Method for producing l-glutamic acid

Description

/ 47 METHOD FOR PRODUCING L-GLUTAMIC ACID Technical Field

[001] This invention relates to the fermentation industry and, in particular, to a method for producing L-glutamic acid and to the bacteria used in this method. Fundamentals of the Technique

[002] L-amino acids are produced industrially, for example, by a fermentation method that uses microorganisms such as bacteria capable of producing L-amino acids (Non-Patent Document 1). For example, strains isolated from nature and their mutant strains are used as such microorganisms. Furthermore, the ability of microorganisms to produce L-amino acids can be improved by recombinant DNA technology.

[003] Serine hydroxymethyltransferase is an enzyme that has the activity of catalyzing the hydroxymethyltransfer reaction of serine and / or glycine (https: / / www.genome.jp / entry / 2.1.2.1), but its contribution to the production of L-glutamic acid is not yet clear. References to Prior Technique Non-Patent Document

[004] Non-Patent Document 1: Kunihiko Akashi et al. Amino Acid Fermentation, Gakkai Shuppan Center, pp. 195-215, 1986 Summary of the Invention Problem to be Solved by the Invention

[005] An objective of the present invention is to develop a new technique to improve the ability of bacteria to produce L-glutamic acid and to provide an efficient method for producing L-glutamic acid and bacteria for use in this method. Ways to Solve the Problem

[006] As a result of intensive studies to solve the Petition 870260059118, dated 06 / 17 / 2026, p. 11 / 106 / 47 Regarding the above problem, the inventors discovered that by modifying the coryneform bacteria so that the serine hydroxymethyltransferase has a specific mutation, the bacteria's ability to produce L-glutamic acid can be improved, thus concluding the present invention.

[007] The invention can be exemplified as follows. [1]

[008] A coryneform bacterium with the ability to produce acid L-glutamic acid, in which the said bacterium was modified to harbor a mutant glyA gene that encodes a mutant serine hydroxymethyltransferase with the substitution of a glycine residue at position 265 in the amino acid sequence of the wild-type serine hydroxymethyltransferase for another amino acid residue. [2]

[009] The coryneform bacterium of [1], in which the other amino acid is lysine, glutamic acid, threonine, serine, aspartic acid, asparagine, glutamine, arginine, cysteine, histidine or tyrosine. [3]

[0010] Coryneform bacteria described in [1] in which the other amino acid is serine. [4]

[0011] The coryneform bacterium as described in any one of (1] to [3], wherein the wild-type serine hydroxymethyltransferase is a protein as described in (a), (b) or (c) below.

[0012] (a) a protein comprising the amino acid sequence shown in SEQ ID No: 2; (b) a protein comprising an amino acid sequence that includes substitutions, deletions, insertions and / or additions of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID No: 2, and with serine hydroxymethyltransferase activity; Petition 870260059118, dated 06 / 17 / 2026, page 12 / 106 / 47 (c) a protein comprising an amino acid sequence with 90% or more identity to the amino acid sequence shown in SEQ ID No: 2, and with serine hydroxymethyltransferase activity. [5]

[0013] The coryneform bacterium as described in any one of [1] to [4], wherein said coryneform bacterium is a bacterium belonging to the genus Corynebacterium. [6]

[0014] The coryneform bacterium as described in any one of [1] to [4], wherein said coryneform bacterium is Corynebacterium glutamicum. [7]

[0015] The coryneform bacterium of any one of [1] to [6], wherein said coryneform bacterium has been modified to harbor a mutant yggB gene, and wherein the mutant yggB gene is a gene encoding a protein with an amino acid sequence that includes substitutions, deletions, insertions and / or additions of one or more amino acids in the amino acid sequence of SEQ ID No: 8. [8]

[0016] The coryneform bacterium described in [7], where the mutant gene yggB is a gene that codes for a protein comprising the amino acid sequence of SEQ ID N°: 10. [9]

[0017] A method for producing L-glutamic acid, comprising cultivating the coryneform bacterium described in any one of [1] to [8] in a culture medium to accumulate L-glutamic acid in said medium and / or in the bacterial cells, and collecting L-glutamic acid from the culture medium and / or from the bacterial cells. Petition 870260059118, dated 06 / 17 / 2026, page 13 / 106 / 47

[10]

[0018] A mutant serine hydroxymethyltransferase with the substitution of a glycine residue at position 265 in the amino acid sequence of wild-type serine hydroxymethyltransferase for another amino acid residue.

[11]

[0019] The mutant serine hydroxymethyltransferase of

[10] , in which the other amino acid is lysine, glutamic acid, threonine, serine, aspartic acid, asparagine, glutamine, arginine, cysteine, histidine or tyrosine.

[12]

[0020] The mutant serine hydroxymethyltransferase of

[10] , in which the other amino acid is serine.

[13]

[0021] The mutant serine hydroxymethyltransferase as described in any one of

[10] to

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

[0022] (a) a protein comprising the amino acid sequence shown in SEQ ID No: 2; (b) a protein comprising an amino acid sequence that includes substitutions, deletions, insertions and / or additions of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID No: 2, and with serine hydroxymethyltransferase activity; (c) a protein comprising an amino acid sequence with 90% or more identity to the amino acid sequence shown in SEQ ID No: 2, and with serine hydroxymethyltransferase activity.

[14]

[0023] A mutant glyA gene encoding mutant serine hydroxymethyltransferase according to any one of

[10] a Petition 870260059118, dated 06 / 17 / 2026, p. 14 / 106 / 47

[13] . Brief Description of the Drawings

[0024] Figure 1: Accumulation of L-glutamic acid in the strain introduced by the wild-type glyA gene and in the strain introduced by the mutant glyA gene.

[0025] Figure 2: Yield of L-glutamic acid per sugar in the strain introduced by the wild-type glyA gene and in the strain introduced by the mutant glyA gene. Methods for Implementing the Invention

[0026] The invention is described in detail below.

[0027] The method of the present invention is a method for producing L-glutamic acid, comprising: cultivating a coryneform bacterium capable of producing L-glutamic acid in a culture medium to accumulate L-glutamic acid in the culture medium and / or in the bacterial cells, and collecting L-glutamic acid from the culture medium and / or from the cells, wherein the coryneform bacterium has been modified to harbor a specific genetic mutation. The bacterium used in the method is also referred to as the “bacterium of the present invention”. <1> Bacteria of the present invention

[0028] The bacterium is a coryneform bacterium that produces L-glutamic acid and has been modified to harbor the specific genetic mutation. <1-1> Coryneform Bacteria Capable of Producing L-Glutamic Acid

[0029] In the present invention, “coryneform bacteria capable of producing L-glutamic acid” refers to a coryneform bacterium that has the ability to produce L-glutamic acid and accumulate it in the medium and / or within the bacterial cells to the point that it can be collected when cultured in the medium. The coryneform bacterium capable of producing L-glutamic acid may be a coryneform bacterium capable of accumulating larger quantities of L-glutamic acid in the medium and / or in the bacterial cells than a non-coryneform strain. Petition 870260059118, dated 06 / 17 / 2026, p. 15 / 106 / 47 modified. The term “unmodified strain” refers to a control strain that has not been modified to harbor the specific genetic mutation. In other words, the unmodified strain includes the wild-type strain and the parental strain. Coryneform bacteria capable of producing L-glutamic acid may be coryneform bacteria capable of accumulating the target L-amino acid in the medium in an amount of 0.5 g / L or more, preferably 1.0 g / L or more.

[0030] The bacteria may produce L-glutamic acid in isolation or as a mixture of L-glutamic acid and one or more amino acids other than L-glutamic acid, such as L-form amino acids (also called L-amino acids).Examples of L-amino acids include, but are not limited to, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, lysoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

[0031] In the present invention, the terms “glutamic acid” and “amino acid” mean L-glutamic acid and L-amino acid, respectively, unless otherwise indicated.

[0032] In the present invention, the terms “L-glutamic acid” and “L-amino acid” mean free L-glutamic acid, free L-amino acid, their salts and / or mixtures thereof, unless otherwise indicated. The salts are explained below.

[0033] Coryneform bacteria include bacteria belonging to genera such as Corynebacterium, Brevibacterium, and Microbacterium.

[0034] Coryneform bacteria specifically include the following species.

[0035] Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Petition 870260059118, dated 17 / 06 / 2026, p. 16 / 106 / 47 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

[0036] Corynebacterium glutamicum (formerly known as Brevibacterium lactofermentum) is a particular example of a coryneform bacterium.

[0037] Coryneform bacteria include, specifically, the following strains.

[0038] Corynebacterium acetoacidophilum ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium alkanolyticum ATCC 21511 Corynebacterium callunae ATCC 15991 Corynebacterium crenatum AS1.542 Petition 870260059118, dated 17 / 06 / 2026, p. 17 / 106 / 47 Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734 Corynebacterium lilium ATCC 15990 Corynebacterium molassecola 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

[0039] A more specific example of a coryneform bacterium is Brevibacterium lactofermentum (updated name: Corynebacterium glutamicum) ATCC 13869.

[0040] Another example of coryneform bacteria is from the strain C. glutamicum 2256ΔsucAΔldhA yggB*, which does not possess the genes ldhA and sucA from Corynebacterium glutamicum ATCC 13869 and has a mutation IS (V419::IS) no gene yggB (WO2014 / 185430). Petition 870260059118, dated 17 / 06 / 2026, p. 18 / 106 / 47

[0041] Coryneform bacteria also include bacteria that were previously classified in the genus Brevibacterium, but are now integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255(1991)). Corynebacterium stationis also includes bacteria that were previously classified as Corynebacterium ammoniagenes, but were reclassified to Corynebacterium stationis based on 16S rRNA sequencing and other analyses (Int. J. Syst. Evol. Microbiol. 60, 874-879 (2010)).

[0042] These strains are available, for example, from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 PO Box 1549, Manassas, VA 20108, United States of America. Each strain is assigned a registration number, which can be used for distribution (http: / / www.atcc.org / ). The registration number corresponding to each strain is listed in the American Type Culture Collection catalog. These strains can also be obtained, for example, from the repository where each strain was deposited.

[0043] The bacteria of the present invention may be those that inherently possess the ability to produce L-glutamic acid or may be modified to obtain the ability to produce L-glutamic acid. The L-glutamic acid-producing bacteria may be obtained by conferring the ability to produce L-glutamic acid to the bacteria, as described above, or by increasing the ability to produce L-glutamic acid in the bacteria, as described above.

[0044] Conferring or increasing the ability to produce L-glutamic acid can be accomplished by methods conventionally employed in the rearing of amino acid-producing bacteria, such as coryneform bacteria or Escherichia coli (Amino Acid Fermentation, Gakkai Publishing Center, Inc. (pages 77 to 100, first published May 30, 1986). Such methods include, for example, obtaining auxotrophic mutant strains of Petition 870260059118, dated 06 / 17 / 2026, page 19 / 106 / 47 nutrients, obtain strains resistant to L-glutamic acid analogs, obtain metabolic control mutant strains, and create recombinant strains with increased enzyme activity in the L-glutamic acid biosynthetic system. Glutamic acid-producing bacteria can be cultivated for one, two, or three or more of the following characteristics: nutrient auxotrophy, analog resistance, and metabolic control mutation, etc. The activity of L-glutamic acid biosynthetic enzymes that are increased in the reproduction of L-glutamic acid-producing bacteria can also be one, two, or three or more. Furthermore, the increased activity of biosynthetic enzymes can be combined with the transmission of properties such as nutrient auxotrophy, analog resistance, and metabolic control mutations.

[0045] Nutrient-resistant or metabolically regulated auxotrophic mutant strains capable of producing L-glutamic acid can be obtained by subjecting parental or wild-type strains to conventional mutagenic treatment and selecting from the resulting mutant strains those that exhibit nutrient-resistant or metabolically regulated auxotrophic mutations and also have the ability to produce L-glutamic acid. Conventional mutagenic treatments include X-ray or ultraviolet irradiation, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and other mutagenic treatments.

[0046] The ability to produce L-glutamic acid can also be conferred or increased by increasing the activity of enzymes involved in L-glutamic acid biosynthesis. For example, enzyme activity can be increased by modifying the bacterium so that the expression of the gene encoding the enzyme is increased. Methods for increasing gene expression are described in documents WO00 / 18935, EP1010755A, etc.

[0047] The ability to produce L-glutamic acid can also be Petition 870260059118, dated 06 / 17 / 2026, p. 20 / 106 / 47 conferred or increased by reducing the activity of enzymes that catalyze branch reactions of the L-glutamic acid biosynthetic pathway to produce compounds other than L-glutamic acid. The term "enzymes that catalyze branch reactions of the L-glutamic acid biosynthetic pathway to produce compounds other than L-glutamic acid" here includes enzymes involved in the degradation of L-glutamic acid.

[0048] The following are specific examples of L-glutamic acid-producing bacteria and methods for conferring or enhancing the ability to produce L-glutamic acid. The properties possessed by L-glutamic acid-producing bacteria and the modifications to confer or enhance the ability to produce L-glutamic acid, as exemplified below, may be used individually or in combination, as appropriate.

[0049] Methods to confer or increase the ability to produce L-glutamic acid include, for example, modifying bacteria to increase the activity of one or more enzymes selected from among the L-glutamic acid biosynthetic enzymes. These enzymes include, but are not limited to, 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), phosphoglycerol mutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triosephosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), glucose phosphate isomerase. (pgi), 6-phosphogluconate dehydratase (edd), 2-keto-3deoxy-6-phosphogluconate aldolase (edd) and transhydrogenase (pntAB).The names in parentheses are examples of genes that encode the enzymes (the same applies to the descriptions that follow). Among these enzymes, it is preferred to increase the activity of one or more selected enzymes from, for example, glutamate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, and so on. Petition 870260059118, dated 06 / 17 / 2026, p. 21 / 106 / 47 methyl citrate synthase.

[0050] Coryneform bacteria modified to increase the expression of the glutamate synthase gene (gltBD) include those described in document WO99 / 07853.

[0051] Methods for conferring or increasing the ability to produce L-glutamic acid include, for example, modifying bacteria so that the activity of one or more enzymes selected from among enzymes that catalyze branch reactions of the L-glutamic acid biosynthetic pathway to produce compounds other than L-glutamic acid is reduced. Such enzymes include, without limitation, isocitrate lyase (aceA), alpha-ketoglutarate dehydrogenase (sucA, odhA), acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), alcohol dehydrogenase (adh), glutamic acid decarboxylase (gadAB), and succinate dehydrogenase (sdhABCD). Among these enzymes, it is preferable, for example, to reduce or eliminate the activity of alpha-ketoglutarate dehydrogenase.

[0052] Coryneform bacteria with reduced or deficient α-ketoglutarate dehydrogenase activity and methods for obtaining them are described in document WO2008 / 075483. Specifically, the following strains are exemplified.

[0053] Strain Corynebacterium glutamicum (Brevibacterium lactofermentum) L30-2 (JP2006-340603A) Strain Corynebacterium glutamicum lactofermentum) AS (WO95 / 34672) Strain Corynebacterium glutamicum (Brevibacterium (Brevibacterium lactofermentum) AJ12821 (FERM BP-4172; French Patent No. 9401748) Corynebacterium glutamicum strain (Brevibacterium flavum) AJ12822 (FERM BP-4173; French Patent No. 9401748) Strain Corynebacterium glutamicum AJ12823 (FERM BP4174; French patent no. 9401748) Petition 870260059118, dated 06 / 17 / 2026, page 22 / 106 / 47

[0054] L-glutamic acid-producing bacteria or parental strains to induce it also include strains with reduced or deficient activities in the enzymes alpha-ketoglutarate dehydrogenase (sucA) and succinate dehydrogenase (sdh) (JP2010-041920A). Such strains specifically include, for example, a doubly odhA, sdhA-deficient strain of Corynebacterium glutamicum ATCC14067 (strain Corynebacterium glutamicum 8L3GΔSDH) (JP2010-041920A).

[0055] For coryneform bacteria, methods to confer or enhance the ability to produce L-glutamic acid include methods to confer resistance to organic acid analogs and respiratory inhibitors, as well as methods to confer sensitivity to cell wall synthesis inhibitors. Such methods include, for example, the method for conferring resistance to monofluoroacetic acid (JP S50-113209 A), the method for conferring resistance to adenine or thymine (JP S57-065198 A), the method for attenuating urease (JP S52-038088 A), the method for conferring resistance to malonic acid (JP S52-038088 A), a method for conferring resistance to benzopyrones or naphthoquinones (JP S56-1889 A), a method for conferring resistance to HOQNO (JP S56-140895 A), a method for conferring resistance to alpha-ketomalonic acid (JP S57-2689 A), resistance to guanidine (JP S56-35981 A) and sensitivity to penicillin (JP H4-88994 A).

[0056] Examples of these resistant or sensitive bacteria include the following onions.

[0057] Corynebacterium glutamicum (Brevibacterium flavum) AJ3949 (FERM BP-2632; JP S50-113209 A) Corynebacterium glutamicum AJ11628 (FERM P-5736; JP S57-065198 A) Corynebacterium glutamicum (Brevibacterium flavum) AJ11355 (FERM P-5007; JP S56-1889 A) Corynebacterium glutamicum AJ11368 (FERM P-5020; JP Petition 870260059118, dated 17 / 06 / 2026, p. 23 / 106 / 47 S56-1889A) Corynebacterium glutamicum (Brevibacterium flavum) AJ11217 (FERM P-4318; JP S57-2689 A) Corynebacterium glutamicum AJ11218 (FERM P-4319; JP S57-2689 A) Corynebacterium glutamicum (Brevibacterium flavum) AJ11564 (FERM P-5472; JP S56-140895 A) Corynebacterium glutamicum (Brevibacterium flavum) AJ11439 (FERM P-5136; JP S56-35981 A) Corynebacterium glutamicum H7684 (FERM BP-3004; JP H04-88994 A) Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ11426 (FERM P-5123; JP S56-048890 A) Corynebacterium glutamicum AJ11440 (FERM P-5137; JP S56-048890 A) Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ11796 (FERM P-6402; JP S58-158192 A)

[0058] The efflux activity of L-glutamic acid from bacterial cells can be increased, for example, by increasing the expression of a gene encoding a protein that effluxes L-glutamic acid. The activity of excreting L-glutamic acid can be increased, for example, by increasing the expression of a gene encoding a protein that excretes L-glutamic acid. For example, the b2682 (ygaZ) gene, the b2683 (ygaH) gene, the b1242 (ychE) gene, and the b3434 (yhgN) gene are examples of genes encoding proteins that excrete various amino acids (JP 2002-300874 A).

[0059] Another method to confer or increase the ability to produce L-glutamic acid is, for example, to modify bacteria so that the activity of proteins involved in sugar metabolism or energy metabolism is increased. Petition 870260059118, dated 06 / 17 / 2026, page 24 / 106 / 47

[0060] The proteins involved in sugar metabolism include proteins involved in sugar absorption and glycolytic enzymes. 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 diphosphate 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 utilization gene (scrAB operon; U.S. Patent No. 7179623).

[0061] Genes encoding proteins involved in energy metabolism include the transhydrogenase gene (pntAB; U.S. Patent No. 5830716) and the cytochrome b-type oxidase gene (cyoB; EP1070376A).

[0062] For coryneform bacteria, methods to confer or enhance the ability to produce L-glutamic acid include increasing the expression of the yggB gene or introducing a mutant yggB gene with a mutation in the coding region (WO2006 / 070944). The bacterium can be modified to increase the expression of the yggB gene or to harbor (have) the mutant yggB gene.

[0063] The yggB gene is a gene that encodes a mechanosensitive channel. Examples of yggB genes include the yggB gene of coryneform bacteria. yggB genes from coryneform bacteria include, for example, the yggB genes of Corynebacterium glutamicum ATCC13869, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC 14967, Corynebacterium melassecola ATCC17965, and Corynebacterium callunae ATCC 15991 (WO2006 / 070944). The yggB gene of the ATCC 13032 strain corresponds to the complementary sequence of sequence 1,336,091-1,337,692 in the genome sequence recorded in the NCBI database under GenBank accession number NC_003450, also called Petition 870260059118, dated 06 / 17 / 2026, p. 25 / 106 / 47 NCgl1221. The YggB protein encoded by the yggB gene of Corynebacterium glutamicum ATCC13032 is registered as GenBank accession number NP_600492. The nucleotide sequence of the yggB gene of Corynebacterium glutamicum 2256 (ATCC 13869) and the amino acid sequence of the YggB protein encoded by the gene are shown in SEQ ID No. 7 and SEQ ID No. 8, respectively.

[0064] In the present invention, the yggB gene possessing the “specific mutation” described below is also called the mutant yggB gene, and the protein encoded by it is called the mutant YggB protein. In the present invention, the yggB gene without the “specific mutation” described below is also called the wild-type yggB gene, and the protein encoded by it is called the wild-type YggB protein. In the case of the YggB protein, the alteration in the amino acid sequence caused by the “specific mutation” in the yggB gene is also called the “specific mutation.” The term “wild-type” here is used for convenience to distinguish it from the “mutant type” and is not limited to those obtained naturally, provided they do not have this “specific mutation.” The wild-type YggB protein includes the YggB protein exemplified above, for example, a protein with the amino acid sequence shown in SEQ ID No. 8.Wild-type YggB protein may also include a conserved variant (a variant in which the original function is maintained) of the YggB protein in the example above that does not have a “specific mutation”. The “original function” of the YggB protein may be, for example, its function as a mechanosensitive channel or its ability to increase L-glutamic acid production in coryneform bacteria when overexpressed in coryneform bacteria.

[0065] Examples of specific mutations are not restricted, provided that the mutation alters the amino acid sequence of the wild-type YggB protein, as described above, and improves the ability of the coryneform bacterium to produce L-glutamic acid. “Specific mutation” includes mutations Petition 870260059118, dated 06 / 17 / 2026, p. 26 / 106 / 47 C-terminal and transmembrane region mutations (WO2006 / 070944). The “specific mutation” can also be a combination of these mutations. (1) C-terminal lateral mutation

[0066] A C-terminal side mutation is a mutation in the wild-type yggB gene region that encodes the amino acid residue at position 419 to 533 of the wild-type YggB protein. C-terminal side mutations can be introduced at one or more locations in the region. C-terminal side mutations can cause, for example, amino acid residue substitutions (missense mutations), amino acid residue insertions, amino acid residue deletions, stop codon occurrences (missense mutations), region-switching mutations, or combinations thereof, and among these, insertion sequences such as insertion sequences (also called “IS”) and transposons are preferred. (1-1) Sequence insertion

[0067] C-terminal side mutations include, for example, mutations that cause the insertion of a sequence into the codon of a valine residue at position 419 of the wild-type YggB protein (2A-1 type mutation). 2A-1 type mutations can cause, for example, deletion or substitution of some or all amino acid residues from 419 to 533 of the wild-type YggB protein. Specifically, a mutant yggB gene with a 2A-1 mutation is, for example, a yggB gene that encodes a mutant YggB protein with an IS inserted after the “G” at position 1255 in SEQ ID No.: 7 resulting in a total length of 423 amino acid residues, which is shorter than the original wild-type YggB protein (SEQ ID No.: 8). The nucleotide sequence of this mutant gene yggB (V419::IS) and the amino acid sequence of the mutant protein YggB (V419::IS) encoded by the 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). A specific example of a bacterium that produces... Petition 870260059118, dated 06 / 17 / 2026, p. 27 / 106 / 47 L-glutamic acid with a mutant yggB gene (V419::IS) is, for example, the strain C. glutamicum 2256AsucAAldhA yggB* (WO2014 / 185430). (1-2) Replacement of proline residue

[0068] Examples of C-terminal side mutations include mutations that replace the proline residue in the amino acid region 419 to 533 of the wild-type YggB protein with another amino acid. These 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. Of these, it is preferable to replace the proline residues at positions 424 and / or 437 with other amino acids. The “other amino acids” are, for example, but not limited to, naturally occurring amino acids other than proline. The other amino acid includes Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, His. For example, the proline residue at position 424 can preferably be replaced by a hydrophobic amino acid (Ala, Gly, Val, Leu or Ile) and more preferably by a branched-chain amino acid (Leu, Val or Ile).For example, the proline residue at position 437 can be replaced by an amino acid preferably with a hydroxyl group in the side chain (Thr, Ser or Tyr) and, more preferably, with Ser. (2) Mutation in transmembrane regions.

[0069] The YggB protein is considered to have five transmembrane regions. The transmembrane regions correspond to amino acid residues 1 to 23 (first transmembrane region), 25 to 47 (second transmembrane region), 62 to 84 (third transmembrane region), 86 to 108 (fourth transmembrane region), and 110 to 132 (fifth transmembrane region) of the wild-type YggB protein, respectively. Mutations in the transmembrane regions are mutations in the regions that encode these transmembrane regions in the wild-type yggB gene. Mutations in the transmembrane regions can be introduced at one or more locations in the same region. Mutations in the transmembrane region can cause one or more Petition 870260059118, dated 06 / 17 / 2026, p. 28 / 106 / 47 substitutions, deletions, additions, insertions or inversions of amino acids, but do not cause region switch mutation or nonsense mutation. “One or more” preferably means from 1 to 20, more preferably from 1 to 10, most preferably from 1 to 5, and especially preferably from 1 to 3. Mutations in the transmembrane region 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 of the wild-type YggB protein, substitution of the alanine residue at position 100 by another amino acid residue (e.g., an amino acid with a hydroxyl group in its side chain, such as Thr, Ser, or Tyr, preferably Thr), and substitution of the alanine residue at position 111 by another amino acid residue (e.g., Val or an amino acid with a hydroxyl group in its side chain, such as Thr, Ser, or Tyr, preferably Val or Thr).

[0070] In the present invention, “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, unless otherwise indicated. “Position X” in the amino acid sequence means position X counting from the N-terminus of the amino acid sequence, where the N-terminal amino acid residue is the amino acid residue at position 1. The position of an amino acid residue indicates its relative position, and its absolute position may change due to the deletion, insertion, or addition of amino acids. For example, “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, and if an amino acid residue on the N-terminal side is deleted from position 419, the amino acid residue 418° from the N-terminal will be “the amino acid residue at position 419 of the wild-type YggB protein”.If an amino acid residue is inserted into the N-terminal side of the protein, the amino acid residue 420° from the N-terminal will be the "residue of." Petition 870260059118, dated 06 / 17 / 2026, page 29 / 106 / 47 amino acid at position 419 of the wild-type YggB protein”. Specifically, for example, in the YggB protein of the Corynebacterium glutamicum ATCC14967 strain, the amino acid residue at position 419-529 corresponds to the amino acid residue at position 419-533 of the wild-type YggB protein. Furthermore, for example, the alanine residue at position 98 in the YggB protein of Corynebacterium callunae corresponds to the alanine residue at position 100 in the wild-type YggB protein.

[0071] In the amino acid sequence of any YggB protein, whose amino acid residue is “the amino acid residue corresponding to the amino acid residue at position X in SEQ ID No: 8”, it can be determined by aligning the amino acid sequence of the YggB protein with that of SEQ ID No: 8. The alignment can be performed, for example, using a known genetic analysis software. Specific software includes DNASIS from Hitachi Solutions and GENETYX from Xenetics (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).

[0072] The mutant yggB gene can be obtained by modifying the wild-type yggB gene to have the “specific mutation” described above. Specifically, for example, a site-directed mutation method to introduce the desired mutation at the target DNA site can be achieved using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P.,. Meth. In Enzymol., 154, 382 (1987)) and methods using phage (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987); Kunkel, TA et al.). The mutant yggB gene can also be obtained by chemical synthesis.

[0073] Modifying a coryneform bacterium to have a mutant yggB gene can be achieved by introducing a mutant yggB gene into the coryneform bacterium. Modifying the coryneform bacterium to have the mutant yggB gene can also be achieved by introducing mutations into the yggB gene. Petition 870260059118, dated 06 / 17 / 2026, page 30 / 106 / 47 possessed by the bacteria through spontaneous mutation or mutagenic treatment. <1-2> Modification with the mutant glyA gene

[0074] The coryneform bacterium was modified to harbor a mutant glyA gene that encodes a mutant serine hydroxymethyltransferase with the glycine residue at position 265 in the wild-type serine hydroxymethyltransferase amino acid sequence replaced by another amino acid residue.

[0075] The coryneform bacterium of the present invention can be modified to harbor the mutant glyA gene in a coryneform bacterium capable of producing L-glutamic acid, as described above, or it can be a coryneform bacterium modified to harbor the mutant glyA gene and thus confer the ability to produce L-glutamic acid. Also included are coryneform bacteria that have been modified to harbor the mutant glyA gene, thus gaining the ability to produce L-glutamic acid.

[0076] The glyA gene is a gene that encodes serine hydroxymethyltransferase. Examples of glyA genes include glyA genes from coryneform bacteria. Specifically, glyA genes from coryneform bacteria include, for example, the glyA gene from Corynebacterium glutamicum ATCC13869, the glyA gene from Corynebacterium glutamicum ATCC13032, the glyA gene from Corynebacterium glutamicum ATCC14967, and the glyA gene from Corynebacterium melassecola ATCC17965. The glyA gene from Corynebacterium glutamicum ATCC13032 is registered in the NCBI database under GenBank accession number NC_003450. The glyA protein encoded by the glyA gene of Corynebacterium glutamicum ATCC13032 is registered as GenBank accession number WP_003856790.1. The nucleotide sequence of the wild-type glyA gene of Corynebacterium glutamicum 2256 (ATCC 13869) and the sequence of Petition 870260059118, dated 06 / 17 / 2026, p. 31 / 106 / 47 amino acids of the GlyA protein encoded by the gene are shown in SEQ ID No.: 1 and SEQ ID No.: 2, respectively.

[0077] “Serine hydroxymethyltransferase” may refer to a protein that has the activity of catalyzing the hydroxymethyltransfer reaction of serine and / or glycine (e.g., EC 2.1.2.1). The activity is also called “serine hydroxymethyltransferase activity”. Serine hydroxymethyltransferase activity may specifically be the activity that catalyzes the reaction that converts L-serine and tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate and / or the reverse reaction of these. Serine hydroxymethyltransferase is also called “glycine hydroxymethyltransferase”. The gene encoding serine hydroxymethyltransferase is the glyA gene. The nucleotide sequences of serine hydroxymethyltransferase genes, such as the glyA gene, possessed by the bacteria to be modified, and the amino acid sequences of serine hydroxymethyltransferases, such as the GlyA protein encoded by them, can be obtained from public databases, such as the NCBI.

[0078] In the present invention, a glyA gene with the “specific genetic mutation” described below is also called a mutant glyA gene, and the protein encoded by it is also called a mutant GlyA protein. In the present invention, a glyA gene that does not possess the “specific genetic mutation” described below is also called a wild-type glyA gene, and the protein encoded by it is also called a wild-type GlyA protein. In the case of the GlyA protein, the alteration in the amino acid sequence caused by a “specific genetic mutation” in the glyA gene is also called a “specific genetic mutation.” The term “wild-type” here is used for convenience to distinguish it from the “mutant type” and is not limited to those obtained naturally, provided they do not have the “specific genetic mutation.” The wild-type GlyA protein includes the GlyA protein exemplified above, for example, a protein that Petition 870260059118, dated 06 / 17 / 2026, page 32 / 106 / 47, includes the amino acid sequence shown in SEQ ID No. 2. The wild-type GlyA protein also includes a conserved variant (a variant in which the original function is maintained) of the GlyA protein from the example above that does not have the “specific genetic mutation”. The “original function” of the GlyA protein includes, for example, its function as a serine hydroxymethyltransferase.

[0079] A “specific genetic mutation” is a mutation that alters the amino acid sequence of the wild-type GlyA protein described above and increases the ability of the coryneform bacterium to produce L-glutamic acid. A “specific genetic mutation” includes a mutation in the wild-type GlyA gene that modifies the amino acid at position 265 of the amino acid sequence of the wild-type GlyA protein.

[0080] Examples of mutations in the amino acid residue at position 265 of the amino acid sequence of the wild-type GlyA protein include substitutions of the amino acid residue (missense mutation).

[0081] For example, a mutation in the amino acid residue at position 265 of the wild-type GlyA protein is a mutation in which the glycine residue at position 265 of the wild-type GlyA protein is replaced by another amino acid residue. The “other amino acid” can be any naturally occurring amino acid other than glycine. The “other amino acids” include Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Pro, Ala, His. For example, the glycine residue at position 265 can be replaced by a hydrophilic amino acid (Lys, Glu, Thr, Ser, Asp, Asn, Gln, Arg, Cys, or His), preferably Ser. For example, the glycine residue at position 265 can be replaced by an amino acid preferably with a hydroxyl group in the side chain (Thr, Ser, or Tyr), and more preferably with Ser.

[0082] In the present invention, “amino acid residue at position X of the wild-type GlyA protein” means an amino acid residue Petition 870260059118, dated 06 / 17 / 2026, page 33 / 106 / 47 corresponding to the amino acid residue at position X in SEQ ID No: 2, unless otherwise indicated. The “X” position in the amino acid sequence means the X position counted from the N-terminus of the same amino acid sequence, where the N-terminal amino acid residue is the amino acid residue at position 1. The position of an amino acid residue indicates its relative position, and its absolute position can change due to the deletion, insertion, or addition of amino acids. For example, “amino acid residue at position 265 of the wild-type GlyA protein” means the amino acid residue corresponding to position 265 in SEQ ID No. 2. If an amino acid residue on the N-terminal side is deleted, the 264th N-terminal amino acid residue will be “the amino acid residue at position 265 of the wild-type GlyA protein”.If an amino acid residue is inserted on the N-terminal side of the protein, the 266th amino acid residue from the N-terminal will be the "amino acid residue at position 265 of the wild-type GlyA protein".

[0083] The amino acid sequence of any GlyA protein, whose 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 GlyA protein with the amino acid sequence of SEQ ID No: 2. The alignment can be performed, for example, using known genetic analysis software. Specific software includes DNASIS from Hitachi Solutions and GENETYX from Xenetics (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).

[0084] The mutant glyA gene can be obtained by modifying the wild-type glyA gene to have the “specific genetic mutation” described above. Specifically, for example, a site-directed mutation method to introduce the target mutation at the target DNA site is a method that uses PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton Petition 870260059118, dated 17 / 06 / 2026, p. 34 / 106 / 47 press (1989); Carter, P., et al. Meth. In Enzymol., 154, 382 (1987)) or using phage (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987); Kunkel, TA et al. 154, 367 (1987)). The mutant glyA gene can also be obtained by chemical synthesis.

[0085] The wild-type glyA gene may be, for example, a gene that has the nucleotide sequence of the glyA gene from the example above (for example, the nucleotide sequence shown in SEQ ID No.: 1). The glyA protein may be, for example, a protein that comprises the amino acid sequence of the glyA protein above (for example, the amino acid sequence shown in SEQ ID No.: 2). The expression “with the sequence (of amino acids or nucleotides)” means “comprising the sequence (of amino acids or nucleotides)” and includes the case “consisting of the sequence (of amino acids or nucleotides)”, unless otherwise specified.

[0086] The wild-type glyA gene can also be a variant of the glyA gene above (e.g., a gene comprising the sequence shown in SEQ ID No.: 1) provided the original function is maintained. Similarly, the GlyA protein can be a variant of the GlyA protein above (e.g., a protein with the amino acid sequence shown in SEQ ID No.: 2) provided the original function is maintained. A variant in which the original function is maintained can be called a “conservative variant.” The term “glyA gene” will encompass such conserved variants as well as the glyA genes exemplified above. Similarly, the term “GlyA protein” should encompass such conserved variants as well as the GlyA proteins exemplified above. Conservative variants include, for example, homologs or artificial variants of the glyA genes and GlyA proteins exemplified above.

[0087] The term “original function is maintained” means that the genetic or protein variant has a function (e.g., activity or property) that corresponds to the function (e.g., activity or property) of the gene or Petition 870260059118, dated 06 / 17 / 2026, page 35 / 106 / 47 of the original protein. “Original function maintained” for a gene means that the genetic variant codes for a protein for which the original function is maintained. In other words, “original function is maintained” for the glyA gene may mean that the variant of the glyA gene codes for a protein that has the activity of the GlyA protein, i.e., serine hydroxymethyltransferase activity. “Original function is maintained” for the GlyA protein may also mean that the variant of the GlyA protein has the activity of the GlyA protein, i.e., serine hydroxymethyltransferase activity.

[0088] The activity of serine hydroxymethyltransferase can be measured, for example, by incubating the enzyme with the corresponding substrates (e.g., L-serine and tetrahydrofolate) and measuring the enzyme- and substrate-dependent production of the corresponding products (e.g., glycine and 5,10-methylenetetrahydrofolate).

[0089] Below are some examples of conservative variants.

[0090] Homologs of the glyA gene or homologs of the GlyA protein can be easily obtained from public databases, for example, through BLAST or FASTA searches using the glyA gene nucleotide sequence above or the GlyA protein amino acid sequence above as a query sequence. GlyA gene homologs can also be obtained, for example, by PCR using chromosomes from various organisms as templates and oligonucleotides made based on these known glyA gene sequences as primers.

[0091] The glyA gene may be a gene encoding a GlyA protein with an amino acid sequence in which one or more amino acids at one or more positions are substituted, deleted, inserted, and / or added to the above amino acid sequence (e.g., amino acid sequence shown in SEQ ID No. 2), provided that the original function is maintained. For example, the encoded protein may have its N- and / or C-terminus extended or shortened. The “one or more” above means, depending on the Petition 870260059118, dated 06 / 17 / 2026, p. 36 / 106 / 47 position and type of amino acid residues in the three-dimensional structure of the protein, specifically, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5 and especially preferably 1 to 3.

[0092] Substitutions, deletions, insertions, and / or additions above one or more amino acids are conservative mutations in which the protein function is usually maintained. A typical conservative mutation is a conservative substitution. Conservative substitutions occur between Phe, Trp, and Tyr when an aromatic amino acid is substituted; between Leu, Ile, and Val when a hydrophobic amino acid is substituted; between Gln and Asn when a polar amino acid is substituted; between Lys, Arg, and His when a basic amino acid is substituted; between Asp and Glu when an acidic amino acid is substituted; and between Ser and Thr when an amino acid with a hydroxyl group is substituted.Substitutions that are considered conservative substitutions are, specifically, substitutions of Ala for Ser or Thr, of Arg for Gln, His or Lys, of Asn for Glu, Gln, Lys, His or Asp, of Asp for Asn, Glu or Gln, of Cys for Ser or Ala; of Gln for Asn, Glu, Lys, His, Asp or Arg; of Glu for Gly, Asn, Gln, Lys or Asp; of Gly for Pro; of His for Asn, Lys, Gln, Arg or Tyr; of Ile for Leu, Met, Val or Phe; of Leu for Ile, Met, Val or Phe; of Lys for Asn, Glu, Gln, His or Arg; of Met for Ile, Leu, Val or Phe; of Phe for Trp, Tyr, Met, Ile or Leu; of Ser for Thr or Ala, of Thr for Ser or Al; From Trp to Phe or Tyr, from Tyr to His, Phe or Trp, and from Val to Met, Ile or Leu. The above amino acid substitutions, deletions, insertions or additions also include those caused by natural mutations (mutants or variants), such as those based on individual differences or species differences of the organism from which the gene is derived.

[0093] The glyA gene may also be a gene encoding a protein comprising an amino acid sequence having, for example, 50% or more, 65% or more, 80% or more, preferably 90% or Petition 870260059118, dated 06 / 17 / 2026, p. 37 / 106 / 47 more, more preferably 95% or more, more preferably 97% or more, especially more preferably 99% or more of sequence identity with the entire amino acid sequence, provided that the original function is maintained.

[0094] The glyA gene can also be a gene (e.g., DNA) that hybridizes under strict conditions with a probe that can be prepared from the above sequence (e.g., the sequence shown in SEQ ID No.: 1), as a sequence complementary to all or part of the above sequence, provided that the original function is maintained. The term “under strict conditions” means under the following conditions. “Strict conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. An example is a condition under which DNAs of high identity hybridize with each other, for example, at least 50%, at least 65%, at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97%, especially most preferably at least 99%, and DNAs of lower identity do not hybridize with each other.Examples of such conditions include hybridization conditions with a normal probe, in which washing at a salt concentration and temperature corresponding to 60°C, 1x SSC, 0.1% SDS, preferably 60°C, 0.1x SSC, 0.1% SDS, more preferably 68°C, 0.1x SSC, 0.1% SDS is performed once, preferably 2 to 3 times can be mentioned.

[0095] As mentioned above, the probe used for the hybridization described above can be part of the complementary sequence of the gene. These probes can be made by PCR using oligonucleotides based on known genetic sequences as primers and DNA fragments containing the gene described above as templates. For example, a DNA fragment approximately 300 bp long can be used as a probe. Petition 870260059118, dated 06 / 17 / 2026, page 38 / 106 / 47 When a DNA fragment of approximately 300 bp in length is used as a probe, the conditions for hybridization washing include 50°C, 2x SSC, and 0.1% SDS.

[0096] The glyA gene can also have any codon alteration with its equivalent codon, since codon degeneracy occurs from host to host. In other words, the glyA gene can be a variant of the glyA gene exemplified above due to the degeneracy of the genetic code. For example, the glyA gene can be modified to have the optimal use of codons according to the frequency of codon use of the host used.

[0097] “Identity” between amino acid sequences means the identity between amino acid sequences calculated by blastp using the default Scoring Parameter settings (Matrix: BLOSUM62; Differential Costs: Existence = 11, Extension = 1; Composition Adjustments: Conditional composition scoring matrix adjustment). “Identity” between nucleotide sequences means the identity between nucleotide sequences calculated by blastn using the default Scoring Parameter settings (Compatibility / Incompatibility Scores = 1, -2; Differential Costs = Linear).

[0098] The above description of conservative variants of genes and proteins can be applied mutatis mutandis to any protein and the genes that encode it. That is, genes and proteins used for breeding L-glutamic acid-producing bacteria may, for example, have the nucleotide and amino acid sequences of known genes and proteins, such as the genes and proteins exemplified above, respectively. The genes and proteins used to create L-glutamic acid-producing bacteria may also be conservative variants of known genes and proteins, such as the genes and proteins exemplified above, respectively. Specifically, for example, a gene used to create L-glutamic acid-producing bacteria Petition 870260059118, dated 06 / 17 / 2026, p. 39 / 106 / 47 glutamic can be a gene that codes for a protein with an amino acid sequence in which one or more amino acids at one or more positions are substituted, deleted, inserted, or added to the amino acid sequence of a known protein, provided that the original function is maintained.

[0099] Methods for modifying the bacterium to harbor the mutant glyA gene include introducing a mutant glyA gene with a mutation in the coding region of the wild-type glyA gene into the coryneform bacterium, introducing a mutation in the coding region of the wild-type glyA gene that the coryneform bacterium possesses, and so on.

[00100] Modification of the coryneform bacterium to have a mutant glyA gene can be achieved by introducing a mutant glyA gene into the coryneform bacterium. Modification of the coryneform bacterium to have the mutant glyA gene can also be achieved by introducing mutations into the glyA gene of the coryneform bacterium by spontaneous mutation or mutagenic treatment.

[00101] The introduction of a mutant glyA gene into a coryneform bacterium can be accomplished by introducing the gene into the host chromosome. The introduction of the gene into the chromosome can be accomplished, for example, by homologous recombination (Miller, JH Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Gene transfer methods that use homologous recombination include, for example, the Red system-driven integration method (Datsenko, KA and Wanner, BL Proc. Natl. Acad. Sci. USA. 97:6640-6645 (USA. 97:6640-645 (2000)), plasmids containing a temperature-sensitive origin of replication, plasmids capable of junctional transmission, suicide vectors without a functioning origin of replication within the host, and phage-based transduction methods are examples. Specifically, the gene can be introduced into the host chromosome transforming the host with recombinant DNA containing the mutant glyA gene that causes Petition 870260059118, dated 06 / 17 / 2026, p. 40 / 106 / 47 homologous recombination with the target site on the host chromosome. The structure of the recombinant DNA used for homologous recombination is not restricted, as long as homologous recombination occurs in the desired manner. For example, a linear DNA containing a mutant glyA gene and having homologous sequences upstream and downstream of the target site on the chromosome at both ends of the gene can be used to transform the host and cause homologous recombination upstream and downstream of the target site, respectively, to replace the target site with the gene. The recombinant DNA used for homologous recombination can be equipped with a marker gene to select transformants. Only one copy of the gene can be inserted, or two or more copies can be inserted.For example, a large number of copies of a mutant glyA gene can be introduced into a chromosome by homologous recombination targeting a sequence with a large number of copies on the chromosome. Sequences with many copies on the chromosome include repetitive DNA sequences (repetitive DNA) and inverted repeats at both ends of the transposon. Homologous recombination can also be performed by targeting appropriate sequences on the chromosome, such as genes that are not required for the production of the target substance. Genes can also be introduced randomly into the chromosome using transposons or Mini-Mu (JP H2109985 A, US Patent No. 5882888, EP805867B1). This method for modifying chromosomes using homologous recombination is not limited to the introduction of mutant glyA genes, but can be used for any chromosome modification, such as modification of expression regulatory sequences.

[00102] Confirmation of the introduction of the mutant glyA gene into the chromosome can be done by probe hybridization using a probe with a sequence complementary to all or part of the gene, or by PCR using primers based on the gene sequence. Petition 870260059118, dated 06 / 17 / 2026, page 41 / 106 / 47

[00103] The introduction of the mutant glyA gene into the coryneform bacterium can also be achieved by introducing a vector containing the gene into the host. For example, a DNA fragment containing the mutant glyA gene can be ligated to a vector that functions in the host to construct an expression vector for the gene, and the gene can be introduced into the coryneform bacterium by transforming the host with the expression vector. DNA fragments containing the mutant glyA gene can be obtained, for example, by PCR using genomic DNA from a microorganism that has the mutant glyA gene as a template. As a vector, a vector capable of autonomous replication in the host cell can be used. The vector can preferably be a multi-copy vector. To select transformants, the vector can have a marker, such as an antibiotic resistance gene. The vector can also have a promoter or terminator for expression of the inserted gene.Vectors can be, for example, vectors derived from bacterial plasmids, yeast plasmids, bacteriophage vectors, cosmids, or phagemids. Vectors capable of autonomous replication in coryneform bacteria include, for example, pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903 (1984)); modified versions of these plasmids with drug resistance genes; pCRY30 (JP H3-210184 A); pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX (JP H2-72876 A, U.S. Patent 5185262); pCRY2 and pCRY3 (JP H01-191686A); pAJ655, pAJ611 and pAJ1844 (JP S58192900A); pCG1 (JP S57-134500A); pCG2 (JP S58-35197A); pCG4 and pCG11 (JP S57-183799A); pVK7 (JP H10-215883 A); pVK9 (U.S. Patent Application Publication No. 2006 / 0141588); pVC7 (JP H9-070291 A); pVS7 (WO2013 / 069634).

[00104] When a gene is introduced, it only needs to be harbored in the host to be expressible. Specifically, the gene can be harbored so that it can be expressed under the control of a promoter that functions Petition 870260059118, dated 06 / 17 / 2026, page 42 / 106 / 47 in the host. The promoter is not limited, as long as it can function in the host. A “host-functioning promoter” refers to a promoter that has promoter activity in the host. The promoter can be a host-origin promoter or a heterologous promoter. The promoter can be a gene-specific promoter or it can be a promoter of another gene.

[00105] A terminator for transcription termination can be placed downstream of the gene. Terminators are not restricted, as long as they are functional in the host. The terminator can be a host-derived terminator or a terminator of heterologous origin. The terminator can be a terminator unique to the gene to be transduced or it can be a terminator from another gene.

[00106] Vectors, promoters, and terminators that can be used in various microorganisms are described in detail, for example, in “Basic Microbiology Course 8: Gene Engineering,” Kyoritsu Shuppan, 1987.

[00107] When introducing two or more genes, each gene can be harbored in the host in an expressible manner. For example, each gene can be held in a single expression vector, or all can be held on a chromosome. Each gene can be held separately in multiple expression vectors and can be held separately in one or multiple expression vectors and on the chromosome. The operon can also be composed of two or more genes and introduced. The “case of introducing two or more genes” includes, for example, the introduction of genes encoding two or more proteins (e.g., enzymes), genes encoding two or more subunits of a single protein complex (e.g., an enzyme complex), and combinations thereof. <2> Method for producing L-glutamic acid

[00108] The method of the present invention is a method for producing L-glutamic acid, comprising cultivating the bacteria of the invention described. Petition 870260059118, dated 06 / 17 / 2026, p. 43 / 106 / 47 in <1> The invention relates to the process of producing L-glutamic acid in a culture medium to accumulate L-glutamic acid in the culture medium and / or in bacterial cells, and to collect L-glutamic acid from the culture medium and / or cells. The L-glutamic acid is as described above. In the present invention, L-glutamic acid can be produced alone, or L-glutamic acid and one or more amino acids other than L-glutamic acid, such as L-amino acids, etc., can be produced.

[00109] The medium used is not restricted, as long as the bacteria can grow and produce L-glutamic acid. For example, common culture media used to cultivate bacteria, such as coryneform bacteria, can be used. For example, a medium containing a carbon source, a nitrogen source, a phosphate source, a sulfur source, and other components selected from various organic and inorganic components, as needed, can be used. The type and concentration of the medium components can be defined according to various conditions, such as the type of bacteria to be used.

[00110] As carbon sources, specifically, sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, maltose, isomaltose, residual molasses, starch hydrolysates, biomass hydrolysates, organic acids such as acetic acid, fumaric acid, citric acid, succinic acid, alcohols such as glycerol, crude glycerol, ethanol, and fatty acids can be used. Carbon sources include, in particular, sugars. More specifically, glucose and fructose are mentioned as carbon sources. Sugars such as glucose and fructose can be used as carbon sources alone or in combination with other carbon sources. For example, a sugar with fructose as a constituent sugar can be used as a carbon source. Fructose, sucrose, and fructooligosaccharides are examples of fructose-based sugars. Fructose-based sugars can be used as a carbon source alone or in combination with other Petition 870260059118, dated 06 / 17 / 2026, page 44 / 106 / 47 carbon sources. Plant-derived materials can be used as carbon sources. Examples of plants include corn, rice, wheat, soybeans, sugarcane, beet, and cotton. Plant-derived raw materials include, for example, organs such as roots, stems, trunks, branches, leaves, flowers, and seeds, plant bodies that include these organs, and decomposition products of these plant organs. Plant-derived raw materials can be used in any form, such as unprocessed products, squeezed juices, crushed products, and refined products. As a carbon source, for example, cane molasses, beet molasses, high-sugar molasses, citrus molasses, or invert sugar, or hydrolyzed products of natural raw materials such as cellulose, starch, corn, cereals, tapioca, and cassava can be used.Pentasaccharides such as xylose, hexasaccharides such as glucose, or mixtures thereof can be obtained from plant biomass, for example. Specifically, these sugars can be obtained by subjecting plant biomass to treatments such as steam treatment, concentrated acid hydrolysis, dilute acid hydrolysis, hydrolysis by enzymes such as cellulase, and alkaline treatment. Since hemicellulose is generally more easily hydrolyzed than cellulose, the hemicellulose in plant biomass can be pre-hydrolyzed to release pentose carbohydrates, and then the cellulose can be hydrolyzed to produce hexoses. Xylose can also be supplied by the conversion of hexa-carbohydrates, for example, when the bacterium possesses a pathway for converting hexacarbohydrates, such as glucose, to xylose. The carbon source can be, for example, glucose alone or a mixture of two carbon sources, such as glucose and fructose or glucose and sucrose in any proportion (e.g., 3:7 to 7:3 by weight).

[00111] As sources of nitrogen, specifically, organic sources of nitrogen, such as ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, etc., peptone, yeast extract, extract of Petition 870260059118, dated 06 / 17 / 2026, page 45 / 106 / 47 Meat, hydrolyzed vegetable protein (HVP; for example, hydrolyzed soy protein, soy sauce, soy and pea sauce, etc.), ammonia, ammonia gas, or ammonia-containing water used for pH adjustment may be used as a nitrogen source. A single nitrogen source or a combination of two or more nitrogen sources may be used.

[00112] Specific examples of phosphoric acid sources include phosphates, such as potassium dihydrogen phosphate, potassium dihydrogen phosphate, and phosphate polymers, such as pyrophosphoric acid. One source of phosphoric acid may be used, or a combination of two or more sources of phosphoric acid may be used.

[00113] Specific examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. One sulfur source may be used, or a combination of two or more sulfur sources may be used.

[00114] Various other organic and inorganic components, specifically, inorganic salts such as sodium chloride, potassium chloride, etc.; trace metals such as iron, manganese, magnesium, calcium, etc.; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinic acid amide, vitamin B12, biotin, folic acid, etc.; amino acids; nucleic acids; organic ingredients such as peptone, casamino acids, yeast extract, hydrolyzed vegetable protein (HVP; for example, soy protein hydrolysates, soy sauce, soy and pea sauce, etc.) containing these ingredients. Other various organic and inorganic ingredients include antifoaming agents, osmotic pressure regulators of the culture medium, osmotic pressure compensating substances, etc. Antifoaming agents include silicone antifoaming agents (oil, solution, oil compound, emulsion, self-emulsifying agent, etc.).), alcohol-based anti-foaming agents, oil-based anti-foaming agents, polyethylene-based anti-foaming agents. Petition 870260059118, dated 06 / 17 / 2026, page 46 / 106 / 47 vegetable oils (cottonseed oil, linseed oil, soybean oil, olive oil, castor oil, coconut oil, etc.). Antifoaming agents can be used in any form, such as liquid, paste, solid, powder, emulsion, wax, etc. Osmotic compensating substances for the culture medium include salts such as sodium chloride and potassium chloride, and polysaccharides (sorbitol, dextrin, etc.) that microorganisms cannot utilize. Osmotic compensating substances include potassium ions, betaine (glycine betaine), residual molasses (especially residual beet molasses), glutamic acid, trehalose, etc.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 are also acceptable components to be added to the culture medium. These various other organic and inorganic components can be used as a single component or in combination with two or more components.

[00115] If an auxotrophic mutant strain of nutrients that requires amino acids or other nutrients for growth is used, it is preferable to supplement the medium with the necessary nutrients.

[00116] Limiting the amount of biotin in the medium and adding surfactants or penicillin to the medium are also preferable.

[00117] Culture conditions are not restricted, as long as the bacteria can grow and produce L-glutamic acid. Cultivation can be carried out under conventional conditions used for culturing bacteria, such as coryneform bacteria. Culture conditions can be appropriately defined according to various factors, such as the type of bacteria used.

[00118] Culture can be performed using liquid medium. For the liquid culture method, one can use, for example, the method described in “Biotechnology Textbook Series 13: Culture Engineering, Toshiomi Yoshida, Corona, 1998”. For liquid culture, it is possible to use, for example, a culture of Petition 870260059118, dated 06 / 17 / 2026, page 47 / 106 / 47 surface, deep culture, membrane (such as dialysis membrane or hollow fiber separate culture) or immobilized microbial culture. Furthermore, culture equipment can be used, for example, aerated agitation type culture equipment, pneumatic agitation type culture equipment, fixed bed type culture equipment or fluidized bed type culture equipment. For culture, the method described in “Fundamentals of Fermentation Engineering, Gakkai Shuppan Center, 1988” can be used. During culture, bacteria cultivated in solid medium, such as agar medium, can be inoculated directly into liquid medium, or bacteria can be cultured as seed in liquid medium and inoculated into liquid medium for the main culture. In other words, the culture can be divided into seed culture and main culture.In these cases, the culture conditions for the seed culture and the main culture may or may not be the same. The amount of bacteria contained in the culture medium at the beginning of the culture is not restricted. The main culture can be carried out, for example, by inoculating 1 to 50% (v / v) of the seed culture medium into the main culture medium. For example, the seed culture process may include two or more seed culture stages to obtain the amount of bacterial cells needed for the main culture process. The seed culture medium may be inoculated only at the beginning of the main culture, or it may be inoculated at the beginning of the main culture and additionally during the main culture.

[00119] The culture can be conducted in batch culture, feed-batch culture, continuous culture, or a combination thereof. For example, two or more stages of feed-batch culture or two or more stages of continuous culture may be used in combination. The medium at the start of the culture is also called the “starter culture medium”. The medium supplied to the culture system (fermenter) in a feed-batch culture or continuous culture is also called the “medium”. Petition 870260059118, dated 06 / 17 / 2026, page 48 / 106 / 47 of feeding”. Furthermore, providing a means of feeding a cropping system in a feed batch or continuous crop is also called “feeding”. When the crop is divided into seed crop and main crop, for example, both the seed crop and the main crop can be carried out in a batch crop. For example, the seed crop can be done in a batch crop, and the main crop can be done in a feed batch or continuous crop. For example, the seed crop can be carried out in a feed batch and the main crop can be carried out in a batch crop.The culture medium can be supplied from a point at the top of the culture tank that is not in contact with the surface of the culture medium, from a point within the culture medium, such as the middle of the culture tank or the bottom of the culture tank, or from the top and middle of the culture tank, respectively. The method for supplying the feed medium from a point within the culture medium is described, for example, in document JP6097869B.

[00120] In the present invention, each component of the culture medium may be contained in the primary culture medium, in the feed medium, or in both. The type of component contained in the initial culture medium may or may not be the same as the type of component contained in the feed medium. The concentration of each component contained in the initial medium may or may not be the same as the concentration of each component contained in the feed medium. Two or more feed media with different types and / or concentrations of the contained components may be used. For example, if several rounds of feeding are carried out intermittently, the type and / or concentration of components contained in each round of feed 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 feed may be sucrose. Petition 870260059118, dated 06 / 17 / 2026, page 49 / 106 / 47

[00121] Sterilization of the culture medium may or may not be performed. Sterilization of the culture medium may be done to prevent bacterial contamination (contamination). Sterilization of the culture medium can be paraphrased as sterilization or sanitization. Sterilization methods include sterilization under high temperature and pressure conditions, UV irradiation sterilization, and sterilization using filters or membranes. Sterilization of the culture medium can be paraphrased as sterilization or sanitization. For example, autoclave sterilization or batch sterilization in a culture tank are examples of methods in which sterilization under high temperature and high pressure conditions is performed discontinuously. For example, the method in which sterilization under high temperature and high pressure conditions is performed continuously includes continuous sterilization equipped with a plate heat exchanger.Sugar sterilization can be performed simultaneously with other components of the culture medium or separately from other components. Preferably, sugar and other components can be sterilized separately.

[00122] The concentration of the carbon source in the medium is not limited, provided the bacteria can grow and produce L-glutamic acid. The concentration of the carbon source in the medium can, for example, be as high as possible, as long as the production of L-glutamic acid is not inhibited. The concentration of the carbon source in the medium can be, for example, 1 to 50% w / v, preferably 1 to 30% w / v, more preferably 3 to 10% w / v, as the initial concentration (concentration in the initial medium). Additional carbon sources can be added to the medium as appropriate. For example, an additional carbon source can be added to the medium as the carbon source is consumed as fermentation progresses. In a continuous or batch feed culture, the amount of carbon source supplied can be the amount that results Petition 870260059118, dated 06 / 17 / 2026, page 50 / 106 / 47 in a sufficiency condition (a condition in which an excessive amount of carbon source is provided in relation to the amount (capacity) of the bacteria of the invention) or a limitation condition (a condition in which an insufficient amount of carbon source is provided compared to the amount (capacity) of carbon utilization of the bacteria of the invention).

[00123] Cultures can be conducted under aerobic or microaerophilic conditions using, for example, a liquid medium. “Aerobic conditions” means that the dissolved oxygen concentration in the liquid medium is 0.33 ppm or more, which is the detection limit of an oxygen membrane electrode, preferably 1.5 ppm or more. The oxygen concentration under aerobic conditions can be controlled to, for example, 5 to 50% of the saturated oxygen concentration, preferably 10%. The term “microaerophilic conditions” can mean conditions in which the dissolved oxygen concentration in the medium is less than 0.33 ppm. For example, the dissolved oxygen concentration in the medium under microaerophilic conditions can be less than 0.30 ppm, 0.25 ppm, 0.20 ppm, 0.15 ppm, 0.10 ppm, or 0.05 ppm. The oxygen concentration under microaerophilic conditions can be controlled, for example, to less than 5%, 3.75%, 3.125%, 2.5%, 1.875%, 1.25%, or 0.8125% of the saturated oxygen concentration.Cultures can be aerated, shaken, agitated, or a combination of these. The pH of the culture medium can 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 needed. The pH of the medium can be adjusted using various alkaline or acidic substances, such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide. Send feedback Petition 870260059118, dated 06 / 17 / 2026, page 51 / 106 / 47

[00124] Press the Tab key to view the actions. The culture temperature can be, for example, 20 to 40°C, preferably 25 to 37°C. In the main culture, the culture temperature can be varied in two or more stages. For example, as described in the Journal of Industrial Microbiology & Biotechnology (2002) 28, 333-337, the culture temperature can be changed to a higher temperature, from 33°C to 37-40°C. The culture period can be, for example, 10 to 120 hours. The culture can continue, for example, until the carbon source in the medium is consumed or until the activity of the bacterium of the invention ceases. Cultivating the bacterium of the present invention under these conditions results in the accumulation of L-glutamic acid in the medium and / or in the bacterial cells.

[00125] The culture can also be carried out during the precipitation of L-glutamic acid in the medium, using a liquid medium adjusted to the conditions under which L-glutamic acid precipitates. Such conditions under which L-glutamic acid precipitates include pH 5.0 to 4.0, preferably pH 4.5 to 4.0, more preferably pH 4.3 to 4.0, and especially preferably pH 4.0 (EP1078989A). When a liquid medium adjusted to the conditions under which L-glutamic acid precipitates is used, pantothenic acid can be added to the medium for more efficient crystallization (WO2004 / 111258). When a liquid medium adjusted to the precipitation conditions of L-glutamic acid is used, L-glutamic acid crystals can be added to the medium as seed crystals for more efficient crystallization (EP1233069A).When a liquid medium adjusted to the precipitation conditions of L-glutamic acid is used, crystals of L-glutamic acid and L-lysine can be added to the medium as seed crystals for more efficient crystallization (EP 1624069A).

[00126] The fermentation liquid can be processed, for example, in a liquid cyclone. The liquid cyclone can be made, for example, of ceramic, stainless steel or resin, with a general cylindrical shape and diameter. Petition 870260059118, dated 06 / 17 / 2026, page 52 / 106 / 47, from 10 to 110 mm. The feed volume of the fermentation liquid into the liquid cyclone can be adjusted, for example, according to the concentration of bacteria and L-glutamic acid in the fermentation liquid. The feed flow rate of the fermentation liquid in relation to the liquid cyclone can be, for example, from 2 to 1,200 L / minute.

[00127] The formation of L-glutamic acid can be confirmed by known methods used to detect or identify the compound. Such methods include, for example, HPLC, LC / MS, GC / MS, and NMR. These methods can be used alone or in combination, as appropriate.

[00128] L-glutamic acid can be collected from fermentation liquid using known methods for compound separation and purification. These methods include, for example, the ion-exchange resin method (Nagai, H. et al., Separation Science and Technology, 39(16), 36913710), precipitation method, membrane separation method (JP H9164323 A, JP H9-173792 A), precipitation by crystallization method (WO2008 / 078448, WO2008 / 078646), and crystallization method (WO2008 / 078448, WO2008 / 078646). These methods can be used alone or in combination, as appropriate. If L-glutamic acid accumulates in bacterial cells, it can be recovered from the supernatant obtained by triturating the bacterial cells with ultrasonic waves and removing the bacterial cells by centrifugation, for example, using an ion-exchange resin method. The recovered L-glutamic acid can be in free form, a salt thereof, or a mixture thereof.Salts include, for example, sulfate, hydrochloride, carbonate, ammonium, sodium, and potassium salts. Specifically, for example, free L-glutamic acid, sodium L-glutamate (e.g., monosodium L-glutamate; MSG), ammonium L-glutamate (e.g., monoammonium L-glutamate), or mixtures thereof. For example, monosodium L-glutamate (MSG) is obtained by crystallizing L-glutamate. Petition 870260059118, dated 06 / 17 / 2026, page 53 / 106 / 47 ammonium glutamate in the fermentation liquid with acid and by adding an equimolar amount of sodium hydroxide to the crystals. Monosodium L-glutamate crystals can be used, for example, as an umami flavoring. Monosodium glutamate crystals can be used as a flavoring when mixed with nucleic acids such as sodium guanylate and sodium inosinate, which also have an umami flavor.

[00129] If L-glutamic acid precipitates in the medium, it can be recovered by centrifugation or filtration. The L-glutamic acid precipitated in the medium can also be isolated after crystallization of the L-glutamic acid dissolved in the medium.

[00130] Recovered L-glutamic acid may contain other components besides L-glutamic acid, such as bacterial cells, culture medium components, water, and bacterial metabolic byproducts, etc. L-glutamic acid can be purified to the desired grade. The purity of the recovered L-glutamic acid may be, for example, greater than 50% (w / w), preferably greater than 85% (w / w) and especially preferably greater than 95% (w / w) (JP1214636B, U.S. Patent No. 5431933, U.S. Patent No. 4956471, U.S. Patent No. 4777051, U.S. Patent No. 4946654, U.S. Patent No. 5840358, U.S. Patent No. 6238714, U.S. Patent Application Publication No. 2005 / 0025878). Examples

[00131] The invention is described more specifically below with reference to non-limiting examples. < 1> Construction of a modified strain of Corynebacterium glutamicum <1-1> Construction of a wild-type glyA gene transfer vector

[00132] Using a chromosomal DNA from C. glutamicum ATCC 13869 (strain 2256) containing the wild-type glyA gene as a template, a DNA fragment containing the wild-type glyA gene was Petition 870260059118, dated 06 / 17 / 2026, page 54 / 106 / 47 amplified by PCR using primers of SEQ ID No: 5 and 6. A vector for the transfer of the wild-type glyA gene (pVK9-glyA (WT)) was obtained by ligating the DNA fragment amplified with pVK9 digested with BamHI and PstI (US Patent Application Publication No. 2006 / 0141588) by an infusion reaction.

[00133] The nucleotide 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 SEQ ID No. 1 and SEQ ID No. 2, respectively. < 1-2> Construction of the mutant glyA gene transfer vector

[00134] Chromosomal DNA from a strain derived from C. glutamicum ATCC 13869 (strain 2256) containing a mutant glyA gene with a mutation that substitutes the glycine residue at position 265 of the wild-type GlyA protein for a serine residue (G265S) was used as a template, and primers with SEQ ID No. 5 and 6 were used to amplify a DNA fragment containing the mutant glyA gene by PCR. A vector to introduce the mutant glyA gene (pVK9-glyA (G265S)) was obtained by ligating the amplified DNA fragment with pVK9 digested with BamHI and PstI by an infusion reaction.

[00135] The nucleotide sequence of the mutant glyA gene and the amino acid sequence of the mutant Gly protein encoded by the gene are shown in SEQ ID No. 3 and 4, respectively. < 1-3> Construction of a modified strain of Corynebacterium glutamicum

[00136] The C. glutamicum strain 2256ΔsucAΔldhA yggB* (WO2014 / 185430) was transformed with each of the constructed mutant transgenic vectors. From the transformants obtained, the strains were selected according to the method described in document WO2006 / 057450 to obtain the wild-type glyA gene-introduced strain and the mutant glyA gene-introduced strain.

[00137] Wild-type glyA gene transfer vector Petition 870260059118, dated 06 / 17 / 2026, p. 55 / 106 / 47 constructed (pVK9-glyA(WT)) or the mutant glyA gene transfer vector (pVK9-glyA(G265S)) was introduced individually into the C. glutamicum strain 2256ΔsucAΔldhA yggB* to obtain the wild-type glyA gene-introduced strain and the mutant glyA gene-introduced strain. The control strain was also obtained by introducing pVK9 alone into the C. glutamicum strain 2256ΔsucAΔldhA yggB*.

[00138] The C. glutamicum 2256ΔsucAΔldhA yggB* strain is an L-glutamic acid-producing strain derived from C. glutamicum 2256 (ATCC 13869), lacking the ldhA and sucA genes and with an IS mutation (V419::IS) in the yggB gene. < 2> Culture for the production of L-glutamic acid

[00139] The L-glutamic acid production culture was conducted using each of the constructed strains (i.e., wild-type glyA gene-introduced strain, mutant glyA gene-introduced strain, and control strain). The composition of the culture medium used is presented in Table 1. [Table 1] Table 1 - Composition of the Medium Glucose (NH4)2SO4 KH2PO4 MgSO4 - 7H2O FeSO4 - 7H2O MnSO4 - 5H2O VB1 Biotin Mameno CaCO3 80 g / L 30 g / L 1 g / L 0.4 g / L 0.01 g / L 0.01 g / L 200 μg / L 60 μg / L 0.48 g / L 50 g / L The medium with the above composition was prepared and adjusted to pH 8.0 with KOH, followed by sterilization in an autoclave (121°C, 20 minutes). Heat-sterilized calcium carbonate (180°C, 6 hours) was added to the sterilized medium at a final concentration of 50 g / L and used for the culture.

[00140] Each strain was inoculated into 20 mL of the above medium (containing g / L of calcium carbonate) contained in a 500 mL Sakaguchi flask and incubated at 31.5°C in a shaker box (ABLE ML-190) with agitation at 120 rpm. After 25 hours of culture, the culture medium was collected. 0.05 mL of the sampled culture medium was diluted 100 times by adding it to 4.95 mL of 0.1 N HCl solution and the absorbance (OD) at 620 nm was measured. The medium of Petition 870260059118, dated 06 / 17 / 2026, page 56 / 106 / 47 The sampled culture was centrifuged at 15,000 rpm for 1 minute and the concentration of L-glutamic acid in the supernatant was determined using a Biotech Analyzer BF7 (Oji Instrument Co., Ltd.) to calculate the yield of L-glutamic acid per sugar.

[00141] The results are shown in Figures 1 and 2. The strain introduced by the mutant glyA gene showed greater accumulation of L-glutamic acid and a higher yield of L-glutamic acid per sugar than the strain introduced by the wild-type glyA gene (Figures 1 and 2). Industrial Applicability

[00142] According to the present invention, the ability of coryneform bacteria to produce L-amino acids can be improved and L-amino acids can be produced efficiently. Sequence Listing Description

[00143] SEQ ID N°: 1: the nucleotide sequence of the wild-type glyA gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID N°: 2: the amino acid sequence of the wild-type GlyA protein from Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID No.: 3: the nucleotide sequence of the mutant glyA gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID N°: 4: the amino acid sequence of the mutant GlyA protein from Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID No. 5 and SEQ ID No. 6: Initiators SEQ ID No.: 7: the nucleotide sequence of the wild-type yggB gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID N°: 8: the amino acid sequence of the wild-type YggB protein from Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID N°: 9: the nucleotide sequence of the mutant gene yggB (V419::IS) SEQ ID N°: 10: the amino acid sequence of the mutant YggB protein (V419::IS) Petition 870260059118, dated 06 / 17 / 2026, p. 57 / 106

Claims

CLAIMS 1. Method for producing L-glutamic acid, the method comprising cultivating Corynebacterium glutamicum in a culture medium to accumulate L-glutamic acid in the medium and / or in the cells of Corynebacterium glutamicum, and collecting L-glutamic acid from the medium and / or from the cells, wherein Corynebacterium glutamicum has the capacity to produce L-glutamic acid, characterized in that Corynebacterium glutamicum has been modified to harbor a mutant glyA gene encoding a mutant serine hydroxymethyltransferase with the substitution of a glycine residue at position 265 in the amino acid sequence of the wild-type serine hydroxymethyltransferase by a serine residue, and the mutant serine hydroxymethyltransferase comprises the amino acid sequence shown in SEQ ID No:

4.

2. Production method according to claim 1, characterized in that Corynebacterium glutamicum has been further modified to harbor a mutant yggB gene, and the mutant yggB gene is a gene encoding a protein comprising the amino acid sequence of SEQ ID No:

10.

3. Production method according to claim 1 or 2, characterized in that the mutant glyA gene comprises the nucleotide sequence shown in SEQ ID No. 3 or a variant thereof due to nucleotide sequence degeneracy. Petition 870260059118, dated 06 / 17 / 2026, p. 58 / 106

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