Manufacturing method of L-glutamic acid

Abstract

A method for producing L-glutamic acid is provided. The present invention relates to a mutant Corynebacterium glutamicum having an ability to produce L-glutamic acid, which has a substitution of the alanine residue at position 364 in the amino acid sequence of wild-type glutamate-ammonia-ligase adenylyltransferase with another amino acid residue. Corynebacterium glutamicum, which has been modified to carry a mutant glnE gene encoding a [glutamate-ammonia-ligase] adenylyltransferase, is cultured in a medium, and L-glutamic acid is collected from the medium and / or the bacterial cells, thereby producing L-glutamic acid.

Description

[Technical Field]

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

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

[0003] [Glutamate-ammonia-ligase] adenylyltransferase is a catalyzer that converts ATP and [glutamine synthetase]-L- It is an enzyme that has the activity of catalyzing the adenylyl transfer reaction using tyrosine as a substrate (Non-Patent Document 2). It has been reported that disrupting the glnE gene encoding this enzyme enhances the ability to produce L-glutamine (Patent Document 1), that modifying the nitrogen metabolism control mechanism centered on the adenylylation of this enzyme enhances the ability to produce basic amino acids such as L-arginine and L-lysine (Patent Document 2), and that in microorganisms belonging to the genus Escherichia that have the ability to produce and accumulate L-glutamine, the glutamine synthetase adenylyltransferase (GlnE protein) and the glutamine synthetase regulatory protein PII (GlnB protein) are expressed. It is known that the reduction or loss of activity of this enzyme enables efficient production of L-glutamine (Patent Document 3). However, the relationship between this enzyme or its amino acid substitution mutants and L-glutamic acid production was unknown. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2002-300887 [Patent Document 2] Japanese Patent Application Laid-Open No. 2004-283167 [Patent Document 3] WO2006 / 001380 [Non-patent literature]

[0005] [Non-Patent Document 1] Kunihiko Akashi et al., Amino Acid Fermentation, Academic Press, pp. 195-215, 1986 [Non-patent document 2] KEGG (Kyoto Encyclopedia of Genes and Genomes) website: https: / / www.genome.jp / entry / 2.7.7.42 Summary of the Invention [Problem to be solved by the invention]

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

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

[0008] That is, the present invention can be exemplified as follows. [1] A coryneform bacterium capable of producing L-glutamic acid, A mutant [glutamate-ammonia-ligase] adenylyltransferase having a substitution of the alanine residue at position 364 in the amino acid sequence of the wild-type [glutamate-ammonia-ligase] adenylyltransferase with another amino acid residue. modified to carry a mutant glnE gene encoding glutamate-ammonia-ligase adenylyltransferase; Corynebacterium. [2] The coryneform bacterium according to [1], wherein the substitution of the alanine residue at position 364 with another amino acid residue is a substitution of the alanine residue at position 364 with a lysine, glutamic acid, threonine, serine, aspartic acid, asparagine, glutamine, arginine, cysteine, or histidine residue. [3] The coryneform bacterium according to [1], wherein the substitution of the alanine residue at position 364 with another amino acid residue is substitution of the alanine residue at position 364 with a threonine residue. [4] The coryneform bacterium according to any one of [1] to [3], wherein the wild-type [glutamate-ammonia-ligase] adenylyltransferase is a protein selected from the group consisting of (a), (b), and (c) below: (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (b) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, but with substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues, and having [glutamate-ammonia-ligase] adenylyltransferase activity; (c) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having [glutamate-ammonia-ligase] adenylyltransferase activity. [5] The coryneform bacterium is a bacterium belonging to the genus Corynebacterium. The coryneform bacterium according to any one of [1] to [4]. [6] The coryneform bacterium according to any one of [1] to [4], wherein the coryneform bacterium is Corynebacterium glutamicum. [7] It has been modified to carry a mutant yggB gene, A coryneform bacterium described in any of [1] to [6], wherein the mutant yggB gene is a gene encoding a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 10. [8] The coryneform bacterium according to [7], wherein the mutant yggB gene is a gene encoding a protein having the amino acid sequence of SEQ ID NO: 12. [9] A method for producing L-glutamic acid, comprising the steps of: Cultivating the coryneform bacterium according to any one of [1] to [8] in a medium and accumulating L-glutamic acid in the medium and / or within the cells of the bacterium; and collecting L-glutamic acid from the medium and / or the bacterial cells; Including, Manufacturing method.

[10] Wild-type glutamate-ammonia-ligase adenylyltransferase Substitution of the alanine residue at position 364 in the amino acid sequence with another amino acid residue, and / or a substitution of the arginine residue at position 393 in the amino acid sequence with another amino acid residue; Mutant glutamate-ammonia-ligase adenylyltransferase.

[11] the substitution of the alanine residue at position 364 with another amino acid residue is substitution of the alanine residue at position 364 with a lysine, glutamic acid, threonine, serine, aspartic acid, asparagine, glutamine, arginine, cysteine, or histidine residue; The mutant [glutamate-ammonia-ligase] adenylyltransferase according to

[10] , wherein the substitution of the arginine residue at position 393 with another amino acid residue is a substitution of the arginine residue at position 393 with a lysine, glutamic acid, threonine, serine, aspartic acid, asparagine, glutamine, cysteine, or histidine residue.

[12] the substitution of the alanine residue at position 364 with another amino acid residue is substitution of the alanine residue at position 364 with a threonine residue; The mutant [glutamate-ammonia-ligase] adenylyltransferase according to

[10] , wherein the substitution of the arginine residue at position 393 with another amino acid residue is a substitution of the arginine residue at position 393 with a histidine residue.

[13] The mutant [glutamate-ammonia-ligase] adenylyltransferase according to any one of

[10] to

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

[14] A mutant glnE gene encoding the mutant glutamate-ammonia ligase adenylyltransferase according to any one of

[10] to

[13] . [Brief explanation of the drawings]

[0009] [Figure 1]FIG. 1 shows the amount of L-glutamic acid accumulated in a wild-type glnE gene-introduced strain and a mutant glnE gene-introduced strain. [Figure 2] FIG. 1 is a graph showing the L-glutamic acid yield relative to sugar in a strain into which the wild-type glnE gene has been introduced and a strain into which the mutant glnE gene has been introduced. [Figure 3] FIG. 1 shows the absorbance at OD620 nm at the time of sampling for a wild-type glnE gene-introduced strain and a mutant glnE gene-introduced strain. DETAILED DESCRIPTION OF THE INVENTION

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

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

[0012] <1> The bacterium of the present invention The bacterium of the present invention is a coryneform bacterium capable of producing L-glutamic acid, which has been modified to carry a specific gene mutation.

[0013] <1-1> Corynebacterium globulinum-producing L-glutamic acid In the present invention, "Coryneform bacteria capable of producing L-glutamic acid" refers to coryneform bacteria that, when cultured in a medium, produce and accumulate L-glutamic acid in the medium and / or intracellularly to an extent that allows recovery. Coryneform bacteria capable of producing L-glutamic acid may be coryneform bacteria that are capable of accumulating greater amounts of L-glutamic acid in the medium and / or intracellularly than unmodified strains. An "unmodified strain" refers to a control strain that has not been modified to carry a specific genetic mutation. Examples of unmodified strains include wild-type strains and parent strains. Furthermore, coryneform bacteria capable of producing L-glutamic acid may be coryneform bacteria that are capable of accumulating the desired L-amino acid in a medium at a concentration of preferably 0.5 g / L or more, more preferably 1.0 g / L or more.

[0014] The bacterium of the present invention can produce L-glutamic acid alone, or L-glutamic acid and one or more amino acids other than L-glutamic acid, for example, L-amino acids (also known as L-amino acids). L-amino acids can be produced as a mixture of L-amino acids, such as 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, but are not limited thereto.

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

[0016] Corynebacterium species include the genus Corynebacterium, Brevibacterium, and Examples of bacteria include those belonging to genera such as Brevibacterium and Microbacterium.

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

[0018] Corynebacterium glutamicum (formerly Brevibacterium lactofermentum) is a particular example of a coryneform bacterium.

[0019] Specific examples of coryneform bacteria include the following strains: Corynebacterium acetoacidophilum ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium alkanolyticum ATCC 21511 Corynebacterium callunae ATCC 15991 Corynebacterium crenatum AS1.542 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

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

[0021] The genus Corynebacterium includes bacteria that were previously classified as Brevibacterium but have now been integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255(1999)). 91)) is also included. Corynebacterium stathionis is also included. This also includes bacteria that were previously classified as Corynebacterium ammoniagenes but were reclassified as Corynebacterium stationis based on 16S rRNA sequence analysis and other factors (Int. J. Syst. Evol. Microbiol., 60, 874-879(2010)).

[0022] These strains can be obtained, for example, from the American Type Culture Collection (Address: 12301 Parklawn Drive, Rockville, Maryland 20852, PO Box 1549, Manassas, VA 20108, United States of America). Each strain has been assigned a registration number, which can be used to obtain a sample (see http: / / www.atcc.org / ). The registration number for each strain is listed in the catalog of the American Type Culture Collection. These strains can also be obtained from, for example, the depository institution where they were deposited.

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

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

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

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

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

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

[0029] Methods for imparting or enhancing L-glutamic acid-producing ability include, for example, modifying bacteria to increase the activity of one or more enzymes selected from L-glutamic acid biosynthesis enzymes, including, but not limited to, glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthase (gltBD), isocitrate dehydrogenase (icdA), aconitate hydratase (aconitate hydratase), and glutamate synthase (gltBD). enzymes (acnA, acnB), citrate synthase (gltA), methylcitrate synthase (prpC), pyruvate carboxylase (pyc), phosphoenolpyruvate carboxylase (ppc), 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-3-deoxy-6-phosphogluconate Examples of enzymes that can be used to enhance the activity of glutamate dehydrogenase include acid aldolase (eda) and transhydrogenase (pntAB). The genes encoding these enzymes are shown in parentheses (the same applies to the following descriptions). Among these enzymes, it is preferable to enhance the activity of one or more enzymes selected from glutamate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, and methylcitrate synthase.

[0030] Corynebacterium coryneum modified to increase expression of the glutamate synthase gene (gltBD) Bacteria include those disclosed in WO99 / 07853.

[0031] Another method for imparting or enhancing L-glutamic acid-producing ability is to modify bacteria so that the activity of one or more enzymes selected from enzymes that catalyze reactions that branch off from the L-glutamic acid biosynthetic pathway to produce compounds other than L-glutamic acid is reduced. Examples of such enzymes include, but are not limited to, isocitrate lyase (aceA), α-ketoglutarate dehydrogenase (sucA, odhA), acetolactate synthase (ilvI), formate acetyltransferase (pfl), and 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.

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

[0033] In addition, L-glutamic acid-producing bacteria or parent strains for deriving them are preferably strains having α-ketoglutarate dehydrogenase (sucA) activity and succinate dehydrogenase (sdh) activity. Strains in which both are reduced or deleted are also included (Japanese Patent Application Laid-Open No. 2010-041920). Specifically, for example, the odhAsdhA double-deficient strain of Corynebacterium glutamicum ATCC14067 (Corynebacterium glutamicum 8L3GΔSDH strain) can be mentioned (Japanese Patent Laid-Open Publication No. 2010-041920). information).

[0034] Methods for imparting or enhancing L-glutamic acid-producing ability to coryneform bacteria include methods for imparting resistance to organic acid analogs or respiratory inhibitors, and methods for imparting sensitivity to cell wall synthesis inhibitors. Specific examples of such methods include a method for imparting monofluoroacetic acid resistance (Japanese Patent Laid-Open Publication No. 113209 / 1975), a method for imparting adenine resistance or a method of imparting thymine resistance (Japanese Patent Laid-Open Publication No. 57-065198), a method of weakening urease activity (Japanese Patent Laid-Open No. 52-038088), a method for imparting resistance to malonic acid (Japanese Patent Laid-Open No. 52-038088), a method for imparting resistance to benzopyrones or naphthoquinones (Japanese Patent Laid-Open No. 56-1889) a method for imparting HOQNO resistance (Japanese Patent Laid-Open Publication No. 56-140895), a method for imparting α-ketomalonic acid resistance a method of imparting guanidine resistance (Japanese Patent Laid-Open No. 57-2689); and a method for imparting sensitivity to penicillin (Japanese Patent Laid-Open Publication No. 4-88994). .

[0035] Specific examples of such resistant or sensitive bacteria include the following strains: Corynebacterium glutamicum (Brevibacterium flavum) AJ3949 (FERM BP-2632; JP 50-113209 A). Corynebacterium glutamicum AJ11628 (FERM P-5736; Japanese Patent Application Laid-Open No. 1983-065198) Corynebacterium glutamicum (Brevibacterium flavum) AJ11355 (FERM P-5007; Japanese Patent Application Laid-Open No. 1889 / 1989) Corynebacterium glutamicum AJ11368 (FERM P-5020; Japanese Patent Application Publication No. 1889 / 1989) Corynebacterium glutamicum (Brvibacterium flavum) AJ11217 (FERM P-4318; Japanese Patent Application Publication No. 57-2689) Corynebacterium glutamicum AJ11218 (FERM P-4319; Japanese Patent Application Laid-Open No. 57-2689) Corynebacterium glutamicum (Brvibacterium flavum) AJ11564 (FERM P-5472; Japanese Patent Application Publication No. 56-140895) Corynebacterium glutamicum (Brvibacterium flavum) AJ11439 (FERM P-5136; Japanese Patent Application Publication No. 56-35981) Corynebacterium glutamicum H7684 (FERM BP-3004; Japanese Patent Publication No. 04-88994) Corynebacterium glutamicum(Brvibacterium lactofermentum)AJ11426(FERM P-5123 ;Tokkaihei No. 56-048890) Corynebacterium glutamicum AJ11440 (FERM P-5137; Japanese Patent Publication No. 56-048890) Corynebacterium glutamicum(Brvibacterium lactofermentum)AJ11796(FERM P-6402 ;Tokkaihei No. 58-158192)

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

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

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

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

[0040] Furthermore, methods for imparting or enhancing L-glutamic acid-producing ability to coryneform bacteria include a method for enhancing the expression of the yggB gene and a method for introducing a mutant yggB gene with a mutation introduced into the coding region (WO2006 / 070944). The strain may be modified to increase gene expression, or may be modified to carry (have) a mutant yggB gene.

[0041] The yggB gene encodes a mechanosensitive channel. Examples of the yggB gene include those of coryneform bacteria. Specific examples of the yggB gene of coryneform bacteria include the yggB genes of Corynebacterium glutamicum ATCC13869, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14967, and Corynebacterium melassecola ATCC17965 (WO2006 / 070944). The yggB genes of Corynebacterium glutamicum ATCC13032 and Corynebacterium callunae ATCC 15991 are also known. The gene is registered in the NCBI database under GenBank Accession No. NC_003450. This sequence corresponds to the complementary sequence of 1,336,091 to 1,337,692 in the NCgl1221 genome sequence, and is also referred to as NCgl1221. The YggB protein encoded by the yggB gene of Corynebacterium glutamicum ATCC13032 has been 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: 9 and SEQ ID NO: 10, respectively.

[0042] In the present invention, a yggB gene having a "specific mutation" described below is also referred to as a mutant yggB gene, and the protein encoded thereby is also referred to as a mutant YggB protein. In the present invention, a yggB gene not having a "specific mutation" described below is also referred to as a wild-type yggB gene, and the protein encoded thereby is also referred to as a wild-type YggB protein. With regard to the YggB protein, a change in amino acid sequence caused by a "specific mutation" in the yggB gene is also referred to as a "specific mutation." The term "wild-type" used here is a convenient description to distinguish it from a "mutant" and is not limited to naturally occurring proteins as long as they do not have the "specific mutation." Examples of wild-type YggB proteins include the YggB proteins exemplified above, such as a protein having the amino acid sequence shown in SEQ ID NO: 10. Wild-type YggB proteins also include conservative variants (variants that maintain the original function) of the above-exemplified YggB proteins that do not have the "specific mutation." The "original function" of the YggB protein may be, for example, its function as a mechanosensitive channel, or it may be a property that improves the L-glutamic acid production ability of coryneform bacteria when its expression is increased in the coryneform bacteria.

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

[0044] (1) C-terminal mutation The C-terminal mutation was located at amino acid positions 419 to 533 of the wild-type YggB protein in the wild-type yggB gene. The C-terminal mutations are mutations in the region encoding the nucleotide residues. The amino acid sequence change caused by the C-terminal mutation can be The type of mutation is not particularly limited. The C-terminal mutation may be, for example, a substitution of an amino acid residue (missense mutation). The C-terminal mutation may be a mutation that causes an insertion of an amino acid residue, a deletion of an amino acid residue, the appearance of a stop codon (nonsense mutation), a frameshift mutation, or a combination thereof. Examples of the C-terminal mutation include an insertion sequence (hereinafter also referred to as "IS") and Insertion of a base sequence such as a transposon is preferred.

[0045] (1-1) Insertion of base sequence An example of a C-terminal mutation is a mutation (2A-1 type mutation) in which a nucleotide sequence is inserted into the site encoding the valine residue at position 419 of the wild-type YggB protein. The 2A-1 type mutation may, for example, result in the partial or complete deletion or substitution of the amino acid residues at positions 419 to 533 of the wild-type YggB protein. Specific examples of mutant yggB genes having a 2A-1 type mutation include those in which an IS is inserted next to the "G" at position 1255 of SEQ ID NO: 9, resulting in the original wild-type YggB protein. The mutant YggB protein (SEQ ID NO: 10) encodes a 423 amino acid residue mutant YggB protein, which is shorter than the full-length YggB protein (SEQ ID NO: 10). The nucleotide sequence of this mutant yggB gene (V419::IS) and the amino acid sequence of the mutant YggB protein (V419::IS) encoded by this gene are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In SEQ ID NO: 11, positions 1 to 1269 correspond to the mutant YggB protein. Specific examples of L-glutamic acid producing bacteria having a mutant yggB gene (V419::IS) include C. glutamicum 2256ΔsucAΔldhA yggB * strain (WO2014 / 185430).

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

[0047] (2) Mutations in the transmembrane domain The YggB protein is predicted to have five transmembrane domains, which are located at positions 1-23 (first transmembrane domain) and 25-47 (second transmembrane domain) of the wild-type YggB protein. , 62-84 (3rd transmembrane region), 86-108 (4th transmembrane region), 110-132 (5th transmembrane region) The mutations in the transmembrane region are mutations in the region of the wild-type yggB gene that encodes these transmembrane regions. Mutations in the transmembrane region may be introduced at one or more positions. Mutations in the transmembrane region preferably cause substitution, deletion, addition, insertion, or inversion of one or several amino acids, and are not accompanied by frameshift mutations or nonsense mutations. "One or several" preferably means 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3. Examples of mutations in the transmembrane region include a mutation that inserts one or several amino acids (e.g., Cys-Ser-Leu) between the leucine residue at position 14 and the tryptophan residue at position 15 of the wild-type YggB protein, a mutation that replaces the alanine residue at position 100 with another amino acid residue (e.g., Mutations that substitute an amino acid having a hydroxyl group in the side chain (Thr, Ser, or Tyr), preferably Thr), or a mutation that substitutes the alanine residue at position 111 with another amino acid residue (e.g., Val or a hydroxyl group in the side chain). Substitution of an amino acid having a hydroxyl group (Thr, Ser, or Tyr, preferably Val or Thr) Examples of such mutations include those that result in the transformation of the nucleotide sequence.

[0048] In the present invention, unless otherwise specified, the "amino acid residue at position X of the wild-type YggB protein" refers to the amino acid residue corresponding to the amino acid residue at position X in SEQ ID NO: 10. The "position X" in an amino acid sequence refers to the Xth position counted from the N-terminus of the amino acid sequence. The amino acid residue at the N-terminus is the first amino acid residue. The absolute position may vary due to amino acid deletion, insertion, or addition. For example, "amino acid residue 419 of the wild-type YggB protein." means the amino acid residue corresponding to the 419th amino acid residue in SEQ ID NO: 10, and when one amino acid residue on the N-terminal side of position 419 is deleted, the 418th amino acid from the N-terminus The residue is defined as "the amino acid residue at position 419 of the wild-type YggB protein." If an amino acid residue is inserted closer to the N-terminus than position 419, the residue is defined as the 420th amino acid residue from the N-terminus. The base is "amino acid residue 419 of the wild-type YggB protein." Specifically, For example, in the YggB protein of Corynebacterium glutamicum ATCC14967 strain, the amino acid residues at positions 419 to 529 correspond to the amino acid residues at positions 419 to 533 in the wild-type YggB protein. 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.

[0049] In the amino acid sequence of any YggB protein, which amino acid residue is "the amino acid residue corresponding to the amino acid residue at position X in SEQ ID NO: 10" can be determined by aligning the amino acid sequence of the YggB protein with the amino acid sequence of SEQ ID NO: 10. Alignment can be performed, for example, using known genetic analysis software. Specific examples of such software include DNASIS manufactured by Hitachi Solutions and GENETYX manufactured by Genetyx (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24(1), 72-96, 1991; Barton GJ et al., Journal of molecular biology, 198(2), 327-37. 1987).

[0050] The mutant yggB gene can be obtained by modifying the wild-type yggB gene so that it has the above-mentioned "specific mutation." DNA modification can be performed by known techniques. Specifically, for example, site-specific mutagenesis, which introduces a target mutation into a target site in DNA, can be performed using PCR. (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton Press (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)) and phage-based methods. Method (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth. In Enzymol., 154, 367 (1987)). The mutant yggB gene can also be obtained by chemical synthesis.

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

[0052] <1-2> Modification of the mutant glnE gene The coryneform bacterium of the present invention has a structure in which the alanine residue at position 364 in the amino acid sequence of wild-type glutamate-ammonia ligase adenylyltransferase is substituted with another amino acid residue. The vector has been modified to carry a mutant glnE gene encoding a mutant [glutamate-ammonia-ligase] adenylyltransferase having the substitution: The coryneform bacterium of the present invention is a coryne having the above-mentioned ability to produce L-glutamic acid. The bacterium may be a bacterium modified to carry a mutant glnE gene, or a bacterium modified to carry a mutant glnE gene and be imparted with L-glutamic acid-producing ability. The coryneform bacterium may be a coryneform bacterium obtained by the method described above. Also included are coryneform bacteria that have been modified to have a mutant glnE gene and thereby have the ability to produce L-glutamic acid.

[0053] The glnE gene encodes glutamate-ammonia-ligase adenylyltransferase. Examples of the glnE gene include the glnE genes of coryneform bacteria. Specific examples of the glnE genes of coryneform bacteria include the glnE genes of Corynebacterium glutamicum ATCC13869, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14967, and Corynebacterium melassecola ATCC17965. The glnE gene of Corynebacterium glutamicum ATCC13032 corresponds to the complementary sequence of the sequence from 2,359,612 to 2,362,749 in the genome sequence registered in the NCBI database under GenBank Accession No. NC_003450, and is also referred to as NCgl2147. The glutamate-ammonia-ligase adenylyltransferase encoded by the glnE gene of Corynebacterium glutamicum ATCC13032 is registered under GenBank Accession No. WP_011014971.1. The nucleotide sequence of the wild-type glnE gene of Corynebacterium glutamicum 2256 (ATCC 13869) and the glutamate-ammonia-ligase adenylyltransferase encoded by the same gene are also registered under GenBank Accession No. WP_011014971.1. The amino acid sequences of the ammonia-ligase adenylyltransferases are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0054] "[Glutamate-ammonia-ligase] adenylyltransferase" refers to the enzyme that binds ATP and glutamine synthetase. The term "glutamic acid-ammonia-ligase" may refer to a protein having an activity of catalyzing an adenylyl transfer reaction using α-L-tyrosine as a substrate (e.g., EC 2.7.7.42). The glutamate-ammonia-ligase adenylyltransferase activity is specifically the enzyme that converts ATP and glutamine synthase. [glutamine synthetase]-L-tyrosine diphosphate and [glutamine synthetase]-O4-(5'-adenosine The activity may be to catalyze the reaction that converts glutamate-ammonia ligase adenylyltransferase into glutamine synthetase (glutamine synthetase adenylyltransferase) and / or the reverse reaction. [Glutamate-ammonia ligase] adenylyltransferase is also called "glutamine synthetase adenylyltransferase." Examples of genes encoding [glutamate-ammonia ligase] adenylyltransferase include the glnE gene. The nucleotide sequences of [glutamate-ammonia ligase] adenylyltransferase genes, such as the glnE gene, contained in the bacterium to be modified, and the amino acid sequences of the [glutamate-ammonia ligase] adenylyltransferases encoded thereby, can be obtained from public databases such as NCBI.

[0055] In the present invention, a glnE gene having a "specific gene mutation" described below is also referred to as a mutant glnE gene, and a protein encoded thereby is also referred to as a mutant [glutamate-ammonia-ligase] adenylyltransferase. Furthermore, in the present invention, a glnE gene not having a "specific gene mutation" described below is also referred to as a wild-type glnE gene, and a protein encoded thereby is also referred to as a wild-type [glutamate-ammonia-ligase] adenylyltransferase. [glutamate-ammonia-ligase] adenylyltransferase In this context, a change in amino acid sequence caused by a "specific genetic mutation" in the glnE gene is also referred to as a "specific genetic mutation." The term "wild-type" used here is a convenient description to distinguish it from a "mutant," and is not limited to naturally occurring ones, as long as they do not have a "specific genetic mutation." Examples of wild-type [glutamate-ammonia-ligase] adenylyltransferases include the above-exemplified [glutamate-ammonia-ligase] adenylyltransferases, such as a protein having the amino acid sequence set forth in SEQ ID NO: 2. Examples of wild-type [glutamate-ammonia-ligase] adenylyltransferases also include conservative variants (variants that maintain the original function) of the above-exemplified [glutamate-ammonia-ligase] adenylyltransferases that do not have a "specific genetic mutation." An example of the "original function" of a [glutamate-ammonia-ligase] adenylyltransferase is its function as a [glutamate-ammonia-ligase] adenylyltransferase.

[0056] The "specific gene mutation" is a mutation that changes the amino acid sequence of the wild-type glutamate-ammonia ligase adenylyltransferase described above, thereby improving the L-glutamate-producing ability of the coryneform bacterium. Specifically, the mutation is a mutation at position 364 of the amino acid sequence of the wild-type glutamate-ammonia ligase adenylyltransferase in the wild-type glnE gene. This is a mutation in the amino acid residue.

[0057] Examples of mutations at the amino acid residue at position 364 of the amino acid sequence of wild-type glutamate-ammonia-ligase adenylyltransferase include substitution of the amino acid residue ( missense mutations).

[0058] Wild-type glutamate-ammonia-ligase adenylyltransferase 364 As an example of a mutation at the amino acid residue at position 364 of the wild-type glutamate-ammonia-ligase adenylyltransferase, the alanine residue at position 364 may be replaced with another amino acid residue. The "other amino acids" are not particularly limited as long as they are naturally occurring amino acids other than alanine. Examples of "other amino acids" include Lys, Glu, Gly, Val, Leu, Ile, and Ser. For example, the alanine residue at position 364 is preferably substituted with a hydrophilic amino acid (Lys, Glu, Thr, Ser, Asp, Asn). , Gln, Arg, Cys or His), more preferably Thr. For example, the alanine residue at position 364 preferably has a hydroxyl group in the side chain. It may be substituted with an amino acid (Thr, Ser, or Tyr), more preferably with Thr.

[0059] In the present invention, unless otherwise specified, the "amino acid residue at position X of wild-type [glutamate-ammonia-ligase] adenylyltransferase" refers to the amino acid residue corresponding to the amino acid residue at position X in SEQ ID NO: 2. The "position X" in an amino acid sequence refers to the Xth position counted from the N-terminus of the amino acid sequence, with the N-terminal amino acid residue being the first amino acid residue. The position of an amino acid residue indicates a relative position, and its absolute position may change due to amino acid deletion, insertion, addition, or the like. For example, the "amino acid residue at position 364 of wild-type [glutamate-ammonia-ligase] adenylyltransferase" refers to the amino acid residue corresponding to the amino acid residue at position 364 in SEQ ID NO: 2. When one amino acid residue N-terminal to position 364 is deleted, the 363rd amino acid residue from the N-terminus is considered to be the "amino acid residue at position 364 of wild-type [glutamate-ammonia-ligase] adenylyltransferase." Furthermore, the "position X" of an amino acid sequence refers to the Xth position counted from the N-terminus of the amino acid sequence, with the N-terminal amino acid residue being the first amino acid residue. If one amino acid residue is inserted at the N-terminus, the 365th amino acid residue from the N-terminus will be the amino acid at position 364 of the wild-type [glutamate-ammonia-ligase] adenylyltransferase. It is assumed that the amino acid residue is "a hydroxyl group."

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

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

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

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

[0064] "Maintaining the original function" means that a variant of a gene or protein has a function (e.g., activity or property) corresponding to the function (e.g., activity or property) of the original gene or protein. "Maintaining the original function" with respect to a gene means that a variant of the gene encodes a protein that maintains the original function. That is, "maintaining the original function" with respect to the glnE gene may mean that a variant of the glnE gene encodes a protein that has [glutamate-ammonia-ligase] adenylyltransferase activity. Also, "maintaining the original function" with respect to [glutamate-ammonia-ligase] adenylyltransferase may mean that a variant of the glnE gene encodes a protein that has [glutamate-ammonia-ligase] adenylyltransferase activity. It may mean that a variant of [glutamate-ammonia-ligase] adenylyltransferase has [glutamate-ammonia-ligase] adenylyltransferase activity.

[0065] [Glutamate-ammonia-ligase] adenylyltransferase activity can be measured, for example, by coupling the enzyme to its corresponding substrate (e.g., ATP and [glutamine synthetase] L-tyrosine). Incubating and measuring the enzyme- and substrate-dependent production of the corresponding product (e.g., diphosphate and [glutamine synthetase]-O4-(5'-adenylyl)-L-tyrosine). It can be measured by:

[0066] Examples of conservative variants are shown below.

[0067] A homolog of the glnE gene or a homolog of the [glutamate-ammonia-ligase] adenylyltransferase can be easily obtained from public databases by, for example, a BLAST search or a FASTA search using the nucleotide sequence of the above-exemplified glnE gene or the amino acid sequence of the above-exemplified [glutamate-ammonia-ligase] adenylyltransferase as a query sequence. Alternatively, a homolog of the glnE gene can be obtained by, for example, PCR using the chromosome of various organisms as a template and oligonucleotides prepared based on the nucleotide sequences of these known glnE genes as primers.

[0068] As long as the original function is maintained, the glnE gene may be a gene encoding a [glutamate-ammonia-ligase] adenylyltransferase having an amino acid sequence obtained by substituting, deleting, inserting, and / or adding one or several amino acids at one or several positions in the amino acid sequence (e.g., the amino acid sequence shown in SEQ ID NO: 2). For example, the encoded protein may be extended or shortened at its N-terminus and / or C-terminus. Note that the term "one or several" varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but specifically means, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0069] The above-mentioned substitution, deletion, insertion and / or addition of one or several amino acids is a conservative mutation that maintains normal protein function. A typical conservative mutation is a conservative substitution. When the substitution site is an aromatic amino acid, a conservative substitution is performed for Phe, Trp, Tyr, etc. If the substitution site is a hydrophobic amino acid, it is between Leu, Ile, and Val. If the substitution site is a polar amino acid, it is between Leu, Ile, and Val. In some cases, between Gln and Asn, and between Lys, Arg, and His, which are basic amino acids, Conservative substitutions are made between Asp and Glu when the amino acid is a hydroxyl group-containing amino acid, and between Ser and Thr when the amino acid is a hydroxyl group-containing amino acid. Specific examples of substitutions that are considered conservative include substitutions of Ala with Ser or Thr, substitution of Arg with Gln, His, or Lys, substitution of Asn with Glu, Gln, Lys, His, or Asp, substitution of Asp with Asn, Glu, or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitution of Glu with Gly, Asn, Gln, Lys, or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg, or Tyr, substitution of Ile with Ile, etc. substitution of Lys with Leu, Met, Val, or Phe; substitution of Leu with Ile, Met, Val, or Phe; substitution of Lys with Asn, Glu, Gln, His, or Arg; substitution of Met with Ile, Leu, Val, or Phe; substitution of Phe with Trp, Tyr, Met, Ile, or Leu; substitution of Ser with Thr or Ala; substitution of Thr with Ser or Ala; substitution of Trp with Phe or Tyr; substitution of Tyr with His, Phe, or Trp; and substitution of Val with Met, Ile, or Leu. The above-mentioned amino acid substitutions, deletions, insertions, or additions may be caused by naturally occurring mutations (mutants or variants) based on individual differences in organisms or differences in species, etc. This also includes those that arise from the following.

[0070] Furthermore, as long as the original function of the glnE gene is maintained, the amino acid sequence of the glnE gene may be, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, based on the entire amino acid sequence. More preferably, the gene may encode a protein having an amino acid sequence with an identity of 97% or more, and particularly preferably 99% or more.

[0071] Furthermore, the glnE gene may be a gene (e.g., DNA) that hybridizes under stringent conditions with a probe that can be prepared from the above-mentioned nucleotide sequence (e.g., the nucleotide sequence shown in SEQ ID NO: 1), such as a sequence complementary to the entire or a part of the above-mentioned nucleotide sequence, as long as the original function is maintained. "Stringent conditions" refers to the condition that allows for the formation of a so-called specific hybrid. For example, DNAs with high identity, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 100% or more, are hybridized under the conditions under which a specific hybrid is formed and no non-specific hybrid is formed. DNAs having an identity of 95% or more, more preferably 97% or more, and particularly preferably 99% or more will hybridize with each other, and DNAs having an identity lower than that will not hybridize with each other, or Alternatively, the washing conditions may include washing once, preferably two to three times, at a salt concentration and temperature equivalent to the washing conditions for conventional Southern hybridization: 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

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

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

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

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

[0076] As a method for modifying the wild-type glnE gene so as to retain the mutant glnE gene, the alanine residue at position 364 and / or the arginine residue at position 393 may be substituted in the coding region of the wild-type glnE gene. Examples of methods for introducing the mutant glnE gene into a coryneform bacterium include introducing the mutation into the coding region of the wild-type glnE gene of the coryneform bacterium.

[0077] Modification of a coryneform bacterium to have a mutant glnE gene can be achieved by introducing the mutant glnE gene into the coryneform bacterium, or by introducing a mutation into the glnE gene of the coryneform bacterium by natural mutation or mutagen treatment.

[0078] The introduction of a mutant glnE gene into a coryneform bacterium can be achieved by introducing the gene into the host chromosome. Introduction of a gene into a chromosome can be achieved, for example, by using homologous recombination (Miller, JH Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Examples of gene introduction methods using homologous recombination include the Red Duck method. Using linear DNA such as the Red-driven integration method (Datsenko, K. A, and Wanner, B. L. Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)), These include a method using a plasmid containing a temperature-sensitive replication origin, a method using a conjugatively transferable plasmid, a method using a suicide vector lacking a replication origin functional in the host, and a transduction method using a phage. Specifically, a host can be transformed with recombinant DNA containing a mutant glnE gene, and the gene can be introduced into the host chromosome by homologous recombination with a target site on the host chromosome. The structure of the recombinant DNA used for homologous recombination is not particularly limited as long as it allows homologous recombination to occur in the desired manner. For example, a host can be transformed with linear DNA containing a mutant glnE gene, with nucleotide sequences at both ends of the gene homologous to those upstream and downstream of the target site on the chromosome, thereby replacing the target site with the gene by homologous recombination upstream and downstream of the target site. The recombinant DNA used for homologous recombination may contain a marker gene for selecting transformants. Only one copy, two or more copies of the gene may be introduced. For example, multiple copies of the mutant glnE gene can be introduced into a chromosome by performing homologous recombination targeting a base sequence that exists in multiple copies on the chromosome. Examples of base sequences that exist in multiple copies on the chromosome include repetitive DNA sequences, inverted linkers at both ends of transposons, and so on. Alternatively, homologous recombination may be performed by targeting an appropriate base sequence on a chromosome, such as a gene that is not required for the production of a target substance. Alternatively, genes may be randomly introduced onto a chromosome using transposons or Mini-Mu (see Japanese Patent Application Laid-Open No. 2-109985, U.S. Patent Application Laid-Open No. 2000-109985, U.S. Patent Application Laid-Open No. 2000-109985). (Specifications of Patent No. 5882888, EP805867B1). This modification technique is not limited to the introduction of a mutant glnE gene, but can be used for any modification of a chromosome, such as modification of an expression regulatory sequence.

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

[0080] Alternatively, the mutant glnE gene can be introduced into a coryneform bacterium by introducing a vector containing the gene into the host. For example, a DNA fragment containing the mutant glnE gene can be ligated to a vector that functions in the host to construct an expression vector for the gene, and the host can be transformed with the expression vector to introduce the gene into the coryneform bacterium. A DNA fragment containing the mutant glnE gene can be obtained, for example, by PCR using the genomic DNA of a microorganism having the mutant glnE gene as a template. A vector that can autonomously replicate within the host cells can be used as the vector. A multicopy vector is preferred. Furthermore, the vector preferably has a marker such as an antibiotic resistance gene to select transformants. The vector may also have a promoter or terminator for expressing the inserted gene. The vector can be used, for example, in bacterial plus. The vector may be a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid. Specific examples of vectors capable of autonomous replication in coryneform bacteria include pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903 (1984)); and drug-resistant vectors obtained by improving these vectors. Plasmids carrying the gene: pCRY30 (Japanese Patent Laid-Open No. 3-210184); pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX (Japanese Patent Laid-Open No. 2-72876, U.S. Pat. No. 5,185,262); pCRY2 and pCRY3 (Japanese Patent Laid-Open No. 1-191686); pAJ655, pAJ611, and pAJ1844 (Patent Literature 1). pCG1 (Japanese Patent Publication No. 58-192900); pCG2 (Japanese Patent Publication No. 58-35197); pCG4 and pCG11 (Japanese Patent Publication No. 57-183799); pVK7 (Japanese Patent Publication No. 10-215883); pVK9 (U.S. Patent Application Publication No. 2006 / 0141588); pVC7 (Japanese Patent Publication No. 9-070291); pVS7 (WO2013 / 069634).

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

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

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

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

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

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

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

[0088] Specific examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, peptone, yeast extract, meat extract, and hydrolyzed vegetable protein (HVP; for example, soy protein hydrolyzate, soybean soy sauce, pea soy sauce, etc.), and organic solvents such as ammonium sulfate, ammonium chloride, and ammonium phosphate. Examples of nitrogen sources include ammonia and urea. Ammonia gas or aqueous ammonia, which is used to adjust pH, may also be used as the nitrogen source. As the nitrogen source, one type of nitrogen source may be used, or two or more types of nitrogen sources may be used in combination.

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

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

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

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

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

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

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

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

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

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

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

[0100] The culture may be carried out, for example, using a liquid medium under aerobic or microaerobic conditions. "Aerobic conditions" refers to a state in which the dissolved oxygen concentration in the liquid medium is 0.33 ppm or more, which is the detection limit of an oxygen membrane electrode, and preferably 1.5 ppm or more. Under aerobic conditions The oxygen concentration is controlled to, for example, 5 to 50% of the saturated oxygen concentration, preferably about 10%. "Microaerobic conditions" may refer to conditions in which the dissolved oxygen concentration in the medium is less than 0.33 ppm. The dissolved oxygen concentration in the medium under microaerobic conditions is, for example, 0.30 ppm or less, 0.25 ppm or less, or The oxygen concentration under microaerobic conditions may be, for example, less than 5%, 3.75%, 3.125%, or less of the saturated oxygen concentration. The pH may be controlled to 0.1% or less, 2.5% or less, 1.875% or less, 1.25% or less, or 0.8125% or less. Specifically, the culture can be performed by aeration culture, shaking culture, agitation culture, or a combination thereof. The pH of the medium may be, for example, 3 to 10, preferably 4.0 to 9.5. During the culture, The pH of the medium can be adjusted as necessary. The pH of the medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, etc. 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 adjusted in two or more stages. For example, see Journal of Industrial Microbiology & Biotechnology (2002) 28, 333-337, the culture temperature was increased from 33°C to 37-40°C. The culture period may be, for example, 10 to 120 hours. For example, the incubation may be continued until the carbon source in the medium is consumed or until the activity of the bacterium of the present invention is lost. By culturing the bacterium of the present invention under such conditions, L-glutamic acid accumulates in the medium and / or within the bacterium.

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

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

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

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

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

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

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

[0108] <1> Construction of engineered strains of Corynebacterium glutamicum <1-1> Construction of a vector for introducing the wild-type glnE gene The chromosomal DNA of C. glutamicum ATCC 13869 (strain 2256) containing the wild-type glnE gene was used as a template. Then, wild-type glnE was isolated by PCR using primers for glnE of SEQ ID NO: 5 and SEQ ID NO: 6. A DNA fragment containing the gene was amplified. PCR was carried out using the above (listed below) as a template and primers for pVK9 of SEQ ID NO: 7 and SEQ ID NO: 8. The DNA fragments were amplified and ligated in an infusion reaction to obtain a vector for introducing the wild-type glnE gene (pVK9-glnE (WT)). The nucleotide sequence of the wild-type glnE gene and the amino acid sequence of the wild-type glutamate-ammonia-ligase adenylyltransferase encoded by the gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0109] <1-2> Construction of a vector for introducing the mutant glnE gene Wild-type glutamate-ammonia-ligase adenylyltransferase 364 A mutant glnE gene with a mutation (A364T) that replaces the alanine residue at position 1 with a threonine residue The chromosomal DNA of a C. glutamicum ATCC 13869 (2256 strain) derivative containing the A DNA fragment containing the mutant glnE gene was amplified by PCR using primers for glnE of SEQ ID NO: 5 and SEQ ID NO: 6. Furthermore, a DNA fragment was amplified by PCR using primers for pVK9 of SEQ ID NO: 7 and SEQ ID NO: 8, using pVK9 as a template. These DNA fragments were used in an infusion reaction. The mutant glnE gene transfer vector (pVK9-glnE (A364T)) was obtained by ligating the Ta. The nucleotide sequence of the mutant glnE gene and the amino acid sequence of the mutant [glutamate-ammonia-ligase] adenylyltransferase encoded by the gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0110] <1-3> Construction of modified strains of Corynebacterium glutamicum The constructed wild-type glnE gene transfer vector (pVK9-glnE (WT)) and mutant glnE gene transfer vector (pVK9-glnE (A364T, R393H)) were introduced into the C. glutamicum 2256ΔsucAΔldhA yggB* strain (WO2014 / 185430) alone to generate wild-type glnE gene transfer strains and mutant glnE gene transfer vectors. A mutant glnE gene-transfected strain was obtained.

[0111] The C. glutamicum 2256ΔsucAΔldhA yggB* strain is an L-glutamic acid-producing strain derived from the C. glutamicum 2256 strain (ATCC 13869), which is deficient in the ldhA and sucA genes and has an IS mutation (V419::IS) in the yggB gene.

[0112] <2> L-glutamic acid producing culture The constructed strains (i.e., wild-type glnE gene-introduced strains and mutant glnE gene-introduced strains) were cultured to produce L-glutamic acid. The composition of the medium used is shown in Table 1.

[0113] [Table 1]

[0114] Each strain was inoculated into 20 mL of the above medium (containing 50 g / L calcium carbonate) placed in a 500 mL Sakaguchi flask, and shaken at 120 rpm in a box shaker (ABLE ML-190) at 31.5°C. The culture was incubated with shaking. 25 hours after the start of cultivation, the culture medium was sampled. 0.05 ml of the sampled culture medium was added to 4.95 ml of 0.1 N HCl solution to dilute it 100-fold, and the optical density (OD) at 620 nm was measured. The sampled culture medium was centrifuged at 15,000 rpm for 1 minute, and the L-glutamic acid concentration in the supernatant was measured using a Biotech Analyzer BF7 (Oji Scientific Instruments Co., Ltd.). The yield of L-glutamic acid relative to sugar was calculated.

[0115] The results are shown in Figures 1 and 2. The absorbance OD 620nm The results are shown in Figure 3. The mutant glnE gene-introduced strain showed higher L-glutamic acid accumulation and higher L-glutamic acid yield per sugar than the wild-type glnE gene-introduced strain (Figures 1 and 2). [Industrial Applicability]

[0116] According to the present invention, the L-amino acid producing ability of a coryneform bacterium can be improved, and L-amino acids can be produced efficiently.

[0117] [Explanation of Sequence Listing] SEQ ID NO: 1: Nucleotide sequence of the wild-type glnE gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 2: Amino acid sequence of wild-type [glutamate-ammonia-ligase] adenylyltransferase from Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 3: Nucleotide sequence of the mutant glnE gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 4: Amino acid sequence of the mutant [glutamate-ammonia-ligase] adenylyltransferase of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 5 and SEQ ID NO: 6: Primers for glnE SEQ ID NO: 7 and SEQ ID NO: 8: Primers for pVK9 SEQ ID NO: 9: The sequence of the wild-type yggB gene of Corynebacterium glutamicum 2256 (ATCC 13869) Base sequence SEQ ID NO: 10: Wild-type YggB protein of Corynebacterium glutamicum 2256 (ATCC 13869) Quality amino acid sequence SEQ ID NO: 11: Nucleotide sequence of mutant yggB gene (V419::IS) SEQ ID NO: 12: Amino acid sequence of mutant YggB protein (V419::IS)

Claims

1. A method for producing L-glutamic acid, comprising: Cultivating Corynebacterium glutamicum in a medium and accumulating L-glutamic acid in the medium and / or within the cells of the Corynebacterium glutamicum; collecting L-glutamic acid from the medium and / or the bacterial cells; Including, the Corynebacterium glutamicum has the ability to produce L-glutamic acid, A mutant [glutamate-ammonia-ligase] adenylyltransferase having a substitution of the alanine residue at position 364 in the amino acid sequence of the wild-type [glutamate-ammonia-ligase] adenylyltransferase with another amino acid residue. modified to carry a mutant glnE gene encoding glutamate-ammonia-ligase adenylyltransferase; the mutant [glutamate-ammonia-ligase] adenylyltransferase comprises the amino acid sequence set forth in SEQ ID NO:4; Manufacturing method.

2. the Corynebacterium glutamicum has been modified to carry a mutant yggB gene; The method according to claim 1 , wherein the mutant yggB gene encodes a protein having the amino acid sequence of SEQ ID NO:

12.

3. The method of claim 1 or 2, wherein the mutant glnE gene comprises the nucleotide sequence shown in SEQ ID NO: 3 or a degenerate variant of the nucleotide sequence.

Images