Method for producing L-amino acids

Abstract

Provided is a method of producing an L-amino acid such as L-glutamic acid. According to this method, an L-amino acid is produced by culturing a coryneform bacterium capable of producing the L-amino acid, said coryneform bacterium having been modified to have one or more of the modifications selected from (A) to (E) below, in a medium and collecting the L-amino acid from the medium and / or the cells of the bacterium: (A) a modification that increases acetate kinase activity; (B) a modification that increases fructose-1,6-bisphosphatase activity; (C) a modification that decreases pyruvate dehydrogenase activity; (D) a modification that decreases aspartate transaminase activity; and (E) a modification that decreases malic enzyme activity.

Description

[Technical Field]

[0001] This invention relates to a method for producing L-amino acids, such as L-glutamic acid, by fermentation using bacteria. L-amino acids are industrially useful as ingredients for seasonings and the like. [Background technology]

[0002] L-amino acids are industrially produced, for example, by fermentation using microorganisms such as bacteria that have the ability to produce L-amino acids (Non-Patent Literature 1). Such microorganisms include, for example, strains isolated from nature and their mutant strains. Furthermore, the L-amino acid production capacity of microorganisms can be improved using recombinant DNA technology. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Kunihiko Akashi et al., Amino Acid Fermentation, Academic Publishing Center, pp. 195-215, 1986. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] The objective of this invention is to develop a novel technology that improves the L-amino acid production capacity of bacteria and to provide an efficient method for producing L-amino acids. [Means for solving the problem]

[0005] As a result of diligent research to solve the above problems, the inventors have discovered that the L-amino acid production capacity of Corynebacterium can be improved by modifying the bacterium to have one or more modifications selected from the following modifications (A) to (E), and have completed the present invention: (A) Modifications that increase the activity of acetate kinase; (B) Modifications that increase the activity of fructose-1,6-bisphosphatase; (C) Modification that reduces pyruvate dehydrogenase activity; (D) Modifications that reduce the activity of aspartate transaminase; (E) Modification that reduces the activity of malic enzyme.

[0006] In other words, the present invention can be illustrated as follows. [1] A method for producing L-amino acids, Culturing Corynebacteria capable of producing L-amino acids in a culture medium, and accumulating L-amino acids in the culture medium and / or within the bacterial cells, and To collect the L-amino acid from the culture medium and / or the bacterial cells, Includes, The aforementioned L-amino acid is a glutamic acid-based L-amino acid, The method, wherein the bacteria have the following modifications (X) and / or (Y): (X) Modifications that improve the availability of carbon sources via the phosphoketolase pathway; (Y) A modification that increases the oxaloacetate pool within cells. [2] The method wherein the bacteria have at least the modification of (X). [3] The aforementioned method, The modification in (X) is one or more modifications selected from the modifications in (A), (B), and (C) below; The modification of (Y) is one or more modifications selected from the modifications of (D) and (E) below, by: (A) Modifications that increase the activity of acetate kinase; (B) Modifications that increase the activity of fructose-1,6-bisphosphatase; (C) Modification that reduces the activity of pyruvate dehydrogenase; (D) Modification that reduces the activity of aspartate transaminase; (E) Modification that reduces the activity of malic enzyme. [4] A method for producing an L - amino acid, comprising: culturing a Corynebacterium bacterium having the ability to produce an L - amino acid in a medium, accumulating the L - amino acid in the medium and / or in the cells of the bacterium, and collecting the L - amino acid from the medium and / or the cells of the bacterium, wherein: the L - amino acid is a glutamic acid - type L - amino acid, the bacterium has one or more modifications selected from the following modifications (A) to (E): (A) Modification that increases the activity of acetate kinase; (B) Modification that increases the activity of fructose - 1,6 - bisphosphatase; (C) Modification that reduces the activity of pyruvate dehydrogenase; (D) Modification that reduces the activity of aspartate transaminase; (E) Modification that reduces the activity of malic enzyme. [5] The method according to claim 1, wherein the bacterium has at least the modification of (A). [6] The method according to claim 1, wherein: the aspartate transaminase is encoded by the aspT gene; the malic enzyme is encoded by the malE gene; the pyruvate dehydrogenase is encoded by the poxB gene; the acetate kinase is encoded by the ack gene; and / or the fructose - 1,6 - bisphosphatase is encoded by the glpX gene. [7] The method according to claim 1, wherein: The activity of the aspartate transaminase is reduced by decreasing the expression of the gene encoding the aspartate transaminase, or by disrupting the gene; The activity of the aforementioned malic enzyme is reduced by reducing the expression of the gene encoding the malic enzyme, or by disrupting said gene; The activity of the pyruvate dehydrogenase is reduced by decreasing the expression of the gene encoding pyruvate dehydrogenase, or by disrupting the gene; The activity of the acetate kinase is increased by increasing the expression of the gene encoding acetate kinase; and / or A method in which the activity of the fructose-1,6-bisphosphatase is increased by increasing the expression of the gene encoding the fructose-1,6-bisphosphatase. [8] The aforementioned method, The expression of the gene encoding acetate kinase is increased by increasing the copy number of the gene and / or by modifying the gene's regulatory sequence; and / or A method in which the expression of a gene encoding fructose-1,6-bisphosphatase is increased by increasing the copy number of the gene and / or modifying the gene's expression regulatory sequence. [9] The aforementioned method, The aspartate transaminase is the protein described in (1a), (1b), or (1c) below: (1a) Proteins containing the amino acid sequence shown in Sequence ID No. 2; (1b) A protein having aspartate transaminase activity, comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 2; (1c) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No. 2, and that has aspartate transaminase activity; The maric enzyme is the protein described in (2a), (2b), or (2c) below: (2a) Proteins containing the amino acid sequence shown in SEQ ID NO: 4; (2b) A protein having a Malic enzyme activity, comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 4; (2c) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No. 4, and that also has Malic enzyme activity; The pyruvate dehydrogenase is the protein described in (3a), (3b), or (3c) below: (3a) Proteins containing the amino acid sequence shown in Sequence ID No. 6; (3b) A protein having pyruvate dehydrogenase activity, comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 6; (3c) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No. 6, and that has pyruvate dehydrogenase activity; The acetate kinase is the protein described in (4a), (4b), or (4c) below: (4a) Proteins containing the amino acid sequence shown in Sequence ID No. 8 or 10; (4b) A protein having acetate kinase activity, comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 8 or 10; (4c) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 8 or 10, and which has acetate kinase activity; and / or The method wherein the fructose-1,6-bisphosphatase is the protein described in (5a), (5b), or (5c) below: (5a) Proteins containing the amino acid sequence shown in Sequence ID No. 12 or 14; (5b) A protein having fructose-1,6-bisphosphatase activity, comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 12 or 14; (5c) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 12 or 14, and that has fructose-1,6-bisphosphatase activity.

[10] The method, wherein the bacterium is further modified to have increased phosphoketolase activity compared to the unmodified strain.

[11] The method wherein the phosphoketolase is D-xylulose-5-phosphate phosphoketolase and / or fructose-6-phosphate phosphoketolase.

[12] The method wherein the bacteria are bacteria of the genus Corynebacterium.

[13] The method wherein the bacterium is Corynebacterium glutamicum.

[14] The method, wherein the glutamate-based L-amino acid is one or more L-amino acids selected from L-glutamic acid, L-glutamine, L-proline, L-arginine, L-citrulline, and L-ornithine.

[15] The method wherein the glutamate-based L-amino acid is L-glutamic acid.

[16] The method wherein the L-glutamic acid is ammonium L-glutamate or sodium L-glutamate. [Brief explanation of the drawing]

[0007] [Figure 1] This figure shows the amount of L-glutamate accumulated in the control strain and the ACK gene-enhanced strain when glucose is used as the carbon source. [Figure 2] This figure shows the L-glutamic acid yield relative to glucose in the control strain and the ACK gene-enhanced strain when glucose is used as the carbon source. [Figure 3] This figure shows the amount of L-glutamate accumulated in the control strain and the glpX gene-enhanced strain when fructose is used as the carbon source. [Figure 4] This figure shows the L-glutamic acid yield relative to glucose in the control strain and the glpX gene-enhanced strain when glucose is used as the carbon source. [Modes for carrying out the invention]

[0008] The present invention will be described in detail below.

[0009] The present invention relates to a method for producing L-amino acids, comprising culturing a Corynebacterium bacterium capable of producing L-amino acids in a culture medium, accumulating L-amino acids in the culture medium and / or within the bacterial cells, and collecting the L-amino acids from the culture medium and / or the bacterial cells, wherein the bacterium is modified to have specific properties. The bacterium used in this method is also referred to as the "bacterium of the present invention."

[0010] <1> The bacteria of the present invention The bacteria of the present invention are Corynebacteria with L-amino acid production ability, modified to possess specific properties.

[0011] <1-1> Bacteria with L-amino acid production ability In the present invention, "bacteria having L-amino acid production ability" refers to bacteria that, when cultured in a culture medium, have the ability to produce a target L-amino acid and accumulate it in the culture medium and / or within the bacterial cell to the extent that it can be recovered. Bacteria having L-amino acid production ability may be bacteria that can accumulate a larger amount of the target L-amino acid in the culture medium and / or within the bacterial cell than an unmodified strain. "Unmodified strain" refers to a control strain that has not been modified to have specific properties. That is, examples of unmodified strains include wild strains and parent strains. Furthermore, bacteria having L-amino acid production ability may be bacteria that can accumulate a quantity of the target L-amino acid in the culture medium preferably of 0.5 g / L or more, more preferably of 1.0 g / L or more.

[0012] The L-amino acids produced in this invention are glutamate-based L-amino acids (L-amino acids of the glutamate family). "Glutamate-based L-amino acids" is a general term for L-glutamic acid and L-amino acids that are biosynthesized using L-glutamic acid as an intermediate. Examples of L-amino acids that are biosynthesized using L-glutamic acid as an intermediate include L-glutamine, L-proline, L-arginine, L-citrulline, and L-ornithine. In particular, L-glutamic acid is an example of a glutamate-based L-amino acid. The bacteria of this invention may have the ability to produce only one type of L-amino acid, or it may have the ability to produce two or more types of L-amino acids.

[0013] In this invention, the term "amino acid" means L-amino acids unless otherwise specified. Furthermore, in this invention, the term "L-amino acid" means free L-amino acids, their salts, or mixtures thereof unless otherwise specified. Salts will be discussed later.

[0014] Examples of coryne-type bacteria include those belonging to genera such as Corynebacterium, Brevibacterium, and Microbacterium.

[0015] Examples of Corynebacteria include the following species: Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Corynebacterium crenatum Corynebacterium glutamicum Corynebacterium lilium Corynebacterium melassecola Corynebacterium thermoaminogenes (Corynebacterium efficiens) Corynebacterium herculis Brevibacterium divaricatum (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium glutamicum) Brevibacterium immariophilum Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium ammoniagenes (Corynebacterium stationis) Brevibacterium album Brevibacterium cerinum Microbacterium ammoniaphilum

[0016] Among the Corynebacterium-type bacteria, Corynebacterium glutamicum (formerly known as Brevibacterium lactofermentum) is a particularly noteworthy example.

[0017] Examples of Corynebacterium strains include the following: Corynebacterium acetoacidophilum ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium alkanolyticum ATCC 21511 Corynebacterium callunae ATCC 15991 Corynebacterium crenatum AS1.542 Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734 Corynebacterium lilium ATCC 15990 Corynebacterium melassecola ATCC 17965 Corynebacterium efficiens (Corynebacterium thermoaminogenes) AJ12340 (FERM BP-1539) Corynebacterium herculis ATCC 13868 Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020 Brevibacterium flavum (Corynebacterium glutamicum) ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205) Brevibacterium immariophilum ATCC 14068 Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC 13869 Brevibacterium roseum ATCC 13825 Brevibacterium saccharolyticum ATCC 14066 Brevibacterium thiogenitalis ATCC 19240 Corynebacterium ammoniagenes (Corynebacterium stationis) ATCC 6871, ATCC 6872 Brevibacterium album ATCC 15111 Brevibacterium cerinum ATCC 15112 Microbacterium ammoniaphilum ATCC 15354

[0018] As the coryneform bacterium, more particularly, Brevibacterium lactofermentum (new name: Corynebacterium glutamicum) ATCC 13869 can be mentioned.

[0019] Furthermore, the genus Corynebacterium includes bacteria that were previously classified under the genus Brevibacterium but have now been integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)). Additionally, the genus Corynebacterium statyonis includes bacteria that were previously classified under Corynebacterium ammoniagenes but have been reclassified as Corynebacterium statyonis based on 16S rRNA sequencing analysis (Int. J. Syst. Evol. Microbiol., 60, 874-879 (2010)).

[0020] These strains can be obtained, for example, from the American Type Culture Collection (address: 12301 Parklawn Drive, Rockville, Maryland 20852 PO Box 1549, Manassas, VA 20108, United States of America). Each strain is assigned a registration number, which can be used to obtain them (see http: / / www.atcc.org / ). The registration numbers for each strain are listed in the American Type Culture Collection catalog. These strains can also be obtained, for example, from the depositary institutions where each strain is deposited.

[0021] The bacteria of the present invention may inherently possess L-amino acid production ability, or they may be modified to possess L-amino acid production ability. Bacteria possessing L-amino acid production ability can be obtained, for example, by conferring L-amino acid production ability to the above-mentioned bacteria, or by enhancing the L-amino acid production ability of the above-mentioned bacteria.

[0022] The conferring or enhancing of L-amino acid production ability can be carried out by methods conventionally employed in the breeding of amino acid-producing bacteria such as Corynebacteria or Escherichia species (see Amino Acid Fermentation, Gakkai Shuppan Center Co., Ltd., first edition published May 30, 1986, pp. 77-100). Such methods include, for example, obtaining nutrient-requiring mutants, obtaining L-amino acid analog-tolerant mutants, obtaining metabolic-regulating mutants, and creating recombinant strains with enhanced activity of L-amino acid biosynthetic enzymes. In the breeding of L-amino acid-producing bacteria, the conferred properties such as nutrient requirements, analog tolerance, and metabolic regulation mutations may be one, two, or three or more. Similarly, in the breeding of L-amino acid-producing bacteria, the enhanced activity of L-amino acid biosynthetic enzymes may also be one, two, or three or more. Furthermore, the conferring of properties such as nutrient requirements, analog tolerance, and metabolic regulation mutations may be combined with the enhancement of biosynthetic enzyme activity.

[0023] Nutritionally dependent mutants, analog-tolerant mutants, or metabolically regulated mutants with L-amino acid production ability can be obtained by subjecting the parental or wild-type strain to a conventional mutagenesis procedure and selecting mutants from the resulting strains that exhibit nutrient dependence, analog tolerance, or metabolic regulation mutations and also possess L-amino acid production ability. Conventional mutagenesis procedures 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).

[0024] Furthermore, the ability to produce L-amino acids can also be enhanced by increasing the activity of enzymes involved in the biosynthesis of the target L-amino acid. Enzyme activity can be enhanced, for example, by modifying bacteria to increase the expression of the gene encoding the enzyme. Methods for enhancing gene expression are described in WO00 / 18935 and EP1010755A, among others. Detailed methods for enhancing enzyme activity will be described later.

[0025] In addition, the imparting or enhancement of the ability to produce L-amino acids can also be achieved by reducing the activity of an enzyme that catalyzes a reaction that branches off from the biosynthetic pathway of the target L-amino acid to produce a compound other than the target L-amino acid. Here, the "enzyme that catalyzes a reaction that branches off from the biosynthetic pathway of the target L-amino acid to produce a compound other than the target L-amino acid" includes enzymes involved in the decomposition of the target amino acid. The methods for reducing enzyme activity will be described later.

[0026] Hereinafter, L-amino acid-producing bacteria and methods for imparting or enhancing the ability to produce L-amino acids will be specifically exemplified. Note that any of the properties possessed by the L-amino acid-producing bacteria and the modifications for imparting or enhancing the ability to produce L-amino acids, as exemplified below, may be used alone or in appropriate combinations.

[0027] <L-glutamic acid-producing bacterium> Methods for conferring or enhancing L-glutamate production ability include, for example, modifying bacteria to increase the activity of one or more enzymes selected from L-glutamate biosynthesis enzymes. Such enzymes are not particularly limited, but include glutamate dehydrogenase (gdhA), glutamate synthase (glnA), glutamate synthase (gltBD), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), methylcitrate synthase (prpC), pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglyceromutase (pgmA, Examples of enzymes include pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), glucose phosphate isomerase (pgi), 6-phosphogluconate dehydratase (edd), 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), and transhydrogenase (pntAB). The names in parentheses are examples of genes encoding these enzymes (the same applies to the following descriptions). Among these enzymes, it is preferable to enhance the activity of one or more enzymes selected from, for example, glutamate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, and methyl citrate synthase.

[0028] Examples of Corynebacteria modified to increase the expression of the glutamate synthase gene (gltBD) include those disclosed in WO99 / 07853.

[0029] Furthermore, methods for conferring or enhancing L-glutamate production ability include, for example, modifying bacteria to reduce the activity of one or more enzymes selected from enzymes that catalyze reactions branching off from the L-glutamate biosynthesis pathway to produce compounds other than L-glutamate. Such enzymes are not particularly limited, but include isocitrate lyase (aceA), α-ketoglutarate dehydrogenase (sucA, odhA), acetolactic acid synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), alcohol dehydrogenase (adh), glutamate decarboxylase (gadAB), and succinate dehydrogenase (sdhABCD). Among these enzymes, for example, it is preferable to reduce or eliminate α-ketoglutarate dehydrogenase activity.

[0030] Corynebacteria with reduced or deficient α-ketoglutarate dehydrogenase activity, and methods for obtaining them, are described in WO2008 / 075483. Specific examples of Corynebacteria with reduced or deficient α-ketoglutarate dehydrogenase activity include the following strains. Corynebacterium glutamicum (Brevibacterium lactofermentum) L30-2 strain (JP 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)

[0031] Furthermore, examples of L-glutamic acid-producing bacteria or parental strains for inducing them include strains in which both α-ketoglutarate dehydrogenase (sucA) activity and succinate dehydrogenase (sdh) activity are reduced or deficient (JP 2010-041920). Specifically, an example of such a strain is the odhAsdhA double knockout strain of Corynebacterium glutamicum ATCC14067 (Corynebacterium glutamicum 8L3GΔSDH strain) (JP 2010-041920).

[0032] Furthermore, methods for conferring or enhancing L-glutamate production ability include, for example, enhancing the expression of L-glutamate efflux genes such as the yhfK gene (WO2005 / 085419) and the ybjL gene (WO2008 / 133161).

[0033] Furthermore, methods for conferring or enhancing L-glutamate production ability in Corynebacteria include methods for conferring resistance to organic acid analogs and respiratory inhibitors, as well as methods for conferring sensitivity to cell wall synthesis inhibitors. Specific examples of such methods include, for instance, a method for conferring resistance to monofluoroacetic acid (JP-A-50-113209), a method for conferring resistance to adenine or thymine (JP-A-57-065198), a method for weakening urease (JP-A-52-038088), a method for conferring resistance to malonic acid (JP-A-52-038088), a method for conferring resistance to benzopyrones or naphthoquinones (JP-A-56-1889), a method for conferring resistance to HOQNO (JP-A-56-140895), a method for conferring resistance to α-ketomalonic acid (JP-A-57-2689), a method for conferring resistance to guanidine (JP-A-56-35981), and a method for conferring sensitivity to penicillin (JP-A-4-88994).

[0034] Specific examples of such resistant or susceptible bacteria include the following strains. Corynebacterium glutamicum (Brevibacterium flavum) AJ3949 (FERM BP-2632; Japanese Patent Application Laid-Open No. 1983-113209) Corynebacterium glutamicum AJ11628 (FERM P-5736; JP-A-57-065198) Corynebacterium glutamicum (Brevibacterium flavum) AJ11355 (FERM P-5007; Japanese Patent Application Laid-open No. 56-1889) Corynebacterium glutamicum AJ11368 (FERM P-5020; Japanese Patent Publication No. 56-1889) Corynebacterium glutamicum (Brevibacterium flavum) AJ11217 (FERM P-4318; Japanese Patent Application Laid-open No. 57-2689) Corynebacterium glutamicum AJ11218 (FERM P-4319; Japanese Patent Publication No. 57-2689) Corynebacterium glutamicum (Brevibacterium flavum) AJ11564 (FERM P-5472; Japanese Patent Application Laid-open No. 56-140895) Corynebacterium glutamicum (Brevibacterium flavum) AJ11439 (FERM P-5136; Japanese Patent Application Laid-open No. 56-35981) Corynebacterium glutamicum H7684 (FERM BP-3004; Japanese Patent Publication No. 04-88994) Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ11426 (FERM P-5123; JP 56-048890) Corynebacterium glutamicum AJ11440 (FERM P-5137; Japanese Patent Publication No. 56-048890) Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ11796 (FERM P-6402; JP 58-158192)

[0035] Furthermore, methods for conferring or enhancing L-glutamate production ability in Corynebacteria include methods for enhancing the expression of the yggB gene and methods for introducing a mutant yggB gene in which a mutation has been introduced into the coding region (WO2006 / 070944). That is, the bacteria of the present invention may be modified to increase the expression of the yggB gene, or may be modified to possess (have) a mutant yggB gene.

[0036] The yggB gene is a gene that codes for a mechanosensitive channel. Examples of yggB genes found in Corynebacteria include those of Corynebacterium glutamicum ATCC13869, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14967, and Corynebacterium melassecola ATCC17965 (WO2006 / 070944). The yggB gene of Corynebacterium glutamicum ATCC13032 corresponds to the complementary sequence of sequences 1,336,091 to 1,337,692 in the genome sequence registered in the NCBI database under GenBank Accession No. NC_003450, and is also called NCgl1221. The YggB protein encoded by the yggB gene of Corynebacterium glutamicum ATCC13032 is registered under GenBank accession No. NP_600492. In addition, the nucleotide sequence of the yggB gene of Corynebacterium glutamicum 2256 (ATCC 13869) and the amino acid sequence of the YggB protein encoded by the same gene are shown in Sequence IDs 15 and 16, respectively.

[0037] In this invention, a yggB gene having the "specific mutation" described later is also called a mutant yggB gene, and the protein encoded thereby is also called a mutant YggB protein. In this invention, a yggB gene that does not have the "specific mutation" described later is also called a wild-type yggB gene, and the protein encoded thereby is also called a wild-type YggB protein. In the case of YggB protein, the change in amino acid sequence caused by the "specific mutation" in the yggB gene is also called the "specific mutation." The term "wild-type" here is a convenient description to distinguish it from the "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, for example, the protein having the amino acid sequence shown in SEQ ID NO: 16. In addition, examples of wild-type YggB proteins include conserved variants (variants that maintain the original function) of the YggB proteins exemplified above 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 the property of improving the L-glutamate production capacity of Corynebacteria when its expression is increased in Corynebacteria.

[0038] A "specific mutation" is not particularly limited as long as it alters the amino acid sequence of the wild-type YggB protein as described above and improves the L-glutamate production capacity of Corynebacteria. Examples of "specific mutations" include C-terminal mutations and transmembrane mutations (WO2006 / 070944). Furthermore, a "specific mutation" may be a combination of these mutations.

[0039] (1) C-terminal mutation A C-terminal mutation is a mutation in the region of the wild-type yggB gene that codes for amino acid residues between positions 419 and 533 of the wild-type YggB protein. A C-terminal mutation may be introduced at one or more locations within this region. The type of amino acid sequence change caused by a C-terminal mutation is not particularly limited. A C-terminal mutation may result in, for example, amino acid residue substitution (missense mutation), amino acid residue insertion, amino acid residue deletion, the appearance of a stop codon (nonsense mutation), a frameshift mutation, or a combination thereof. For example, insertion of base sequences such as insertion sequences (hereinafter also referred to as "IS") or transposons is preferred as a C-terminal mutation.

[0040] (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 at the site encoding the valine residue at position 419 of the wild-type YggB protein. A 2A-1 type mutation may, for example, cause the deletion or substitution of some or all of the amino acid residues between positions 419 and 533 of the wild-type YggB protein. Specifically, an example of a mutant yggB gene having a 2A-1 type mutation is a yggB gene in which IS is inserted after the "G" at position 1255 of SEQ ID NO: 15, encoding a mutant YggB protein with a total length of 423 amino acids, which is shorter than the original wild-type YggB protein (SEQ ID NO: 16). 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 (SEQ ID NO: 17 and 18) are shown, respectively. In sequence number 17, positions 1-1269 are the CDS of the mutant YggB protein (V419::IS). An example of an L-glutamic acid-producing bacterium possessing the mutant yggB gene (V419::IS) is, for instance, C. glutamicum 2256ΔsucAΔldhA yggB. * One example is the stock (WO2014 / 185430).

[0041] (1-2) Substitution of proline residues C-terminal mutations include, for example, mutations that replace proline residues located at positions 419-533 of the wild-type YggB protein with other amino acids. Such proline residues include those at positions 424, 437, 453, 457, 462, 469, 484, 489, 497, 515, 529, and 533 of the wild-type YggB protein. In particular, it is preferable to replace the proline residue at position 424 and / or 437 with another amino acid. The "other amino acid" is not particularly limited as long as it is a native 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), and more preferably with a branched-chain amino acid (Leu, Val, or Ile). Also, for example, the proline residue at position 437 may preferably be substituted with an amino acid having a hydroxyl group in its side chain (Thr, Ser, or Tyr), and more preferably with Ser.

[0042] (2) Transmembrane region mutations The YggB protein is presumed to have five transmembrane regions. Each transmembrane region corresponds to the amino acid residues at positions 1-23 (first transmembrane region), 25-47 (second transmembrane region), 62-84 (third transmembrane region), 86-108 (fourth transmembrane region), and 110-132 (fifth transmembrane region) of the wild-type YggB protein. Transmembrane region mutations are mutations in the regions of the wild-type YggB gene that encode these transmembrane regions. Transmembrane region mutations may be introduced at one or more locations within the same region. Transmembrane region mutations preferably result in the substitution, deletion, addition, insertion, or inversion of one or more amino acids, and preferably do not involve 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. Transmembrane region mutations include mutations that insert one or more amino acids (e.g., Cys-Ser-Leu) between the leucine residue at position 14 and the tryptophan residue at position 15 of the wild-type YggB protein, mutations that replace the alanine residue at position 100 with another amino acid residue (e.g., an amino acid with a hydroxyl group in its side chain (Thr, Ser, or Tyr), preferably Thr), and mutations that replace the alanine residue at position 111 with another amino acid residue (e.g., Val or an amino acid with a hydroxyl group in its side chain (Thr, Ser, or Tyr), preferably Val or Thr).

[0043] In this invention, "the amino acid residue at position X of the wild-type YggB protein" means the amino acid residue corresponding to the amino acid residue at position X in SEQ ID NO: 16, unless otherwise specified. In an amino acid sequence, "position X" means the Xth position counting from the N-terminus of the amino acid sequence, with the N-terminal amino acid residue being the amino acid residue at position 1. Note that the position of an amino acid residue indicates a relative position, and its absolute position may change due to deletion, insertion, or addition of amino acids. For example, "the amino acid residue at position 419 of the wild-type YggB protein" means the amino acid residue corresponding to the amino acid residue at position 419 in SEQ ID NO: 16. If one amino acid residue is deleted N-terminally from position 419, the 418th amino acid residue from the N-terminus shall be considered "the amino acid residue at position 419 of the wild-type YggB protein." Also, if one amino acid residue is inserted N-terminally from position 419, the 420th amino acid residue from the N-terminus shall be considered "the amino acid residue at position 419 of the wild-type YggB protein." Specifically, for example, in the YggB protein of Corynebacterium glutamicum strain ATCC14967, the amino acid residues at positions 419-529 correspond to the amino acid residues at positions 419-533 in the wild-type YggB protein.

[0044] The amino acid residue that corresponds to the amino acid residue at position X in SEQ ID NO: 16 in the amino acid sequence of any given YggB protein can be determined by aligning the amino acid sequence of the YggB protein with the amino acid sequence of SEQ ID NO: 16. Alignment can be performed, for example, using known gene analysis software. Specific examples of such software include DNASIS from Hitachi Solutions and GENETYX from Genetics (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).

[0045] The mutant yggB gene can be obtained by modifying the wild-type yggB gene to have the "specific mutation" described above. DNA modification can be performed using known methods. Specifically, site-directed mutagenesis methods for introducing the desired mutation to a target site in the DNA include methods using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and methods using phages (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)). The mutant yggB gene can also be obtained by chemical synthesis.

[0046] Modifying bacteria to possess a mutant yggB gene can be achieved by introducing the mutant yggB gene into the bacteria. Alternatively, this can also be achieved by introducing mutations into the yggB gene already present in the bacteria through natural mutation or mutagenic treatment.

[0047] Furthermore, methods for conferring or enhancing the ability to produce L-glutamic acid may also be effective for conferring or enhancing the ability to produce L-amino acids (e.g., L-glutamine, L-proline, L-arginine, L-citrulline, L-ornithine) that are biosynthesized using L-glutamic acid as an intermediate. In other words, bacteria that have the ability to produce these L-amino acids that are biosynthesized using L-glutamic acid as an intermediate may appropriately possess the properties of L-glutamic acid-producing bacteria as described above. For example, bacteria that have the ability to produce these L-amino acids that are biosynthesized using L-glutamic acid as an intermediate may be modified to reduce the activity of α-ketoglutarate dehydrogenase and / or succinate dehydrogenase.

[0048] <L-Glutamine-producing bacterium> As a method for conferring or enhancing the ability to produce L-glutamine, for example, a method of modifying bacteria so that the activity of one or more enzymes selected from L-glutamine biosynthetic enzymes is increased can be mentioned. Such enzymes include, but are not particularly limited to, glutamate dehydrogenase (gdhA) and glutamine synthetase (glnA). The activity of glutamine synthetase may also be enhanced by disruption of the glutamine adenylyltransferase gene (glnE) or disruption of the PII regulatory protein gene (glnB) (EP1229121).

[0049] In addition, as a method for conferring or enhancing the ability to produce L-glutamine, for example, a method of modifying bacteria so that the activity of one or more enzymes selected from enzymes that catalyze reactions that branch from the L-glutamine biosynthetic pathway to produce compounds other than L-glutamine is decreased can also be mentioned. Such enzymes include, but are not particularly limited to, glutaminase.

[0050] As an L-glutamine-producing bacterium or a parent strain for deriving the same, specifically, for example, coryneform bacteria with enhanced activities of glutamate dehydrogenase (gdhA) and / or glutamine synthetase (glnA) (EP1229121, EP1424398), and coryneform bacteria with decreased glutaminase activity (JP 2004-187684) can be mentioned.

[0051] In addition, for coryneform bacteria, as a method for conferring or enhancing the ability to produce L-glutamine, a method of conferring 6-diazo-5-oxo-norleucine resistance (JP 3-232497), a method of conferring purine analog resistance and methionine sulfoxide resistance (JP 61-2,026,94), and a method of conferring α-ketomalonic acid resistance (JP 56-151495) can be mentioned. As coryneform bacteria having the ability to produce L-glutamine, specifically, for example, the following strains can be mentioned. Corynebacterium glutamicum (Brevibacterium flavum) AJ11573 (FERM P-5492; Japanese Patent Laid-Open No. 56-151495) Corynebacterium glutamicum (Brevibacterium flavum) AJ11576 (FERM BP-10381; Japanese Patent Laid-Open No. 56-151495) Corynebacterium glutamicum (Brevibacterium flavum) AJ12212 (FERM P-8123; Japanese Patent Laid-Open No. 61-202694)

[0052] <L-proline-producing bacterium> As a method for imparting or enhancing L-proline-producing ability, for example, a method of modifying bacteria so that the activity of one or more enzymes selected from L-proline biosynthetic enzymes increases can be mentioned. Such enzymes include glutamate-5-kinase (proB), γ-glutamyl-phosphate reductase, and pyrroline-5-carboxylate reductase (putA). For enhancing enzyme activity, for example, the proB gene encoding glutamate-5-kinase whose feedback inhibition by L-proline is released (German Patent No. 3127361) can be preferably used.

[0053] In addition, as a method for imparting or enhancing L-proline-producing ability, for example, a method of modifying bacteria so that the activity of enzymes involved in L-proline degradation decreases can be mentioned. Such enzymes include proline dehydrogenase and ornithine aminotransferase.

[0054] <L-arginine-producing bacterium> Methods for conferring or enhancing L-arginine production ability include, for example, modifying bacteria to increase the activity of one or more enzymes selected from the L-arginine biosynthesis system enzymes. Such enzymes are not particularly limited, but include N-acetylglutamate synthase (argA), N-acetylglutamate kinase (argB), N-acetylglutamyl phosphate reductase (argC), acetylornithine transaminase (argD), acetylornithine deacetylase (argE), ornithine carbamoyltransferase (argF, argI), argininosuccinate synthase (argG), argininosuccinate lyase (argH), ornithine acetyltransferase (argJ), and carbamoyl phosphate synthase (carAB). As the N-acetylglutamate synthase (argA) gene, it is preferable to use, for example, a gene encoding a mutant N-acetylglutamate synthase in which amino acid residues corresponding to positions 15-19 of the wild type are substituted, thereby releasing feedback inhibition by L-arginine (EP1170361A).

[0055] Furthermore, other examples of Corynebacteria that can be used as L-arginine-producing bacteria or parental strains for inducing L-arginine production include strains lacking the arginine repressor ArgR (US2002-0045223A) and strains with increased intracellular glutamine synthetase activity (US2005-0014236A).

[0056] In addition, examples of the L-arginine-producing bacterium or the parent strain for inducing the same include mutant strains of Corynebacterium bacteria having resistance to amino acid analogs and the like. Examples of such strains include, for example, strains having L-histidine, L-proline, L-threonine, L-isoleucine, L-methionine, or L-tryptophan auxotrophy in addition to 2-thiazolealanine resistance (Japanese Patent Laid-Open No. 54-44096); strains having resistance to ketomalonic acid, fluoromalonic acid, or monofluoroacetic acid (Japanese Patent Laid-Open No. 57-18989); strains having resistance to argininol (Japanese Patent Publication No. 62-24075); strains having resistance to X-guanidine (X is an aliphatic chain or its derivative) (Japanese Patent Laid-Open No. 2-186995); and strains having resistance to arginine hydroxamate and 6-azauracil (Japanese Patent Laid-Open No. 57-150381). Specific examples of Corynebacterium bacteria having the ability to produce L-arginine include the following strains. Corynebacterium glutamicum (Brevibacterium flavum) AJ11169 (FERM BP-6892) Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ12092 (FERM BP-6906) Corynebacterium glutamicum (Brevibacterium flavum) AJ11336 (FERM BP-6893) Corynebacterium glutamicum (Brevibacterium flavum) AJ11345 (FERM BP-6894) Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ12430 (FERM BP-2228)

[0057] <L-citrulline-producing bacterium and L-ornithine-producing bacterium> L-citrulline and L-ornithine are intermediates in the L-arginine biosynthesis pathway. Therefore, methods for conferring or enhancing the ability to produce L-citrulline and / or L-ornithine include, for example, modifying bacteria to increase the activity of one or more enzymes selected from the L-arginine biosynthesis enzymes. Such enzymes are not particularly limited, but for L-citrulline, they include N-acetylglutamate synthase (argA), N-acetylglutamate kinase (argB), N-acetylglutamyl phosphate reductase (argC), acetylornithine transaminase (argD), acetylornithine deacetylase (argE), ornithine carbamoyltransferase (argF, argI), ornithine acetyltransferase (argJ), and carbamoyl phosphate synthase (carAB). Furthermore, while there are no particular limitations on such enzymes, examples of enzymes for L-ornithine include N-acetylglutamate synthase (argA), N-acetylglutamate kinase (argB), N-acetylglutamyl phosphate reductase (argC), acetylornithine transaminase (argD), acetylornithine deacetylase (argE), and ornithine acetyltransferase (argJ).

[0058] Furthermore, L-citrulline-producing bacteria can be easily obtained, for example, from any L-arginine-producing bacteria by reducing the activity of argininosuccinate synthase encoded by the argG gene. Similarly, L-ornithine-producing bacteria can be easily obtained, for example, from any L-arginine-producing bacteria by reducing the activity of ornithine carbamoyltransferase encoded by both the argF and argI genes.

[0059] Furthermore, methods for conferring or enhancing L-amino acid production ability include, for example, modifying bacteria to increase their activity in effluxing L-amino acids from bacterial cells. This activity can be increased, for example, by increasing the expression of genes encoding proteins that efflux L-amino acids. Examples of genes encoding proteins that efflux various amino acids include the b2682 gene (ygaZ), the b2683 gene (ygaH), the b1242 gene (ychE), and the b3434 gene (yhgN) (Japanese Patent Publication No. 2002-300874).

[0060] Furthermore, methods for conferring or enhancing L-amino acid production capacity include, for example, modifying bacteria to increase the activity of proteins involved in glucose metabolism or energy metabolism.

[0061] 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), pyruvate carboxylase gene (pyc; WO99 / 18228, EP1092776A), phosphoglucomutase gene (pgm; WO03 / 04598), fructose diphosphate aldolase gene (pfkB, fbp; WO03 / 04664), trans aldolase gene (talB; WO03 / 008611), fumarase gene (fum; WO01 / 02545), non-PTS sucrose uptake gene (csc; EP1149911A), and sucrose assimilation gene (scrAB operon; U.S. Patent No. 7,179,623).

[0062] Genes encoding proteins involved in energy metabolism include the transhydrogenase gene (pntAB; U.S. Patent No. 5,830,716) and the cytochrome bo type oxidase gene (cyoB; EP1070376A).

[0063] Furthermore, as a method for conferring or enhancing the ability to produce useful substances such as L-amino acids, one example is to modify the bacteria so that the activity of phosphoketolase is increased (WO2006 / 016705). That is, the bacteria of the present invention may be modified so that the activity of phosphoketolase is increased. This method may be particularly effective for conferring or enhancing the ability to produce glutamate-based L-amino acids such as L-glutamic acid. Examples of phosphoketolases include D-xylulose-5-phosphate phosphoketolase and fructose-6-phosphate phosphoketolase. Either the activity of D-xylulose-5-phosphate phosphoketolase or the activity of fructose-6-phosphate phosphoketolase may be enhanced, or both may be enhanced.

[0064] D-xylulose-5-phosphate phosphoketolase activity refers to the activity of consuming phosphate to convert xylulose-5-phosphate into glyceraldehyde-3-phosphate and acetyl phosphate, releasing one molecule of H2O. This activity can be measured by the methods described in the literature by Goldberg, M. et al. (Methods Enzymol., 9, 515-520 (1966)) or L. Meile (J. Bacteriol. (2001) 183; 2929-2936). Examples of D-xylulose-5-phosphate phosphoketolase include D-xylulose-5-phosphate phosphoketolases from bacteria belonging to the genera Acetobacter, Bifidobacterium, Lactobacillus, Thiobacillus, Streptococcus, Methylococcus, Butyrivibrio, or Fibrobacter, and D-xylulose-5-phosphate phosphoketolases from yeasts belonging to the genera Candida, Rhodotorula, Rhodosporidium, Pichia, Yarrowia, Hansenula, Kluiveromyces, Saccharomyces, Trichosporon, or Wingea. Specific examples of D-xylulose-5-phosphate phosphoketolase and the genes encoding it are disclosed in WO2006 / 016705.

[0065] Furthermore, fructose-6-phosphate phosphoketolase activity refers to the activity of consuming phosphate to convert fructose-6-phosphate into erythrose-4-phosphate and acetyl phosphate, releasing one molecule of H2O. This activity can be measured by the methods described in the literature by Racker, E. (Methods Enzymol., 5, 276-280 (1962)) or L. Meile (J. Bacteriol. (2001) 183; 2929-2936). Examples of fructose-6-phosphate phosphoketolase include those from bacteria belonging to the genera Acetobacter, Bifidobacterium, Chlorobium, Brucella, Methylococcus, or Gardnerella, as well as yeasts belonging to genera such as Rhodotorula, Candida, and Saccharomyces. Specific examples of fructose-6-phosphate phosphoketolase and the gene encoding it are disclosed in WO2006 / 016705.

[0066] In some cases, both phosphoketolase activities may be maintained by a single enzyme (D-xylulose-5-phosphate / fructose-6-phosphate phosphoketolase).

[0067] The genes and proteins used in breeding L-amino acid-producing bacteria may each have the base sequence and amino acid sequence of known genes and proteins, such as those exemplified above. Furthermore, the genes and proteins used in breeding L-amino acid-producing bacteria may each be conserved variants of known genes and proteins, such as those exemplified above. Specifically, for example, the genes used in breeding L-amino 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. Regarding conserved variants of genes and proteins, the description of conserved variants of target genes and target proteins described later may be applied mutatis mutandis.

[0068] <1-2> Specific properties The bacteria of the present invention have been modified to possess specific properties. The bacteria of the present invention can be obtained by modifying bacteria that have the ability to produce L-amino acids to possess specific properties. Alternatively, the bacteria of the present invention can also be obtained by modifying bacteria to possess specific properties and then conferring or enhancing their L-amino acid production ability. In addition, the bacteria of the present invention may acquire L-amino acid production ability as a result of being modified to possess specific properties. In addition to being modified to possess specific properties, the bacteria of the present invention may appropriately possess, for example, the properties of L-amino acid producing bacteria as described above. The modifications for constructing the bacteria of the present invention can be carried out in any order.

[0069] By modifying bacteria to possess specific properties, it is possible to improve their L-amino acid production capacity, that is, to increase L-amino acid production by those bacteria. An example of "increased L-amino acid production" is an improvement (increase) in the accumulation of L-amino acids in the culture medium.

[0070] One specific property is a modification that increases the intracellular oxaloacetate (OAA) pool. "Increased intracellular oxaloacetate (OAA) pool" can be interpreted as an increase in the amount of oxaloacetate (OAA) accumulated within the cell.

[0071] Furthermore, a specific property is a modification that improves the availability of carbon sources via the phosphoketolase pathway. "Improved availability of carbon sources via the phosphoketolase pathway" can mean an increase in the flux of the phosphoketolase pathway (i.e., the metabolic rate of carbon sources via the phosphoketolase pathway) and / or a decrease in the accumulation of by-products that can be produced from carbon sources metabolized via the phosphoketolase pathway. Acetic acid is an example of a by-product. Acetic acid can accumulate, for example, due to an increase in the flux of the phosphoketolase pathway. Modifications that improve the availability of carbon sources via the phosphoketolase pathway include modifications that increase the flux of the phosphoketolase pathway and modifications that improve the assimilation of acetic acid.

[0072] Furthermore, specific properties include the following modifications (A) to (E): (A) Modifications that increase the activity of acetate kinase; (B) Modifications that increase the activity of fructose-1,6-bisphosphatase; (C) Modification that reduces pyruvate dehydrogenase activity; (D) Modifications that reduce the activity of aspartate transaminase; (E) Modification that reduces the activity of malic enzyme.

[0073] The modifications described in (D) and (E) above may be examples of modifications that increase the intracellular oxaloacetate (OAA) pool. The modifications described in (A), (B), and (C) above may be examples of modifications that improve the availability of carbon sources via the phosphoketolase pathway. Specifically, the modifications described in (A) and (C) above may be examples of modifications that improve the assimilation of acetic acid. Specifically, the modification described in (B) above may be an example of a modification that increases the flux of the phosphoketolase pathway.

[0074] The bacteria of the present invention may have one or more properties selected from the properties exemplified above.

[0075] The bacteria of the present invention may have, for example, modifications that increase the intracellular oxaloacetate (OAA) pool and / or modifications that improve the availability of carbon sources via the phosphoketolase pathway. The bacteria of the present invention may have, for example, at least modifications that improve the availability of carbon sources via the phosphoketolase pathway. Specifically, the bacteria of the present invention may have, for example, only the modification that improves the availability of carbon sources via the phosphoketolase pathway among the modifications that increase the intracellular oxaloacetate (OAA) pool and modifications that improve the availability of carbon sources via the phosphoketolase pathway, or a combination of the modification that improves the availability of carbon sources via the phosphoketolase pathway and the modification that increases the intracellular oxaloacetate (OAA) pool.

[0076] The bacteria of the present invention may have, for example, modifications that increase the intracellular oxaloacetate (OAA) pool, modifications that increase the flux of the phosphoketolase pathway, and / or modifications that improve acetic acid assimilation. The bacteria of the present invention may have, for example, at least modifications that improve acetic acid assimilation. Specifically, the bacteria of the present invention may have, for example, only the modification that improves acetic acid assimilation among the modifications that increase the intracellular oxaloacetate (OAA) pool, modifications that increase the flux of the phosphoketolase pathway, and modifications that improve acetic acid assimilation, or they may have a combination of modifications that improve acetic acid assimilation and modifications that increase the intracellular oxaloacetate (OAA) pool and / or modifications that increase the flux of the phosphoketolase pathway.

[0077] The bacteria of the present invention may have, for example, one or more modifications selected from the modifications (A) to (E) above, for example, one, two, three, four, or all five modifications. Specifically, the bacteria of the present invention may have, for example, (A), (B), (C), (D), (E), (A)+(B), (A)+(C), (A)+(D), (A)+(E), (B)+(C), (B)+(D), (B)+(E), (C)+(D), (C)+(E), (D)+(E), (A)+(B)+(C), (A)+(B)+(D), (A)+(B)+(E), (A)+(C)+(D), (A The bacteria of the present invention may have modifications of (A)+(D)+(E), (B)+(C)+(D), (B)+(C)+(E), (B)+(D)+(E), (C)+(D)+(E), (A)+(B)+(C)+(D), (A)+(B)+(C)+(E), (A)+(B)+(D)+(E), (A)+(C)+(D)+(E), (B)+(C)+(D)+(E), and (A)+(B)+(C)+(D)+(E). "Modification of (A)+(B)" means a combination of modification of (A) and modification of (B). This explanation can be applied mutatis mutandis to other combinations. The bacteria of the present invention may have, for example, at least the above modification of (A). In other words, the bacteria of the present invention may, specifically, have only the modification of (A) among the modifications of (A) to (E) above, or it may have a combination of the modification of (A) and one or more modifications selected from the modifications of (B) to (E), for example, one, two, three, or all four modifications. Furthermore, the bacteria of the present invention may have, for example, at least the modification of (B) above.

[0078] "Aspartate transaminase" may refer to a protein that has the activity to catalyze the amino group transfer reaction of aspartic acid and / or glutamic acid (e.g., EC 2.6.1.1). This activity is also called "aspartate transaminase activity." Specifically, aspartate transaminase activity may refer to the activity to catalyze the reaction that converts L-aspartic acid and α-ketoglutarate to oxaloacetate and / or L-glutamic acid, and / or the reverse reaction. Aspartate transaminase is also called "glutamic-oxaloacetic transaminase." The gene that encodes aspartate transaminase is also called the "aspartate transaminase gene." An example of an aspartate transaminase gene is the aspT gene. The nucleotide sequences of aspartate transaminase genes, such as the aspT gene, possessed by the modified bacteria, and the amino acid sequences of the aspartate transaminases encoded by them, such as the AspT protein, can be obtained, for example, from publicly available databases such as NCBI. The nucleotide sequence of the aspT gene (CGBL_0102840) of Corynebacterium glutamicum ATCC 13869 and the amino acid sequence of the protein encoded by the same gene are shown in Sequence ID No. 1 and No. 2, respectively.

[0079] The term "malic enzyme" can refer to a protein that has the activity to catalyze the oxidative decarboxylation of malic acid (for example, EC 1.1.1.38, EC 1.1.1.39, or EC 1.1.1.40). This activity is also called "malic enzyme activity." Specifically, malic enzyme activity may refer to the activity that catalyzes the reaction in which malic acid is decarboxylated to produce pyruvate in the presence of an electron acceptor. The electron acceptor is NAD. + naDP +Examples include: Malic enzymes only need to be able to utilize at least one electron acceptor. Malic enzymes may also have the activity to catalyze the decarboxylation of oxaloacetate (specifically, the reaction that converts oxaloacetate to pyruvate and carbon dioxide). Malic enzymes are also called "malate dehydrogenases." The gene that codes for a Malic enzyme is also called a "Malic enzyme gene." An example of a Malic enzyme gene is the malE gene. The nucleotide sequences of Malic enzyme genes such as the malE gene possessed by the modified bacteria, and the amino acid sequences of Malic enzymes such as the MalE protein encoded by them, can be obtained, for example, from public databases such as NCBI. The nucleotide sequence of the malE gene (CGBL_0129850) of Corynebacterium glutamicum ATCC 13869 and the amino acid sequence of the protein encoded by the same gene are shown in SEQ ID NOs. 3 and 4, respectively.

[0080] "Pyruvate dehydrogenase" can refer to a protein that has the activity to catalyze the oxidative decarboxylation of pyruvate (e.g., EC 1.2.5.1). This activity is also called "pyruvate dehydrogenase activity." Specifically, pyruvate dehydrogenase activity may refer to the activity that catalyzes the reaction in which pyruvate is decarboxylated to produce acetic acid in the presence of an electron acceptor. Examples of electron acceptors include quinones such as ubiquinone. Pyruvate dehydrogenase only needs to be able to utilize at least one electron acceptor. The gene that encodes pyruvate dehydrogenase is also called the "pyruvate dehydrogenase gene." An example of a pyruvate dehydrogenase gene is the poxB gene. The base sequences of pyruvate dehydrogenase genes such as the poxB gene possessed by the bacteria to be modified, and the amino acid sequences of pyruvate dehydrogenase such as the PoxB protein encoded by them, can be obtained from public databases such as NCBI. The nucleotide sequence of the poxB gene (CGBL_0125410) of Corynebacterium glutamicum ATCC 13869 and the amino acid sequence of the protein encoded by this gene are shown in Sequence IDs 5 and 6, respectively.

[0081] "Acetate kinase" can refer to a protein that has the activity to catalyze the phosphorylation reaction of acetic acid (e.g., EC 2.7.2.1). This activity is also called "acetate kinase activity." Specifically, acetate kinase activity may refer to the activity of catalyzing the reaction in which acetic acid is phosphorylated to produce acetyl phosphate in the presence of a phosphate group donor. ATP is an example of a phosphate group donor. Acetate kinase only needs to be able to utilize at least one phosphate group donor. The gene that codes for acetate kinase is also called the "acetate kinase gene." An example of an acetate kinase gene is the ack gene. Examples of acetate kinase genes such as the ack gene include genes from various organisms, such as Corynebacteria and bacteria belonging to the Enterobacteriaceae family. Specific examples of ack genes include the ack genes of Corynebacterium glutamicum and E. coli. The base sequences of acetate kinase genes such as the ack gene from various organisms and the amino acid sequences of acetate kinases such as the Ack protein encoded by them can be obtained from publicly available databases such as NCBI. The nucleotide sequence of the ack gene (CGBL_0126910) of Corynebacterium glutamicum ATCC 13869 and the amino acid sequence of the protein encoded by this gene are shown in SEQ ID NOs. 7 and 8, respectively. The nucleotide sequence of the ack gene of E. coli MG1655 and the amino acid sequence of the protein encoded by this gene are shown in SEQ ID NOs. 9 and 10, respectively.

[0082] "Fructose-1,6-bisphosphatase" can refer to a protein that has the activity to catalyze the dephosphorylation reaction of fructose-1,6-bisphosphate (e.g., EC 3.1.3.11). This activity is also called "fructose-1,6-bisphosphatase activity." Specifically, fructose-1,6-bisphosphatase activity may refer to the activity that catalyzes the reaction that converts fructose-1,6-bisphosphate to fructose-6-phosphate. The gene that encodes fructose-1,6-bisphosphatase is also called the "fructose-1,6-bisphosphatase gene." An example of a fructose-1,6-bisphosphatase gene is the glpX gene. Examples of fructose-1,6-bisphosphatase genes such as the glpX gene can be found in genes from various organisms, such as Corynebacteria and bacteria belonging to the Enterobacteriaceae family. Specific examples of glpX genes include the glpX genes of Corynebacterium glutamicum and E. coli. The nucleotide sequences of fructose-1,6-bisphosphatase genes, such as the glpX gene, of various organisms, and the amino acid sequences of the fructose-1,6-bisphosphatases such as the GlpX protein encoded by them can be obtained from publicly available databases such as NCBI. The nucleotide sequence of the glpX gene (NCgl0976) of Corynebacterium glutamicum ATCC 13032 and the amino acid sequence of the protein encoded by this gene are shown in SEQ ID NOs. 11 and 12, respectively. The nucleotide sequence of the glpX gene of E. coli MG1655 and the amino acid sequence of the protein encoded by this gene are shown in SEQ ID NOs. 13 and 14, respectively.

[0083] Methods for reducing protein activity will be described later. Protein activity can be reduced, for example, by decreasing the expression of the gene encoding the protein, or by disrupting the gene. Such methods for reducing protein activity can be used individually or in appropriate combinations.

[0084] Methods for increasing protein activity will be described later. Protein activity can be increased, for example, by increasing the expression of the gene encoding the protein. Gene expression can be increased, for example, by increasing the copy number of the gene or by modifying the gene expression regulatory sequence. Such methods for increasing protein activity can be used individually or in appropriate combinations.

[0085] Proteins whose activity is reduced or increased in specific properties are collectively called "target proteins." Genes that code for target proteins are collectively called "target genes."

[0086] The target gene may be, for example, a gene having the nucleotide sequence of the target gene exemplified above (for example, the nucleotide sequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, or 13). The target protein may be, for example, a protein having the amino acid sequence of the target protein exemplified above (for example, the amino acid sequence shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 14). Unless otherwise specified, the expression "having an (amino acid or nucleotide) sequence" means "containing the (amino acid or nucleotide) sequence" and also includes cases where "consisting of the (amino acid or nucleotide) sequence".

[0087] The target gene may be a variant of the target gene exemplified above (for example, a gene having the nucleotide sequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, or 13), as long as the original function is maintained. Similarly, the target protein may be a variant of the target protein exemplified above (for example, a protein having the amino acid sequence shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 14), as long as the original function is maintained. Such variants that maintain the original function are sometimes referred to as "conserved variants." The terms "aspT gene," "malE gene," "poxB gene," "ack gene," and "glpX gene" shall include the aspT gene, malE gene, poxB gene, ack gene, and glpX gene exemplified above, as well as their conserved variants, respectively. Similarly, the terms “AspT protein,” “MalE protein,” “PoxB protein,” “Ack protein,” and “GlpX protein” shall encompass the AspT protein, MalE protein, PoxB protein, Ack protein, and GlpX protein exemplified above, as well as their conserved variants. Conserved variants include, for example, homologs and artificially modified versions of the target genes and target proteins exemplified above.

[0088] "Maintaining the original function" means that a variant of a gene or protein has a function (e.g., activity or properties) that corresponds to the function (e.g., activity or properties) of the original gene or protein. For a gene, "maintaining the original function" means that the variant codes for a protein whose original function is maintained. In other words, for each target gene, "maintaining the original function" may mean that the variant codes for a protein that has the activity of each target protein: aspartate transaminase activity; malic enzyme activity; pyruvate dehydrogenase activity; acetate kinase activity; fructose-1,6-bisphosphatase activity. Furthermore, "maintaining the original function" for each target protein may mean that the protein variant possesses the activity of each target protein: aspartate transaminase activity for aspartate transaminase; maric enzyme activity for maric enzyme; pyruvate dehydrogenase activity for pyruvate dehydrogenase; acetate kinase activity for acetate kinase; and fructose-1,6-bisphosphatase activity for fructose-1,6-bisphosphatase.

[0089] Aspartate transaminase activity can be measured, for example, by incubating the enzyme with the corresponding substrate (e.g., L-aspartate and α-ketoglutarate) and measuring the enzyme- and substrate-dependent production of the corresponding products (e.g., oxaloacetate and L-glutamate).

[0090] Malic enzyme activity can be measured, for example, by incubating the enzyme with a corresponding substrate (e.g., malate) in the presence of an electron acceptor and measuring the enzyme- and substrate-dependent production of the corresponding product (e.g., pyruvate).

[0091] Pyruvate dehydrogenase activity can be measured, for example, by incubating the enzyme with a corresponding substrate (e.g., pyruvate) in the presence of an electron acceptor and measuring the enzyme and substrate-dependent production of the corresponding product (e.g., acetic acid).

[0092] Acetate kinase activity can be measured, for example, by incubating the enzyme with a corresponding substrate (e.g., acetic acid) in the presence of a phosphate group donor and measuring the enzyme and substrate-dependent production of the corresponding product (e.g., acetyl phosphate).

[0093] Fructose-1,6-bisphosphatase activity can be measured, for example, by incubating the enzyme with the corresponding substrate (e.g., fructose-1,6-bisphosphate) and measuring the enzyme and substrate-dependent production of the corresponding product (e.g., fructose-6-phosphate).

[0094] The following are examples of conservative variants.

[0095] Homologs of target genes or target proteins can be easily obtained from public databases, for example, by BLAST or FASTA searches using the nucleotide sequence of the target gene or the amino acid sequence of the target protein exemplified above as query sequences. Alternatively, homologs of target genes can be obtained, for example, by PCR using oligonucleotides prepared based on the nucleotide sequences of known target genes, with the chromosomes of various organisms as templates.

[0096] The target gene may be a gene encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added at one or several positions in the above amino acid sequence (for example, the amino acid sequence shown in SEQ ID NOs. 2, 4, 6, 8, 10, 12, or 14), as long as the original function is maintained. For example, the encoded protein may have its N-terminus and / or C-terminus elongated or shortened. The term "one or several" above 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.

[0097] The above substitutions, deletions, insertions, and / or additions of one or more amino acids are conservative mutations that maintain normal protein function. A typical example of a conservative mutation is a conservative substitution. A conservative substitution is a mutation in which the substitution site is between Phe, Trp, and Tyr if the substitution site is an aromatic amino acid; between Leu, Ile, and Val if the substitution site is a hydrophobic amino acid; between Gln and Asn if the substitution site is a polar amino acid; between Lys, Arg, and His if the substitution site is a basic amino acid; between Asp and Glu if the substitution site is an acidic amino acid; and between Ser and Thr if the amino acid has a hydroxyl group. Substitutions considered conservative include, specifically, substitutions from Ala to Ser or Thr, from Arg to Gln, His or Lys, from Asn to Glu, Gln, Lys, His or Asp, from Asp to Asn, Glu or Gln, from Cys to Ser or Ala, from Gln to Asn, Glu, Lys, His, Asp or Arg, from Glu to Gly, Asn, Gln, Lys or Asp, from Gly to Pro, from His to Asn, Lys, Gln, Arg or Tyr, and Il Examples of substitutions include e to Leu, Met, Val, or Phe; Leu to Ile, Met, Val, or Phe; Lys to Asn, Glu, Gln, His, or Arg; Met to Ile, Leu, Val, or Phe; Phe to Trp, Tyr, Met, Ile, or Leu; Ser to Thr or Ala; Thr to Ser or Ala; Trp to Phe or Tyr; Tyr to His, Phe, or Trp; and Val to Met, Ile, or Leu. Furthermore, such amino acid substitutions, deletions, insertions, or additions may also result from naturally occurring mutations (mutants or variants) based on individual differences or species differences in the organisms from which the genes originate.

[0098] Furthermore, the target gene may be a gene encoding a protein having an amino acid sequence that has, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identity with respect to the entire amino acid sequence, as long as the original function is maintained.

[0099] Furthermore, the target gene may also be a gene (e.g., DNA) that hybridizes under stringent conditions with a probe prepared from the above-mentioned base sequence (e.g., the base sequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, or 13), such as a complementary sequence to all or part of the above-mentioned base sequence, as long as the original function is maintained. "Stringent conditions" refers to conditions under which so-called specific hybrids are formed and nonspecific hybrids are not formed. For example, one example is a condition in which DNAs with high identity, for example, DNAs with 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identity, hybridize, and DNAs with lower identity do not hybridize. Alternatively, one example is a condition in which the DNA is washed once, preferably two to three times, at a salt concentration and temperature equivalent to the washing conditions for normal 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.

[0100] As described above, the probe used in the hybridization may be a part of the complementary sequence of the gene. Such probes can be prepared by PCR using oligonucleotides prepared based on known gene sequences as primers and a DNA fragment containing the gene as a template. For example, a DNA fragment approximately 300 bp in length can be used as a probe. When using a DNA fragment approximately 300 bp in length as a probe, the conditions for hybridization washing may be 50°C, 2×SSC, and 0.1% SDS.

[0101] Furthermore, since codon degeneracy differs depending on the host, the target gene may be one in which any codon is replaced with an equivalent codon. In other words, the target gene may be a variant of the target gene exemplified above due to the degeneracy of the genetic code. For example, the target gene may be modified to have the optimal codon depending on the codon usage frequency of the host being used.

[0102] Note that "identity" between amino acid sequences refers to the identity between amino acid sequences calculated using the default Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence=11, Extension=1; Compositional Adjustments: Conditional compositional score matrix adjustment) by blastp. Furthermore, "identity" between nucleotide sequences refers to the identity between nucleotide sequences calculated using the default Scoring Parameters (Match / Mismatch Scores=1,-2; Gap Costs=Linear) by blastn.

[0103] Furthermore, the above descriptions regarding conserved variants of genes and proteins can also be applied mutatis mutandis to any protein, such as L-amino acid biosynthesis enzymes, and the genes that encode them.

[0104] <1-3> Methods to increase protein activity The following describes methods for increasing protein activity.

[0105] "Increased protein activity" means that the activity of the protein increases compared to the unmodified strain. Specifically, "increased protein activity" means that the activity of the protein per cell increases compared to the unmodified strain. Here, "unmodified strain" refers to a control strain that has not been modified to increase the activity of the target protein. Examples of unmodified strains include wild-type strains and parental strains. Specifically, examples of unmodified strains include the type strains of each bacterial species. Also, specific examples of unmodified strains include the bacterial strains exemplified in the description of the bacteria. That is, in one embodiment, the protein activity may increase compared to the type strain (i.e., the type strain of the species to which the bacteria of the present invention belongs). In another embodiment, the protein activity may increase compared to C. glutamicum ATCC 13869 strain. In yet another embodiment, the protein activity may increase compared to C. glutamicum ATCC 13032 strain. In another embodiment, the protein activity may be increased compared to C. glutamicum AJ12036 (FERM BP-734). In yet another embodiment, the protein activity may be increased compared to C. glutamicum YDK010 strain. Note that "increased protein activity" is also referred to as "enhanced protein activity." More specifically, "increased protein activity" may mean that the number of molecules of the protein per cell is increased and / or the function per molecule of the protein is increased compared to the unmodified strain. That is, "activity" in the context of "increased protein activity" is not limited to the catalytic activity of the protein, but may also mean the amount of transcription (mRNA amount) or translation (amount of protein) of the gene encoding the protein. "Number of molecules of the protein per cell" may mean the average value of the number of molecules of the protein per cell. Furthermore, "increased protein activity" encompasses not only increasing the activity of the target protein in strains that already possess that activity, but also conferring the activity of the target protein to strains that do not originally possess that activity.Furthermore, as long as the resulting increase in protein activity is achieved, the activity of the host's naturally occurring target protein may be reduced or eliminated, and the activity of a suitable target protein may be conferred.

[0106] The degree of increase in protein activity is not particularly limited, as long as the protein activity is increased compared to the unmodified strain. For example, the protein activity may increase by 1.5 times or more, 2 times or more, or 3 times or more compared to the unmodified strain. Also, if the unmodified strain does not have the activity of the target protein, it is sufficient that the protein is produced by introducing the gene encoding the protein, for example, the protein may be produced to a degree that allows its activity to be measured.

[0107] Modifications that increase the activity of a protein can be achieved, for example, by increasing the expression of the gene encoding that protein. "Increased gene expression" means that the expression of the gene increases compared to unmodified strains such as wild-type or parental strains. Specifically, "increased gene expression" means that the amount of gene expressed per cell increases compared to unmodified strains. "Genes expressed per cell" may mean the average amount of gene expression per cell. More specifically, "increased gene expression" may mean an increase in the amount of gene transcription (mRNA) and / or an increase in the amount of gene translation (protein). Note that "increased gene expression" is also called "enhanced gene expression." Gene expression may increase by, for example, 1.5 times or more, 2 times or more, or 3 times or more compared to unmodified strains. Furthermore, "increased gene expression" includes not only increasing the expression level of the target gene in strains that originally express the target gene, but also expressing the target gene in strains that did not originally express the target gene. In other words, "increased gene expression" may mean, for example, introducing the target gene into a bacterial strain that does not possess the target gene and causing it to express.

[0108] Increased gene expression can be achieved, for example, by increasing the copy number of a gene.

[0109] An increase in the copy number of a gene can be achieved by introducing the gene into the host chromosome. Gene introduction into chromosomes can be done, for example, using homologous recombination (Miller, JH Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Examples of gene introduction methods using homologous recombination include methods using linear DNA such as Red-driven integration (Datsenko, K. A, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97:6640-6645 (2000)), methods using plasmids containing temperature-sensitive origins of replication, methods using conjugate-transferable plasmids, methods using suicide vectors that do not have origins of replication that function in the host, and phage-based transduction methods. Specifically, the host can be transformed with recombinant DNA containing the target gene, and the gene can be introduced into the host chromosome by inducing homologous recombination with the target site on the host chromosome. The structure of recombinant DNA used for homologous recombination is not particularly limited as long as homologous recombination occurs in the desired manner. For example, a target site can be replaced with the gene by transforming a host with linear DNA containing a target gene, wherein homologous base sequences are present at both ends of the gene, upstream and downstream of the target site on the chromosome. Homologous recombination can then occur upstream and downstream of the target site. The recombinant DNA used for homologous recombination may include a marker gene for selecting the transformant. Only one copy of the gene may be introduced, or two or more copies may be introduced. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination targeting a base sequence that has multiple copies on the chromosome. Examples of base sequences that have multiple copies on the chromosome include repetitive DNA sequences and inverted repeats present at both ends of transposons. Alternatively, homologous recombination may be performed targeting appropriate base sequences on the chromosome, such as genes that are not necessary for the production of the target substance.Furthermore, genes can also be randomly introduced onto chromosomes using transposons or Mini-Mu (Japanese Patent Publication No. 2-109985, US5,882,888, EP805867B1). It should be noted that this method of chromosome modification using homologous recombination is not limited to the introduction of target genes, but can be used for any modification of chromosomes, such as the modification of expression regulatory sequences.

[0110] The introduction of a target gene onto a chromosome can be confirmed by Southern hybridization using a probe with a sequence complementary to all or part of the gene, or by PCR using primers created based on the gene's sequence.

[0111] Furthermore, increasing the copy number of a gene can also be achieved by introducing a vector containing the gene into a host. For example, the copy number of the gene can be increased by constructing an expression vector for the gene by ligating a DNA fragment containing the target gene with a vector that functions in the host, and then transforming the host with this expression vector. The DNA fragment containing the target gene can be obtained, for example, by PCR using the genomic DNA of a microorganism having the target gene as a template. As the vector, a vector capable of autonomous replication within the host cell can be used. The vector is preferably a multicopy vector. In addition, it is preferable that the vector has markers such as antibiotic resistance genes in order to select transformants. The vector may also be equipped with a promoter or terminator for expressing the inserted gene. The vector may be, for example, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a bacteriophage-derived vector, a cosmid, or a phagemid. Specifically, examples of vectors capable of autonomous replication in Corynebacteria include pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903 (1984)); plasmids containing drug resistance genes that are improved versions of these; pCRY30 (JP-A-3-210184); pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX (JP-A-2-72876, U.S. Patent No. 5,185,262); pCRY2 and pCRY3 (JP-A-1-191686); pAJ655, pAJ6 Examples include 11, and pAJ1844 (JP-A-58-192900); pCG1 (JP-A-57-134500); pCG2 (JP-A-58-35197); pCG4 and pCG11 (JP-A-57-183799); pVK7 (JP-A-10-215883); pVK9 (US2006-0141588); pVC7 (JP-A-9-070291); and pVS7 (WO2013 / 069634).

[0112] When introducing a gene, it is sufficient that the gene is held in the host in an expressible state. Specifically, the gene should be held in a state where it can be 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" means a promoter that has promoter activity in the host. The promoter may be a promoter derived from the host or a promoter derived from a different species. The promoter may be the specific promoter of the gene being introduced or a promoter of another gene. As a promoter, for example, a more potent promoter, such as those described later, may be used.

[0113] A terminator for transcription termination 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 of host origin or of heterologous origin. The terminator may be specific to the gene being introduced or it may be a terminator of another gene.

[0114] Vectors, promoters, and terminators usable in various microorganisms are described in detail in, for example, "Basic Microbiology Course 8: Genetic Engineering," Kyoritsu Shuppan, 1987, and these can be utilized.

[0115] Furthermore, when introducing two or more genes, each gene only needs to be retained in the host in an expressible state. For example, each gene may be retained entirely on a single expression vector, or entirely on a chromosome. Alternatively, each gene may be retained separately on multiple expression vectors, or separately on one or more expression vectors and on a chromosome. Alternatively, two or more genes may be introduced as an operon. 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 constituting a single protein complex (e.g., an enzyme complex), and combinations thereof.

[0116] The introduced gene is not particularly limited as long as it codes for a protein that functions in the host. The introduced gene may be of host origin or of heterologous origin. The introduced gene can be obtained, for example, by PCR using primers designed based on the gene's base sequence, and using the genomic DNA of an organism containing the gene or a plasmid containing the gene as a template. Alternatively, the introduced gene may be totally synthesized based on the gene's base sequence (Gene, 60(1), 115-127 (1987)). The obtained gene can be used as is or modified as appropriate. That is, variants can be obtained by modifying the gene. Gene modification can be carried out by known methods. For example, site-directed mutagenesis can be used to introduce a desired mutation at a desired site in the DNA. That is, for example, site-directed mutagenesis can be used to modify the coding region of a gene so that the encoded protein includes substitution, deletion, insertion, and / or addition of amino acid residues at a specific site. Site-directed mutagenesis methods include those using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and those using phages (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)). Alternatively, gene variants may be totally synthesized.

[0117] Furthermore, when a protein functions as a complex composed of multiple subunits, all of these subunits may be modified, or only some of them may be modified, as long as the protein's activity increases as a result. That is, for example, when increasing protein activity by increasing gene expression, the expression of all of the genes encoding those subunits may be enhanced, or only some of them may be enhanced. Usually, it is preferable to enhance the expression of all of the genes encoding those subunits. Also, each subunit constituting the complex may originate from one organism, or from two or more different organisms, as long as the complex has the function of the target protein. That is, for example, a gene from the same organism encoding multiple subunits may be introduced into the host, or genes from different organisms may be introduced into the host.

[0118] Furthermore, increased gene expression can be achieved by improving the transcription efficiency of the gene. Increased gene expression can also be achieved by improving the translation efficiency of the gene. Improvements in gene transcription and translation efficiency can be achieved, for example, by modifying gene expression regulatory sequences. "Generic expression sequences" are a general term for regions that influence gene expression. Examples of gene expression regulatory sequences include promoters, Shine-Dalgano (SD) sequences (also called ribosome-binding sites (RBS)), and the spacer region between the RBS and the start codon. Gene expression regulatory sequences can be determined using promoter search vectors or gene analysis software such as GENETYX. Modification of these gene expression regulatory sequences can be performed, for example, by methods using temperature-sensitive vectors or by the Red-driven integration method (WO2005 / 010175).

[0119] Improving gene transcription efficiency can be achieved, for example, by replacing the promoter of a gene on a chromosome with a stronger promoter. A "stronger promoter" refers to a promoter that improves gene transcription compared to the originally present wild-type promoter. Examples of stronger promoters available in Corynebacteria include the artificially modified P54-6 promoter (Appl. Microbiol. Biotechnol., 53, 674-679(2000)), the pta, aceA, aceB, adh, and amyE promoters that can be induced in Corynebacteria with acetic acid, ethanol, pyruvate, etc., and the cspB, SOD, and tuf(EF-Tu) promoters, which are strong promoters with high expression levels in Corynebacteria (Journal of Biotechnology 104 (2003) 311-323, Appl Environ Microbiol. 2005 Dec;71(12):8587-96.), the lac promoter, the tac promoter, and the trc promoter. Furthermore, to obtain more powerful promoters, highly active versions of conventional promoters can be obtained by using various reporter genes. For example, promoter activity can be increased by bringing the -35 and -10 regions within the promoter region closer to the consensus sequence (International Publication No. 00 / 18935). Examples of highly active promoters include various tac-like promoters (Katashkina JI et al. Russian Federation Patent application 2006134574) and the pnlp8 promoter (WO2010 / 027045). Methods for evaluating promoter strength and examples of powerful promoters are described in Goldstein et al.'s paper (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)), etc.

[0120] Improving gene translation efficiency can be achieved, for example, by replacing the Shine-Dalgano (SD) sequence (also called the ribosome binding site (RBS)) of a gene on a chromosome with a stronger SD sequence. A "stronger SD sequence" refers to an SD sequence that improves mRNA translation compared to the originally present wild-type SD sequence. An example of a stronger SD sequence is the RBS of gene 10 from phage T7 (Olins PO et al, Gene, 1988, 73, 227-235). Furthermore, it is known that substitutions, insertions, or deletions of a few nucleotides in the spacer region between the RBS and the start codon, particularly the sequence immediately upstream of the start codon (5'-UTR), have a significant impact on mRNA stability and translation efficiency, and modifying these regions can also improve gene translation efficiency.

[0121] Improving the translation efficiency of genes can also be achieved, for example, by modifying codons. For instance, replacing rare codons in a gene with more frequently used synonymous codons can improve the translation efficiency of a gene. That is, the introduced gene may be modified to have optimal codons according to the codon usage frequency of the host organism. Codon substitution can be performed, for example, by site-directed mutagenesis. Alternatively, a gene fragment with substituted codons may be totally synthesized. The codon usage frequencies in various organisms are disclosed in the "Codon Usage Database" (http: / / www.kazusa.or.jp / codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000)).

[0122] Furthermore, increased gene expression can also be achieved by amplifying regulators that increase gene expression, or by deleting or weakening regulators that decrease gene expression.

[0123] The methods for increasing gene expression described above may be used individually or in any combination.

[0124] Furthermore, modifications that increase protein activity can also be achieved, for example, by enhancing the specific activity of the protein. Enhancement of specific activity includes desensitization to feedback inhibition. Proteins with enhanced specific activity can be obtained, for example, by searching for various organisms. Alternatively, a highly active form may be obtained by introducing mutations into a conventional protein. The introduced mutation may be, for example, the substitution, deletion, insertion, and / or addition of one or more amino acids at one or more positions in the protein. Mutation can be introduced, for example, by site-directed mutagenesis as described above. Alternatively, mutation can be introduced, for example, by mutagenesis treatment. Mutagenesis treatments include irradiation with X-rays, irradiation with ultraviolet light, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS). Alternatively, random mutations may be induced by directly treating DNA with hydroxylamine in vitro. The enhancement of specific activity may be used alone, or it may be used in any combination with the gene expression enhancement methods described above.

[0125] The transformation method is not particularly limited, and conventionally known methods can be used. Examples of methods for transforming Corynebacteria include the protoplast method (Gene, 39, 281-286 (1985)), the electroporation method (Bio / Technology, 7, 1067-1070 (1989)), and the electrical pulse method (Japanese Patent Publication No. Hei 2-207791).

[0126] The increase in protein activity can be confirmed by measuring the activity of that protein.

[0127] Increased protein activity can also be confirmed by observing an increase in the expression of the gene encoding that protein. Increased gene expression can be confirmed by observing an increase in the transcription level of that gene or by observing an increase in the amount of protein expressed by that gene.

[0128] An increase in gene transcription can be confirmed by comparing the amount of mRNA transcribed from the gene with that of an unmodified strain such as the wild-type or parental strain. Methods for evaluating mRNA levels include Northern hybridization, RT-PCR, microarrays, and RNA-seq (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of mRNA (e.g., number of molecules per cell) may increase by, for example, 1.5 times or more, 2 times or more, or 3 times or more compared to an unmodified strain.

[0129] The increase in protein levels can be confirmed by Western blotting using antibodies (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The protein level (e.g., number of molecules per cell) may increase by, for example, 1.5 times, 2 times, or 3 times compared to the unmodified strain.

[0130] The methods described above for increasing protein activity can be used to enhance the activity of any protein or to enhance the expression of any gene.

[0131] <1-4> Methods to reduce protein activity The following describes methods for reducing protein activity.

[0132] "Decreased protein activity" means that the activity of the protein is reduced compared to the unmodified strain. Specifically, "decreased protein activity" means that the activity of the protein per cell is reduced compared to the unmodified strain. "Unmodified strain" here refers to a control strain that has not been modified to reduce the activity of the target protein. Examples of unmodified strains include wild-type strains and parental strains. Specifically, examples of unmodified strains include the type strains of each bacterial species. Also, specific examples of unmodified strains include the bacterial strains exemplified in the description of the bacteria. That is, in one embodiment, the protein activity may be reduced compared to the type strain (i.e., the type strain of the species to which the bacteria of the present invention belongs). In another embodiment, the protein activity may be reduced compared to C. glutamicum ATCC 13869 strain. In another embodiment, the protein activity may be reduced compared to C. glutamicum ATCC 13032 strain. In another embodiment, the protein activity may be reduced compared to C. glutamicum AJ12036 (FERM BP-734). In another embodiment, the protein activity may be reduced compared to the C. glutamicum YDK010 strain. Note that "reduced protein activity" includes cases where the protein activity is completely absent. More specifically, "reduced protein activity" may mean that the number of molecules of the protein per cell is reduced, and / or the function per molecule of the protein is reduced, compared to the unmodified strain. That is, "activity" in the context of "reduced protein activity" is not limited to the catalytic activity of the protein, but may also mean the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. "Number of molecules of the protein per cell" may mean the average number of molecules of the protein per cell. Note that "reduced number of molecules of the protein per cell" includes cases where the protein is completely absent. Also, "reduced function per molecule of the protein" includes cases where the function per molecule of the protein is completely absent.The degree of reduction in protein activity is not particularly limited, as long as the protein activity is reduced compared to the unmodified strain. For example, the protein activity may be reduced to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of the unmodified strain.

[0133] Modifications that reduce protein activity can be achieved, for example, by reducing the expression of the gene encoding the protein. "Reduced gene expression" means that the expression of the gene is lower compared to unmodified strains such as the wild type or parental strain. Specifically, "reduced gene expression" means that the amount of gene expressed per cell is lower compared to unmodified strains. "Genes expressed per cell" may mean the average amount of gene expression per cell. More specifically, "reduced gene expression" may mean that the amount of gene transcription (mRNA) is reduced, and / or the amount of gene translation (protein) is reduced. "Reduced gene expression" also includes cases where the gene is not expressed at all. Note that "reduced gene expression" is also called "weakening of gene expression." Gene expression may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to unmodified strains.

[0134] A decrease in gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination of both. A decrease in gene expression can be achieved, for example, by modifying the gene's promoter, the Shine-Dalgano (SD) sequence (also called the ribosome binding site (RBS)), or the spacer region between the RBS and the start codon. When modifying the gene expression regulatory sequence, preferably one or more bases, more preferably two or more bases, and particularly preferably three or more bases are modified. A decrease in gene transcription efficiency can be achieved, for example, by replacing the gene promoter on the chromosome with a weaker promoter. A "weaker promoter" means a promoter that weakens gene transcription compared to the originally present wild-type promoter. An example of a weaker promoter is an inductive promoter. That is, an inductive promoter can function as a weaker promoter under non-inductive conditions (for example, in the absence of an inductive substance). Alternatively, part or all of the gene expression regulatory sequence may be deleted (deleted). Furthermore, a decrease in gene expression can also be achieved, for example, by manipulating factors involved in gene expression regulation. Factors involved in gene expression regulation include small molecules (inducers, inhibitors, etc.), proteins (transcription factors, etc.), and nucleic acids (siRNA, etc.) involved in transcription and translation control. Furthermore, a decrease in gene expression can be achieved, for example, by introducing mutations in the gene's coding region that reduce gene expression. For instance, gene expression can be reduced by replacing a codon in the gene's coding region with a synonymous codon that is used less frequently in the host. Additionally, gene expression itself can be reduced by disrupting the gene, as described later.

[0135] Furthermore, modifications that reduce protein activity can be achieved, for example, by disrupting the gene that codes for the protein. "Disruption of a gene" means that the gene is modified so as not to produce a normally functioning protein. "Failure to produce a normally functioning protein" includes cases where no protein is produced at all from the gene, or where the gene produces a protein with reduced or lost per-molecule function (e.g., activity or properties).

[0136] Gene disruption can be achieved, for example, by deleting (removing) a gene on a chromosome. "Genetic deletion" refers to the deletion of part or all of the gene's coding region. Furthermore, the entire gene may be deleted, including the sequences before and after the gene's coding region on the chromosome. The sequences before and after the gene's coding region may include, for example, gene expression regulatory sequences. The region to be deleted can be any region, such as the N-terminal region (the region that codes for the N-terminus of the protein), the internal region, or the C-terminal region (the region that codes for the C-terminus of the protein), as long as a reduction in protein activity is achieved. Generally, a longer deleted region ensures more reliable gene inactivation. The deleted region may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the total length of the gene's coding region. It is also preferable that the sequences before and after the deleted region do not have matching reading frames. A mismatch in reading frames can cause frame shifts downstream of the region to be deleted.

[0137] Furthermore, gene disruption can also be achieved, for example, by introducing amino acid substitutions (missense mutations), stop codons (nonsense mutations), or the addition or deletion of one or two bases (frameshift mutations) into the coding region of a gene on a chromosome (Journal of Biological Chemistry 272:8611-8617 (1997), Proceedings of the National Academy of Sciences, USA 95 5511-5515 (1998), Journal of Biological Chemistry 26 116, 20833-20839 (1991)).

[0138] Furthermore, gene disruption can also be achieved, for example, by inserting another base sequence into the coding region of a gene on a chromosome. The insertion site can be any region of the gene, but a longer base sequence ensures more reliable gene inactivation. It is also preferable that the sequences before and after the insertion site do not have matching reading frames. Mismatch in reading frames can cause a frameshift downstream of the insertion site. Other base sequences are not particularly limited as long as they reduce or eliminate the activity of the encoded protein, but examples include marker genes such as antibiotic resistance genes and genes useful for the production of target substances.

[0139] Gene disruption may be carried out in a manner that specifically results in the deletion (deletion) of the amino acid sequence of the encoded protein. In other words, modifications that reduce protein activity can be achieved, for example, by deleting the amino acid sequence (part or all of the amino acid sequence) of a protein, specifically by modifying the gene to encode a protein with a deleted amino acid sequence (part or all of the amino acid sequence). Note that "deletion of the amino acid sequence of a protein" refers to the deletion of part or all of the amino acid sequence of a protein. Furthermore, "deletion of the amino acid sequence of a protein" means that the original amino acid sequence is no longer present in the protein, and includes cases where the original amino acid sequence is changed to a different amino acid sequence. That is, for example, a region that has been changed to a different amino acid sequence by frameshift may be considered a deleted region. Typically, the total length of a protein is shortened by the deletion of the amino acid sequence of a protein, but the total length of the protein may not change or may even be lengthened. For example, by deleting part or all of the coding region of a gene, the region encoded by the deleted region can be deleted in the amino acid sequence of the encoded protein. Furthermore, for example, by introducing a stop codon into the coding region of a gene, the region encoded by the region downstream of the introduction site in the amino acid sequence of the encoded protein can be deleted. Also, for example, by frameshifting the coding region of a gene, the region encoded by the frameshift site can be deleted. The location and length of the region to be deleted in amino acid sequence deletions can be described by analogy to the explanation of the location and length of the region to be deleted in gene deletions.

[0140] Modifying a gene on a chromosome as described above can be achieved, for example, by creating a disruption gene that is modified not to produce a normally functioning protein, transforming the host with recombinant DNA containing the disruption gene, and inducing homologous recombination between the disruption gene and the wild-type gene on the chromosome, thereby replacing the wild-type gene on the chromosome with the disruption gene. In this case, it is easier to manipulate if the recombinant DNA contains marker genes according to the host's nutritional requirements and other traits. Examples of disruption genes include genes in which part or all of the coding region is deleted, genes with missense mutations introduced, genes with nonsense mutations introduced, genes with frameshift mutations introduced, and genes with insertion sequences such as transposons or marker genes inserted. Proteins encoded by disruption genes, even if produced, will have a different three-dimensional structure from wild-type proteins and will have reduced or lost function. The structure of the recombinant DNA used for homologous recombination is not particularly limited as long as homologous recombination occurs in the desired manner. For example, by transforming a host with linear DNA containing a disruptive gene, which has the upstream and downstream sequences of the wild-type gene on the chromosome at both ends, homologous recombination can be induced upstream and downstream of the wild-type gene, thereby replacing the wild-type gene with the disruptive gene.Gene disruption by gene substitution using homologous recombination is already well-established and includes methods using linear DNA such as "Red-driven integration" (Datsenko, K. A, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97:6640-6645 (2000)), a method combining Red-driven integration with a lambda phage-derived excision system (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002)) (see WO2005 / 010175), methods using plasmids containing temperature-sensitive origins of replication, methods using conjugate-transferable plasmids, and methods using suicide vectors that do not have origins of replication that function in the host (U.S. Patent No. 6303383, Japanese Patent Publication No. 05-007491). Furthermore, this type of chromosome modification technique using homologous recombination is not limited to disrupting target genes, but can be used for any arbitrary modification of chromosomes, such as altering gene expression regulatory sequences.

[0141] Furthermore, modifications that reduce protein activity may be carried out, for example, by mutagenesis. Mutagenesis methods include irradiation with X-rays, irradiation with ultraviolet light, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0142] Furthermore, if a protein functions as a complex composed of multiple subunits, all of these subunits may be modified, or only some of them may be modified, as long as the resulting decrease in protein activity. That is, for example, all of the genes encoding those subunits may be destroyed, or only some of them may be destroyed. Also, if a protein contains multiple isozymes, the activity of all of the isozymes may be reduced, or only some of them may be reduced, as long as the resulting decrease in protein activity. That is, for example, all of the genes encoding those isozymes may be destroyed, or only some of them may be destroyed.

[0143] The methods described above for reducing protein activity may be used individually or in any combination.

[0144] A decrease in protein activity can be confirmed by measuring the activity of that protein.

[0145] A decrease in protein activity can also be confirmed by observing a decrease in the expression of the gene encoding that protein. A decrease in gene expression can be confirmed by observing a decrease in the transcription level of that gene or by observing a decrease in the amount of protein expressed by that gene.

[0146] The decrease in gene transcription can be confirmed by comparing the amount of mRNA transcribed from the gene with that of an unmodified strain. Methods for evaluating mRNA levels include Northern hybridization, RT-PCR, microarrays, and RNA-seq (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The mRNA level (e.g., number of molecules per cell) may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of an unmodified strain.

[0147] The decrease in protein levels can be confirmed by Western blotting using antibodies (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The protein level (e.g., number of molecules per cell) may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of the unmodified strain.

[0148] The disruption of a gene can be confirmed by determining the base sequence, restriction enzyme map, or full length of the gene, depending on the method used for disruption.

[0149] The methods described above for reducing the activity of proteins can be used to reduce the activity of any protein or the expression of any gene.

[0150] <2> The present invention provides a method for producing L-amino acids. The present invention provides a method for producing L-amino acids, comprising culturing the bacteria of the present invention in a culture medium, accumulating L-amino acids in the culture medium and / or within the bacterial cells, and collecting the L-amino acids from the culture medium and / or the bacterial cells. The L-amino acids are as described above. In the present invention, one type of L-amino acid may be produced, or two or more types of L-amino acids may be produced.

[0151] The culture medium used is not particularly limited, as long as it allows the bacteria of the present invention to grow and produces the target L-amino acid. For example, a conventional culture medium used for culturing bacteria such as Corynebacteria can be used. For example, a culture medium containing, as needed, a carbon source, nitrogen source, phosphate source, sulfur source, and other components selected from various organic and inorganic components can be used. The types and concentrations of culture medium components may be appropriately set according to various conditions, such as the type of bacteria used.

[0152] Specific examples of carbon sources include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, 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. Sugars are particularly suitable as carbon sources. More specifically, glucose and fructose are suitable as carbon sources. Sugars such as glucose and fructose may be used alone or in combination with other carbon sources. For example, sugars with fructose as a constituent sugar may be used as a carbon source. Examples of sugars with fructose as a constituent sugar include fructose, sucrose, and fructooligosaccharides. Sugars with fructose as a constituent sugar may be used alone or in combination with other carbon sources. Plant-derived raw materials can be suitably used as carbon sources. Examples of plants include corn, rice, wheat, soybeans, sugarcane, beets, and cotton. Examples of plant-derived raw materials include organs such as roots, stems, trunks, branches, leaves, flowers, and seeds, plant bodies containing these organs, and decomposition products of these plant organs. The form in which plant-derived raw materials are used is not particularly limited and can be used in any form, such as unprocessed products, juices, pulverized products, or refined products. In addition, pentose sugars such as xylose, hexose sugars such as glucose, or mixtures thereof can be obtained and used, for example, from plant biomass. Specifically, these sugars can be obtained by subjecting plant biomass to treatments such as steam treatment, concentrated acid hydrolysis, dilute acid hydrolysis, hydrolysis with enzymes such as cellulase, or alkaline treatment. Since hemicellulose is generally more easily hydrolyzed than cellulose, hemicellulose in plant biomass may be hydrolyzed beforehand to release pentose sugars, and then cellulose may be hydrolyzed to produce hexose sugars. Furthermore, xylose may be supplied, for example, by providing the bacteria of the present invention with a conversion pathway from hexoses such as glucose to xylose, and converting from hexoses. As a carbon source, one carbon source may be used, or two or more carbon sources may be used in combination.

[0153] Specific examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; peptone; organic nitrogen sources such as yeast extract, meat extract, and soy protein hydrolysate; ammonia; and urea. Ammonia gas or ammonia water used for pH adjustment may also be used as a nitrogen source. One nitrogen source may be used, or two or more nitrogen sources may be used in combination.

[0154] Examples of phosphate sources include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. One phosphate source may be used, or two or more phosphate sources may be used in combination.

[0155] 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. A single sulfur source may be used, or a combination of two or more sulfur sources may be used.

[0156] Other various organic and inorganic components include, specifically, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components containing these, such as peptone, casamino acid, yeast extract, and soy protein hydrolysate. These other organic and inorganic components may be used individually, or in combination of two or more components.

[0157] Furthermore, when using nutrient-dependent mutant strains that require amino acids or other nutrients for growth, it is preferable to supplement the culture medium with the necessary nutrients.

[0158] Furthermore, it is preferable to limit the amount of biotin in the culture medium or to add a surfactant or penicillin to the culture medium.

[0159] The culture conditions are not particularly limited, as long as the bacteria of the present invention can grow and the target L-amino acid can be produced. Culture can be carried out under the usual conditions used for culturing bacteria such as Corynebacteria. The culture conditions may be set appropriately depending on various conditions such as the type of bacteria used.

[0160] Culturing can be carried out using a liquid culture medium. During culturing, the bacteria of the present invention may be cultured on a solid medium such as agar medium and directly inoculated into the liquid culture medium, or the bacteria of the present invention may be cultured as a seed culture on a liquid medium and then inoculated into the liquid culture medium for the main culture. In other words, culturing may be carried out separately as a seed culture and the 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 bacteria of the present invention contained in the culture medium at the start of culturing is not particularly limited. The main culture may be carried out, for example, by inoculating the culture medium for the main culture with 1 to 50% (v / v) of the seed culture solution.

[0161] Culture can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. The culture medium used at the start of culture is also called the "initial culture medium." The culture medium supplied to the culture system (fermenter) in fed-batch or continuous culture is also called the "fed-batch medium." The act of supplying the fed-batch medium to the culture system in fed-batch or continuous culture is also called "fed-batch." When culture is carried out separately as seed culture and main culture, for example, both seed culture and main culture may be carried out by batch culture. Alternatively, for example, seed culture may be carried out by batch culture and main culture by fed-batch or continuous culture.

[0162] In the present invention, each culture medium component may be contained in the initial medium, the fed-batch medium, or both. The types of components contained in the initial medium may be the same as, or different from, the types of components contained in the fed-batch medium. Furthermore, the concentrations of each component contained in the initial medium may be the same as, or different from, the concentrations of each component contained in the fed-batch medium. In addition, two or more types of fed-batch media with different types and / or concentrations of components may be used. For example, if multiple feedings are performed intermittently, the types and / or concentrations of components contained in each feeding medium may be the same or different.

[0163] The concentration of the carbon source in the culture medium is not particularly limited, as long as the bacteria of the present invention can grow and L-amino acids can be produced. The concentration of the carbon source in the culture medium may be as high as possible, for example, as long as the production of L-amino acids is not inhibited. The concentration of the carbon source in the culture medium may be, for example, 1 to 30 w / v%, preferably 3 to 10 w / v%, as the initial concentration (concentration in the initial culture medium). In addition, additional carbon sources may be added to the culture medium as appropriate. For example, additional carbon sources may be added to the culture medium in accordance with the consumption of carbon sources as fermentation progresses.

[0164] Culturing can be carried out, for example, under aerobic conditions. Aerobic conditions mean that the dissolved oxygen concentration in the liquid medium is 0.33 ppm or higher, which is the detection limit of the oxygen membrane electrode, and preferably 1.5 ppm or higher. The oxygen concentration may be controlled, for example, to 5-50%, preferably about 10%, relative to the saturated oxygen concentration. Culturing under aerobic conditions can be carried out by aeration culture, shaking culture, stirring culture, or a combination thereof. The pH of the medium may be, for example, pH 3-10, preferably pH 4.0-9.5. The pH of the medium can be adjusted as needed during cultivation. The pH of the medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide. The culture temperature may be, for example, 20-40°C, preferably 25-37°C. The culture period may be, for example, 10-120 hours. The culture may be continued, for example, until the carbon source in the culture medium is consumed or until the activity of the bacteria of the present invention is lost. By culturing the bacteria of the present invention under such conditions, L-amino acids accumulate in the culture medium and / or within the bacterial cells.

[0165] Furthermore, when producing L-glutamic acid, it is also possible to culture it while precipitating L-glutamic acid in the medium using a liquid medium adjusted to conditions for L-glutamic acid precipitation. Examples of conditions for L-glutamic acid precipitation include 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). In addition, when using a liquid medium adjusted to conditions for L-glutamic acid precipitation, crystallization can be performed more efficiently by adding pantothenic acid to the medium (WO2004 / 111258). Furthermore, when using a liquid medium adjusted to conditions for L-glutamic acid precipitation, crystallization can be performed more efficiently by adding L-glutamic acid crystals to the medium as seed crystals (EP1233069A). Furthermore, when using a liquid culture medium adjusted to conditions for L-glutamic acid precipitation, crystallization can be performed more efficiently by adding L-glutamic acid crystals and L-lysine crystals to the medium as seed crystals (EP1624069A).

[0166] Furthermore, when producing basic amino acids, the culture process (fermentation process) may be carried out so that bicarbonate ions and / or carbonate ions become counterions for the basic amino acids. Such a form of fermentation is also called "carbonate fermentation." Carbonate fermentation allows for the fermentation and production of basic amino acids while reducing the amount of sulfate ions and / or chloride ions that were conventionally used as counterions for basic amino acids. Carbonate fermentation can be carried out as described, for example, in US2002-025564A, EP1813677A, and JP 2002-65287.

[0167] The fermentation liquid can be processed, for example, in a liquid cyclone. A liquid cyclone of a general shape with a cylindrical diameter of 10 to 110 mm, made of ceramic, stainless steel, or resin, can be used. The feed rate of the fermentation liquid to the liquid cyclone can be set, for example, according to the microbial cell concentration and L-amino acid concentration in the fermentation liquid. The feed rate of the fermentation liquid to the liquid cyclone may be, for example, 2 to 1200 L / min.

[0168] The formation of L-amino acids can be confirmed by known methods used for the detection or identification of compounds. Such methods include, for example, HPLC, LC / MS, GC / MS, and NMR. These methods can be used individually or in appropriate combinations.

[0169] L-amino acids can be recovered from fermentation broth using known methods used for the separation and purification of 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, JP-A-9-173792), and crystallization (WO2008 / 078448, WO2008 / 078646). These methods can be used individually or in appropriate combinations. If L-amino acids accumulate within the bacterial cells, for example, the bacterial cells can be crushed using ultrasound, and the L-amino acids can be recovered from the supernatant obtained by removing the bacterial cells by centrifugation using the ion exchange resin method. The recovered L-amino acids may be in the form of free amino acids, their salts, or mixtures thereof. Examples of salts include sulfates, hydrochlorides, carbonates, ammonium salts, sodium salts, and potassium salts. When producing L-glutamic acid, the recovered L-glutamic acid may be, specifically, free L-glutamic acid, sodium L-glutamate (e.g., monosodium L-glutamate; MSG), ammonium L-glutamate (e.g., monoammonium L-glutamate), or a mixture thereof. For example, sodium L-glutamate (MSG) can be obtained by crystallizing ammonium L-glutamate in the fermentation liquid with acid, and then adding equimolar sodium hydroxide to the crystals. Decolorization may be performed by adding activated carbon before or after crystallization (see "Industrial Crystallization of Sodium Glutamate," Journal of the Japan Society of Marine Science, Vol. 56, No. 5, Tetsuya Kawakita). Sodium L-glutamate crystals can be used, for example, as a flavor enhancer. Sodium L-glutamate crystals may also be mixed with nucleic acids such as sodium guanylate or sodium inosinate, which similarly have a umami flavor, and used as a seasoning.

[0170] Furthermore, if L-amino acids precipitate in the culture medium, they can be recovered by centrifugation or filtration. Alternatively, the L-amino acids precipitated in the culture medium may be isolated together with the L-amino acids dissolved in the culture medium after crystallization.

[0171] Furthermore, the recovered L-amino acids may contain components other than L-amino acids, such as bacterial cells, culture medium components, water, and metabolic by-products of bacteria. The L-amino acids may be purified to a desired degree. The purity of the recovered L-amino acids may be, for example, 50% (w / w) or higher, preferably 85% (w / w) or higher, and particularly preferably 95% (w / w) or higher (JP1214636B, USP5,431,933, USP4,956,471, USP4,777,051, USP4,946,654, USP5,840,358, USP6,238,714, US2005 / 0025878). [Examples]

[0172] The present invention will be described in more detail below with reference to non-limiting embodiments.

[0173] <1> Construction of a modified strain of Corynebacterium glutamicum <1-1> Construction of a vector for aspT gene deletion Using chromosomal DNA from C. glutamicum ATCC 13869 as a template, a DNA fragment of approximately 1 kbp upstream (5') and approximately 1 kbp downstream (3') of the aspT gene (CGBL_0102840), which encodes aspartate transaminase, is amplified by PCR using appropriately designed primers. The amplified DNA fragments are then inserted into the SmaI region of pBS5T (WO2006 / 057450) by infusion to obtain an aspT gene deletion vector.

[0174] <1-2> Construction of a vector for malE gene deletion Using chromosomal DNA from C. glutamicum ATCC 13869 as a template, a DNA fragment of approximately 1 kbp upstream (5') and approximately 1 kbp downstream (3') of the malE gene (CGBL_0129850), which encodes the malic enzyme, is amplified by PCR using appropriately designed primers. The amplified DNA fragments are then inserted into the SmaI site of pBS5T (WO2006 / 057450) by infusion to obtain a malE gene deletion vector.

[0175] <1-3> Construction of a vector for poxB gene deletion Using chromosomal DNA from C. glutamicum ATCC 13869 as a template, a DNA fragment of approximately 1 kbp upstream (5') and approximately 1 kbp downstream (3') of the poxB gene (CGBL_0125410), which encodes pyruvate dehydrogenase, is amplified by PCR using appropriately designed primers. The amplified DNA fragments are inserted into the SmaI region of pBS5T (WO2006 / 057450) by infusion to obtain a poxB gene deletion vector.

[0176] <1-4> Construction of an ACK gene expression vector Using chromosomal DNA from C. glutamicum ATCC 13869 as a template, a DNA fragment containing the ack gene (CGBL_0126910), which encodes acetate kinase, was amplified by PCR using primers SEQ ID NOs. The amplified DNA fragment was then ligated with pVK9 (US2006-0141588), which had been cleaved with bamHI and pstI, by an infusion reaction to obtain the ack gene expression vector pVK9-Plac ackA.

[0177] Furthermore, using chromosomal DNA from C. glutamicum ATCC 13869 as a template, PCR was performed using primers SEQ ID NOs. 21 and 22 to amplify a DNA fragment containing the upstream sequence (including the promoter region, 369 bp) of the msrA gene derived from C. glutamicum. Separately, using chromosomal DNA from C. glutamicum ATCC 13869 as a template, PCR was performed using primers SEQ ID NOs. 23 and 20 to amplify a DNA fragment containing the ack gene (CGBL_0126910). By ligating both amplified DNA fragments with pVK9 (US2006-0141588) cleaved with bamHI and pstI via an infusion reaction, the ack gene expression vector pVK9-PmsrA ackA was obtained.

[0178] <1-5> Construction of glpX gene expression vector Using chromosomal DNA from E. coli K-12 MG1655 (ATCC 47076) as a template, a DNA fragment containing the glpX gene encoding fructose-1,6-bisphosphatase was amplified by PCR using primers SEQ ID NOs. 24 and 25. The amplified DNA fragment was then ligated with pVK9 (US2006-0141588), which had been cleaved with bamHI and pstI, via an infusion reaction to obtain the glpX gene expression vector pVK9-Plac glpX.

[0179] <1-6> Construction of modified strains of Corynebacterium glutamicum Each of the constructed gene deletion vectors was used for C. glutamicum 2256ΔsucAΔldhA yggB * The strain (WO2014 / 185430) is transformed. From the resulting transformants, strains are selected according to the method described in WO2006 / 057450 to obtain aspT gene-deficient strains, malE gene-deficient strains, and poxB gene-deficient strains.

[0180] The constructed ackA gene expression vectors, pVK9-Plac ackA or pVK9-PmsrA ackA, can be used alone or in combination with pVS7-xfp (US2018-0282773) to express C. glutamicum 2256ΔsucAΔldhA yggB. * By introducing it into strain (WO2014 / 185430), we obtained a strain with enhanced ack gene expression. pVS7-xfp is an expression vector for the phosphoketolase gene (xfp) derived from B. longum JCM1217 (US2018-0282773).

[0181] The constructed glpX gene expression vectors pVK9-Plac glpX and pVS7-xfp (US2018-0282773) were used in C. glutamicum 2256ΔsucAΔldhA yggB * By introducing it into strain (WO2014 / 185430), we obtained a strain with enhanced glpX gene expression.

[0182] pVK9 alone or in combination with pVS7-xfp (US2018-0282773) is used in C. glutamicum 2256ΔsucAΔldhA yggB * The strain (WO2014 / 185430) was introduced to obtain a control strain.

[0183] C. glutamicum 2256ΔsucAΔldhA yggB * The strain is an L-glutamic acid-producing strain derived from C. glutamicum strain 2256 (ATCC 13869), lacking the ldhA and sucA genes, and possessing an IS mutation (V419::IS) in the yggB gene. The nucleotide sequence of this mutant yggB gene (V419::IS) and the amino acid sequence of the mutant YggB protein (V419::IS) encoded by the same gene are shown in Sequence IDs 17 and 18, respectively.

[0184] <2> L-glutamic acid production culture L-glutamic acid production cultures were carried out using each of the constructed strains (i.e., the control strain and the strains with enhanced expression of the ack gene and the glpX gene). The composition of the medium used is shown in Table 1. The medium with glucose as the carbon source is also referred to as the "Glc medium", and the medium with fructose as the carbon source is also referred to as the "Frc medium".

[0185]

Table 1

[0186] Each strain was inoculated into 5 mL of the above medium (containing 50 g / L calcium carbonate) placed in a large test tube, and shake-cultured at 120 rpm using a box shaker (ABLE ML-190) at 31.5 °C. Sampling of the culture broth was carried out 25 or 30 hours after the start of the culture. The L-glutamic acid concentration in the culture broth was quantified using a biotech analyzer AS-310 (Sakura ES AI), and the sugar yield of L-glutamic acid was calculated.

[0187] The results are shown in Figs. 1 to 4. The strains with enhanced expression of the ack gene (2256ΔsucAΔldhA yggB * / pVK9-Plac ackA / pVS7-xfp, 2256ΔsucAΔldhA yggB * / pVK9-PmsrA ackA / pVS7-xfp, 2256ΔsucAΔldhA yggB * / pVK9-Plac ackA, and 2256ΔsucAΔldhA yggB * / pVK9-PmsrA ackA) showed that when glucose was used as the carbon source, the corresponding control strains (2256ΔsucAΔldhA yggB * / pVK9-Plac ackA / pVS7-xfp and 2256ΔsucAΔldhA yggB * / pVK9-PmsrA ackA / pVS7-xfp for 2256ΔsucAΔldhA yggB * / pVK9 / pVS7-xfp; 2256ΔsucAΔldhA yggB * / pVK9-Plac ackA and 2256ΔsucAΔldhA yggB * / pVK9-PmsrA ackAについて2256ΔsucAΔldhA yggB * It showed higher L-glutamate accumulation and L-glutamate-to-sugar yield than the / pVK9 strain (Figures 1 and 2). Furthermore, it exhibited enhanced glpX gene expression (2256ΔsucAΔldhA yggB). * / pVK9-Plac glpX / pVS7-xfp) is different from the corresponding control strain (2256ΔsucAΔldhA yggB) when fructose is used as the carbon source. * It showed a higher accumulation of L-glutamic acid than / pVK9 / pVS7-xfp (Figure 3), and when glucose was used as the carbon source, it was compared with the corresponding control strain (2256ΔsucAΔldhA yggB * It showed a higher L-glutamic acid-to-sugar yield than / pVK9 / pVS7-xfp (Figure 4).

[0188] Furthermore, improvements in L-glutamate production were confirmed by performing L-glutamate production culture using the same procedure for aspT gene-deficient strains, malE gene-deficient strains, and poxB gene-deficient strains. [Industrial applicability]

[0189] According to the present invention, the L-amino acid production capacity of Corynebacteria can be improved, and L-amino acids can be produced efficiently.

[0190] [Explanation of Sequence Listing] Sequence ID 1: Base sequence of the aspT gene in Corynebacterium glutamicum 2256 (ATCC 13869) Sequence ID 2: Amino acid sequence of the AspT protein of Corynebacterium glutamicum 2256 (ATCC 13869) Sequence ID 3: Base sequence of the malE gene in Corynebacterium glutamicum 2256 (ATCC 13869) Sequence ID 4: Amino acid sequence of MalE protein from Corynebacterium glutamicum 2256 (ATCC 13869) Sequence ID 5: Base sequence of the poxB gene of Corynebacterium glutamicum 2256 (ATCC 13869) Sequence ID 6: Amino acid sequence of the PoxB protein of Corynebacterium glutamicum 2256 (ATCC 13869) Sequence ID 7: Base sequence of the ack gene in Corynebacterium glutamicum 2256 (ATCC 13869) Sequence ID 8: Amino acid sequence of the Ack protein from Corynebacterium glutamicum 2256 (ATCC 13869) Sequence ID 9: Base sequence of the ack gene in Escherichia coli K-12 MG1655 Sequence ID 10: Amino acid sequence of the Ack protein of Escherichia coli K-12 MG1655 Sequence ID 11: Base sequence of the glpX gene of Corynebacterium glutamicum ATCC 13032 Sequence ID 12: Amino acid sequence of GlpX protein from Corynebacterium glutamicum ATCC 13032 Sequence ID 13: Base sequence of the glpX gene of Escherichia coli K-12 MG1655 Sequence ID 14: Amino acid sequence of the GlpX protein of Escherichia coli K-12 MG1655 Sequence ID 15: Base sequence of the yggB gene in Corynebacterium glutamicum 2256 (ATCC 13869) Sequence ID 16: Amino acid sequence of the YggB protein of Corynebacterium glutamicum 2256 (ATCC 13869) Sequence ID 17: Nucleotide sequence of the mutant yggB gene (V419::IS) of Corynebacterium glutamicum 2256 (ATCC 13869) Sequence ID 18: Amino acid sequence of the mutant YggB protein (V419::IS) of Corynebacterium glutamicum 2256 (ATCC 13869) Sequence IDs 19-25: Primers

Claims

1. A method for producing L-amino acids, Culturing Corynebacterium-type bacteria capable of producing L-amino acids in a culture medium, and accumulating L-amino acids in the culture medium and / or within the bacterial cells, and To collect the L-amino acid from the culture medium and / or the bacterial cells, Includes, The aforementioned L-amino acid is a glutamic acid-based L-amino acid, The bacteria have at least the following modification (A): (A) Modifications that increase the activity of acetate kinase, The aforementioned bacteria have been further modified to exhibit increased phosphoketolase activity compared to the unmodified strain. The aforementioned glutamic acid-based L-amino acid is L-glutamic acid. A method wherein the bacterium is Corynebacterium glutamicum.

2. The method according to claim 1, A method wherein the acetate kinase is encoded by the ACK gene.

3. A method according to claim 1 or 2, A method in which the activity of the acetate kinase is increased by increasing the expression of the gene encoding acetate kinase.

4. The method according to claim 3, A method in which the expression of a gene encoding acetate kinase is increased by increasing the copy number of the gene and / or modifying the gene's expression regulatory sequence.

5. A method according to any one of claims 1 to 4, The method wherein the acetate kinase is the protein described in (4a), (4b), or (4c) below: (4a) Proteins containing the amino acid sequence shown in Sequence ID No. 8 or 10; (4b) A protein having acetate kinase activity, comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 8 or 10; (4c) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No. 8 or 10, and which has acetate kinase activity.

6. The method according to any one of claims 1 to 5, wherein the phosphoketolase is D-xylulose-5-phosphate phosphoketolase and / or fructose-6-phosphate phosphoketolase.

7. The method according to any one of claims 1 to 6, wherein the L-glutamic acid is ammonium L-glutamate or sodium L-glutamate.

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