene into the coryneform bacterium. Modification of a coryneform bacterium to have a mutant acetyl-CoA hydrolase gene can also be achieved by introducing a mutation into the acetyl-CoA hydrolase gene of the coryneform bacterium by natural mutation or mutagen treatment.

[0078] Introduction of a mutant acetyl-CoA hydrolase gene into a coryneform bacterium can be achieved by introducing the gene into the host chromosome. Introduction of a gene into a chromosome can be achieved, for example, by homologous recombination (Miller, JH, Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Examples of gene introduction methods using homologous recombination include methods using linear DNA, such as Red-driven integration (Datsenko, K. A., and Wanner, BL, Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)), methods using plasmids containing a temperature-sensitive replication origin, methods using conjugatively transferable plasmids, methods using suicide vectors lacking a replication origin functional in the host, and transduction methods using phages. Specifically, the gene can be introduced into the host chromosome by transforming the host with recombinant DNA containing the mutant acetyl-CoA hydrolase gene and causing homologous recombination with a target site on the host chromosome. The structure of the recombinant DNA used for homologous recombination is not particularly limited as long as it allows homologous recombination to occur in the desired manner. For example, a host can be transformed with linear DNA containing a mutant acetyl-CoA hydrolase gene, with base sequences at both ends of the gene that are homologous to the target site on the chromosome, respectively, and homologous recombination can occur upstream and downstream of the target site, thereby replacing the target site with the gene. The recombinant DNA used for homologous recombination may contain a marker gene for selecting transformants. Only one copy of the gene may be introduced, or two or more copies may be introduced. For example, multiple copies of the mutant acetyl-CoA hydrolase gene can be introduced into a chromosome by performing homologous recombination targeting a base sequence that exists in multiple copies on the chromosome.Examples of base sequences present in multiple copies on a chromosome include repetitive DNA sequences and inverted repeats at both ends of transposons. Homologous recombination may also be performed by targeting an appropriate base sequence on a chromosome, such as a gene not required for the production of a target substance. Genes can also be randomly introduced into a chromosome using transposons or Mini-Mu (see JP-A-2-109985, U.S. Pat. No. 5,882,888, and EP805867B1). This method of chromosome modification using homologous recombination is not limited to the introduction of mutant acetyl-CoA hydrolase genes, but can also be used for any other chromosomal modification, such as modification of expression regulatory sequences.

[0079] The introduction of the mutant acetyl-CoA hydrolase gene into the chromosome can be confirmed by Southern hybridization using a probe having a sequence complementary to all or part of the gene, or by PCR using primers prepared based on the sequence of the gene.

[0080] Alternatively, a mutant acetyl-CoA hydrolase gene can be introduced into a coryneform bacterium by introducing a vector containing the gene into the host. For example, a DNA fragment containing the mutant acetyl-CoA hydrolase gene can be ligated to a vector functional in the host to construct an expression vector for the gene, and the host can be transformed with the expression vector to introduce the gene into the coryneform bacterium. A DNA fragment containing the mutant acetyl-CoA hydrolase gene can be obtained, for example, by PCR using the genomic DNA of a microorganism harboring the mutant acetyl-CoA hydrolase gene as a template. A vector capable of autonomous replication within host cells can be used. A multicopy vector is preferred. Furthermore, the vector preferably contains a marker such as an antibiotic resistance gene for the selection of transformants. The vector may also contain a promoter or terminator for expressing the inserted gene. The vector may be, for example, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a bacteriophage-derived vector, a cosmid, or a phagemid.Specific examples of vectors capable of autonomous replication in coryneform bacteria include pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903 (1984)); plasmids having drug resistance genes improved from these; pCRY30 (Japanese Patent Laid-Open No. 3-210184); pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX (Japanese Patent Laid-Open No. 2-72876, U.S. Pat. No. 5,185,262); pCRY2 and pCRY3 (Japanese Patent Laid-Open No. 1-191686); pAJ655, pAJ611, and pAJ 1844 (Japanese Patent Publication No. 58-192900); pCG1 (Japanese Patent Publication No. 57-134500); pCG2 (Japanese Patent Publication No. 58-35197); pCG4 and pCG11 (Japanese Patent Publication No. 57-183799); pVK7 (Japanese Patent Publication No. 10-215883); pVK9 (U.S. Patent Application Publication No. 2006 / 0141588); pVC7 (Japanese Patent Publication No. 9-070291); and pVS7 (WO2013 / 069634).

[0081] When a gene is introduced, it is sufficient that the gene is retained in the host in an expressible manner. Specifically, it is sufficient that the gene is retained so that it is expressed under the control of a promoter that functions in the host. The promoter is not particularly limited as long as it functions in the host. A "promoter that functions in the host" refers to a promoter that has promoter activity in the host. The promoter may be a promoter derived from the host or a heterologous promoter. The promoter may be a promoter native to the gene to be introduced or a promoter of another gene.

[0082] A terminator for terminating transcription can be placed downstream of the gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be a terminator derived from the host or a heterologous terminator. The terminator may be a terminator inherent to the gene to be introduced or a terminator of another gene.

[0083] Vectors, promoters, and terminators that can be used in various microorganisms are described in detail in, for example, "Basic Microbiology Lectures 8: Genetic Engineering, Kyoritsu Shuppan, 1987," and they can be used.

[0084] Furthermore, when two or more genes are introduced, it is sufficient that each gene is retained in an expressible state in the host. For example, all of the genes may be retained on a single expression vector, or all may be retained on a chromosome. Furthermore, each gene may be retained separately on multiple expression vectors, or may be retained separately on a single or multiple expression vectors and on a chromosome. Furthermore, two or more genes may constitute an operon and be introduced. Examples of "introducing two or more genes" include introducing genes that each encode two or more proteins (e.g., enzymes), introducing genes that each encode two or more subunits that constitute a single protein complex (e.g., an enzyme complex), and combinations thereof.

[0085] <2> Method for Producing L-Glutamic Acid of the Present Invention The method of the present invention is a method for producing L-glutamic acid, comprising culturing the bacterium of the present invention described in <1> in a medium, accumulating L-glutamic acid in the medium and / or within the bacterial cells of the bacterium, and collecting L-glutamic acid from the medium and / or the bacterial cells. L-glutamic acid is as described above. In the present invention, L-glutamic acid may be produced alone, or L-glutamic acid and one or more amino acids other than L-glutamic acid, such as L-amino acids (also referred to as L-amino acids), may also be produced.

[0086] The medium used is not particularly limited as long as it allows the bacterium of the present invention to grow and produces L-glutamic acid. For example, a conventional medium used for culturing bacteria such as coryneform bacteria can be used. For example, a medium containing a carbon source, a nitrogen source, a phosphate source, a sulfur source, and components selected from various other organic and inorganic components as needed can be used. The types and concentrations of medium components can be appropriately determined depending on various conditions, such as the type of bacterium used.

[0087] Specific examples of carbon sources include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, maltose, isomaltose, blackstrap molasses, starch hydrolysates, and biomass hydrolysates; organic acids such as acetic acid, fumaric acid, citric acid, and succinic acid; alcohols such as glycerol, crude glycerol, and ethanol; and fatty acids. Examples of carbon sources include sugars. Examples of carbon sources include glucose and fructose. Sugars such as glucose and fructose may be used alone or in combination with other carbon sources. Examples of carbon sources include sugars containing fructose as a constituent sugar. Examples of sugars containing fructose include fructose, sucrose, and fructooligosaccharides. Sugars containing fructose may be used alone or in combination with other carbon sources. Plant-derived materials are preferably used as carbon sources. Examples of plants include corn, rice, wheat, soybeans, sugarcane, beets, and cotton. Examples of plant-derived materials include organs such as roots, stems, trunks, branches, leaves, flowers, and seeds, plants containing these, and decomposition products of these plant organs. The form of use of plant-derived materials is not particularly limited, and they can be used in any form, such as raw products, squeezed juice, crushed products, or purified products. Examples of carbon sources include cane molasses, beet molasses, high-test molasses, citrus molasses, or invert sugar, as well as hydrolysates of natural materials such as cellulose, starch, corn, cereals, tapioca, and cassava. Furthermore, pentoses such as xylose, hexoses such as glucose, or mixtures thereof can be obtained from plant biomass and used. Specifically, these sugars can be obtained by subjecting plant biomass to treatments such as steam treatment, concentrated acid hydrolysis, dilute acid hydrolysis, hydrolysis with enzymes such as cellulase, and alkali treatment. In addition, since hemicellulose is generally more easily hydrolyzed than cellulose, the hemicellulose in the plant biomass may be hydrolyzed in advance to liberate pentoses, and then the cellulose may be hydrolyzed to produce hexoses.Xylose may also be supplied by conversion of a hexose such as glucose, for example, by imparting a conversion pathway from the hexose to xylose to the bacterium of the present invention. Specifically, the carbon source may be, for example, glucose alone, or a mixture of two carbon sources, such as glucose and fructose or glucose and sucrose, in any ratio (for example, a weight ratio of 3:7 to 7:3).

[0088] Specific examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen sources such as peptone, yeast extract, meat extract, and hydrolyzed vegetable protein (HVP; for example, soy protein hydrolyzate, soy sauce, and pea sauce); ammonia; and urea. Ammonia gas or aqueous ammonia, which is used for pH adjustment, may also be used as a nitrogen source. A single nitrogen source may be used, or two or more nitrogen sources may be used in combination.

[0089] Specific examples of the phosphate source include phosphate salts such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. As the phosphate source, one type of phosphate source may be used, or two or more types of phosphate sources may be used in combination.

[0090] Specific examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. As the sulfur source, one type of sulfur source may be used, or two or more types of sulfur sources may be used in combination.

[0091] Other organic and inorganic components include, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, vitamin B12, biotin, and folic acid; amino acids; nucleic acids; and organic components containing these, such as peptone, casamino acids, yeast extract, and hydrolyzed vegetable protein (HVP; e.g., soy protein hydrolyzate, soy sauce, and pea sauce). Other organic and inorganic components include antifoaming agents, medium osmotic pressure regulators, and osmotic pressure compensation substances. Examples of antifoaming agents include silicone-based antifoaming agents (oil-type, solution-type, oil-compound-type, emulsion-type, self-emulsifying type, etc.), alcohol-based antifoaming agents, oil-based antifoaming agents, polyether-based antifoaming agents, and vegetable oils (cottonseed oil, linseed oil, soybean oil, olive oil, castor oil, coconut oil, etc.). Antifoaming agents can be used in any form, including liquid, paste, solid, powder, emulsion, and wax. Osmotic pressure regulators for the medium include salts such as sodium chloride and potassium chloride, and polysaccharides that cannot be assimilated by microorganisms (e.g., sorbitol and dextrin). Osmotic pressure compensation substances include potassium ions, betaine (glycine betaine), blackstrap molasses (particularly sugar beet blackstrap molasses), glutamic acid, and trehalose. Other components that may be added to the medium include polymers selected from the group consisting of water-soluble cellulose derivatives, water-soluble polyvinyl compounds, polar organic solvent-soluble polyvinyl compounds, water-soluble starch derivatives, alginates, and polyacrylates. These and other various organic and inorganic components may be used alone or in combination of two or more.

[0092] When using an auxotrophic mutant strain that requires amino acids or the like for growth, it is preferable to supplement the required nutrients in the medium.

[0093] It is also preferable to limit the amount of biotin in the medium or to add a surfactant or penicillin to the medium.

[0094] The culture conditions are not particularly limited as long as the bacterium of the present invention can grow and produce L-glutamic acid. The culture can be carried out under standard conditions used for culturing bacteria such as coryneform bacteria. The culture conditions may be appropriately set depending on various factors such as the type of bacterium used.

[0095] Culturing can be carried out using a liquid medium. Examples of liquid culture methods include those described in "Biotechnology Textbook Series 13: Culture Engineering" by Toshiomi Yoshida, published by Corona Publishing in 1998. Specifically, liquid culture can be performed using methods such as surface culture, submerged culture, membrane (e.g., dialysis membrane or for-ferber) isolation culture, or immobilized microbial culture. Cultivation devices include aeration and agitation culture devices, airlift culture devices, packed-bed culture devices, and fluidized-bed culture devices. Cultivation can be performed using methods described in "Fermentation Engineering Fundamentals" published by the Academic Press in 1988. During cultivation, the bacterium of the present invention may be cultured in a solid medium such as an agar medium and then directly inoculated into a liquid medium, or a seed culture of the bacterium of the present invention in a liquid medium may be inoculated into a liquid medium for main culture. That is, cultivation can be performed separately in two stages: seed culture and main culture. In this case, the culture conditions for the seed culture and main culture may be the same or different. The amount of the bacterium of the present invention contained in the medium at the start of cultivation is not particularly limited. The main culture may be carried out, for example, by inoculating the seed culture solution into the medium for the main culture at 1 to 50% (v / v). Furthermore, for example, the seed culture step may include two or more seed culture steps to obtain the amount of bacteria required for the main culture step. The seed culture solution may be inoculated only at the start of the main culture, or may be inoculated at the start of the main culture and then additionally during the main culture.

[0096] Cultivation can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. Examples of combinations include two or more stages of connected fed-batch culture and two or more stages of connected continuous culture. The medium at the start of culture is also called the "initial medium." The medium supplied to the culture system (fermentor) in fed-batch culture or continuous culture is also called the "fed-batch medium." Supplying a fed-batch medium to the culture system in fed-batch culture or continuous culture is also called "fed-batch." When culturing is divided into seed culture and main culture, both the seed culture and the main culture may be performed by batch culture, for example. Alternatively, the seed culture may be performed by batch culture, and the main culture may be performed by fed-batch or continuous culture. Alternatively, the seed culture may be performed by fed-batch culture, and the main culture may be performed by batch culture. The feed medium may be supplied, for example, from a location in the upper part of the culture tank that is not in contact with the liquid surface of the culture medium, or from a location inside the culture tank such as the middle or lower part of the culture tank, or from both the upper and middle parts of the culture tank. An embodiment in which the feed medium is supplied from a location inside the culture tank is disclosed, for example, in Japanese Patent No. 6097869.

[0097] In the present invention, each medium component may be contained in the initial medium, the feed medium, or both. The type of component contained in the initial medium may or may not be the same as the type of component contained in the feed medium. Furthermore, the concentration of each component contained in the initial medium may or may not be the same as the concentration of each component contained in the feed medium. Furthermore, two or more feed media containing different types and / or concentrations of components may be used. For example, when multiple feedings are performed intermittently, the type and / or concentration of components contained in the feed medium for each feeding may or may not be the same. For example, the carbon source of the initial medium may be glucose, and the carbon source of the feed medium may be sucrose.

[0098] The medium may or may not be sterilized. The medium may be sterilized for the purpose of preventing contamination. Sterilization of the medium can also be referred to as sterilization or sterilization. Methods for sterilizing the medium include sterilization under high temperature and high pressure conditions, sterilization by UV irradiation, and sterilization using a filter or membrane. The medium may be sterilized batchwise or continuously. For example, methods for batchwise sterilization under high temperature and high pressure conditions include autoclave sterilization and batch sterilization in a culture tank. For example, methods for continuous sterilization under high temperature and high pressure conditions include continuous sterilization using a plate-type heat exchanger. Furthermore, sterilization of the sugar may be performed simultaneously with other medium components, or may be performed separately from the other components. Preferably, the sugar and the other components may be sterilized separately.

[0099] The concentration of the carbon source in the medium is not particularly limited, as long as the bacterium of the present invention can grow and L-glutamic acid can be produced. The concentration of the carbon source in the medium may be as high as possible, for example, within a range that does not inhibit L-glutamic acid production. The initial concentration (initial concentration in the medium) of the carbon source may be, for example, 1 to 50 w / v%, preferably 1 to 30 w / v%, and more preferably 3 to 10 w / v%. Additional carbon source may be added to the medium as needed. For example, additional carbon source may be added to the medium depending on the consumption of the carbon source as fermentation progresses. In fed-batch culture or continuous culture, the amount of carbon source supplied may be sufficient (a condition in which an amount in excess of the carbon assimilation capacity of the bacterium of the present invention is supplied) or limiting (a condition in which an amount insufficient to the carbon assimilation capacity of the bacterium of the present invention is supplied).

[0100] Cultivation may be carried out, for example, using a liquid medium under aerobic or microaerobic conditions. "Aerobic conditions" refers to a liquid medium in which the dissolved oxygen concentration is 0.33 ppm or higher, which is the detection limit using an oxygen membrane electrode, and preferably 1.5 ppm or higher. The oxygen concentration under aerobic conditions may be controlled, for example, to 5 to 50%, preferably about 10%, of the saturated oxygen concentration. "Microaerobic conditions" may refer to conditions in which the dissolved oxygen concentration in the medium is less than 0.33 ppm. The dissolved oxygen concentration in the medium under microaerobic conditions may be, for example, 0.30 ppm or lower, 0.25 ppm or lower, 0.20 ppm or lower, 0.15 ppm or lower, 0.10 ppm or lower, or 0.05 ppm or lower. The oxygen concentration under microaerobic conditions may be controlled, for example, to less than 5%, 3.75% or less, 3.125% or less, 2.5% or less, 1.875% or less, 1.25% or less, or 0.8125% or less of the saturated oxygen concentration. Specifically, the culture can be performed by aerobic culture, shaking culture, agitation culture, or a combination thereof. The pH of the medium may be, for example, 3 to 10, preferably 4.0 to 9.5. The pH of the medium can be adjusted as needed during culture. The pH of the medium can be adjusted using various alkaline or acidic substances, such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, or magnesium hydroxide. The culture temperature may be, for example, 20 to 40°C, preferably 25 to 37°C. During the main culture, the culture temperature may be changed in two or more stages. For example, as disclosed in Journal of Industrial Microbiology & Biotechnology (2002) 28, 333-337, the culture temperature may be shifted to a higher temperature, from 33°C to 37-40°C. The culture period may be, for example, 10 to 120 hours. Culture may be continued, for example, until the carbon source in the medium is consumed or until the activity of the bacterium of the present invention is lost. Culturing the bacterium of the present invention under such conditions results in accumulation of L-glutamic acid in the medium and / or within the bacterial cells.

[0101] Alternatively, a liquid medium adjusted to conditions for L-glutamic acid precipitation can be used to culture the L-glutamic acid while precipitating it in the medium. Conditions for L-glutamic acid precipitation include, for example, pH 5.0 to 4.0, preferably pH 4.5 to 4.0, more preferably pH 4.3 to 4.0, and particularly preferably pH 4.0 (EP1078989A). When using a liquid medium adjusted to conditions for L-glutamic acid precipitation, adding pantothenic acid to the medium can increase the efficiency of crystallization (WO2004 / 111258). When using a liquid medium adjusted to conditions for L-glutamic acid precipitation, adding L-glutamic acid crystals as seed crystals can increase the efficiency of crystallization (EP1233069A). Furthermore, when a liquid medium adjusted to conditions for the precipitation of L-glutamic acid is used, crystallization can be more efficient by adding L-glutamic acid crystals and L-lysine crystals to the medium as seed crystals (EP1624069A).

[0102] The fermentation liquor can be treated, for example, with a hydrocyclone. The hydrocyclone can be, for example, a generally shaped one with a cylindrical diameter of 10 to 110 mm and made of ceramic, stainless steel, or resin. The amount of the fermentation liquor fed to the hydrocyclone can be set depending on, for example, the bacterial cell concentration and L-glutamic acid concentration in the fermentation liquor. The amount of the fermentation liquor fed to the hydrocyclone can be, for example, 2 to 1200 L / min.

[0103] The production of L-glutamic acid can be confirmed by known techniques used for detecting or identifying compounds. Such techniques include, for example, HPLC, LC / MS, GC / MS, and NMR. These techniques can be used alone or in appropriate combination.

[0104] L-glutamic acid can be recovered from the fermentation broth by known methods used for separating and purifying compounds. Examples of such methods include the ion exchange resin method (Nagai, H. et al., Separation Science and Technology, 39(16), 3691-3710), precipitation, membrane separation (JP-A-9-164323 and JP-A-9-173792), and crystallization (WO2008 / 078448 and WO2008 / 078646). These methods can be used alone or in appropriate combination. When L-glutamic acid accumulates within the cells, for example, the cells can be disrupted by ultrasound or the like, and then removed by centrifugation. From the resulting supernatant, L-glutamic acid can be recovered by the ion exchange resin method or the like. The recovered L-glutamic acid may be in the free form, a salt thereof, or a mixture thereof. Examples of salts include sulfates, hydrochlorides, carbonates, ammonium salts, sodium salts, and potassium salts. Specifically, free L-glutamic acid, monosodium L-glutamate (e.g., monosodium L-glutamate; MSG), ammonium L-glutamate (e.g., monoammonium L-glutamate), or mixtures thereof may be used. For example, monosodium L-glutamate (MSG) can be obtained by adding acid to ammonium L-glutamate in a fermentation broth to crystallize it, and then adding an equimolar amount of sodium hydroxide to the crystals. Activated carbon may be added before or after crystallization to decolorize the product (see "Industrial Crystallization of Monosodium Glutamate," Journal of the Society of Sea Water Science of Japan, Vol. 56, No. 5, Tetsuya Kawakita). Monosodium L-glutamate crystals can be used, for example, as an umami seasoning. Monosodium L-glutamate crystals may also be used as a seasoning by mixing them with nucleic acids, such as sodium guanylate or sodium inosinate, which also have an umami flavor.

[0105] When L-glutamic acid precipitates in the medium, it can be recovered by centrifugation, filtration, etc. Alternatively, L-glutamic acid precipitated in the medium may be isolated together with L-glutamic acid dissolved in the medium after crystallization.

[0106] The recovered L-glutamic acid may contain, in addition to L-glutamic acid, bacterial cells, medium components, water, bacterial metabolic by-products, and other components. The L-glutamic acid may be purified to a desired degree. The purity of the recovered L-glutamic acid may be, for example, 50% (w / w) or more, preferably 85% (w / w) or more, and particularly preferably 95% (w / w) or more (see Japanese Patent No. 1214636, U.S. Patent No. 5431933, U.S. Patent No. 4956471, U.S. Patent No. 4777051, U.S. Patent No. 4946654, U.S. Patent No. 5840358, U.S. Patent No. 6238714, and U.S. Patent Application Publication No. 2005 / 0025878).

[0107] The present invention will now be described in more detail with reference to the following non-limiting examples.

[0108] <1> Construction of Modified Corynebacterium glutamicum Strains <1-1> Construction of a Vector for Introducing the Wild-Type Acetyl-CoA Hydrolase Gene A DNA fragment containing the wild-type acetyl-CoA hydrolase gene was amplified by PCR using the chromosomal DNA of C. glutamicum ATCC 13869 (strain 2256) harboring the wild-type acetyl-CoA hydrolase gene as a template and the primers set forth in SEQ ID NOs: 5 and 6. The amplified DNA fragment was ligated by infusion reaction with pVK9 (U.S. Patent Application Publication No. 2006 / 0141588) digested with bamHI and pstI to obtain a vector for introducing the wild-type acetyl-CoA hydrolase gene (pVK9-Acetyl-CoA hydrolase (WT)). The nucleotide sequence of the wild-type acetyl-CoA hydrolase gene and the amino acid sequence of the wild-type acetyl-CoA hydrolase encoded by the gene are set forth in SEQ ID NOs: 1 and 2, respectively.

[0109] <1-2> Construction of a Vector for Introducing a Mutant Acetyl-CoA Hydrolase Gene. A DNA fragment containing the mutant acetyl-CoA hydrolase gene was amplified by PCR using primers set forth in SEQ ID NOs: 5 and 6, with the chromosomal DNA of a C. glutamicum ATCC 13869 (2256) derivative containing a mutant acetyl-CoA hydrolase gene with a mutation (S383C) substituting the serine residue at position 383 of wild-type acetyl-CoA hydrolase with a cysteine ​​residue. The amplified DNA fragment was ligated to pVK9 digested with bamHI and pstI in an infusion reaction to obtain a vector for introducing the mutant acetyl-CoA hydrolase gene (pVK9-Acetyl-CoA hydrolase (S383C)). The nucleotide sequence of the mutant acetyl-CoA hydrolase gene and the amino acid sequence of the mutant acetyl-CoA hydrolase encoded by the gene are set forth in SEQ ID NOs: 3 and 4, respectively.

[0110] <1-3> Construction of modified Corynebacterium glutamicum strains The C. glutamicum 2256ΔsucAΔldhA yggB* strain (WO2014 / 185430) was transformed with each of the constructed mutant gene transfer vectors. From the resulting transformants, strains were selected according to the method described in WO2006 / 057450 to obtain wild-type acetyl-CoA hydrolase gene-introduced strains and mutant acetyl-CoA hydrolase gene-introduced strains.

[0111] The wild-type acetyl-CoA hydrolase gene vector (pVK9-Acetyl-CoA hydrolase (WT)) and the mutant acetyl-CoA hydrolase gene vector (pVK9-Acetyl-CoA hydrolase (S383C)) were introduced into C. glutamicum 2256ΔsucAΔldhA yggB* to obtain wild-type acetyl-CoA hydrolase gene-transfected strains and mutant acetyl-CoA hydrolase gene-transfected strains, respectively. Furthermore, a control strain was obtained by introducing pVK9 alone into C. glutamicum 2256ΔsucAΔldhA yggB*.

[0112] The C. glutamicum 2256ΔsucAΔldhA yggB* strain is an L-glutamic acid-producing strain derived from the C. glutamicum 2256 strain (ATCC 13869), which is deficient in the ldhA and sucA genes and has an IS mutation (V419::IS) in the yggB gene.

[0113] <2> L-Glutamic Acid Production Culture L-glutamic acid production culture was carried out using each of the constructed strains (i.e., the wild-type acetyl-CoA hydrolase gene-introduced strain, the mutant acetyl-CoA hydrolase gene-introduced strain, and the control strain). The composition of the medium used is shown in Table 1.

[0114]

[0115] Each strain was inoculated into 20 mL of the above medium (containing 50 g / L calcium carbonate) in a 500 mL Sakaguchi flask and cultured at 31.5°C with shaking at 120 rpm in a box shaker (ABLE ML-190). Culture samples were taken 25 hours after the start of culture. A 0.05 mL aliquot of the sampled culture was diluted 100-fold by adding it to 4.95 mL of 0.1 N HCl solution, and the optical density (OD) at 620 nm was measured. The sampled culture was centrifuged at 15,000 rpm for 1 minute, and the L-glutamic acid concentration in the supernatant was quantified using a Biotech Analyzer BF7 (Oji Instruments Co., Ltd.). The L-glutamic acid yield per sugar was calculated.

[0116] The results are shown in Figures 1 and 2. The mutant acetyl-CoA hydrolase gene-introduced strain showed a higher amount of accumulated L-glutamic acid and a higher L-glutamic acid yield per sugar than the wild-type acetyl-CoA hydrolase gene-introduced strain (Figures 1 and 2).

[0117] According to the present invention, the L-amino acid-producing ability of a coryneform bacterium can be improved, and L-amino acids can be produced efficiently.

[0118] [Explanation of the Sequence Listing] SEQ ID NO: 1: Nucleotide sequence of the wild-type acetyl-CoA hydrolase gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 2: Amino acid sequence of the wild-type acetyl-CoA hydrolase of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 3: Nucleotide sequence of the mutant acetyl-CoA hydrolase gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 4: Amino acid sequence of the mutant acetyl-CoA hydrolase of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 5 and SEQ ID NO: 6: Primers SEQ ID NO: 7: Nucleotide sequence of the wild-type yggB gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 8: Amino acid sequence of the mutant acetyl-CoA hydrolase of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 9: Nucleotide sequence of mutant yggB gene (V419::IS) SEQ ID NO: 10: Amino acid sequence of mutant YggB protein (V419::IS)

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