L-Glutamic acid-producing bacterium and method for producing L-glutamic acid

Abstract

Provided are a method for producing L-glutamic acid and bacteria used therefor. Coryneform bacteria having the ability to produce L-glutamic acid are modified to retain a mutant acetyl-CoA hydrolase gene encoding a mutant acetyl-CoA hydrolase having a substitution of another amino acid residue for the serine residue at position 383 in the amino acid sequence of the wild-type acetyl-CoA hydrolase. The L-glutamic acid is produced by culturing the coryneform bacteria in a medium and collecting L-glutamic acid from the medium and / or the cells thereof.

Description

Technical Field

[0001] The present invention relates to the fermentation industry, and more particularly to a method for producing L-glutamic acid and bacteria used therefor. L-glutamic acid is industrially useful as a seasoning raw material and the like.

Background Art

[0002] L-amino acids are industrially produced, for example, by a fermentation method using microorganisms such as bacteria having L-amino acid-producing ability (Non-Patent Document 1). As such microorganisms, for example, strains isolated from nature and their mutant strains are used. In addition, the L-amino acid-producing ability of microorganisms can be improved by recombinant DNA technology.

[0003] Acetyl-CoA hydrolase is an enzyme having an activity of catalyzing a reaction of hydrolyzing acetyl-CoA to produce coenzyme A and acetic acid and / or its reverse reaction (Non-Patent Document 2), and by reducing the enzyme activity, It is known that the production abilities of L-glutamic acid, L-valine and L-alanine are enhanced (Patent Document 1). However, it has not been known that specific mutations within the amino acid sequence of the enzyme contribute to the enhancement of L-glutamic acid production ability.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object 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 used therefor.

Means for Solving the Problems

[0007] As a result of intensive studies to solve the above problems, the present inventors have found that by modifying coryneform bacteria so that acetyl-CoA hydrolase has specific mutations, the L-glutamic acid-producing ability of the bacteria can be improved, and thus completed the present invention.

[0008] That is, the present invention can be exemplified as follows. [1] A coryneform bacterium having an L-glutamic acid-producing ability, which is modified to retain a mutant acetyl-CoA hydrolase gene encoding a mutant acetyl-CoA hydrolase having a substitution of another amino acid residue for the serine residue at position 383 in the amino acid sequence of wild-type acetyl-CoA hydrolase, coryneform bacterium. [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 one of [1] to [3], 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 shown in SEQ ID NO: 2; (b) A protein comprising an amino acid sequence containing substitution, deletion, insertion and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 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 one of [1] to [4], wherein the coryneform bacterium belongs to the genus Corynebacterium. [6] The coryneform bacterium according to any one of [1] to [4], wherein the coryneform bacterium is Corynebacterium glutamicum. [7] It is modified to retain the mutant yggB gene, The coryneform bacterium according to any one of [1] to [6], wherein the mutant yggB gene is a gene encoding a protein having an amino acid sequence in which one or several amino acids are 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: [1] Culturing the coryneform bacterium according to any one of [1] to [8] in a medium to accumulate L-glutamic acid in the medium and / or in the cells of the bacterium, and [2] Collecting L-glutamic acid from the medium and / or the cells, and a production method.

[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 one 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 shown in SEQ ID NO: 2; (b) A protein comprising an amino acid sequence containing substitution, deletion, insertion and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 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.

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

[10] to

[13] .

Brief Description of Drawings

[0009]

Figure 1

Figure 2

BEST MODE FOR CARRYING OUT THE INVENTION

[0010] Hereinafter, the present invention will be described in detail.

[0011] The method of the present invention comprises culturing a coryneform bacterium having the ability to produce L-glutamic acid in a medium, accumulating L-glutamic acid in the medium and / or in the cells of the bacterium, and collecting L-glutamic acid from the medium and / or the cells, and is a method for producing L-glutamic acid, wherein the coryneform bacterium is modified to retain a specific gene mutation. The bacterium used in the method is also referred to as "the bacterium of the present invention".

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

[0013] <1-1>Coryneform bacterium having the ability to produce L-glutamic acid In the present invention, the "coryneform bacterium having the ability to produce L-glutamic acid" refers to a coryneform bacterium that has the ability to produce and accumulate L-glutamic acid in the medium and / or in the cells to such an extent that it can be recovered when cultured in a medium. The coryneform bacterium having the ability to produce L-glutamic acid may be a coryneform bacterium that can accumulate a larger amount of L-glutamic acid in the medium and / or in the cells than the unmodified strain. The "unmodified strain" refers to a control strain that has not been modified to retain a specific gene mutation. That is, examples of the unmodified strain include wild strains and parental strains. Further, the coryneform bacterium having the ability to produce L-glutamic acid may preferably be a coryneform bacterium that can accumulate the target L-amino acid in the medium in an amount of 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 with one or more amino acids other than L-glutamic acid, such as L-form amino acids (also referred to as L-amino acids). The L-amino acids are not particularly limited, and examples include 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. In the present invention, the terms "L-glutamic acid" and "L-amino acid" mean free L-glutamic acid, free L-amino acid, their salts, 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 thiogenitalis Corynebacterium ammoniagenes (Corynebacterium stationis) Brevibacterium album Brevibacterium cerinum Microbacterium ammoniaphilum

[0018] Examples of coryneform bacteria include, in particular, Corynebacterium glutamicum (former name: Brevibacterium lactofermentum).

[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 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] As coryneform bacteria, more particularly, Brevibacterium lactofermentum (new name: Corynebacterium glutamicum) ATCC 13869 can be mentioned. In addition, examples of coryneform bacteria include the C. glutamicum 2256ΔsucAΔldhA yggB* strain, which lacks the ldhA gene and the sucA gene of Corynebacterium glutamicum ATCC 13869 and has an IS mutation (V419::IS) in the yggB gene (WO2014 / 185430).

[0021] Note that coryneform bacteria include bacteria that were formerly classified in the genus Brevibacterium but are now integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)). Also, Corynebacterium stationis includes bacteria that were formerly classified as Corynebacterium ammoniagenes but have been reclassified as Corynebacterium stationis by analysis of the nucleotide sequence of 16S rRNA and the like (Int. J. Syst. Evol. Microbiol., 60, 874-879 (2010)).

[0022] These strains can be obtained, for example, from the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852 P.O. Box 1549, Manassas, VA 20108, United States of America). That is, each strain is assigned a registration number, and this registration number can be used to obtain the strain (see http: / / www.atcc.org / ). The registration number corresponding to each strain is described in the catalog of the American Type Culture Collection. Also, these strains can be obtained, for example, from the depository institution where each strain is deposited.

[0023] The bacterium of the present invention may inherently have the ability to produce L-glutamic acid, or may be modified to retain the ability to produce L-glutamic acid. Bacteria having the ability to produce L-glutamic acid can be obtained, for example, by conferring the ability to produce L-glutamic acid on the above-described bacteria or by enhancing the ability of the above-described bacteria to produce L-glutamic acid.

[0024] The conferring or enhancement of the ability to produce L-glutamic acid can be carried out by a method conventionally employed for breeding amino acid-producing bacteria such as coryneform bacteria or Escherichia bacteria (see Amino Acid Fermentation, The Society Publishing Center, first edition published on May 30, 1986, pages 77 to 100). Such methods include, for example, obtaining auxotrophic mutants, obtaining analog-resistant mutants of L-glutamic acid, obtaining metabolic control mutants, and creating recombinant strains in which the activity of L-glutamic acid biosynthetic enzymes is enhanced. In the breeding of L-glutamic acid-producing bacteria, the properties such as auxotrophy, analog resistance, and metabolic control mutations to be conferred may be single, or two or more. Also, in the breeding of L-glutamic acid-producing bacteria, the L-glutamic acid biosynthetic enzymes whose activity is enhanced may be single, or two or more. Furthermore, the conferring of properties such as auxotrophy, analog resistance, and metabolic control mutations may be combined with the enhancement of the activity of biosynthetic enzymes.

[0025] An auxotrophic mutant, analog-resistant mutant, or metabolic control mutant having the ability to produce L-glutamic acid can be obtained by subjecting a parent strain or wild strain to ordinary mutagenesis treatment and selecting from the obtained mutants those that show auxotrophy, analog resistance, or metabolic control mutation and have the ability to produce L-glutamic acid. Examples of ordinary mutagenesis treatment include irradiation with X-rays or ultraviolet rays, treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0026] In addition, imparting or enhancing the ability to produce L-glutamic acid can also be achieved by enhancing the activity of enzymes involved in the biosynthesis of L-glutamic acid. Enhancement of enzyme activity can be carried out, for example, by modifying bacteria so that the expression of the gene encoding the enzyme is enhanced. Methods for enhancing gene expression are described in WO00 / 18935, EP1010755A, and the like.

[0027] In addition, imparting or enhancing the ability to produce L-glutamic acid can also be achieved by reducing the activity of enzymes that catalyze reactions branching from the L-glutamic acid biosynthesis pathway to produce compounds other than L-glutamic acid. Herein, the "enzymes that catalyze reactions branching from the L-glutamic acid biosynthesis pathway to produce compounds other than L-glutamic acid" also include enzymes involved in the decomposition of L-glutamic acid.

[0028] Hereinafter, L-glutamic acid-producing bacteria and methods for imparting or enhancing the ability to produce L-glutamic acid will be specifically exemplified. Note that any of the properties of L-glutamic acid-producing bacteria and the modifications for imparting or enhancing the ability to produce L-glutamic acid as exemplified below may be used alone or in appropriate combinations.

[0029] As a method for imparting or enhancing the ability to produce L-glutamic acid, for example, there can be mentioned a method of modifying bacteria so that the activity of one or more enzymes selected from L-glutamic acid biosynthetic enzymes increases. Such enzymes are not particularly limited, but 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), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglyceromutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triosephosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), glucose phosphate isomerase (pgi), 6-phosphogluconate dehydrogenase (edd), 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), transhydrogenase (pntAB). The contents in parentheses are examples of the genes encoding the enzymes (the same applies in the following descriptions). Among these enzymes, for example, it is preferable to enhance the activity of one or more enzymes selected from glutamate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, and methyl citrate synthase. <> <>

[0030] <> Examples of coryneform bacteria modified so that the expression of the glutamate synthase gene (gltBD) increases include those disclosed in WO99 / 07853. <> <>

[0031] <> In addition, as a method for imparting or enhancing the ability to produce L-glutamic acid, for example, there is also a method of modifying bacteria so that the activity of one or more enzymes selected from enzymes that catalyze reactions that branch off from the biosynthetic pathway of L-glutamic acid to produce compounds other than L-glutamic acid is reduced. Such enzymes are not particularly limited, but include 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, for example, it is preferable to reduce or eliminate the α-ketoglutarate dehydrogenase activity.

[0032] Coryneform bacteria with reduced or deficient α-ketoglutarate dehydrogenase activity, and methods for obtaining them, are described in WO2008 / 075483. Specific examples of coryneform bacteria with reduced or deficient α-ketoglutarate dehydrogenase activity include the following strains. Corynebacterium glutamicum (Brevibacterium lactofermentum) L30-2 strain (Japanese Patent Application Laid-Open No. 2006-340603) Corynebacterium glutamicum (Brevibacterium lactofermentum) ΔS strain (WO95 / 34672) Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ12821 (FERM BP-4172; French Patent Invention No. 9401748 Specification) Corynebacterium glutamicum (Brevibacterium flavum) AJ12822 (FERM BP-4173; French Patent Invention No. 9401748 Specification) Corynebacterium glutamicum AJ12823 (FERM BP-4174; French Patent Invention No. 9401748)

[0033] In addition, as L-glutamic acid-producing bacteria or parental strains for inducing them, strains in which both α-ketoglutarate dehydrogenase (sucA) activity and succinate dehydrogenase (sdh) activity are reduced or deficient can also be mentioned (Japanese Patent Application Laid-Open No. 2010-041920). As such a strain, specifically, for example, the odhAsdhA double-deficient strain of Corynebacterium glutamicum ATCC14067 (Corynebacterium glutamicum 8L3GΔSDH strain) can be mentioned (Japanese Patent Application Laid-Open No. 2010-041920).

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

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

[0036] As a method for imparting or enhancing the ability to produce L-glutamic acid, for example, a method of modifying bacteria so that the activity of discharging L-glutamic acid from the cells of the bacteria increases can be mentioned. The activity of discharging L-glutamic acid can be increased, for example, by increasing the expression of a gene encoding a protein that discharges L-glutamic acid. Examples of genes encoding proteins that discharge various amino acids include the b2682 gene (ygaZ), the b2683 gene (ygaH), the b1242 gene (ychE), and the b3434 gene (yhgN) (Japanese Patent Application Laid-Open No. 2002-300874).

[0037] Also, as a method for imparting or enhancing the ability to produce L-glutamic acid, for example, a method of modifying bacteria so that the activity of a protein involved in sugar metabolism or a protein involved in energy metabolism increases can be mentioned.

[0038] Examples of proteins involved in sugar metabolism include proteins involved in sugar uptake and glycolytic enzymes. Examples of genes encoding proteins involved in sugar metabolism include the glucose 6-phosphate isomerase gene (pgi; WO01 / 02542), the pyruvate carboxylase gene (pyc; WO99 / 18228, EP1092776A), the phosphoglucomutase gene (pgm; WO03 / 04598), the fructose bisphosphate aldolase gene (pfkB, fbp; WO03 / 04664), the transaldolase gene (talB; WO03 / 008611), the fumarase gene (fum; WO01 / 02545), the non-PTS sucrose uptake gene (csc; EP1149911A), and the sucrose-assimilating gene (scrAB operon; U.S. Patent No. 7179623).

[0039] Genes encoding proteins involved in energy metabolism include the transhydrogenase gene (pntAB; U.S. Patent No. 5,830,716) and the cytochrome bo type oxidase gene (cyoB; EP1070376A).

[0040] In addition, for coryneform bacteria, methods for conferring or enhancing the ability to produce L-glutamic acid include a method of enhancing the expression of the yggB gene and a method of introducing a mutant yggB gene having a mutation introduced into the coding region (WO2006 / 070944). That is, the bacterium of the present invention may be modified so that the expression of the yggB gene is increased, or may be modified to retain (have) the mutant yggB gene.

[0041] The yggB gene is a gene encoding a mechanosensitive channel. Examples of the yggB gene include the yggB gene of coryneform bacteria. Specific examples of the yggB gene of coryneform bacteria include, for example, 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 the sequence from 1,336,091 to 1,337,692 in the genomic sequence registered in the NCBI database under GenBank Accession No. NC_003450 and are also called NCgl1221. The YggB protein encoded by the yggB gene of Corynebacterium glutamicum ATCC13032 is registered as GenBank accession No. NP_600492. Further, the nucleotide sequence of the yggB gene of Corynebacterium glutamicum 2256 (ATCC 13869) and the amino acid sequence of the YggB protein encoded by the same gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0042] In the present invention, the yggB gene having the "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. Further, in the present invention, the yggB gene not having the "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. In the case of the YggB protein, the change in the amino acid sequence caused by the "specific mutation" in the yggB gene is also referred to as the "specific mutation". The "wild type" mentioned here is a description for convenience in distinguishing from the "mutant type", and is not limited to those obtained naturally as long as it does not have the "specific mutation". Examples of the wild-type YggB protein include the YggB proteins exemplified above, for example, a protein having the amino acid sequence shown in SEQ ID NO: 8. Further, examples of the wild-type YggB protein also include conservative variants of the YggB proteins exemplified above (variants in which the original function is maintained) that do not have the "specific mutation". The "original function" of the YggB protein may be, for example, a function as a mechanosensitive channel, or a property of improving the L-glutamic acid-producing ability of coryneform bacteria when its expression is increased in coryneform bacteria.

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

[0044] (1) Mutations on the C-terminal side The C-terminal side mutation is a mutation in the region encoding amino acid residues 419 to 533 of the wild-type YggB protein in the wild-type yggB gene. The C-terminal side mutation may be introduced at one or more positions in the same region. The type of change in the amino acid sequence caused by the C-terminal side mutation is not particularly limited. The C-terminal side mutation may cause, for example, substitution of amino acid residues (missense mutation), insertion of amino acid residues, deletion of amino acid residues, appearance of a stop codon (nonsense mutation), frameshift mutation, or a combination thereof. As the C-terminal side mutation, for example, insertion of a base sequence such as an insertion sequence (hereinafter also referred to as "IS") or a transposon is preferable.

[0045] (1-1) Insertion of base sequence Examples of the C-terminal side mutation include a mutation (type 2A-1 mutation) in which a base sequence is inserted at the position encoding the valine residue at position 419 of the wild-type YggB protein. The type 2A-1 mutation may cause, for example, deletion or substitution of some or all of the amino acid residues at positions 419 to 533 of the wild-type YggB protein. As a mutant yggB gene having the type 2A-1 mutation, specifically, for example, an IS is inserted after the "G" at position 1255 of SEQ ID NO: 7, and a yggB gene encoding a mutant YggB protein having a total length of 423 amino acid residues shorter than the original wild-type YggB protein (SEQ ID NO: 8) can be mentioned. The base sequence of this mutant yggB gene (V419::IS) and the amino acid sequence of the mutant YggB protein (V419::IS) encoded by the same gene are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. In SEQ ID NO: 9, positions 1 to 1269 are the CDS of the mutant YggB protein (V419::IS). As an L-glutamic acid-producing bacterium having the mutant yggB gene (V419::IS), specifically, for example, the C. glutamicum 2256ΔsucAΔldhA yggB * strain (WO2014 / 185430) can be mentioned.

[0046] (1-2) Substitution of proline residue Examples of the C-terminal side mutations include mutations that substitute proline residues present at positions 419 to 533 of the wild-type YggB protein with other amino acids. Such proline residues include the proline residues at positions 424, 437, 453, 457, 462, 469, 484, 489, 497, 515, 529, and 533 of the wild-type YggB protein. Among them, it is preferable to substitute the proline residues at positions 424 and / or 437 with other amino acids. The "other amino acids" are not particularly limited as long as they are natural-type amino acids other than proline. Examples of the "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 preferably be substituted with a hydrophobic amino acid (Ala, Gly, Val, Leu, or Ile), and more preferably with a branched-chain amino acid (Leu, Val, or Ile). Also, for example, the proline residue at position 437 may preferably be substituted with an amino acid having a hydroxyl group in the side chain (Thr, Ser, or Tyr), and more preferably with Ser.

[0047] (2) Mutations in the transmembrane region The YggB protein is presumed to have five transmembrane regions. Each transmembrane region corresponds to the amino acid residues at positions 1 - 23 (the first transmembrane region), 25 - 47 (the second transmembrane region), 62 - 84 (the third transmembrane region), 86 - 108 (the fourth transmembrane region), and 110 - 132 (the fifth transmembrane region) of the wild-type YggB protein. Mutations in the transmembrane regions are mutations in the regions of the wild-type yggB gene that encode these transmembrane regions. Mutations in the transmembrane regions may be introduced at one or more positions within the region. Mutations in the transmembrane regions preferably cause substitution, deletion, addition, insertion, or inversion of one or several amino acids and do not involve frameshift mutations and nonsense mutations. "One or several" preferably means 1 - 20, more preferably 1 - 10, still more preferably 1 - 5, and particularly preferably 1 - 3. Examples of mutations in the transmembrane regions include a mutation in which one or several amino acids (e.g., Cys-Ser-Leu) are inserted between the leucine residue at position 14 and the tryptophan residue at position 15 of the wild-type YggB protein; a mutation in which the alanine residue at position 100 is substituted with another amino acid residue (e.g., an amino acid having a hydroxyl group in the side chain (Thr, Ser, or Tyr), preferably Thr); and a mutation in which the alanine residue at position 111 is substituted with another amino acid residue (e.g., Val or an amino acid having a hydroxyl group in the 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" means an amino acid residue corresponding to the amino acid residue at position X in SEQ ID NO: 8. The "position X" in the amino acid sequence means the Xth position counted from the N-terminus of the amino acid sequence, and the amino acid residue at the N-terminus is the amino acid residue at the 1st position. Note that the position of the amino acid residue indicates a relative position, and its absolute position may shift due to amino acid deletion, insertion, addition, etc. For example, the "amino acid residue at position 419 of the wild-type YggB protein" means an amino acid residue corresponding to the amino acid residue at position 419 in SEQ ID NO: 8. When one amino acid residue on the N-terminal side of position 419 is deleted, the 418th amino acid residue from the N-terminus shall be regarded as the "amino acid residue at position 419 of the wild-type YggB protein". Also, when one amino acid residue is inserted on the N-terminal side of position 419, the 420th amino acid residue from the N-terminus shall be regarded as 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 of 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 of the wild-type YggB protein.

[0049] In the amino acid sequence of any YggB protein, which amino acid residue is 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. The alignment can be performed, for example, using known gene analysis software. Specific software includes DNASIS manufactured by Hitachi Solutions, GENETYX manufactured by Genetics, etc. (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] The mutant yggB gene can be obtained by modifying the wild-type yggB gene to have the above-mentioned "specific mutation". The modification of DNA can be performed by known methods. Specifically, for example, as a site-directed mutagenesis method for introducing a target mutation into a target site of DNA, there are a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, H. A. Eds., Stockton press (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)) and a method using phage (Kramer, W. and Frits, H. J., Meth. In Enzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth. In Enzymol., 154, 367 (1987)). In addition, the mutant yggB gene can also be obtained by chemical synthesis.

[0051] Modifying a coryneform bacterium to have a mutant yggB gene can be achieved by introducing the mutant yggB gene into the coryneform bacterium. Further, modifying a coryneform bacterium to have a mutant yggB gene can also be achieved by introducing a mutation into the yggB gene that the bacterium has by natural mutation or mutagenesis treatment.

[0052] <1-2>Modification regarding the mutant acetyl-CoA hydrolase gene The coryneform bacterium of the present invention is modified to retain a mutant acetyl-CoA hydrolase gene encoding a mutant acetyl-CoA hydrolase having a substitution of another amino acid residue for the serine residue at position 383 in the amino acid sequence of wild-type acetyl-CoA hydrolase. Note that the coryneform bacterium of the present invention may be a coryneform bacterium modified to retain the mutant acetyl-CoA hydrolase gene in a coryneform bacterium having the ability to produce L-glutamic acid as described above, or may be a coryneform bacterium obtained by imparting the ability to produce L-glutamic acid to a coryneform bacterium modified to retain the mutant acetyl-CoA hydrolase gene. Further included are coryneform bacteria that have become capable of producing L-glutamic acid by being modified to retain the mutant acetyl-CoA hydrolase gene.

[0053] The acetyl-CoA hydrolase gene is a gene encoding acetyl-CoA hydrolase. Examples of the acetyl-CoA hydrolase gene include the acetyl-CoA hydrolase gene of coryneform bacteria. Specific examples of the acetyl-CoA hydrolase gene of coryneform bacteria include, for example, 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 the sequence from 2,729,376 to 2,730,884 in the genomic sequence registered in the NCBI database under GenBank Accession No. NC_003450 and is also called NCgl2480. The acetyl-CoA hydrolase encoded by the acetyl-CoA hydrolase gene of Corynebacterium glutamicum ATCC13032 is registered as GenBank accession No. WP_003858947.1. In addition, 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 mean a protein having an activity of catalyzing a reaction of hydrolyzing acetyl-CoA to produce coenzyme A and acetic acid and / or its reverse reaction (for example, EC 3.1.2.1). The same activity is also referred to as "acetyl-CoA hydrolase activity". Specifically, the acetyl-CoA hydrolase activity may be an activity of catalyzing a reaction of converting acetyl-CoA and H2O into coenzyme A and acetic acid and / or its reverse reaction. Acetyl-CoA hydrolase is also referred to as "Acetyl-CoA deacylase". Examples of the gene encoding acetyl-CoA hydrolase include the NCgl2480 gene. The nucleotide sequences of the acetyl-CoA hydrolase genes such as the NCgl2480 gene possessed by the bacterium to be modified and the amino acid sequences of the acetyl-CoA hydrolase encoded thereby can be obtained from public databases such as NCBI, for example.

[0055] In the present invention, the acetyl-CoA hydrolase gene having the "specific gene mutation" described below is also referred to as a mutant acetyl-CoA hydrolase gene, and the protein encoded thereby is also referred to as a mutant acetyl-CoA hydrolase. In the present invention, the acetyl-CoA hydrolase gene not having the "specific gene mutation" described below is also referred to as a wild-type acetyl-CoA hydrolase gene, and the protein encoded thereby is also referred to as a wild-type acetyl-CoA hydrolase. In the case of acetyl-CoA hydrolase, the change in the amino acid sequence caused by the "specific gene mutation" in the acetyl-CoA hydrolase gene is also referred to as the "specific gene mutation". The "wild type" referred to herein is a description for convenience in distinguishing from the "mutant type", and is not limited to those obtained naturally as long as they do not have the "specific gene mutation". Examples of the wild-type acetyl-CoA hydrolase include the acetyl-CoA hydrolase exemplified above, for example, a protein having the amino acid sequence shown in SEQ ID NO: 2. In addition, examples of the wild-type acetyl-CoA hydrolase include conservative variants of the acetyl-CoA hydrolase exemplified above (variants in which the original function is maintained) that do not have the "specific gene mutation".

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

[0057] Examples of the mutation at the amino acid residue at position 383 of the amino acid sequence of the wild-type acetyl-CoA hydrolase include, for example, substitution of the amino acid residue (missense mutation).

[0058] Examples of mutations at the 383rd amino acid residue of wild-type acetyl-CoA hydrolase include mutations that substitute the serine residue at the 383rd position of wild-type acetyl-CoA hydrolase with other amino acid residues. "Other amino acids" are not particularly limited as long as they are natural amino acids other than serine. Examples of "other amino acids" 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 the 383rd position may preferably be substituted with a hydrophilic amino acid (Lys, Glu, Thr, Asp, Asn, Gln, Arg, Cys, or His), and more preferably with Cys. Also, for example, the serine residue at the 383rd position may preferably be substituted with a sulfur-containing amino acid (Cys or Met), and more preferably with Cys.

[0059] In the present invention, the "amino acid residue at the Xth position of wild-type acetyl-CoA hydrolase" means, unless otherwise specified, the amino acid residue corresponding to the amino acid residue at the Xth position in SEQ ID NO: 2. The "Xth position" in the amino acid sequence means the Xth position counted from the N-terminus of the same amino acid sequence, and the amino acid residue at the N-terminus is the 1st amino acid residue. Note that the position of the amino acid residue indicates a relative position, and its absolute position may shift due to amino acid deletions, insertions, additions, etc. For example, the "amino acid residue at the 383rd position of wild-type acetyl-CoA hydrolase" means the amino acid residue corresponding to the amino acid residue at the 383rd position in SEQ ID NO: 2. When one amino acid residue on the N-terminal side of the 383rd position is deleted, the 382nd amino acid residue from the N-terminus shall be regarded as the "amino acid residue at the 383rd position of wild-type acetyl-CoA hydrolase". Also, when one amino acid residue is inserted on the N-terminal side of the 383rd position, the 384th amino acid residue from the N-terminus shall be regarded as the "amino acid residue at the 383rd position of wild-type acetyl-CoA hydrolase".

[0060] In the amino acid sequence of any acetyl-CoA hydrolase, which amino acid residue is the "amino acid residue corresponding 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. The alignment can be performed, for example, using known gene analysis software. Specific software includes DNASIS manufactured by Hitachi Solutions, GENETYX manufactured by Genetics, etc. (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] The mutant acetyl-CoA hydrolase gene can be obtained by modifying the wild-type acetyl-CoA hydrolase gene to have the above-mentioned "specific gene mutation". The modification of DNA can be carried out by known methods. Specifically, for example, as a site-directed mutagenesis method for introducing a desired mutation into a target site of DNA, a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, H. A. Eds., Stockton press (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)) and a method using phage (Kramer, W. and Frits, H. J., Meth. In Enzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth. In Enzymol., 154, 367 (1987)) can be mentioned. In addition, the mutant acetyl-CoA hydrolase gene can also 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 (for example, the nucleotide sequence shown in SEQ ID NO: 1). Further, acetyl-CoA hydrolase may be, for example, a protein having the amino acid sequence of the acetyl-CoA hydrolase exemplified above (for example, the amino acid sequence shown in SEQ ID NO: 2). In addition, the expression "having the (amino acid or base) sequence" means "including the (amino acid or base) sequence" unless otherwise specified, and also includes the case of "consisting of the (amino acid or base) sequence".

[0063] The wild-type acetyl-CoA hydrolase gene may also be a variant of the acetyl-CoA hydrolase gene exemplified above (for example, a gene having the nucleotide sequence shown in SEQ ID NO: 1) as long as its original function is maintained. Similarly, acetyl-CoA hydrolase may be a variant of the acetyl-CoA hydrolase exemplified above (for example, a protein having the amino acid sequence shown in SEQ ID NO: 2) as long as its original function is maintained. Such variants with the original function maintained are sometimes referred to as "conservative variants". The term "acetyl-CoA hydrolase gene" shall include, in addition to the acetyl-CoA hydrolase genes exemplified above, their conservative variants. Similarly, the term "acetyl-CoA hydrolase" shall include, in addition to the acetyl-CoA hydrolases exemplified above, their conservative variants. Examples of conservative variants include homologs and artificial variants of the acetyl-CoA hydrolase genes and acetyl-CoA hydrolases exemplified above.

[0064] "The original function is maintained" means that a variant of a gene or protein has a function (such as activity or property) corresponding to the function (such as activity or property) of the original gene or protein. "The original function is maintained" for a gene means that a variant of the gene encodes a protein in which the original function is maintained. That is, "the original function is maintained" for the acetyl-CoA hydrolase gene may mean that a variant of the acetyl-CoA hydrolase gene encodes a protein having acetyl-CoA hydrolase activity. Also, "the original function is maintained" for acetyl-CoA hydrolase may mean that a variant of 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 (such as acetyl-CoA and H2O) and measuring the production of the corresponding product (such as coenzyme A and acetic acid) that is enzyme- and substrate-dependent.

[0066] Examples of conservative variants are shown below.

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

[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 (for example, the amino acid sequence shown in SEQ ID NO: 2), as long as the original function is maintained. For example, the N-terminus and / or C-terminus of the encoded protein may be extended or shortened. The above "one or several" varies depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, but specifically, for example, it means 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, and particularly preferably 1 to 3.

[0069] The above substitution, deletion, insertion, and / or addition of one or several amino acids are conservative mutations that maintain the normal function of the protein. Representative conservative mutations are conservative substitutions. A conservative substitution means that when the substitution site is an aromatic amino acid, it is between Phe, Trp, and Tyr; when the substitution site is a hydrophobic amino acid, it is between Leu, Ile, and Val; when it is a polar amino acid, it is between Gln and Asn; when it is a basic amino acid, it is between Lys, Arg, and His; when it is an acidic amino acid, it is between Asp and Glu; and when it is an amino acid with a hydroxyl group, it is between Ser and Thr. Specific substitutions considered conservative substitutions include substitution of Ala with Ser or Thr, substitution of Arg with Gln, His, or Lys, substitution of Asn with Glu, Gln, Lys, His, or Asp, substitution of Asp with Asn, Glu, or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitution of Glu with Gly, Asn, Gln, Lys, or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg, or Tyr, substitution of Ile 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. In addition, the above amino acid substitutions, deletions, insertions, or additions also include those caused by naturally occurring mutations (mutant or variant) such as those based on individual differences or species differences of the organism from which the gene is derived.

[0070] In addition, as long as the original function is maintained, the acetyl-CoA hydrolase gene may be a gene encoding a protein having an amino acid sequence having, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more identity to the entire amino acid sequence.

[0071] In addition, as long as the original function is maintained, 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 base sequence (e.g., the base sequence shown in SEQ ID NO: 1), for example, a complementary sequence to the whole or part of the above base sequence. "Stringent conditions" refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. By way of example, conditions under which DNAs with high identity, for example, DNAs having 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more identity hybridize with each other and DNAs with lower identity do not hybridize with each other, or conditions for washing in ordinary Southern hybridization, that is, washing once, preferably 2 to 3 times, at a salt concentration and temperature corresponding to 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 can be mentioned.

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

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

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

[0075] The description regarding the conservative variants of the above-mentioned genes and proteins can also be applied mutatis mutandis to any protein and the genes encoding them. That is, the genes and proteins used for breeding L-glutamic acid-producing bacteria may each have, for example, the nucleotide sequences and amino acid sequences of known genes and proteins such as the genes and proteins exemplified above. Further, the genes and proteins used for breeding L-glutamic acid-producing bacteria may each be conservative variants of known genes and proteins such as the genes and proteins exemplified above. Specifically, for example, as long as the original function is maintained, the gene used for breeding L-glutamic acid-producing bacteria may be a gene encoding a protein 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.

[0076] As a method for modifying so as to retain the mutant acetyl-CoA hydrolase gene, there may be mentioned a method of introducing into a coryneform bacterium a mutant acetyl-CoA hydrolase gene in which a mutation for substituting the serine residue at the 383rd position is introduced into the coding region of the wild-type acetyl-CoA hydrolase gene, a method of introducing the same mutation into the coding region of the wild-type acetyl-CoA hydrolase gene possessed by the coryneform bacterium, and the like.

[0077] Modifying 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. Incidentally, modifying 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 possessed by the coryneform bacterium by natural mutation or mutagen treatment.

[0078] The introduction of the mutant acetyl-CoA hydrolase gene into a coryneform bacterium can be achieved by introducing the gene into the host chromosome. The introduction of the gene into the chromosome can be carried out, for example, by using homologous recombination (Miller, J. H. 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, B. L. Proc. Natl. Acad. Sci. U S A. 97:6640-6645 (2000)), methods using a plasmid containing a temperature-sensitive origin of replication, methods using a conjugative plasmid, methods using a suicide vector having no origin of replication functioning in the host, and transduction methods using phages. Specifically, a host can be transformed with recombinant DNA containing the mutant acetyl-CoA hydrolase gene, and the gene can be introduced onto the host chromosome by 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 homologous recombination occurs in a desired manner. For example, a linear DNA containing the mutant acetyl-CoA hydrolase gene and having base sequences homologous to the upstream and downstream of the target site on the chromosome at both ends of the gene is used to transform the host, and homologous recombination is caused at the upstream and downstream of the target site, respectively, so that the target site can be replaced with the gene. The recombinant DNA used for homologous recombination may be provided with 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, by performing homologous recombination targeting a base sequence present in multiple copies on the chromosome, multiple copies of the mutant acetyl-CoA hydrolase gene can be introduced into the chromosome.Examples of nucleotide sequences that exist in multiple copies on a chromosome include repetitive DNA sequences and inverted repeats present at both ends of transposons. Homologous recombination may also be performed targeting an appropriate nucleotide sequence on the chromosome, such as a gene unnecessary for the production of the target substance. Genes can also be randomly introduced onto the chromosome using transposons or Mini-Mu (Japanese Patent Application Laid-Open No. 2-109985, U.S. Patent No. 5,882,888, EP805867B1). Note that the method for modifying a chromosome using such homologous recombination is not limited to the introduction of a mutant acetyl-CoA hydrolase gene, and can be used for any modification of a chromosome, such as modification of an expression regulatory sequence.

[0079] Confirmation that a mutant acetyl-CoA hydrolase gene has been introduced onto the chromosome can be made 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] In addition, the introduction of the mutant acetyl-CoA hydrolase gene into coryneform bacteria can also be achieved by introducing a vector containing the gene into the host. For example, a DNA fragment containing the mutant acetyl-CoA hydrolase gene is ligated with a vector that functions in the host to construct an expression vector for the gene, and the host is transformed with the expression vector, whereby the gene can be introduced into coryneform bacteria. The DNA fragment containing the mutant acetyl-CoA hydrolase gene can be obtained, for example, by PCR using the genomic DNA of a microorganism having the mutant acetyl-CoA hydrolase gene as a template. As the vector, a vector capable of autonomous replication in the host cell can be used. The vector is preferably a multicopy vector. In addition, in order to select the transformant, the vector preferably has a marker such as an antibiotic resistance gene. The vector may also be provided with a promoter and a terminator for expressing the inserted gene. The vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid, etc.Examples of vectors capable of autonomous replication in Corynebacterium include, specifically, pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903 (1984)); plasmids having drug resistance genes obtained by modifying 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. Patent No. 5185262); pCRY2 and pCRY3 (Japanese Patent Laid-Open No. 1-191686); pAJ655, pAJ611, and pAJ1844 (Japanese Patent Laid-Open No. 58-192900); pCG1 (Japanese Patent Laid-Open No. 57-134500); pCG2 (Japanese Patent Laid-Open No. 58-35197); pCG4 and pCG11 (Japanese Patent Laid-Open No. 57-183799); pVK7 (Japanese Patent Laid-Open No. 10-215883); pVK9 (U.S. Patent Application Publication No. 2006 / 0141588); pVC7 (Japanese Patent Laid-Open No. 9-070291); pVS7 (WO2013 / 069634).

[0081] When introducing a gene, the gene only needs to be retained in the host so that it can be expressed. Specifically, the gene only needs to be 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. The "promoter that functions in the host" refers to a promoter having promoter activity in the host. The promoter may be a promoter derived from the host or a promoter derived from a heterologous source. The promoter may be the specific promoter of the gene to be introduced or the promoter of another gene.

[0082] A terminator for transcription termination can be arranged 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 terminator derived from a heterologous source. The terminator may be the specific terminator of the gene to be introduced or the terminator of another gene.

[0083] Regarding vectors, promoters, and terminators that can be used in various microorganisms, they are described in detail, for example, in "Basic Microbiology Course 8 Genetic Engineering, Kyoritsu Shuppan, 1987", and it is possible to use them.

[0084] Also, when introducing two or more genes, each gene may be retained in the host in an expressible manner. For example, all the genes may be retained on a single expression vector, or all may be retained on the chromosome. Also, each gene may be separately retained on a plurality of expression vectors, or may be separately retained on a single or a plurality of expression vectors and on the chromosome. Further, an operon may be constituted by two or more genes and introduced. Examples of "when introducing two or more genes" include cases where genes encoding two or more proteins (for example, enzymes) are introduced, cases where genes encoding two or more subunits constituting a single protein complex (for example, an enzyme complex) are introduced, and combinations thereof.

[0085] <2> Method for producing L-glutamic acid of the present invention The method of the present invention comprises culturing the bacterium of the present invention described in item <1> in a medium, accumulating L-glutamic acid in the medium and / or in the cells of the bacterium, and collecting L-glutamic acid from the medium and / or the cells. This is a method for producing L-glutamic acid. 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-form amino acids (also referred to as L-amino acids), etc. may be produced.

[0086] The medium to be used is not particularly limited as long as the bacterium of the present invention can grow and L-glutamic acid can be produced. As the medium, for example, a normal medium used for culturing bacteria such as coryneform bacteria can be used. As the medium, for example, a medium containing components selected from a carbon source, a nitrogen source, a phosphate source, a sulfur source, and other various organic and inorganic components as required can be used. The types and concentrations of the medium components may be appropriately set according to various conditions such as the type of bacterium used.

[0087] As the carbon source, specifically, for example, sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, maltose, isomaltose, molasses, starch hydrolyzate, biomass hydrolyzate, etc., organic acids such as acetic acid, fumaric acid, citric acid, succinic acid, etc., alcohols such as glycerol, crude glycerol, ethanol, etc., and fatty acids can be mentioned. As the carbon source, in particular, sugars can be mentioned. As the carbon source, more particularly, glucose and fructose can be mentioned. Sugars such as glucose and fructose may be used alone or in combination with other carbon sources as the carbon source. As the carbon source, for example, a sugar having fructose as a constituent sugar may be used. Examples of the sugar having fructose as a constituent sugar include fructose, sucrose, and fructooligosaccharide. The sugar having fructose as a constituent sugar may be used alone or in combination with other carbon sources as the carbon source. Incidentally, as the carbon source, plant-derived raw materials can be preferably used. Examples of the plant include corn, rice, wheat, soybeans, sugarcane, beet, and cotton. Examples of the plant-derived raw material include organs such as roots, stems, trunks, branches, leaves, flowers, and seeds, plant bodies containing them, and decomposition products of these plant organs. The utilization form of the plant-derived raw material is not particularly limited, and it can be used in any form such as raw products, squeezed juices, pulverized products, and purified products. As the carbon source, for example, cane molasses, beet molasses, high-test molasses, citrus molasses, or invert sugar may be used, or hydrolyzates of natural raw materials such as cellulose, starch, corn, cereal, tapioca, and cassava may be used. Also, pentoses such as xylose, hexoses such as glucose, or mixtures thereof can be obtained and used from, for example, plant biomass. 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. Since hemicellulose is generally more easily hydrolyzed than cellulose, hemicellulose in plant biomass may be hydrolyzed in advance to liberate pentoses, and then cellulose may be hydrolyzed to produce hexoses.Alternatively, xylose may be supplied, for example, by causing the bacterium of the present invention to have a conversion pathway from hexose such as glucose to xylose and converting it from hexose. Specifically, as the carbon source, for example, glucose may be used alone, or a mixture of two carbon sources such as glucose and fructose or glucose and sucrose may be used at an arbitrary ratio (for example, 3:7 to 7:3 by weight).

[0088] Specifically, as the nitrogen source, for example, ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract, and plant protein hydrolysates (HVP; for example, soybean protein hydrolyzate, soy sauce, pea soy sauce, etc.), ammonia, and urea can be mentioned. Ammonia gas or aqueous ammonia used for pH adjustment may be used as the nitrogen source. As the nitrogen source, one kind of nitrogen source may be used, or two or more kinds of nitrogen sources may be used in combination.

[0089] Specifically, as the phosphate source, for example, phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate can be mentioned. As the phosphate source, one kind of phosphate source may be used, or two or more kinds of phosphate sources may be used in combination.

[0090] Specifically, as the sulfur source, for example, inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione can be mentioned. As the sulfur source, one kind of sulfur source may be used, or two or more kinds of sulfur sources may be used in combination.

[0091] As other various organic and inorganic components, specifically, 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 such as peptone, casamino acid, yeast extract, and plant protein hydrolysates (HVP; for example, soybean protein hydrolysate, soy sauce, pea soy sauce, etc.) containing these. Other various organic and inorganic components include antifoaming agents, medium osmotic pressure regulators, and osmotic pressure compensators. 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.). As antifoaming agents, those in any form such as liquid, paste, solid, powder, emulsion, and wax can be used. Examples of medium osmotic pressure regulators include salts such as sodium chloride and potassium chloride, and polysaccharides (such as sorbitol and dextrin) that cannot be assimilated by microorganisms. Examples of osmotic pressure compensators include potassium ions, betaine (glycine betaine), molasses (especially sugar beet molasses), glutamic acid, and trehalose. Also, polymers selected from the group consisting of water-soluble cellulose derivatives, water-soluble polyvinyl compounds, polyvinyl compounds soluble in polar organic solvents, water-soluble starch derivatives, alginates, and polyacrylates can be mentioned as components that may be added to the medium. These other various organic and inorganic components may be used singly or in combination of two or more components.

[0092] In addition, when using auxotrophic mutants that require amino acids or the like for growth, it is preferable to supplement the nutrients required by the medium.

[0093] Also, it is 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 bacteria of the present invention can grow and L-glutamic acid is produced. The culture can be carried out under normal conditions used for culturing bacteria such as coryneform bacteria. The culture conditions may be appropriately set according to various conditions such as the type of bacteria used.

[0095] The culture can be carried out using a liquid medium. As a method of liquid culture, for example, the methods described in "Biotechnology Textbook Series 13 Culture Engineering, Toshitomi Yoshida, Corona Publishing Co., Ltd., 1998" can be utilized. That is, as liquid culture, for example, surface culture, deep culture, membrane (dialysis membrane, follow fiber, etc.) separation type culture, or immobilized microorganism culture can be utilized. Also, as a culture apparatus, for example, an aeration agitation type culture apparatus, an air lift type culture apparatus, a packed bed type culture apparatus, or a fluidized bed type culture apparatus can be utilized. During the culture, the methods described in "Fundamentals of Fermentation Engineering, The Society Publishing Center, 1988" can be utilized. During the culture, the bacteria of the present invention cultured on a solid medium such as an agar medium may be directly inoculated into the liquid medium, or the bacteria of the present invention subcultured in a liquid medium may be inoculated into the liquid medium for main culture. That is, the culture may be divided into subculture and main culture. In that case, the culture conditions for subculture and main culture may be the same or different. The amount of the bacteria of the present invention contained in the medium at the start of the culture is not particularly limited. The main culture may be carried out, for example, by inoculating 1 to 50% (v / v) of the subculture solution into the medium for main culture. Also, for example, the subculture process may include two or more subculture steps in order to obtain the amount of bacteria required for the main culture process. Also, the subculture solution may be inoculated only at the start of the main culture, or in addition to the start of the main culture, it may be additionally inoculated during the main culture.

[0096] The cultivation can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. Examples of the combination include a culture in which fed-batch cultures are connected in two or more stages, a culture in which continuous cultures are connected in two or more stages, etc. The medium at the start of cultivation is also referred to as the "initial medium". Also, the medium supplied to the culture system (fermenter) in fed-batch culture or continuous culture is also referred to as the "fed medium". Further, supplying the fed medium to the culture system in fed-batch culture or continuous culture is also referred to as "feeding". When the cultivation is carried out separately into a seed culture and a main culture, for example, both the seed culture and the main culture may be carried out by batch culture. Also, for example, the seed culture may be carried out by batch culture and the main culture may be carried out by fed-batch culture or continuous culture. Also, for example, the seed culture may be carried out by fed-batch culture and the main culture may be carried out by batch culture. The fed medium may be supplied, for example, from a location not in contact with the liquid surface of the medium in the upper part of the culture tank, from a location inside the medium such as the middle part or the lower part of the culture tank, or from both the upper part and the middle part of the culture tank. The mode of supplying the fed medium from a location inside the medium 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 fed medium, or both. The types of components contained in the initial medium may or may not be the same as the types of components contained in the fed medium. Also, 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 fed medium. Also, two or more fed media having different types and / or concentrations of contained components may be used. For example, when feeding is carried out intermittently a plurality of times, the types and / or concentrations of components contained in each fed medium 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 fed medium may be sucrose.

[0098] Sterilization of the medium may or may not be performed. The sterilization of the medium may be performed for the purpose of preventing contamination by miscellaneous bacteria. The sterilization of the medium can be paraphrased as sterilization or disinfection. Examples of the method for sterilizing the medium include sterilization under high temperature and high pressure conditions, sterilization by UV irradiation, disinfection using a filter or a membrane, etc. The sterilization of the medium may be performed batchwise or continuously. For example, as a method of performing sterilization under high temperature and high pressure conditions batchwise, autoclave sterilization, batch sterilization performed in a culture tank, etc. can be mentioned. Also, for example, as a method of continuously performing sterilization under high temperature and high pressure conditions, continuous sterilization equipped with a plate type heat exchanger can be mentioned. Also, the sterilization of sugar may be performed simultaneously with other medium components or separately from other components. Preferably, sugar and other components may be sterilized separately.

[0099] The concentration of the carbon source in the medium is not particularly limited as long as the bacteria of the present invention can grow and L-glutamic acid is produced. The concentration of the carbon source in the medium may be, for example, as high as possible within the range where the production of L-glutamic acid is not inhibited. The concentration of the carbon source in the medium may be, for example, as an initial concentration (concentration in the initial medium), 1 to 50 w / v%, preferably 1 to 30 w / v%, more preferably 3 to 10 w / v%. Also, a carbon source may be appropriately added to the medium additionally. For example, a carbon source may be added to the medium additionally according to the consumption of the carbon source accompanying the progress of fermentation. Also, in fed-batch culture or continuous culture, the supply amount of the carbon source may be an amount that becomes a sufficient condition (a condition of supplying an amount in excess of the carbon assimilation amount (ability) of the bacteria of the present invention) or a limiting condition (a condition of supplying an amount insufficient compared to the carbon assimilation amount (ability) of the bacteria of the present invention).

[0100] Cultivation may be carried out, for example, using a liquid medium under aerobic or microaerobic conditions. "Aerobic conditions" means that the dissolved oxygen concentration in the liquid medium is 0.33 ppm or more, which is the detection limit by an oxygen membrane electrode, and preferably may be 1.5 ppm or more. The oxygen concentration under aerobic conditions may be controlled, for example, to 5 to 50% of the saturated oxygen concentration, preferably about 10%. "Microaerobic conditions" may mean conditions where 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 less, 0.25 ppm or less, 0.20 ppm or less, 0.15 ppm or less, 0.10 ppm or less, or 0.05 ppm or less. 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, cultivation can be carried out by aerated cultivation, shaking cultivation, stirring cultivation or a combination thereof. The pH of the medium may be, for example, pH 3 to 10, preferably pH 4.0 to 9.5. During cultivation, the pH of the medium can be adjusted as necessary. The pH of the medium can be adjusted using various alkaline or acidic substances such as ammonia gas, aqueous ammonia, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, etc. The cultivation temperature may be, for example, 20 to 40°C, preferably 25°C to 37°C. In this cultivation, the cultivation temperature may be changed in two or more stages. For example, as disclosed in Journal of Industrial Microbiology & Biotechnology (2002) 28, 333-337, the cultivation temperature may be shifted to the higher temperature side from 33°C to 37 to 40°C. The cultivation period may be, for example, 10 hours to 120 hours. Cultivation may be continued, for example, until the carbon source in the medium is consumed or until the activity of the bacteria of the present invention disappears. By culturing the bacteria of the present invention under such conditions, L-glutamic acid accumulates in the medium and / or in the bacterial cells.

[0101] In addition, using a liquid medium adjusted to conditions under which L-glutamic acid precipitates, cultivation can be carried out while precipitating L-glutamic acid in the medium. Examples of the conditions under which L-glutamic acid precipitates 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). Further, when using a liquid medium adjusted to conditions under which L-glutamic acid precipitates, by adding pantothenic acid to the medium, crystallization can be carried out more efficiently (WO2004 / 111258). Further, when using a liquid medium adjusted to conditions under which L-glutamic acid precipitates, by adding crystals of L-glutamic acid as seed crystals to the medium, crystallization can be carried out more efficiently (EP1233069A). Further, when using a liquid medium adjusted to conditions under which L-glutamic acid precipitates, by adding crystals of L-glutamic acid and crystals of L-lysine as seed crystals to the medium, crystallization can be carried out more efficiently (EP1624069A).

[0102] The fermentation broth can be treated, for example, with a hydrocyclone. As the hydrocyclone, for example, those made of ceramic, stainless steel or resin in a general shape with a cylindrical part diameter of 10 to 110 mm can be used. The feed amount of the fermentation broth to the hydrocyclone can be set, for example, according to the cell concentration and L-glutamic acid concentration in the fermentation broth. The feed amount of the fermentation broth to the hydrocyclone can be, for example, 2 to 1200 L / min.

[0103] The production of L-glutamic acid can be confirmed by known methods used for the detection or identification of compounds. Such methods include, for example, HPLC, LC / MS, GC / MS, and NMR. These methods can be used alone or in appropriate combinations.

[0104] The recovery of L-glutamic acid from the fermentation broth can be carried out by known methods used for the separation and purification of compounds. Such methods include, for example, the ion exchange resin method (Nagai, H. et al., Separation Science and Technology, 39(16), 3691-3710), the precipitation method, the membrane separation method (Japanese Patent Laid-Open No. 9-164323, Japanese Patent Laid-Open No. 9-173792), and the crystallization method (WO2008 / 078448, WO2008 / 078646). These methods can be used alone or in appropriate combinations. When L-glutamic acid accumulates in the cells, for example, the cells are disrupted by ultrasonic waves or the like, and L-glutamic acid can be recovered from the supernatant obtained by removing the cells by centrifugation by the ion exchange resin method or the like. The recovered L-glutamic acid may be in the free form, its salt, or a mixture thereof. Examples of the salt include sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, and potassium salt. Specifically, for example, it may be free L-glutamic acid, sodium L-glutamate (e.g., monosodium L-glutamate; MSG), ammonium L-glutamate (e.g., monoammonium L-glutamate), or a mixture thereof. For example, ammonium L-glutamate in the fermentation broth is crystallized by adding an acid, and sodium L-glutamate (MSG) can be obtained by adding an equimolar amount of sodium hydroxide to the crystals. Activated carbon may be added for decolorization before and after crystallization (see Tetsuya Kawakita, "Industrial Crystallization of Sodium Glutamate," Journal of the Japan Society of Seawater Science, Vol. 56, No. 5). Sodium L-glutamate crystals can be used, for example, as a umami seasoning. Sodium L-glutamate crystals may also be mixed with nucleic acids such as sodium guanylate and sodium inosinate, which also have umami, and used as a seasoning.

[0105] Also, when L-glutamic acid precipitates in the medium, it can be recovered by centrifugation, filtration, or the like. Further, the L-glutamic acid precipitated in the medium may be isolated together after crystallizing the L-glutamic acid dissolved in the medium.

[0106] In addition, the recovered L-glutamic acid may contain components such as bacterial cells, culture medium components, moisture, and bacterial metabolic by-products, in addition to L-glutamic acid. 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, particularly preferably 95% (w / w) or more (Patent No. 1214636, US Patent No. 5431933, US Patent No. 4956471, US Patent No. 4777051, US Patent No. 4946654, US Patent No. 5840358, US Patent No. 6238714, US Patent Application Publication No. 2005 / 0025878).

Examples

[0107] Hereinafter, the present invention will be described more specifically with reference to non-limiting examples.

[0108] <1>Construction of a modified strain of Corynebacterium glutamicum <1-1>Construction of a vector for introducing the wild-type acetyl-CoA hydrolase gene Using the chromosomal DNA of C. glutamicum ATCC 13869 (strain 2256) having the wild-type acetyl-CoA hydrolase gene as a template, a DNA fragment containing the wild-type acetyl-CoA hydrolase gene was amplified by PCR using the primers of SEQ ID NO: 5 and SEQ ID NO: 6. The amplified DNA fragment and pVK9 (US Patent Application Publication No. 2006 / 0141588) cut with bamHI and pstI were ligated by an infusion reaction 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 shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0109] <1-2>Construction of Vector for Introducing Mutant Acetyl-CoA Hydrolase Gene Using the chromosomal DNA of a derivative strain of C. glutamicum ATCC 13869 (strain 2256) containing a mutant acetyl-CoA hydrolase gene having a mutation (S383C) in which the 383rd serine residue of wild-type acetyl-CoA hydrolase is replaced with a cysteine residue as a template, a DNA fragment containing the mutant acetyl-CoA hydrolase gene was amplified by PCR using the primers of SEQ ID NO: 5 and SEQ ID NO: 6. By ligating the amplified DNA fragment and pVK9 digested with bamHI and pstI by an infusion reaction, a vector for introducing the mutant acetyl-CoA hydrolase gene (pVK9-Acetyl-CoA hydrolase (S383C)) was obtained. 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 shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0110] <1-3>Construction of Modified Strains of Corynebacterium glutamicum Transform the C. glutamicum 2256ΔsucAΔldhA yggB* strain (WO2014 / 185430) with each constructed vector for introducing a mutant gene. Select strains from the obtained transformants according to the method described in WO2006 / 057450 to obtain a wild-type acetyl-CoA hydrolase gene-introduced strain and a mutant acetyl-CoA hydrolase gene-introduced strain.

[0111] The constructed vectors for introducing wild-type acetyl-CoA hydrolase gene (pVK9-Acetyl-CoA hydrolase (WT)) and mutant acetyl-CoA hydrolase gene (pVK9-Acetyl-CoA hydrolase (S383C)) were each introduced into the C. glutamicum 2256ΔsucAΔldhA yggB* strain alone to obtain a wild-type acetyl-CoA hydrolase gene-introduced strain and a mutant acetyl-CoA hydrolase gene-introduced strain. In addition, a control strain was obtained by introducing pVK9 alone into the C. glutamicum 2256ΔsucAΔldhA yggB* strain.

[0112] Note that the C. glutamicum 2256ΔsucAΔldhA yggB* strain is an L-glutamic acid-producing strain derived from the C. glutamicum 2256 strain (ATCC 13869), lacking the ldhA gene and sucA gene and having 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]

Table 1

[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 with shaking at 120 rpm using a box shaker (ABLE ML-190) at 31.5°C. Sampling of the culture broth was performed 25 hours after the start of the culture. 0.05 ml of the sampled culture broth was added to 4.95 ml of 0.1N HCl solution for 100-fold dilution, and the absorbance (OD) at 620 nm was measured. Also, after centrifuging the sampled culture broth at 15,000 rpm for 1 minute, the L-glutamic acid concentration in the supernatant was quantified using a biotech analyzer BF7 (Oji Scientific Instruments Co., Ltd.), and the sugar yield of L-glutamic acid was calculated.

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

Industrial Applicability

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

[0118] 〔Explanation of 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) Accession No. 5 and Accession No. 6: Primer Accession No. 7: Nucleotide sequence of the wild-type yggB gene of Corynebacterium glutamicum 2256 (ATCC 13869) Accession No. 8: Amino acid sequence of the wild-type YggB protein of Corynebacterium glutamicum 2256 (ATCC 13869) Accession No. 9: Nucleotide sequence of the mutant yggB gene (V419::IS) Accession No. 10: Amino acid sequence of the mutant YggB protein (V419::IS)

Claims

1. Corynebacterium glutamicum having the ability to produce L-glutamic acid, which is modified to retain 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 the wild-type acetyl-CoA hydrolase with a cysteine residue, wherein the mutant acetyl-CoA hydrolase comprises the amino acid sequence shown in SEQ ID NO: 4, Corynebacterium glutamicum.

2. which is modified to retain a mutant yggB gene, wherein the mutant yggB gene is a gene encoding a protein having the amino acid sequence of SEQ ID NO: 10, Corynebacterium glutamicum according to claim 1.

3. The mutant acetyl-CoA hydrolase gene according to claim 1 or 2, wherein the mutant acetyl-CoA hydrolase gene comprises the nucleotide sequence shown in SEQ ID NO: 3 or a variant due to degeneracy of the same nucleotide sequence, Corynebacterium glutamicum.

4. A method for producing L-glutamic acid, comprising: culturing Corynebacterium glutamicum according to claim 1 or 2 in a medium, accumulating L-glutamic acid in the medium and / or in the bacterial cells of the bacterium, and collecting L-glutamic acid from the medium and / or the bacterial cells. Production method.

5. A method for producing L-glutamic acid, comprising: culturing Corynebacterium glutamicum according to claim 3 in a medium, accumulating L-glutamic acid in the medium and / or in the bacterial cells of the bacterium, and collecting L-glutamic acid from the medium and / or the bacterial cells. Production method.

6. A mutant acetyl-CoA hydrolase comprising the amino acid sequence shown in SEQ ID NO:

4.

7. A mutant acetyl-CoA hydrolase gene encoding the mutant acetyl-CoA hydrolase according to claim 6.

8. ​ ​ ​ The variant acetyl-CoA hydrolase gene according to claim 7, comprising the nucleotide sequence shown in SEQ ID NO: 3 or a variant due to degeneracy of the nucleotide sequence.

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