L-glutamic acid-producing strain and method for producing l-glutamic acid

Abstract

The present application provides a method for producing L-glutamic acid and a bacterium used in the method. L-glutamic acid is produced by culturing in a culture medium a coryneform bacterium having an L-glutamic acid-producing ability, which is modified in a manner that a mutant glyA gene encoding a mutant serine hydroxymethyltransferase having a substitution of a glycine residue at position 265 in the amino acid sequence of a wild-type serine hydroxymethyltransferase to another amino acid residue, and collecting L-glutamic acid from the culture medium and / or the bacterial cells.

Description

Technical Field

[0001] This invention relates to the fermentation industry, and more specifically to a method for producing L-glutamic acid and the bacteria used in this method. L-glutamic acid is industrially useful as a seasoning ingredient, etc. Background Technology

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

[0003] Serine hydroxymethyltransferase is an enzyme that catalyzes the hydroxymethyl transfer reaction of serine and / or glycine (https: / / www.genome.jp / entry / 2.1.2.1), but its relationship with L-glutamate production is unknown.

[0004] Existing technical documents

[0005] Non-patent literature

[0006] Non-patent literature 1: Akashi Kunihiko et al., Amino Acid Fermentation, Academic Society Publications Center, pp. 195-215, 1986.

[0007] Content of the invention

[0008] The problem that the invention aims to solve

[0009] The subject of this invention is to develop new technologies to improve the L-glutamic acid production capacity of bacteria, and to provide an effective method for producing L-glutamic acid and bacteria for use in the method.

[0010] Methods for solving problems

[0011] In order to solve the above-mentioned problems, the inventors conducted in-depth research and found that by modifying rod-shaped bacteria in a way that induces a specific mutation in serine hydroxymethyltransferase, the L-glutamic acid production capacity of the bacteria can be improved, thus completing the present invention.

[0012] That is, the present invention is as follows. [1]

[0014] A rod-shaped bacterium that has the ability to produce L-glutamic acid.

[0015] The rod-shaped bacteria were modified to retain the mutant glyA gene encoding a mutant serine hydroxymethyltransferase, which has a substitution of the glycine residue at position 265 in the amino acid sequence of the wild-type serine hydroxymethyltransferase for other amino acid residues. [2]

[0017] According to the rod-shaped bacteria described in [1], the other amino acids mentioned above are lysine, glutamic acid, threonine, serine, aspartic acid, asparagine, glutamine, arginine, cysteine, histidine, or tyrosine. [3]

[0019] According to the rod-shaped bacteria described in [1], the other amino acid mentioned above is serine. [4]

[0021] The rod-shaped bacteria according to any one of [1] to [3], wherein the above-mentioned wild-type serine hydroxymethyltransferase is the protein described in (a), (b) or (c) below.

[0022] (a) A protein containing the amino acid sequence shown in sequence number 2;

[0023] (b) Proteins containing an amino acid sequence with 1 to 10 amino acid residues of substitution, deletion, insertion and / or addition in the amino acid sequence shown in Serial No. 2 and having serine hydroxymethyltransferase activity.

[0024] (c) A protein containing an amino acid sequence that is more than 90% identical to the amino acid sequence shown in sequence number 2 and has serine hydroxymethyltransferase activity. [5]

[0026] The rod-shaped bacteria according to any one of [1] to [4], wherein the rod-shaped bacteria are bacteria belonging to the genus Corynebacterium. [6]

[0028] The rod-shaped bacteria according to any one of [1] to [4], wherein the rod-shaped bacteria is Corynebacterium glutamicum. [7]

[0030] The rod-shaped bacteria according to any one of [1] to [6] are modified in a manner that preserves the mutant yggB gene.

[0031] The aforementioned mutant yggB gene is a gene that encodes a protein with an amino acid sequence consisting of one or more amino acid substitutions, deletions, insertions, and / or additions in the amino acid sequence at sequence number 8. [8]

[0033] According to the rod-shaped bacteria described in [7], the mutant yggB gene is a gene encoding a protein with an amino acid sequence having sequence number 10. [9]

[0035] A method for producing L-glutamic acid, the method comprising:

[0036] Culturing the rod-shaped bacteria described in any one of [1] to [8] in a culture medium, so that L-glutamate accumulates in the culture medium and / or in the cells of the bacteria; and

[0037] L-glutamic acid was collected from the above-mentioned culture medium and / or the above-mentioned bacterial cells.

[10]

[0039] A mutant serine hydroxymethyltransferase having a substitution of glycine residue at position 265 in the amino acid sequence of wild-type serine hydroxymethyltransferase with other amino acid residues.

[11]

[0041] According to the mutant serine hydroxymethyltransferase described in

[10] , the other amino acids mentioned above are lysine, glutamic acid, threonine, serine, aspartic acid, asparagine, glutamine, arginine, cysteine, histidine, or tyrosine.

[12]

[0043] According to the mutant serine hydroxymethyltransferase described in

[10] , the other amino acids mentioned above are serine.

[13]

[0045] The mutant serine hydroxymethyltransferase according to any one of

[10] to

[12] , wherein the wild-type serine hydroxymethyltransferase is the protein described in (a), (b) or (c) below.

[0046] (a) A protein containing the amino acid sequence shown in sequence number 2;

[0047] (b) Proteins containing an amino acid sequence with 1 to 10 amino acid residues of substitution, deletion, insertion and / or addition in the amino acid sequence shown in Serial No. 2 and having serine hydroxymethyltransferase activity.

[0048] (c) A protein containing an amino acid sequence that is more than 90% identical to the amino acid sequence shown in sequence number 2 and has serine hydroxymethyltransferase activity.

[14]

[0050] A mutant glyA gene that encodes any of the mutant serine hydroxymethyltransferases described in any of

[10] to

[13] . Attached Figure Description

[0051] Figure 1 This is a graph showing the accumulation of L-glutamic acid in wild-type glyA gene-introduced strains and mutant glyA gene-introduced strains.

[0052] Figure 2 This is a graph showing the yield of L-glutamic acid relative to sugar in wild-type glyA gene-introduced strains and mutant glyA gene-introduced strains. Detailed Implementation

[0053] The present invention will now be described in detail.

[0054] The method of the present invention is a method for producing L-glutamic acid, comprising: culturing rod-shaped bacteria capable of producing L-glutamic acid in a culture medium, accumulating L-glutamic acid in the culture medium and / or within the bacterial cells; and collecting L-glutamic acid from the culture medium and / or the bacterial cells, wherein the rod-shaped bacteria are modified in a manner that maintains a specific gene mutation. The bacteria used in this method are referred to as "the bacteria of the present invention".

[0055] <1> The bacteria of the present invention

[0056] The bacteria of the present invention are rod-shaped bacteria that have been modified in a manner that maintains a specific gene mutation and are capable of producing L-glutamic acid.

[0057] <1-1> Cork-shaped bacteria with L-glutamate production capacity

[0058] In this invention, "rod-shaped bacteria capable of producing L-glutamic acid" refers to rod-shaped bacteria that, when cultured in a culture medium, produce L-glutamic acid and accumulate it in the culture medium and / or within the bacterial cells to a recoverable extent. The rod-shaped bacteria capable of producing L-glutamic acid can be rod-shaped bacteria that can accumulate a greater amount of L-glutamic acid in the culture medium and / or within the bacterial cells than unmodified strains. "Unmodified strain" refers to a control strain that has not been modified in a manner that maintains a specific gene mutation. That is, wild-type strains and parental strains can be examples of unmodified strains. Furthermore, the rod-shaped bacteria capable of producing L-glutamic acid can be rod-shaped bacteria capable of accumulating, preferably, 0.5 g / L or more, and more preferably 1.0 g / L or more, of the target L-amino acid in the culture medium.

[0059] The bacteria of the present invention can produce L-glutamic acid alone, or in the form of a mixture of L-glutamic acid and one or more amino acids other than L-glutamic acid, such as L-somatic amino acids (also called L-amino acids). There are no particular limitations on the L-amino acids, 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.

[0060] In this invention, unless otherwise specified, the terms "glutamic acid" and "amino acid" refer to L-glutamic acid and L-amino acid, respectively.

[0061] Furthermore, in this invention, unless otherwise specified, the terms "L-glutamic acid" and "L-amino acid" refer to free L-glutamic acid, free L-amino acids, their salts, or mixtures thereof. Salts will be explained later.

[0062] Examples of rod-shaped bacteria include those belonging to genera such as Corynebacterium, Brevibacterium, and Microbacterium.

[0063] The following are specific examples of rod-shaped bacteria.

[0064] Corynebacterium acetoacidophilum

[0065] Corynebacterium acetoglutamicum

[0066] Corynebacterium alkanolyticum

[0067] Corynebacterium callunae

[0068] Corynebacterium crenatum

[0069] Corynebacterium glutamicum

[0070] Corynebacterium lilium

[0071] Corynebacterium melassecola

[0072] Corynebacterium thermoaminogenes (Corynebacterium efficiens)

[0073] Corynebacterium herculis

[0074] Brevibacterium divaricatum (Corynebacterium glutamicum)

[0075] Brevibacterium flavum (Corynebacterium glutamicum)

[0076] Brevibacterium immariophilum

[0077] Brevibacterium lactofermentum (Corynebacterium glutamicum)

[0078] Brevibacterium roseum

[0079] Brevibacterium saccharolyticum

[0080] Brevibacterium thiogenitalis

[0081] Corynebacterium ammoniagenes (Corynebacterium stationis)

[0082] Brevibacterium album

[0083] Brevibacterium cerinum

[0084] Microbacterium ammoniaphilum

[0085] As a rod-shaped bacterium, Corynebacterium glutamicum (formerly known as Brevibacterium lactofermentum) is a particularly notable example.

[0086] The following strains are examples of rod-shaped bacteria.

[0087] Corynebacterium acetoacetate ATCC 13870

[0088] Corynebacterium acetylglucosamine ATCC 15806

[0089] Corynebacterium alkylate ATCC 21511

[0090] Corynebacterium tumefaciens ATCC 15991

[0091] Corynebacterium obliterans AS1.542

[0092] Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734

[0093] Corynebacterium lilyense ATCC 15990

[0094] Corynebacterium molasses ATCC 17965

[0095] Effective Corynebacterium (thermogenic ammonia-producing rod-shaped bacteria) AJ12340 (FERM BP-1539)

[0096] Corynebacterium tumefaciens ATCC 13868

[0097] Corynebacterium glutamicum (ATCC 14020)

[0098] Corynebacterium glutamicum (Yellow Short Bacterium) ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205)

[0099] Marine breve ATCC 14068

[0100] Corynebacterium lactis (glutamate-producing bacteria) ATCC 13869

[0101] Rose-colored short bacillus ATCC 13825

[0102] Brevibacterium saccharolyticum ATCC 14066

[0103] Thiobacillus thiocyanate ATCC 19240

[0104] Corynebacterium ammoniagenes (Stasisobacterium tarda) ATCC 6871, ATCC 6872

[0105] Leukobacter pylori ATCC 15111

[0106] Short bacillus cereus ATCC 15112

[0107] Ammonia-loving microbes ATCC 15354

[0108] As a rod-shaped bacterium, *Bryophyte lactis* (new name: *Corynebacterium glutamicum*) ATCC13869 can be specifically cited as an example.

[0109] In addition, as a rod-shaped bacterium, the strain 2256ΔsucAΔldhA yggB* (WO2014 / 185430) of Corynebacterium glutamicum ATCC 13869, which has the deletion of the ldhA and sucA genes and has an IS mutation (V419::IS) in the yggB gene, can be cited.

[0110] It should be noted that the genus *Corynebacterium* also includes bacteria that were previously classified as *Brevibacteria* but are now incorporated into the genus *Corynebacterium* (Int. J. Syst. Bacteriol., 41, 255(1991)). Additionally, the genus *Corynebacterium tarda* also includes bacteria that were previously classified as *Corynebacterium ammonia-producing* but have been reclassified as *Corynebacterium tarda* based on 16S rRNA base sequence analysis, etc. (Int. J. Syst. Evol. Microbiol., 60, 874-879(2010)).

[0111] These strains can be obtained, for example, from the American Type Culture Collection (ATCC) (address: 12301 Parklawn Drive, Rockville, Maryland 20852; POBox 1549, Manassas, VA 20108, United States of America). Each strain is assigned a corresponding accession number, which can be used to obtain the strain (see http: / / www.atcc.org / ). The accession numbers corresponding to each strain are listed in the ATCC catalog. Alternatively, these strains can be obtained, for example, from institutions that hold the respective strains.

[0112] The bacteria of the present invention can be bacteria that originally possess the ability to produce L-glutamate, or bacteria that have been modified in a manner that maintains the ability to produce L-glutamate. Bacteria with the ability to produce L-glutamate can be obtained, for example, by imparting the ability to produce L-glutamate to the bacteria described above, or by enhancing the L-glutamate production ability of the bacteria described above.

[0113] The ability to produce L-glutamate can be endowed or enhanced using methods previously employed in the breeding of amino acid-producing bacteria such as Corynebacterium or Escherichia (see Amino Acid Fermentation, Gakuen Publishing Center, May 30, 1986, pp. 77-100). Examples of such methods include: obtaining auxotrophic mutants, obtaining L-glutamate analog-resistant strains, obtaining metabolic control mutants, and creating recombinant strains with enhanced activity of L-glutamate biosynthetic enzymes. In the breeding of L-glutamate-producing bacteria, the endowed properties such as auxotrophicity, analog resistance, and metabolic control mutations can be a single property, or two or more. Furthermore, in the breeding of L-glutamate-producing bacteria, the enhanced activity of L-glutamate biosynthetic enzymes can also be a single property, or two or more. In addition, the endowment of properties such as nutritional deficiencies, analog resistance, and metabolic control mutations can be combined with the enhancement of the activity of biosynthetic enzymes.

[0114] Auxotrophic mutants, analog-resistant mutants, or metabolic control mutants capable of producing L-glutamate can be obtained by subjecting parental or wild-type strains to conventional mutagen treatment, and selecting from the resulting mutants those exhibiting auxotrophic, analog-resistant, or metabolic control mutations and possessing L-amino acid production capabilities. Common mutagen treatments include X-ray or ultraviolet irradiation, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0115] Furthermore, the ability to produce L-glutamate can also be enhanced by increasing the activity of enzymes involved in the biosynthesis of L-glutamate. Enzyme activity can be enhanced, for example, by modifying bacteria to increase the expression of the gene encoding the enzyme. Methods for enhancing gene expression are described in WO00 / 18935, EP1010755A, etc.

[0116] Furthermore, the ability to produce L-glutamate can also be enhanced by reducing the activity of enzymes that catalyze reactions that branch off from the L-glutamate biosynthetic pathway to produce compounds other than L-glutamate. It should be noted that the term "enzymes that catalyze reactions that branch off from the L-glutamate biosynthetic pathway to produce compounds other than L-glutamate" also includes enzymes involved in the breakdown of L-glutamate.

[0117] The following provides specific examples of L-glutamic acid producing bacteria and methods for imparting or enhancing L-glutamic acid production capacity. It should be noted that the properties of the L-glutamic acid producing bacteria and the modifications used to impart or enhance L-glutamic acid production capacity described below can be used alone or in appropriate combinations.

[0118] As a method for conferring or enhancing the ability to produce L-glutamate, one example is modifying bacteria in a manner that increases the activity of one or more enzymes selected from L-glutamate biosynthetic enzymes. Such enzymes are not particularly limited and can be listed as follows: glutamate dehydrogenase (gdhA), glutamine synthase (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), and phosphoenol. Pyruvate synthase (ppsA), enolase (eno), phosphoglycerate mutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose diphosphate aldolase (fbp), glucose phosphate isomerase (pgi), 6-phosphogluconate dehydratase (edd), 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), and transhydrogenase (pntAB). It should be noted that the parentheses are examples of genes encoding the enzyme (and the same applies in the following description). 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.

[0119] As an example of rod-shaped bacteria that have been modified to increase the expression of the glutamate synthase gene (gltBD), the bacteria disclosed in WO99 / 07853 can be cited.

[0120] Furthermore, as a method for conferring or enhancing the ability to produce L-glutamate, one example is the modification of bacteria by reducing the activity of one or more enzymes selected from those that catalyze reactions that branch off from the biosynthetic pathway of L-glutamate to produce compounds other than L-glutamate. There are no particular limitations on such enzymes, and examples include isocitrate lyase (aceA), α-ketoglutarate dehydrogenase (sucA,odhA), acetyllactate 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 activity of α-ketoglutarate dehydrogenase.

[0121] Rod-shaped bacteria with reduced or absent α-ketoglutarate dehydrogenase activity, and methods for obtaining them, are described in WO2008 / 075483. Specific examples of rod-shaped bacteria with reduced or absent α-ketoglutarate dehydrogenase activity include the following strains.

[0122] Corynebacterium glutamicum (Lactobacillus brevis) strain L30-2 (Japanese Patent Application Publication No. 2006-340603)

[0123] Corynebacterium glutamicum (Lactobacillus brevis) ΔS strain (WO95 / 34672)

[0124] Corynebacterium glutamicum (Lactobacillus brevis) AJ12821 (FERM BP-4172; French Patent No. 9401748)

[0125] Corynebacterium glutamicum (Yellow Short Bacterium) AJ12822 (FERM BP-4173; French Patent No. 9401748)

[0126] Corynebacterium glutamicum AJ12823 (FERM BP-4174; French Patent No. 9401748)

[0127] In addition, strains with reduced or absent α-ketoglutarate dehydrogenase (sucA) and succinate dehydrogenase (sdh) activities can be cited as L-glutamate producing bacteria or parental strains used to induce it (Japanese Patent Application Publication No. 2010-041920). A specific example of such a strain is the odhAsdhA double-deleted strain of *Corynebacterium glutamicum* ATCC14067 (*Corynebacterium glutamicum* 8L3GΔSDH strain) (Japanese Patent Application Publication No. 2010-041920).

[0128] In addition, as a method to confer or enhance the L-glutamate production capacity of rod-shaped bacteria, methods to confer resistance to organic acid analogs, respiratory inhibitors, etc., or methods to confer sensitivity to cell wall synthesis inhibitors can be cited. Specifically, examples of such methods include, for instance, methods for conferring resistance to monofluoroacetic acid (Japanese Patent Application Publication No. 50-113209), methods for conferring resistance to adenine or thymine (Japanese Patent Application Publication No. 57-065198), methods for weakening urease (Japanese Patent Application Publication No. 52-038088), methods for conferring resistance to malonic acid (Japanese Patent Application Publication No. 52-038088), methods for conferring resistance to benzopyranones or naphthoquinones (Japanese Patent Application Publication No. 56-1889), methods for conferring resistance to HOQNO (Japanese Patent Application Publication No. 56-140895), methods for conferring resistance to α-ketomalonic acid (Japanese Patent Application Publication No. 57-2689), methods for conferring resistance to guanidine (Japanese Patent Application Publication No. 56-35981), and methods for conferring sensitivity to penicillin (Japanese Patent Application Publication No. 4-88994).

[0129] The following strains can be cited as specific examples of such resistant or susceptible bacteria.

[0130] Corynebacterium glutamicum (Yellow Short Bacterium) AJ3949 (FERM BP-2632; Japanese Patent Application Publication No. 50-113209)

[0131] Corynebacterium glutamicum AJ11628 (FERM P-5736; Japanese Patent Application Publication No. 57-065198)

[0132] Corynebacterium glutamicum (Yellow Short Bacterium) AJ11355 (FERM P-5007; Japanese Patent Application Publication No. 56-1889)

[0133] Corynebacterium glutamicum AJ11368 (FERM P-5020; Japanese Patent Application Publication No. 56-1889)

[0134] Corynebacterium glutamicum (Yellow Short Bacterium) AJ11217 (FERM P-4318; Japanese Patent Application Publication No. 57-2689)

[0135] Corynebacterium glutamicum AJ11218 (FERM P-4319; Japanese Patent Application Publication No. 57-2689)

[0136] Corynebacterium glutamicum (Yellow Short Bacterium) AJ11564 (FERM P-5472; Japanese Patent Application Publication No. 56-140895)

[0137] Corynebacterium glutamicum (Yellow Short Bacterium) AJ11439 (FERM P-5136; Japanese Patent Application Publication No. 56-35981)

[0138] Corynebacterium glutamicum H7684 (FERM BP-3004; Japanese Patent Application Publication No. 04-88994)

[0139] Corynebacterium glutamicum (Lactobacillus brevis) AJ11426 (FERM P-5123; Japanese Patent Application Publication No. 56-048890)

[0140] Corynebacterium glutamicum AJ11440 (FERM P-5137; Japanese Patent Application Publication No. 56-048890)

[0141] Corynebacterium glutamicum (Lactobacillus brevis) AJ11796 (FERM P-6402; Japanese Patent Application Publication No. 58-158192)

[0142] As a method to endow or enhance the ability to produce L-glutamate, one example is the modification of bacteria in a way that increases the activity of expelling L-glutamate from bacterial cells. The activity of expelling L-glutamate can be increased, for example, by increasing the expression of genes encoding proteins that expel L-glutamate. Examples of genes encoding proteins that expel various amino acids include, for example, the b2682 gene (ygaZ), b2683 gene (ygaH), b1242 gene (ychE), and b3434 gene (yhgN) (Japanese Patent Application Publication No. 2002-300874).

[0143] In addition, as a method to endow or enhance the ability to produce L-glutamate, one example is to modify bacteria in a way that increases the activity of proteins involved in sugar metabolism and proteins involved in energy metabolism.

[0144] Examples of proteins involved in carbohydrate metabolism include those involved in glucose uptake and glycolytic enzymes. Genes encoding proteins involved in carbohydrate metabolism include: glucose-6-phosphate isomerase gene (pgi; WO01 / 02542), pyruvate carboxylase gene (pyc; WO99 / 18228, EP1092776A), phosphogluconomutase gene (pgm; WO03 / 04598), fructose-1,2-bisphosphate aldolase gene (pfkB, fbp; WO03 / 04664), transaldolase gene (talB; WO03 / 008611), fumarate enzyme gene (fum; WO01 / 02545), non-PTS sucrose uptake gene (csc; EP1149911A), and sucrose assimilation gene (scrAB operon; US Patent No. 7179623).

[0145] Genes that encode proteins involved in energy metabolism include: transhydrogenase gene (pntAB; US Patent No. 5,830,716) and cytochrome bo type oxidase gene (cyoB; EP1070376A).

[0146] In addition, as a method for conferring or enhancing the L-glutamate production capacity of rod-shaped bacteria, other methods include enhancing the expression of the yggB gene and introducing a mutant yggB gene with a mutation inserted into the coding region (WO2006 / 070944). That is, the bacteria of the present invention can be modified in a way that increases the expression of the yggB gene, or in a way that maintains (possesses) the mutant yggB gene.

[0147] The yggB gene encodes a mechanosensitive channel. Examples of yggB genes include those found in rod-shaped bacteria. Specific examples include the yggB genes of *Corynebacterium glutamicum* ATCC13869, *Corynebacterium glutamicum* ATCC13032, *Corynebacterium glutamicum* ATCC14967, *Corynebacterium molasses* ATCC17965, and *Corynebacterium tumefaciens* ATCC 15991 (WO2006 / 070944). The yggB gene of *Corynebacterium glutamicum* ATCC13032 corresponds to the complementary sequence of sequences 1336091–1337692 in the genome sequence registered in the NCBI database with GenBank Accession No. NC_003450, also known as NCgl1221. The YggB protein encoded by the yggB gene of Corynebacterium glutamicum ATCC13032 is registered in GenBank accession No. NP_600492. Additionally, the base sequence of the yggB gene of Corynebacterium glutamicum 2256 (ATCC 13869) and the amino acid sequence of the YggB protein encoded by this gene are shown in Serial No. 7 and Serial No. 8, respectively.

[0148] In this invention, the yggB gene having the "specific mutation" described later is also called the mutant yggB gene, and the protein encoded by it is also called the mutant YggB protein. Furthermore, in this invention, the yggB gene not having the "specific mutation" described later is also called the wild-type yggB gene, and the protein encoded by it is also called the wild-type YggB protein. It should be noted that, for YggB proteins, changes in the amino acid sequence caused by the "specific mutation" in the yggB gene are also called "specific mutations." The term "wild-type" here is used for convenience of distinguishing it from "mutant," and it is not limited to naturally occurring proteins as long as they do not have the "specific mutation." Examples of wild-type YggB proteins include the YggB proteins exemplified above, such as proteins having the amino acid sequence shown in Serial Number 8. Additionally, examples of wild-type YggB proteins include conserved variants (variants that retain the original function) of the YggB proteins exemplified above that do not have the "specific mutation." The "original function" of the YggB protein could refer, for example, to its function as a mechanosensitive channel, or to its property of increasing L-glutamate production in rod-shaped bacteria when its expression increases.

[0149] For the term "specific mutation," any mutation that alters the amino acid sequence of the wild-type YggB protein to enhance L-glutamate production in rod-shaped bacteria is acceptable, with no particular restrictions. Examples of "specific mutations" include C-terminal mutations and transmembrane mutations (WO2006 / 070944). Furthermore, a combination of these mutations can also be considered a "specific mutation."

[0150] (1) C-terminal mutation

[0151] C-terminal mutations are mutations in the region of amino acid residues 419–533 of the wild-type yggB gene encoding the wild-type YggB protein. C-terminal mutations can be introduced into one or more sites within this region. There are no particular limitations on the types of amino acid sequence changes caused by C-terminal mutations. C-terminal mutations can cause, for example, substitutions of amino acid residues (missense mutations), insertions of amino acid residues, deletions of amino acid residues, the appearance of stop codons (speechless mutations), frameshift mutations, or combinations thereof. As C-terminal mutations, insertions of base sequences such as insertion sequences (hereinafter also referred to as "IS") and transposons are preferred.

[0152] (1-1) Insertion of base sequence

[0153] Examples of C-terminal mutations include, for instance, the insertion of a base sequence at position 419 of the wild-type YggB protein (type 2A-1 mutation). Type 2A-1 mutations can, for example, cause partial or complete deletion or substitution of amino acid residues from positions 419 to 533 of the wild-type YggB protein. A specific example of a mutant yggB gene with a type 2A-1 mutation is a yggB gene in sequence 7 with an IS sequence inserted after the "G" at position 1255, encoding a shorter 423 amino acid residue protein than the original wild-type YggB protein (sequence 8). The base sequence of this mutant yggB gene (V419::IS) and the amino acid sequence of the mutant YggB protein (V419::IS) encoded by this gene are shown in sequences 9 and 10, respectively. In sequence number 9, positions 1-1269 represent the CDS of the mutant YggB protein (V419::IS). Examples of L-glutamate-producing bacteria possessing the mutant yggB gene (V419::IS) include *Corynebacterium glutamicum* 2256ΔsucAΔldhA yggB. * strain (WO2014 / 185430).

[0154] (1-2) Substitution of proline residues

[0155] Examples of C-terminal mutations include, for instance, replacing proline residues at positions 419-533 of the wild-type YggB protein with other amino acids. Examples of such proline residues include those at positions 424, 437, 453, 457, 462, 469, 484, 489, 497, 515, 529, and 533 of the wild-type YggB protein. Preferably, the proline residues at positions 424 and / or 437 are replaced with other amino acids. "Other amino acids" can be any naturally occurring amino acid other than proline, without particular limitation. Examples of "other amino acids" include Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, and His. For example, the proline residue at position 424 can preferably be replaced with a hydrophobic amino acid (Ala, Gly, Val, Leu, or Ile), and more preferably with a branched-chain amino acid (Leu, Val, or Ile). Additionally, for example, the proline residue at position 437 can preferably be replaced with an amino acid having a hydroxyl group on its side chain (Thr, Ser, or Tyr), and more preferably with Ser.

[0156] (2) Mutations in the transmembrane region

[0157] It can be inferred that the YggB protein has five transmembrane regions. These transmembrane regions correspond to amino acid residues at positions 1-23 (first transmembrane region), 25-47 (second transmembrane region), 62-84 (third transmembrane region), 86-108 (fourth transmembrane region), and 110-132 (fifth transmembrane region) of the wild-type YggB protein, respectively. Mutations in the transmembrane regions are mutations in the regions encoding these transmembrane regions in the wild-type yggB gene. These mutations can be introduced into one or more sites within the region. Preferably, the transmembrane region mutations result in substitutions, deletions, additions, insertions, or inversions of one or more amino acids, without accompanying frameshift or nonsense mutations. "One or more" means preferably 1-20, more preferably 1-10, further preferably 1-5, and particularly preferably 1-3. Examples of transmembrane region mutations include the insertion of one or more amino acids (e.g., Cys-Ser-Leu) between the leucine residue at position 14 and the tryptophan residue at position 15 in the wild-type YggB protein; the replacement of the alanine residue at position 100 with other amino acid residues (e.g., amino acids with a hydroxyl group on the side chain (Thr, Ser, or Tyr), preferably Thr); and the replacement of the alanine residue at position 111 with other amino acid residues (e.g., Val or amino acids with a hydroxyl group on the side chain (Thr, Ser, or Tyr), preferably Val or Thr).

[0158] In this invention, unless otherwise specified, "the amino acid residue at position X of the wild-type YggB protein" refers to the amino acid residue corresponding to position X in sequence number 8. The "X position" in the amino acid sequence refers to the position counting from the N-terminus of the amino acid sequence; the amino acid residue at the N-terminus is the first amino acid residue. It should be noted that the position of the amino acid residue indicates a relative position, and its absolute position may change due to the deletion, insertion, or addition of amino acids. For example, "the amino acid residue at position 419 of the wild-type YggB protein" refers to the amino acid residue corresponding to position 419 in sequence number 8. If one amino acid residue closer to the N-terminus than position 419 is deleted, the amino acid residues from the N-terminus to position 418 are "the amino acid residue at position 419 of the wild-type YggB protein." Conversely, if one amino acid residue is inserted closer to the N-terminus than position 419, the amino acid residues from the N-terminus to position 420 are "the amino acid residue at position 419 of the wild-type YggB protein." Specifically, for example, in the YggB protein of *Corynebacterium glutamicum* strain ATCC14967, amino acid residues at positions 419–529 are equivalent to amino acid residues at positions 419–533 of the wild-type YggB protein. Additionally, for example, in the YggB protein of *Corynebacterium heliotropium*, the alanine residue at position 98 is equivalent to the alanine residue at position 100 of the wild-type YggB protein.

[0159] In any YggB protein amino acid sequence, which amino acid residue corresponds to the amino acid residue at position X in sequence number 8 can be determined by comparing the amino acid sequence of the YggB protein with the amino acid sequence of sequence number 8. This comparison can be performed, for example, using well-known gene analysis software. Specific examples of such software include DNASIS (Hitachi Solutions) and GENETYX (GENETYX, GENETYX, 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).

[0160] The mutant yggB gene can be obtained by modifying the wild-type yggB gene to give it the aforementioned "specific mutation". DNA modification can be performed using known methods. Specifically, for example, site-specific mutagenesis methods for introducing a target mutation into a target site of DNA include PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)) and phage methods (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. InEnzymol., 154, 367 (1987)). Alternatively, the mutant yggB gene can also be obtained through chemical synthesis.

[0161] Modifying rod-shaped bacteria to possess a mutant yggB gene can be achieved by introducing the mutant yggB gene into the bacteria. Alternatively, modification of rod-shaped bacteria to possess a mutant yggB gene can also be achieved by introducing a mutation into the yggB gene already present in the bacteria using natural mutation or mutant treatment.

[0162] <1-2> Modifications associated with the mutant glyA gene

[0163] The rod-shaped bacteria of the present invention are modified to retain the mutant glyA gene encoding a mutant serine hydroxymethyltransferase, wherein the mutant serine hydroxymethyltransferase has a substitution of the glycine residue at position 265 in the amino acid sequence of the wild-type serine hydroxymethyltransferase for other amino acid residues.

[0164] It should be noted that the rod-shaped bacteria of the present invention can be rod-shaped bacteria modified in a manner that retains the mutant glyA gene in rod-shaped bacteria with L-glutamate production capacity as described above, or rod-shaped bacteria obtained by conferring L-glutamate production capacity in rod-shaped bacteria modified in a manner that retains the mutant glyA gene. Additionally, it also includes rod-shaped bacteria that have become capable of L-glutamate production through modification in a manner that retains the mutant glyA gene.

[0165] The glyA gene encodes a serine hydroxymethyltransferase. Examples of glyA genes include those found in rod-shaped bacteria. Specific examples include the glyA genes of *Corynebacterium glutamicum* ATCC13869, *Corynebacterium glutamicum* ATCC13032, *Corynebacterium glutamicum* ATCC14967, and *Corynebacterium molasses* ATCC17965. The glyA gene of *Corynebacterium glutamicum* ATCC13032 corresponds to the complementary sequence of sequences 1050624–1051928 in the genome sequence registered in the NCBI database with GenBank Accession No. NC_003450, also known as NCgl0954. The GlyA protein encoded by the glyA gene of *Corynebacterium glutamicum* ATCC13032 is registered with GenBank accession No. WP_003856790.1. In addition, the base sequence of the wild-type glyA gene of Corynebacterium glutamicum 2256 (ATCC 13869) and the amino acid sequence of the GlyA protein encoded by the gene are shown in Serial No. 1 and Serial No. 2, respectively.

[0166] "Serine hydroxymethyltransferase" can refer to a protein with activity catalyzing the hydroxymethyl transfer reaction of serine and / or glycine (e.g., EC 2.1.2.1). This activity is also referred to as "serine hydroxymethyltransferase activity." Specifically, serine hydroxymethyltransferase activity can be the activity of catalyzing the conversion of L-serine and tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate and / or the reverse reaction. Serine hydroxymethyltransferase is also referred to as "glycine hydroxymethyltransferase." The glyA gene is an example of a gene encoding serine hydroxymethyltransferase. The base sequences of serine hydroxymethyltransferase genes such as the glyA gene possessed by modified bacteria, and the amino acid sequences of serine hydroxymethyltransferases such as the GlyA protein encoded by them, can be obtained from public databases such as NCBI.

[0167] In this invention, the glyA gene having the "specific gene mutation" described later is also called a mutant glyA gene, and the protein encoded by it is also called a mutant GlyA protein. Conversely, in this invention, the glyA gene not having the "specific gene mutation" described later is also called a wild-type glyA gene, and the protein encoded by it is also called a wild-type GlyA protein. It should be noted that, for GlyA proteins, changes in the amino acid sequence caused by the "specific gene mutation" in the glyA gene are also called "specific gene mutations." The term "wild-type" here is used for ease of distinction from "mutant," and it is not limited to naturally occurring proteins as long as they do not have the "specific gene mutation." Examples of wild-type GlyA proteins include the GlyA proteins exemplified above, and proteins having the amino acid sequence shown in Serial Number 2. Furthermore, examples of wild-type GlyA proteins include conserved variants (variants that retain the original function) of the GlyA proteins exemplified above that do not have the "specific gene mutation." One example of the "original function" of the GlyA protein is its function as a serine hydroxymethyltransferase.

[0168] For the term "specific gene mutation," any mutation that alters the amino acid sequence of the wild-type GlyA protein to enhance L-glutamate production in Corynebacterium tumefaciens is acceptable; there are no particular restrictions. An example of a "specific gene mutation" is a mutation at amino acid residue 265 of the wild-type GlyA protein's amino acid sequence in the wild-type glyA gene.

[0169] Mutations in the amino acid residue at position 265 of the amino acid sequence of the wild-type GlyA protein can be exemplified by, for example, substitutions of amino acid residues (missense mutations).

[0170] Mutations at amino acid residue 265 of the wild-type GlyA protein can include, for example, replacing the glycine residue at position 265 with another amino acid residue. "Other amino acids" can be any naturally occurring amino acid other than glycine, without particular limitation. Examples of "other amino acids" include Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Pro, Ala, and His. For example, the glycine residue at position 265 can preferably be replaced with a hydrophilic amino acid (Lys, Glu, Thr, Ser, Asp, Asn, Gln, Arg, Cys, or His), and more preferably with Ser. Furthermore, the glycine residue at position 265 can preferably be replaced with an amino acid having a hydroxyl group on its side chain (Thr, Ser, or Tyr), and more preferably with Ser.

[0171] In this invention, unless otherwise specified, "the amino acid residue at position X of the wild-type GlyA protein" refers to the amino acid residue corresponding to position X in sequence number 2. The "X position" in the amino acid sequence refers to the position counting from the N-terminus of the amino acid sequence; the amino acid residue at the N-terminus is the first amino acid residue. It should be noted that the positions of amino acid residues are relative, and their absolute positions may vary due to deletions, insertions, or additions of amino acids. For example, "the amino acid residue at position 265 of the wild-type GlyA protein" refers to the amino acid residue corresponding to position 265 in sequence number 2. If one amino acid residue closer to the N-terminus than position 265 is deleted, the amino acid residues from the N-terminus to position 264 are considered "the amino acid residue at position 265 of the wild-type GlyA protein." Conversely, if one amino acid residue is inserted closer to the N-terminus than position 265, the amino acid residues from the N-terminus to position 266 are considered "the amino acid residue at position 265 of the wild-type GlyA protein."

[0172] In any GlyA protein amino acid sequence, which amino acid residue corresponds to the amino acid residue at position X in sequence number 2 can be determined by comparing the amino acid sequence of the GlyA protein with the amino acid sequence of sequence number 2. This comparison can be performed, for example, using well-known gene analysis software. Specific examples include DNASIS from Hitachi Solutions and GENTYX from GENTYX (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).

[0173] The mutant glyA gene can be obtained by modifying the wild-type glyA gene to possess the aforementioned "specific gene mutation". DNA modification can be performed using known methods. Specifically, for example, site-specific mutagenesis methods for introducing a target mutation into a target site of DNA include PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)) and phage methods (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. In Enzymol., 154, 367 (1987)). Alternatively, the mutant glyA gene can also be obtained through chemical synthesis.

[0174] The wild-type glyA gene can be, for example, a gene having the base sequence of the glyA gene exemplified above (e.g., the base sequence shown in Serial No. 1). Similarly, the GlyA protein can be, for example, a protein having the amino acid sequence of the GlyA protein exemplified above (e.g., the amino acid sequence shown in Serial No. 2). It should be noted that, unless otherwise specified, the expression "having (amino acid or base) sequence" refers to including both the inclusion of that "(amino acid or base) sequence" and cases where the protein is composed of that "(amino acid or base) sequence".

[0175] Furthermore, the wild-type glyA gene, as long as it retains its original function, can also be a variant of the glyA gene exemplified above (e.g., the gene with the base sequence shown in SEQ ID NO: 1). Similarly, the GlyA protein, as long as it retains its original function, can also be a variant of the GlyA protein exemplified above (e.g., the protein with the amino acid sequence shown in SEQ ID NO: 2). It should be noted that such variants that retain their original function are sometimes referred to as "conserved variants." The term "glyA gene" includes not only the glyA genes exemplified above but also their conserved variants. Similarly, the term "GlyA protein" includes not only the GlyA proteins exemplified above but also their conserved variants. Examples of conserved variants include, for example, the glyA genes exemplified above, homologs of GlyA proteins, and artificially modified forms.

[0176] "Retaining the original function" means that a variant of a gene or protein has a function (e.g., activity, properties) corresponding to the original gene or protein. Regarding genes, "retaining the original function" means that the gene variant encodes a protein that retains the original function. That is, regarding the glyA gene, "retaining the original function" means that a variant of the glyA gene encodes a protein with the activity of the GlyA protein, i.e., serine hydroxymethyltransferase activity. Furthermore, regarding the GlyA protein, "retaining the original function" means that a variant of the GlyA protein possesses the activity of the GlyA protein, i.e., serine hydroxymethyltransferase activity.

[0177] Serine hydroxymethyltransferase activity can be determined, for example, by incubating the enzyme with the corresponding substrate (e.g., L-serine and tetrahydrofolate) and measuring the production of the enzyme and the corresponding substrate-dependent products (e.g., glycine and 5,10-methylenetetrahydrofolate).

[0178] The following are examples of conservative variants.

[0179] Homologs of the glyA gene or the GlyA protein can be readily obtained from public databases, for example, through BLAST or FASTA searches using the example base sequences of the glyA gene or the example amino acid sequences of the GlyA protein as search sequences. Alternatively, homologs of the glyA gene can be obtained, for example, through PCR using chromosomes of various organisms as templates and oligonucleotides prepared based on these known glyA gene base sequences as primers.

[0180] The glyA gene only needs to retain its original function and can be a gene encoding a GlyA protein having an amino acid sequence formed by substitution, deletion, insertion, and / or addition of one or more amino acids at one or more positions in the above-mentioned amino acid sequence (e.g., the amino acid sequence shown in sequence number 2). For example, the N-terminus and / or C-terminus of the encoded protein may be elongated or shortened. It should be noted that the term "one or more" varies depending on the position and type of amino acid residues in the protein's three-dimensional structure, specifically referring to, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, further preferably 1 to 5, and particularly preferably 1 to 3.

[0181] The substitution, deletion, insertion, and / or addition of one or more of the aforementioned amino acids are conserved mutations that maintain the normal function of the protein. A representative example of a conserved mutation is a conserved substitution. For a conserved substitution, in the case of an aromatic amino acid, it refers to a mutation that substitutes for each other between Phe, Trp, and Tyr; in the case of a hydrophobic amino acid, it refers to a mutation that substitutes for each other between Leu, Ile, and Val; in the case of a polar amino acid, it refers to a mutation that substitutes for each other between Gln and Asn; in the case of a basic amino acid, it refers to a mutation that substitutes for each other between Lys, Arg, and His; in the case of an acidic amino acid, it refers to a mutation that substitutes for each other between Asp and Glu; and in the case of an amino acid with a hydroxyl group, it refers to a mutation that substitutes for each other between Ser and Thr. As substitutions that can be considered conservative, specific examples include substitutions from Ala to Ser or Thr, from Arg to Gln, His, or Lys, from Asn to Glu, Gln, Lys, His, or Asp, from Asp to Asn, Glu, or Gln, from Cys to Ser or Ala, from Gln to Asn, Glu, Lys, His, Asp, or Arg, from Glu to Gly, Asn, Gln, Lys, or Asp, from Gly to Pro, and from His to Asn, Lys, Gln, Arg, or Tyr. Substitutions from Ile to Leu, Met, Val, or Phe; substitutions from Leu to Ile, Met, Val, or Phe; substitutions from Lys to Asn, Glu, Gln, His, or Arg; substitutions from Met to Ile, Leu, Val, or Phe; substitutions from Phe to Trp, Tyr, Met, Ile, or Leu; substitutions from Ser to Thr or Ala; substitutions from Thr to Ser or Ala; substitutions from Trp to Phe or Tyr; substitutions from Tyr to His, Phe, or Trp; and substitutions from Val to Met, Ile, or Leu. Furthermore, the amino acid substitutions, deletions, insertions, or additions described above also include substitutions, deletions, insertions, or additions resulting from naturally occurring mutations (mutants or variants) based on individual differences in the organism from which the gene originates, species differences, etc.

[0182] In addition, the glyA gene only needs to maintain its original function. It can be a gene encoding a protein that has an amino acid sequence with an overall identity of, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, and particularly preferably 99% or more, relative to the above-mentioned amino acid sequence.

[0183] Furthermore, the glyA gene only needs to maintain its original function. It can be a gene (e.g., DNA) that hybridizes under strict conditions with a probe prepared from the aforementioned base sequence (e.g., the base sequence shown in Serial No. 1), or with a complementary sequence to the entirety or a portion of the aforementioned base sequence. "Strict conditions" refer to conditions that form so-called specific hybrids without forming non-specific hybrids. As an example, conditions can be given where DNAs with high identity hybridize with each other, for example, DNAs with identity of 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, and particularly preferably 99% or more, while DNAs with identity lower than this do not hybridize with each other; or conditions equivalent to the washing conditions of ordinary Southern hybridization, i.e., washing once at a salt concentration and temperature of 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, preferably washing 2 to 3 times.

[0184] As described above, the probe used in the hybridization can be a portion of the complementary sequence of a gene. Such a probe can be prepared by PCR using oligonucleotides prepared based on a known gene sequence as primers and a DNA fragment containing the aforementioned gene as a template. For example, a DNA fragment approximately 300 bp in length can be used as the probe. When using a DNA fragment approximately 300 bp in length as the probe, washing conditions for hybridization can include 50°C, 2×SSC, and 0.1% SDS.

[0185] Furthermore, since codon degeneracy varies depending on the host, the glyA gene can be a gene obtained by replacing any codon with its equivalent. That is, the glyA gene can be a variant of the glyA gene exemplified above, based on the degeneracy of the genetic code. For example, the glyA gene can be modified to have the most suitable codons based on the frequency of codon usage in the host.

[0186] It should be noted that the "identity" between amino acid sequences refers to the identity between amino acid sequences calculated by BLASTP using the default Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence=11, Extension=1; Compositional Adjustments: Conditional compositional score matrix adjustment). Conversely, the "identity" between base sequences refers to the identity between base sequences calculated by BLASTN using the default Scoring Parameters (Match / Mismatch Scores=1,-2; Gap Costs=Linear).

[0187] The description of conserved variants of genes and proteins described above can also be applied to any protein and the gene encoding it. That is, the genes and proteins used in the breeding of L-glutamate-producing bacteria can each have known gene and protein base sequences and amino acid sequences, such as those exemplified above. Furthermore, the genes and proteins used in the breeding of L-glutamate-producing bacteria can be conserved variants of known genes and proteins, such as those exemplified above. Specifically, for example, the gene used in the breeding of L-glutamate-producing bacteria, as long as it retains its original function, can be a gene encoding a protein having an amino acid sequence formed by substitution, deletion, insertion, or addition of one or more amino acids at one or more positions in the amino acid sequence of a known protein.

[0188] Examples of methods for modifying the glyA gene in a manner that preserves the aforementioned mutant glyA gene include introducing a mutant glyA gene with a mutation introduced into the coding region of the wild-type glyA gene into rod-shaped bacteria, and introducing a mutation into the coding region of the wild-type glyA gene present in rod-shaped bacteria.

[0189] Modifying rod-shaped bacteria to possess a mutant glyA gene can be achieved by introducing the mutant glyA gene into the bacteria. It should be noted that modifying rod-shaped bacteria to possess a mutant glyA gene can also be achieved by introducing a mutation into the glyA gene already present in rod-shaped bacteria using natural mutations or mutant treatment.

[0190] The introduction of the mutant glyA gene into rod-shaped bacteria can be achieved by introducing the gene into the host's chromosome. Gene introduction into the chromosome can be achieved, for example, using homologous recombination (Miller, JH Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Examples of gene introduction methods utilizing homologous recombination include red-driven integration (Datsenko, K. A, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97: 6640-6645 (2000)), methods using straight-stranded DNA, methods using temperature-sensitive origins of replication, methods using plasmids capable of conjugation, methods using suicide vectors lacking origins of replication that function within the host, and phage transduction. Specifically, the gene can be introduced into the host chromosome by transforming the host with recombinant DNA containing the mutant glyA gene, resulting in homologous recombination with a target site on the host chromosome. The structure of the recombinant DNA used in homologous recombination is not particularly limited, as long as homologous recombination can occur in the desired manner. For example, a host can be transformed with linear DNA containing a mutant glyA gene, and having base sequences at both ends of the gene that are identical to those upstream and downstream of the target site on the chromosome. Homologous recombination occurs upstream and downstream of the target site, thereby replacing the target site with the gene. The recombinant DNA used in homologous recombination may contain a marker gene for selecting transformants. Only one copy of the gene can be introduced, or two or more copies can be introduced. For example, homologous recombination can be performed using a base sequence that exists in multiple copies on the chromosome as a target, thereby introducing multiple copies of the mutant glyA gene into the chromosome. Examples of base sequences that exist in multiple copies on the chromosome include repetitive DNA sequences and inverted repeat sequences located at both ends of transposons. Alternatively, appropriate base sequences on the chromosome, such as genes not needed in the production of the target substance, can be used as targets for homologous recombination. In addition, genes can also be randomly introduced into chromosomes using transposons or Mini-Mu (Japanese Patent Application Publication No. 2-109985, US Patent No. 5882888, EP805867B1). It should be noted that chromosome modification methods utilizing homologous recombination are not limited to the introduction of mutant glyA genes; they can be used for arbitrary chromosome modifications, such as modifications to expression regulatory sequences.

[0191] The introduction of a mutant glyA gene into a chromosome can be confirmed by Southern hybridization using probes with sequences that are fully or partially complementary to the gene, or by PCR using primers prepared based on the gene sequence.

[0192] Alternatively, the introduction of the mutant glyA gene into rod-shaped bacteria can also be achieved by introducing a vector containing the gene into the host. For example, an expression vector for the gene can be constructed by linking a DNA fragment containing the mutant glyA gene to a vector that functions in the host, and the host can be transformed using this expression vector to introduce the gene into rod-shaped bacteria. The DNA fragment containing the mutant glyA gene can be obtained, for example, by PCR using genomic DNA of a microorganism possessing the mutant glyA gene as a template. As the vector, a vector capable of autonomous replication within the host cell can be used. A multi-copy vector is preferred. Furthermore, to select transformants, the vector preferably has a marker such as an antibiotic resistance gene. Additionally, the vector may have a promoter and a terminator for expressing the inserted gene. The vector can be, for example, a vector derived from bacterial plasmids, a vector derived from yeast plasmids, a vector derived from bacteriophages, a granule, or a phage particle. Examples of vectors capable of autonomous replication in rod-shaped bacteria include: pHM1519 (Agric. Biol. Chem., 48, 2901-2903(1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903(1984)); and pAM330 (Agric. Biol. Chem., 48, 2901-2903(1984)). 2901-2903 (1984)); plasmids containing drug resistance genes that have been modified from them; pCRY30 (Japanese Patent Application Publication No. 3-210184); pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX (Japanese Patent Application Publication No. 2-72876, US Patent No. 5185262); pCRY2 and pCRY3 (Japanese Patent Application Publication No. 1-191686); pAJ655, pAJ611 and pAJ1844 ( Japanese Patent Application Publication No. 58-192900; pCG1 (Japanese Patent Application Publication No. 57-134500); pCG2 (Japanese Patent Application Publication No. 58-35197); pCG4 and pCG11 (Japanese Patent Application Publication No. 57-183799); pVK7 (Japanese Patent Application Publication No. 10-215883); pVK9 (U.S. Patent Application Publication No. 2006 / 0141588); pVC7 (Japanese Patent Application Publication No. 9-070291); pVS7 (WO2013 / 069634).

[0193] When introducing a gene, the gene only needs to be maintained in the host in a manner that allows for expression. Specifically, the gene only needs to be maintained in a manner controlled by a promoter that functions within the host. The promoter only needs to function within the host; there are no particular restrictions. "A promoter that functions within the host" refers to a promoter that has promoter activity within the host. The promoter can be of host origin or of foreign origin. The promoter can be an intrinsic promoter of the gene to be introduced or a promoter of another gene.

[0194] Downstream of the gene, a terminator can be configured to terminate transcription. There are no particular restrictions as long as the terminator functions within the host. The terminator can be of host origin or of heterologous origin. It can be an intrinsic terminator of the gene to be introduced or a terminator of another gene.

[0195] Detailed information about vectors, promoters, and terminators that can be used in various microorganisms is available in publications such as "Lectures on the Fundamentals of Microbiology 8: Genetic Engineering," Kyoritsu Publishing, 1987.

[0196] Furthermore, when introducing two or more genes, each gene only needs to be maintained in the host in a manner that allows for expression. For example, all genes can be maintained on a single expression vector or on the chromosome. Alternatively, each gene can be maintained on multiple expression vectors, or on one or more expression vectors and on the chromosome. Additionally, the introduction can be performed by forming an operon from two or more genes. Examples of "introducing two or more genes" include, for instance, introducing genes that encode two or more proteins (e.g., enzymes), introducing genes that encode two or more subunits of a single protein complex (e.g., an enzyme complex), and combinations thereof.

[0197] <2> Method for producing L-glutamic acid of the present invention

[0198] The method of the present invention is a method for producing L-glutamic acid, which includes: culturing the bacteria of the present invention described in section <1> in a culture medium, accumulating L-glutamic acid in the culture medium and / or in the bacterial cells; and collecting L-glutamic acid from the culture medium and / or the bacterial cells. Regarding L-glutamic acid, as described above. In the present invention, L-glutamic acid can be produced alone, or one or more amino acids other than L-glutamic acid, such as L-form amino acids (also called L-amino acids), can be produced.

[0199] The culture medium used is not particularly limited as long as it enables the bacteria of the present invention to proliferate and produce L-glutamic acid. As the culture medium, a conventional culture medium used for culturing bacteria such as corynebacteria can be used. For example, a culture medium containing components selected from carbon sources, nitrogen sources, phosphate sources, sulfur sources, and various other organic and inorganic components can be used as needed. The types and concentrations of the culture medium components can be appropriately set according to the type of bacteria used and other conditions.

[0200] Specific examples of carbon sources include: glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, maltose, isomaltose, molasses, starch hydrolysate, biomass hydrolysate, and other sugars; organic acids such as acetic acid, fumaric acid, citric acid, and succinic acid; alcohols such as glycerol, crude glycerol, and ethanol; and fatty acids. Sugars are particularly noteworthy as carbon sources. Glucose and fructose are particularly noteworthy examples. Glucose and fructose can be used alone or in combination with other carbon sources. For example, sugars composed of fructose can be used as carbon sources. Examples of sugars composed of fructose include fructose, sucrose, and fructooligosaccharides. Sugars composed of fructose can be used alone or in combination with other carbon sources. It should be noted that plant-derived raw materials can be appropriately used as carbon sources. Examples of plants that can be used include corn, rice, wheat, soybeans, sugarcane, sugar beets, and cotton. Examples of plant-derived raw materials include organs such as roots, stems, trunks, branches, leaves, flowers, and seeds; the plant body containing them; and the decomposition products of these plant organs. There are no particular restrictions on the utilization of plant-derived raw materials; they can be used in any way, such as as unprocessed products, juices, pulverized products, or purified products. As carbon sources, for example, sugarcane molasses, sugar beet molasses, high-test molasses, citrus molasses, or invert sugar can be used, as well as hydrolysates of natural raw materials such as cellulose, starch, corn, grains, tapioca starch, and cassava. Furthermore, pentoses such as xylose, hexoses such as glucose, or mixtures thereof can be obtained and utilized from plant biomass. Specifically, these sugars can be obtained by treating plant biomass with steam, concentrated acid hydrolysis, dilute acid hydrolysis, hydrolysis based on enzymes such as cellulase, or alkali treatment. It should be noted that since hemicellulose is generally more readily hydrolyzed than cellulose, hemicellulose in plant biomass can be pre-hydrolyzed to release pentoses, followed by cellulose hydrolysis to generate hexoses. Furthermore, regarding xylose, for example, the bacteria of this invention can maintain a pathway for converting hexoses such as glucose to xylose, and supply it through conversion starting from hexoses. Specifically, as a carbon source, glucose can be used alone, or a mixture of two carbon sources, such as glucose and fructose, or glucose and sucrose, in any ratio (e.g., 3:7 to 7:3 by weight).

[0201] Specific examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; peptone; yeast extract; meat extract; organic nitrogen sources such as plant protein hydrolysates (HVP; for example, soybean protein hydrolysates, soy sauce, pea soy sauce, etc.); ammonia; and urea. Ammonia gas or ammonia water used for pH adjustment can also be used as a nitrogen source. One nitrogen source can be used, or two or more nitrogen sources can be used in combination.

[0202] Examples of phosphoric acid sources include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and other phosphates, as well as phosphoric acid polymers such as pyrophosphate. One phosphoric acid source can be used, or two or more phosphoric acid sources can be used in combination.

[0203] Specific examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, as well as sulfur-containing amino acids such as cysteine, cystine, and glutathione. One sulfur source can be used, or two or more sulfur sources can be used in combination.

[0204] Other organic and inorganic components include, for example: inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, niacin, nicotinamide, vitamin B12, biotin, and folic acid; amino acids; nucleic acids; and organic components containing them, such as peptones, casein amino acids, yeast extracts, and plant protein hydrolysates (HVP; for example, soybean protein decomposition products, soybean soy sauce, pea soy sauce, etc.). Other examples of organic and inorganic components include defoamers, osmotic pressure adjusting substances for culture media, and osmotic pressure compensating substances. Examples of defoamers include organosilicon defoamers (oil-based, solution-based, oil-compound-based, emulsion-based, self-emulsifying, etc.), alcohol-based defoamers, oil-based defoamers, polyether-based defoamers, and vegetable oils (cottonseed oil, flaxseed oil, soybean oil, olive oil, castor oil, coconut oil, etc.). As defoamers, defoamers in any form, such as liquid, paste, solid, powder, emulsion, or wax, can be used. As osmotic pressure adjusters for culture media, examples include salts such as sodium chloride and potassium chloride, and polysaccharides that microorganisms cannot assimilate (sorbitol, dextrin, etc.). As osmotic pressure compensators, examples include potassium ions, betaine (glycine betaine), waste molasses (especially beet waste molasses), glutamic acid, and trehalose. Additionally, as optional components added to the culture medium, examples include polymers selected from water-soluble cellulose derivatives, water-soluble polyvinyl compounds, polyvinyl compounds soluble in polar organic solvents, water-soluble starch derivatives, alginates, and polyacrylates. These other various organic and inorganic components can be used individually or in combination of two or more components.

[0205] In addition, when using auxotrophic mutants that require amino acids or other nutrients for growth, it is preferable to supplement the required nutrients in the culture medium.

[0206] In addition, it is also preferable to limit the amount of biotin in the culture medium, or to add surfactants or penicillin to the culture medium.

[0207] The culture conditions are not particularly limited as long as they enable the bacteria of the present invention to proliferate and produce L-glutamic acid. Culture can be carried out, for example, under the usual conditions used for culturing bacteria such as rod-shaped bacteria. The culture conditions can be appropriately set according to various factors such as the type of bacteria used.

[0208] Cultivation can be carried out using liquid culture media. For example, the methods described in "Biotechnology Textbook Series 13: Cultivation Engineering, Toshiomi Yoshida, Corona Publishing, 1998" can be used for liquid cultivation. That is, for example, surface cultivation, deep cultivation, membrane (dialysis membrane, hollow fiber, etc.) separation cultivation, or immobilized microbial cultivation can be used for liquid cultivation. Furthermore, for cultivation devices, for example, aerated and stirred cultivation devices, airlift cultivation devices, packed bed cultivation devices, or fluidized bed cultivation devices can be used. During cultivation, the methods described in "Fundamentals of Fermentation Engineering, Academic Publishing Center, 1988" can be used. During cultivation, bacteria cultured in a solid culture medium such as agar can be directly inoculated into the liquid culture medium, or bacteria seeded in a liquid culture medium can be inoculated into the liquid culture medium for the main culture. That is, cultivation can be divided into seed culture and main culture. In this case, the cultivation conditions for seed culture and main culture can be the same or different. There is no particular limitation on the amount of bacteria of the present invention contained in the culture medium at the start of the culture. The main culture can be carried out, for example, by inoculating the main culture medium with 1-50% (v / v) of seed culture. Alternatively, for example, to obtain the amount of bacteria necessary for the main culture process, the seed culture process may include two or more seed culture stages. Furthermore, the seed culture may be inoculated only at the start of the main culture, or it may be further inoculated during the main culture process in addition to the inoculation at the start of the main culture.

[0209] Culture can be carried out through batch culture, fed-batch culture, continuous culture, or a combination thereof. Examples of combinations include connecting fed-batch culture to two or more stages, or connecting continuous culture to two or more stages. It should be noted that the culture medium at the beginning of the culture is also called the "initial culture medium." Furthermore, the culture medium supplied to the culture system (fermentation tank) in fed-batch or continuous culture is also called "fed-batch medium." It should be noted that when the culture is divided into seed culture and master culture, for example, both the seed culture and master culture can be carried out in batch culture. Alternatively, for example, the seed culture can be carried out in batch culture, and the master culture can be carried out in fed-batch or continuous culture. Alternatively, for example, the seed culture can be carried out in fed-batch culture, and the master culture in batch culture. Feed-on culture medium can be supplied from a portion of the culture tank at the top that is not in contact with the liquid surface, or from a portion inside the culture tank, such as the middle or bottom of the tank, or from both the top and middle portions of the tank. A method of supplying feed-on culture medium from a portion inside the culture tank is disclosed, for example, in Japanese Patent No. 6097869.

[0210] In this invention, each culture medium component may be contained in an initial culture medium, a fed-batch culture medium, or both. The types of components contained in the initial culture medium may be the same as or different from those contained in the fed-batch culture medium. Furthermore, the concentrations of each component in the initial culture medium may be the same as or different from those in the fed-batch culture medium. Additionally, two or more fed-batch culture media containing different types and / or concentrations of components may be used. For example, in the case of intermittent multiple fed-batch operations, the types and / or concentrations of components contained in each fed-batch culture medium may be the same or different. For example, glucose may be used as the carbon source for the initial culture medium, and sucrose may be used as the carbon source for the fed-batch culture medium.

[0211] Culture media can be sterilized, or not. Sterilization can be performed to prevent contamination. Sterilization of culture media can also be referred to as sterilization or disinfection. Methods for sterilizing culture media include sterilization under high temperature and pressure (HTHP), sterilization based on UV irradiation, and sterilization using filters or membranes. Sterilization of culture media can be performed in batches or continuously. For example, methods for batch sterilization under HTHP include autoclave sterilization and batch sterilization in a culture tank. Alternatively, methods for continuous sterilization under HTHP include continuous sterilization using a plate heat exchanger. Furthermore, sugar sterilization can be performed simultaneously with other culture media components or separately. It is preferable to sterilize sugar separately from other components.

[0212] The concentration of the carbon source in the culture medium is not particularly limited as long as it enables the bacteria of the present invention to proliferate and produce L-glutamic acid. The concentration of the carbon source in the culture medium can be as high as possible, for example, within a range that does not impair L-glutamic acid production. The concentration of the carbon source in the culture medium, for example, can be 1-50 w / v%, preferably 1-30 w / v%, more preferably 3-10 w / v%, based on the initial concentration (concentration in the initial culture medium). Furthermore, additional carbon source can be added to the culture medium as appropriate. For example, additional carbon source can be added to the culture medium in response to the consumption of carbon source accompanying fermentation. In addition, in fed-batch culture or continuous culture, the amount of carbon source supplied can be either sufficient (supplying an amount exceeding the carbon assimilation capacity of the bacteria of the present invention) or limiting (supplying an amount less than the carbon assimilation capacity of the bacteria of the present invention).

[0213] Culture can be carried out, for example, using liquid culture medium under aerobic or microaerobic conditions. "Aerobic conditions" refers to a dissolved oxygen concentration in the liquid culture medium that is 0.33 ppm or higher, preferably 1.5 ppm or higher, which is the detection limit for the oxygen membrane electrode. The oxygen concentration under aerobic conditions can be controlled, for example, to be 5-50% relative to the saturated oxygen concentration, preferably around 10%. "Microaerobic conditions" refers to conditions where the dissolved oxygen concentration in the culture medium is less than 0.33 ppm. The dissolved oxygen concentration in the culture medium under microaerobic conditions can be, for example, less than 0.30 ppm, less than 0.25 ppm, less than 0.20 ppm, less than 0.15 ppm, less than 0.10 ppm, or less than 0.05 ppm. The oxygen concentration under microaerobic conditions can be controlled, for example, to be less than 5% relative to the saturated oxygen concentration, less than 3.75%, less than 3.125%, less than 2.5%, less than 1.875%, less than 1.25%, or less than 0.8125%. Specifically, the culture can be carried out by aeration, shaking, stirring, or a combination thereof. The pH of the culture medium can be, for example, pH 3 to 10, preferably pH 4.0 to 9.5. During the culture, the pH of the culture medium can be adjusted as needed. The pH of the culture medium can be adjusted using various alkaline or acidic substances such as ammonia, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide. The culture temperature can be, for example, 20 to 40°C, preferably 25°C to 37°C. In the main culture, the culture temperature can be varied in two or more stages. For example, as disclosed in Journal of Industrial Microbiology & Biotechnology (2002) 28, 333-337, the culture temperature can be moved from 33°C to a higher temperature side, such as 37 to 40°C. The culture period can be, for example, 10 hours to 120 hours. The culture can continue, for example, until the carbon source in the culture 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 is accumulated in the culture medium and / or within the bacteria.

[0214] Alternatively, a liquid culture medium adjusted to conditions for L-glutamic acid precipitation can be used, allowing L-glutamic acid to precipitate in the medium while culturing. Examples of conditions for L-glutamic acid precipitation include pH 5.0–4.0, preferably pH 4.5–4.0, more preferably pH 4.3–4.0, and particularly preferably pH 4.0 (EP1078989A). Furthermore, when using a liquid culture medium adjusted to conditions for L-glutamic acid precipitation, adding pantothenic acid to the medium can further enhance crystallization efficiency (WO2004 / 111258). Additionally, when using a liquid culture medium adjusted to conditions for L-glutamic acid precipitation, adding L-glutamic acid crystals as seed crystals to the medium can further enhance crystallization efficiency (EP1233069A). In addition, when using a liquid culture medium with conditions adjusted to allow L-glutamic acid to precipitate, crystallization can be performed more efficiently by adding L-glutamic acid crystals and L-lysine crystals as seed crystals to the culture medium (EP1624069A).

[0215] The fermentation broth can be treated, for example, using a hydrocyclone. As a hydrocyclone, a ceramic, stainless steel, or resin device with a general shape and a cylindrical section diameter of 10-110 mm can be used. The feed rate 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 rate of the fermentation broth to the hydrocyclone can be, for example, 2-1200 L / min.

[0216] The formation of L-glutamic acid can be confirmed using well-known methods used for the detection or identification of compounds. Examples of such methods include HPLC, LC / MS, GC / MS, and NMR. These methods can be used individually or in appropriate combinations.

[0217] The recovery of L-glutamic acid from fermentation broth can be carried out using known methods used for the separation and purification of compounds. Examples of such methods include ion exchange resin methods (Nagai, H. et al., Separation Science and Technology, 39(16), 3691-3710), precipitation methods, membrane separation methods (Japanese Patent Application Publication No. 9-164323, Japanese Patent Application Publication No. 9-173792), and crystallization methods (WO2008 / 078448, WO2008 / 078646). These methods can be used alone or in appropriate combinations. It should be noted that when L-glutamic acid accumulates within the bacterial cells, for example, the cells can be broken up using ultrasound, and the cells can be removed by centrifugation to obtain a supernatant. L-glutamic acid can then be recovered from this supernatant using methods such as ion exchange resin methods. The recovered L-glutamic acid can be in its free form, its salt, or a mixture thereof. Examples of salts include sulfates, hydrochlorides, carbonates, ammonium salts, sodium salts, and potassium salts. Specifically, for example, it can be free L-glutamic acid, monosodium L-glutamate (e.g., monosodium L-glutamate; MSG), ammonium L-glutamate (e.g., monoammonium L-glutamate), or mixtures thereof. For example, monosodium glutamate (MSG) can be obtained by adding acid to crystallize ammonium L-glutamate from the fermentation broth and then adding an equimolar amount of sodium hydroxide to the crystals. It should be noted that activated carbon can also be added before or after crystallization for decolorization (see *Industrial Crystallization of Monosodium Glutamate*, *Journal of the Japanese Marine Science Society*, Vol. 56, No. 5, by Tetsuya Kawakita). Monosodium glutamate crystals can be used, for example, as a umami seasoning. Monosodium glutamate crystals can also be mixed with nucleic acids such as sodium guanylate and sodium inosinate, which also have umami flavor, for use as a seasoning.

[0218] In addition, if L-glutamic acid precipitates in the culture medium, it can be recovered by centrifugation or filtration. Alternatively, the L-glutamic acid precipitated in the culture medium can be separated together with the precipitated L-glutamic acid after crystallization.

[0219] It should be noted that the recovered L-glutamic acid may contain not only L-glutamic acid but also bacterial cells, culture medium components, moisture, and bacterial metabolic byproducts. L-glutamic acid can be purified to the desired degree. The purity of the recovered L-glutamic acid can be, for example, 50% (w / w) or more, preferably 85% (w / w) or more, and particularly preferably 95% (w / w) or more (Japanese 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, and US Patent Application Publication No. 2005 / 0025878).

[0220] Example

[0221] The present invention will now be described in further detail with reference to non-limiting embodiments.

[0222] <1> Construction of modified strains of Corynebacterium glutamicum

[0223] <1-1> Construction of vector for wild-type glyA gene introduction

[0224] A DNA fragment containing the wild-type glyA gene was amplified by PCR using chromosomal DNA from Corynebacterium glutamicum ATCC 13869 (strain 2256) containing the wild-type glyA gene as a template and primers SEQ ID NO: 5 and SEQ ID NO: 6. The amplified DNA fragment was then ligated with pVK9 (US Patent Application Publication No. 2006 / 0141588), which was cut with bamHI and pstI, via an in-fusion reaction to obtain the wild-type glyA gene delivery vector (pVK9-glyA (WT)).

[0225] The base sequence of the wild-type glyA gene and the amino acid sequence of the wild-type GlyA protein encoded by the gene are shown in Serial No. 1 and Serial No. 2, respectively.

[0226] <1-2> Construction of the vector for introducing the mutant glyA gene

[0227] A DNA fragment containing the mutant glyA gene was amplified by PCR using chromosomal DNA from a Corynebacterium glutamicum ATCC 13869 (strain 2256) derivative containing the mutant glyA gene as a template and primers SEQ ID NO: 5 and SEQ ID NO: 6. The mutant glyA gene has a mutation (G265S) in which a glycine residue at position 265 of the wild-type GlyA protein is replaced with a serine residue. The amplified DNA fragment was ligated to pVK9, which was cleaved with bamHI and pstI, via an In-Fusion reaction to obtain the mutant glyA gene delivery vector (pVK9-glyA (G265S)).

[0228] The base sequence of the mutant glyA gene and the amino acid sequence of the mutant Gly protein encoded by the gene are shown in Serial No. 3 and Serial No. 4, respectively.

[0229] <1-3> Construction of modified strains of Corynebacterium glutamicum

[0230] Corynebacterium glutamicum strain 2256ΔsucAΔldhA yggB* (WO2014 / 185430) was transformed using the constructed mutant gene transfer vectors. Following the method described in WO2006 / 057450, strains were selected from the transformed organisms to obtain wild-type glyA gene-transferred strains and mutant glyA gene-transferred strains.

[0231] Wild-type and mutant glyA gene-introducing strains were obtained by separately introducing the constructed wild-type glyA gene-introducing vector (pVK9-glyA(WT)) and mutant glyA gene-introducing vector (pVK9-glyA(G265S)) into *Corynebacterium glutamicum* strain 2256ΔsucAΔldhA yggB*. Additionally, a control strain was obtained by separately introducing pVK9 into *Corynebacterium glutamicum* strain 2256ΔsucAΔldhA yggB*.

[0232] It should be noted that Corynebacterium glutamicum 2256ΔsucAΔldhA yggB* strain is an L-glutamate producing strain derived from Corynebacterium glutamicum 2256 (ATCC 13869), which lacks the ldhA and sucA genes and has an IS mutation (V419::IS) in the yggB gene.

[0233] <2> L-Glutamic Acid Production Cultivation

[0234] L-glutamic acid production was carried out using the constructed strains (i.e., wild-type glyA gene-introduced strain, mutant glyA gene-introduced strain, and control strain). The composition of the culture media used is shown in Table 1.

[0235]

[0236] Each strain was inoculated into 20 mL of the above-mentioned culture medium (containing 50 g / L calcium carbonate) in a 500 mL Sakaguchi flask and cultured using a shaker (ABLE ML-190) at 31.5 °C and 120 rpm. The culture medium was sampled 25 hours after the start of incubation. The absorbance (OD) at 620 nm was measured by diluting 0.05 mL of the sampled culture medium 100-fold with 4.95 mL of 0.1 N HCl solution. Furthermore, the concentration of L-glutamic acid in the supernatant was quantified using a Biotech Analyzer BF7 (Oji Analyzer Co., Ltd.), and the yield of L-glutamic acid relative to sugar was calculated.

[0237] The results are shown in Figure 1 and Figure 2 The mutant glyA gene-introduced strain showed higher accumulation of L-glutamate and higher L-glutamate yield relative to sugar compared to the wild-type glyA gene-introduced strain. Figure 1 and Figure 2 ).

[0238] Industrial applicability

[0239] According to the present invention, the L-amino acid production capacity of rod-shaped bacteria can be improved, and L-amino acids can be produced efficiently.

[0240] [Explanation of sequence lists]

[0241] Sequence number 1: Base sequence of the wild-type glyA gene of Corynebacterium glutamicum 2256 (ATCC 13869)

[0242] Sequence No. 2: Amino acid sequence of wild-type GlyA protein from Corynebacterium glutamicum 2256 (ATCC 13869)

[0243] Sequence number 3: Base sequence of the mutant glyA gene in Corynebacterium glutamicum 2256 (ATCC 13869)

[0244] Sequence No. 4: Amino acid sequence of mutant GlyA protein from Corynebacterium glutamicum 2256 (ATCC 13869)

[0245] Serial numbers 5 and 6: Primers

[0246] Sequence number 7: Base sequence of the wild-type yggB gene in Corynebacterium glutamicum 2256 (ATCC 13869)

[0247] Serial number 8: Amino acid sequence of wild-type YggB protein from Corynebacterium glutamicum 2256 (ATCC 13869)

[0248] Sequence number 9: Base sequence of the mutant yggB gene (V419::IS)

[0249] Sequence number 10: Amino acid sequence of mutant YggB protein (V419::IS)

Claims

1. A method for producing L-glutamic acid, the method comprising: Culturing Corynebacterium glutamicum in a culture medium allows L-glutamate to accumulate in the culture medium and / or within the bacteria. as well as L-glutamic acid was collected from the culture medium and / or the bacterial cells. The *Corynebacterium glutamicum* strain described herein possesses L-glutamate production capabilities and has been modified to retain the mutant *glyA* gene encoding a mutant serine hydroxymethyltransferase, wherein the mutant serine hydroxymethyltransferase exhibits a substitution of a glycine residue at position 265 in the amino acid sequence of the wild-type serine hydroxymethyltransferase for a serine residue. The mutant serine hydroxymethyltransferase contains the amino acid sequence of sequence number 4.

2. The manufacturing method according to claim 1, wherein, The Corynebacterium glutamicum strain was modified to maintain the mutant yggB gene. The mutant yggB gene is a gene that encodes a protein with an amino acid sequence having sequence number 10.

3. The manufacturing method according to claim 1 or 2, wherein, The mutant glyA gene contains the base sequence shown in sequence number 3 or a degenerate variant based on that base sequence.

Images