JP2007097573A - L-glutamic acid-producing microorganism and method for producing l-glutamic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new technology enhancing L-glutamic acid-producing ability in producing L-glutamic acid using a coryneform bacterium. <P>SOLUTION: The invention relates to the technology comprising use of a coryneform bacterium that is modified by using a yggB gene so that L-glutamic acid-producing ability is enhanced as compared to a non-modified strains, in culturing the bacterium in a medium to cause accumulation of L-glutamic acid in the medium or bacterial cells, and collecting the L-glutamic acid from the medium or cells. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to the fermentation industry, and in particular, to a method for producing L-glutamic acid and a bacterium used therefor. L-glutamic acid is widely used as a seasoning material.

  Conventionally, L-glutamic acid is industrially produced by fermentation using coryneform bacteria belonging to the genus Brevibacterium or Corynebacterium having the ability to produce L-glutamic acid. In order to improve the productivity of these coryneform bacteria, strains isolated from nature or artificial mutants of the strains are used.

  Wild strains of coryneform bacteria generally do not produce glutamic acid under conditions where biotin is present. Therefore, glutamic acid production by coryneform bacteria is performed in a state in which glutamic acid production is induced by biotin restriction, addition of a surfactant, addition of penicillin, or the like (Non-patent Document 1). In addition, as a strain capable of producing L-glutamic acid under conditions where biotin is sufficiently present without applying these methods, a surfactant temperature sensitive strain (Patent Document 1), a penicillin sensitive strain (Patent Document 2), A cerulenin sensitive strain (Patent Document 3), a lysozyme sensitive strain (Patent Document 4) and the like have been developed.

  However, L-glutamic acid-producing bacteria developed by these methods are often accompanied by a decrease in the ability to synthesize fatty acids and the ability to synthesize cell walls. In exchange for the production of L-glutamic acid, the ability to adapt to environmental changes is reduced. Is likely to cause. Therefore, considerable effort has been required to develop a strain capable of accumulating a significant amount of L-glutamic acid using these methods.

  On the other hand, a strain that produces L-glutamic acid under sufficient biotin conditions can also be achieved by deleting a gene encoding α-ketoglutarate dehydrogenase (Patent Document 5). However, α-ketoglutarate dehydrogenase-deficient strains that block the TCA cycle in the middle have problems such as difficulty in securing the amount of cells due to slow growth.

The yggB gene of the coryneform bacterium is a homolog of the yggB gene of Escherichia coli (Non-patent Documents 2 and 3), and has been analyzed as a kind of mechanosensitive channel (Non-patent Document 4). The effect on L-glutamic acid was not known.
International publication 99/02692 pamphlet Japanese Unexamined Patent Publication No. 04-088994 JP 55-124492 A International Publication No. 00/14241 Pamphlet International Publication No. 95/34672 Pamphlet Biosci.Biotech.Biochem., 61 (7), 1109-1112, 1997 FEMS Microbiol Lett. 2003 Jan 28; 218 (2): 305-9. Mol Microbiol. 2004 Oct; 54 (2): 420-38. EMBO J. 1999, 18 (7): 1730-7

  An object of the present invention is to provide a novel technique for improving L-glutamic acid production ability in the production of L-glutamic acid using coryneform bacteria.

    In order to solve the above-mentioned problems, the present researchers conducted intensive studies. As a result, it was clarified that the yggB gene is involved in L-glutamine production of coryneform bacteria, and the ability to produce L-glutamic acid is greatly improved by modifying the coryneform bacteria using the yggB gene. As a result, the present invention has been completed.

That is, the present invention is as follows.
(1) A coryneform bacterium having L-glutamic acid-producing ability, wherein the coryneform bacterium has improved L-glutamic acid-producing ability as compared with an unmodified strain by being modified using the yggB gene.
(2) The coryneform bacterium according to (1), wherein the yggB gene is DNA selected from any of the following (a) to (h):
(a) DNA comprising the nucleotide sequence of nucleotide numbers 1437 to 3035 of SEQ ID NO: 5,
(b) The coryneform bacterium by hybridizing under a stringent condition with a complementary base sequence of base numbers 1437 to 3035 of SEQ ID NO: 5 or a probe that can be prepared from the base sequence and introducing it into the coryneform bacterium. DNA which improves L-glutamic acid production ability of.
(c) DNA comprising the nucleotide sequence of base numbers 507 to 2093 of SEQ ID NO: 61,
(d) the coryneform bacterium by hybridizing with a complementary base sequence of base numbers 507 to 2093 of SEQ ID NO: 61 or a probe that can be prepared from the base sequence under stringent conditions and introducing it into the coryneform bacterium DNA which improves L-glutamic acid production ability of.
(e) DNA comprising the base sequence of base numbers 403 to 2001 of SEQ ID NO: 67,
(f) the coryneform bacterium by hybridizing under a stringent condition with a complementary base sequence of base numbers 403 to 2001 of SEQ ID NO: 67 or a probe that can be prepared from the base sequence and introducing it into the coryneform bacterium DNA which improves L-glutamic acid production ability of.
(g) DNA comprising the base sequence of base numbers 501 to 2099 of SEQ ID NO: 83,
(h) The coryneform bacterium by hybridizing under a stringent condition with a complementary base sequence of base numbers 501 to 2099 of SEQ ID NO: 83 or a probe that can be prepared from the base sequence and introducing it into the coryneform bacterium DNA which improves L-glutamic acid production ability of.
(3) The yggB gene is one or a number in the amino acid sequence selected from SEQ ID NO: 6, 62, 68, 84, or 85, or the amino acid sequence selected from SEQ ID NO: 6, 62, 68, 84, or 85 A DNA that encodes a protein having an amino acid sequence in which one amino acid is substituted, deleted, inserted or added, and is introduced into a coryneform bacterium, thereby improving the L-glutamic acid-producing ability of the coryneform bacterium (1 ) Coryneform bacteria.
(4) The coryneform bacterium according to (1), which has been modified so that the expression level of the yggB gene is increased as compared with the unmodified strain.
(5) The coryneform bacterium according to (4), which has been modified to increase the expression level of the gene by increasing the copy number of the yggB gene or modifying the expression regulatory sequence of the gene.
(6) The coryneform bacterium according to (1), into which a mutant yggB gene has been introduced.
(7) The mutant yggB gene has a mutation in a region encoding the sequence of amino acid numbers 419-533 of SEQ ID NO: 6, 68, 84 or 85, or the sequence of amino acid numbers 419-529 of SEQ ID NO: 62. 6) Coryneform bacteria.
(8) The coryneform bacterium according to (7), wherein the mutation is a deletion of the region.
(9) The coryneform bacterium according to (7), wherein the mutation is an insertion sequence or transposon insertion into the region.
(10) The coryneform bacterium according to (7), wherein the mutation is a mutation that substitutes other amino acids for proline present in the region.
(11) The mutant yggB gene is a mutation that substitutes other amino acids for proline at position 424 and / or 437 in the amino acid sequence of SEQ ID NO: 6, 62, 68, 84, or 85. Coryneform bacteria.
(12) The coryneform bacterium according to (6), wherein the mutant yggB gene is a gene having a mutation in a transmembrane region of a protein encoded by the yggB gene.
(13) In the amino acid sequence of SEQ ID NO: 6, 62, 68, 84 or 85, the transmembrane region is a group consisting of amino acid numbers 1-23, 25-47, 62-84, 86-108, and 110-132 The coryneform bacterium according to (12), which is a region selected from
(14) The coryneform bacterium according to (12), wherein the mutation is introduced without accompanying a frame shift.
(15) The mutant yggB gene is a gene having a mutation that substitutes an alanine at position 100 and / or alanine at position 111 with another amino acid in the amino acid sequence of SEQ ID NO: 6, 62, 68, 84, or 85 (12) Coryneform bacterium.
(16) The mutant yggB gene is a gene having a mutation that inserts one or several amino acids between leucine at position 14 and tryptophan at position 15 in the amino acid sequence of SEQ ID NO: 6, 62, 68, 84, or 85. A coryneform bacterium according to (12).
(17) The coryneform bacterium according to any one of (6) to (16), wherein resistance to L-glutamic acid analog is improved by introduction of the mutant yggB gene.
(18) The coryneform bacterium according to any one of (6) to (17), further modified so that a gene that suppresses the function of the mutant yggB gene is inactivated.
(19) The coryneform bacterium according to (18), wherein the gene that suppresses the function of the mutant yggB gene is a symA gene, and the symA gene is DNA selected from the following (i) or (j):
(i) DNA comprising the base sequence of base numbers 585 to 1121 of SEQ ID NO: 86,
(j) It hybridizes under a stringent condition with a complementary nucleotide sequence of nucleotide numbers 585 to 1121 of SEQ ID NO: 86 or a probe that can be prepared from the nucleotide sequence, and suppresses the function of the mutant yggB gene in coryneform bacteria DNA to do.
(20) The coryneform bacterium according to any one of (1) to (19), which is further modified so that the α-ketoglutarate dehydrogenase activity is decreased.
(21) The coryneform bacterium according to any one of (1) to (20), which belongs to the genus Corynebacterium or Brevibacterium.
(22) The coryneform bacterium of any one of (1) to (21) is cultured in a medium, and L-glutamic acid is produced and accumulated in the medium or in the microbial cells, and L-glutamic acid is recovered from the medium or the microbial cells. A process for producing L-glutamic acid characterized by the above.
(23) A mutant yggB gene selected from the following (a) to (p):
(a) DNA encoding the amino acid sequence of SEQ ID NO: 8,
(b) An excess amount of biotin is encoded by encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 8 and introducing the protein into a coryneform bacterium. DNA which improves the L-glutamic acid-producing ability of the coryneform bacterium when cultured in a medium containing
(c) DNA encoding the amino acid sequence of SEQ ID NO: 20,
(d) an excess amount of biotin by encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 20 and introducing the protein into a coryneform bacterium. DNA which improves the L-glutamic acid-producing ability of the coryneform bacterium when cultured in a medium containing
(e) DNA encoding the amino acid sequence of SEQ ID NO: 22,
(f) An excess amount of biotin is encoded by encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 22 and introducing the protein into a coryneform bacterium. DNA which improves the L-glutamic acid-producing ability of the coryneform bacterium when cultured in a medium containing
(g) DNA encoding the amino acid sequence of SEQ ID NO: 24,
(h) An excess amount of biotin is encoded by encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 24 and introducing the protein into a coryneform bacterium. L of the coryneform bacterium when cultured in a medium containing
-DNA which improves glutamic acid production ability.
(i) DNA encoding the amino acid sequence of SEQ ID NO: 64,
(j) An excess amount of biotin is encoded by encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 64, and introducing the protein into a coryneform bacterium. DNA which improves the L-glutamic acid-producing ability of the coryneform bacterium when cultured in a medium containing
(k) DNA encoding the amino acid sequence of SEQ ID NO: 70,
(l) An excess amount of biotin is encoded by encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 70 and introducing the protein into a coryneform bacterium. DNA which improves the L-glutamic acid-producing ability of the coryneform bacterium when cultured in a medium containing
(m) DNA encoding the amino acid sequence of SEQ ID NO: 74,
(n) In the amino acid sequence of SEQ ID NO: 74, an excess amount of biotin is encoded by encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added and introduced into a coryneform bacterium. DNA which improves the L-glutamic acid-producing ability of the coryneform bacterium when cultured in a medium containing
(24) A method for producing a coryneform bacterium having a mutant yggB gene, wherein a coryneform bacterium lacking a gene encoding α-ketoglutarate dehydrogenase is inoculated into a medium containing an excess amount of biotin, And obtaining a strain capable of accumulating L-glutamic acid.
(25) A method for producing a coryneform bacterium having a mutated yggB gene, wherein a coryneform bacterium into which a yggB gene into which a mutation has been randomly introduced in vitro is introduced is inoculated into a medium containing an excess amount of biotin. A method for obtaining a strain capable of accumulating L-glutamic acid in a medium.
(26) A method for producing a coryneform bacterium having a mutant yggB gene, wherein a coryneform bacterium in which a transposable element is randomly introduced on a chromosome is inoculated into a medium containing an excessive amount of biotin, and L- A method of obtaining a strain capable of accumulating glutamic acid.
(27) A method for producing a coryneform bacterium having a mutant yggB gene, wherein the coryneform bacterium is cultured in a medium containing an L-glutamic acid analog, and a strain capable of growing in the same medium is obtained. Method.
(28) The method for producing a coryneform bacterium according to any one of (24) to (27), wherein the strain having the mutant yggB gene is the coryneform bacterium according to any one of (6) to (16).
(29) The method for producing a coryneform bacterium according to any one of (24) to (28), wherein the medium containing an excessive amount of biotin is a medium containing 30 μg / L or more of biotin.

By using a coryneform bacterium modified using the yggB gene of the present invention, L-glutamic acid can be efficiently produced.

Hereinafter, the present invention will be described in detail.
<1> Coryneform bacterium of the present invention The coryneform bacterium of the present invention is a coryneform bacterium having L-glutamic acid-producing ability, and has been modified by using the yggB gene. -Coryneform bacterium with improved glutamate production ability.

In the present invention, the “coryneform bacterium” has been conventionally classified into the genus Brevibacterium, but includes bacteria that are currently classified into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41
, 255 (1991)), and Brevibacterium spp. Closely related to the genus Corynebacterium. Examples of such coryneform bacteria include the following.
Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium carnae Corynebacterium glutamicum Corynebacterium lylium Corynebacterium merasecola Corynebacterium thermoaminogenes ( Corynebacterium efficiens)
Corynebacterium herculis Brevibacterium divaricatam Brevibacterium flavum Brevibacterium immariophyllum Brevibacterium lactofermentum (Corynebacterium glutamicum)
Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium ammoniagenes Brevibacterium album Brevibacterium cerinum Microbacteria ammonia filam

Specifically, the following strains can be exemplified.
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium alkanolyticum ATCC21511
Corynebacterium carnae ATCC15991
Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13060
Corynebacterium lilium ATCC15990
Corynebacterium melasecola ATCC17965
Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539)
Corynebacterium herculis ATCC13868
Brevibacterium divaricatam ATCC14020
Brevibacterium flavum ATCC13826, ATCC14067 (Corynebacterium glutamicum ATCC14067)
Brevibacterium flavum ATCC13826, ATCC14067
Brevibacterium immariophilum ATCC14068
Brevibacterium lactofermentum ATCC13869 (Corynebacterium glutamicum ATCC 13869)
Brevibacterium rose ATCC13825
Brevibacterium saccharolyticum ATCC14066
Brevibacterium thiogenitalis ATCC19240
Corynebacterium ammoniagenes ATCC6871, ATCC6872
Brevibacterium album ATCC15111
Brevibacterium cerinum ATCC15112
Microbacterium ammonia film ATCC15354

You can get them from, for example, the American Type Culture Collection. That is, a corresponding registration number is assigned to each strain, and distribution can be received using this registration number. The registration number corresponding to each strain is described in the catalog of American Type Culture Collection. In addition, AJ12340 shares were issued on October 27, 1987, from the Ministry of International Trade and Industry, Institute of Industrial Science, Biotechnology Institute of Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Microorganism Depositary Center) (Ibaraki, 305-5466, Japan) It is deposited under the Budapest Treaty under the accession number of FERM BP-1539 in 1st, 1st East, 1st Street, Tsukuba City.

In the present invention, “L-glutamic acid-producing ability” refers to the ability to accumulate L-glutamic acid in the medium or in the cells when the coryneform bacterium of the present invention is cultured. Since many coryneform bacteria can produce L-glutamic acid under the “L-glutamic acid production conditions” described later, “L-glutamic acid producing ability” is a property of a wild strain of coryneform bacteria. Also good. However, the property may be imparted or enhanced by breeding, or may be imparted with L-glutamic acid-producing ability by modification using the yggB gene as described later.
“L-glutamic acid-producing ability is improved” means that L-glutamic acid-producing ability is increased as compared to an unmodified strain such as a wild-type strain. Here, examples of wild strains of coryneform bacteria include Corynebacterium glutamicum ATCC13032 strain, 13869 strain, 14067 strain, Corynebacterium melacecola ATCC17965 strain, and the like. The “non-modified strain” includes strains exhibiting the same level of expression of the yggB gene as the wild strain as described above, and strains in which no mutation is introduced into the coding region of the yggB gene.

  Examples of a method for imparting or enhancing L-glutamic acid-producing ability by breeding include a method of modifying so that expression of a gene encoding an enzyme involved in L-glutamic acid biosynthesis is enhanced. Examples of enzymes involved in L-glutamate biosynthesis include glutamate dehydrogenase, glutamine synthetase, glutamate synthase, isocitrate dehydrogenase, aconitate hydratase, citrate synthase, phosphoenolpyruvate carboxylase, pyruvate carboxylase, pyruvate dehydrogenase, and pyruvin. Acid kinase, phosphoenolpyruvate synthase, enolase, phosphoglycermutase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, furtose bisphosphate aldolase, phosphofructokinase, glucose phosphate isomerase Etc.

  The expression of these genes can be enhanced in the same manner as the expression enhancement of the yggB gene described later.

  Examples of microorganisms modified to enhance expression of citrate synthase gene, isocitrate dehydrogenase, pyruvate dehydrogenase gene, and / or glutamate dehydrogenase gene include International Publication No. 00/18935, Japanese Patent Application Laid-Open No. 2000-232890, etc. Can be exemplified.

The modification for imparting L-glutamic acid-producing ability may be performed by reducing or eliminating the activity of an enzyme that catalyzes a reaction that branches off from the biosynthetic pathway of L-glutamic acid to produce another compound. Examples of enzymes that catalyze reactions that branch off from the biosynthetic pathway of L-glutamic acid to produce compounds other than L-glutamic acid include isocitrate lyase, α-ketoglutarate dehydrogenase, phosphate acetyltransferase, acetate kinase, acetohydroxyacid synthase And acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, and the like. Among them, strains with reduced α-ketoglutarate dehydrogenase activity are particularly desirable. For example, the following strains may be mentioned as strains having reduced α-ketoglutarate dehydrogenase activity.
Brevibacterium lactofermentum strain ΔS (International pamphlet No. 95/34672)
Brevibacterium lactofermentum AJ12821 (FERM BP-4172; see French Patent Publication 9401748)
Brevibacterium flavum AJ12822 (FERM BP-4173; refer to French Patent Publication 9401748)
Corynebacterium glutamicum AJ12823 (FERM BP-4174; see French Patent Publication 9401748)

  In order to reduce or eliminate the activity of the enzyme as described above, a mutation that reduces or eliminates the activity of the enzyme in the cell is introduced into the gene of the enzyme on the chromosome by a usual mutation treatment method. Good. For example, it can be achieved by deleting a gene encoding an enzyme on a chromosome by genetic recombination or by modifying an expression regulatory sequence such as a promoter or Shine-Dalgarno (SD) sequence. Also, introducing amino acid substitutions (missense mutations) into regions encoding enzymes on chromosomes, introducing stop codons (nonsense mutations), and introducing frameshift mutations that add or delete 1-2 bases. It can also be achieved by deleting a part or the entire region of the gene. (Journal of biological Chemistry 272: 8611-8617 (1997)) Also, construct a gene that encodes a mutant enzyme whose coding region has been deleted, and replace the normal gene on the chromosome with the gene by homologous recombination. The enzyme activity can also be reduced or eliminated by introducing a transposon or IS (insertion sequence) factor into the gene.

For example, in order to introduce a mutation that reduces or eliminates the activity of the above enzyme by genetic recombination, the following method is used. A partial gene of the target gene is modified to produce a mutant gene that does not produce a normally functioning enzyme, a coryneform bacterium is transformed with the DNA containing the gene, and the mutant gene and the gene on the chromosome are transformed. By causing recombination, the target gene on the chromosome can be replaced with a mutant type. Such site-directed mutagenesis by gene replacement using homologous recombination has already been established, and includes a method using linear DNA and a method using a plasmid containing a temperature-sensitive replication origin (US Pat. No. 6,303,383). Or Japanese Patent Laid-Open No. 05-007491). Examples of temperature-sensitive plasmids for coryneform bacteria include p48K and pSFKT2 (see US Pat. No. 6,303,383), pHSC4 (see French Patent Publication No. 2667875, Japanese Patent Laid-Open No. 5-7491), and the like. These plasmids can replicate autonomously in coryneform bacteria at least at 25 ° C, but not at 37 ° C. Escherichia coli AJ12571, which holds pHSC4, was established on October 11, 1990 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Microorganism Depositary Center) (〒305-5466 Japan) FERM in Tsukuba City, Ibaraki Prefecture
Deposited as P-11763, transferred to an international deposit under the Budapest Treaty on August 26, 1991, and deposited under the deposit number FERM BP-3524. Moreover, site-directed mutagenesis by gene replacement using homologous recombination as described above can also be performed using a plasmid that does not have replication ability on the host. A plasmid that does not have replication ability in coryneform bacteria is preferably a plasmid that has replication ability in Escherichia coli, and examples thereof include pHSG299 (manufactured by Takara Bio Inc.), pHSG399 (manufactured by Takara Bio Inc.), and the like.

  In order to replace the target gene of the mutant type with the target gene on the host chromosome, for example, the following may be performed. First, a recombination plasmid is prepared by inserting a temperature-sensitive replication origin, a mutant target gene, a sacB gene encoding levanshuclease, and a marker gene exhibiting drug resistance such as chloramphenicol.

Here, the sacB gene encoding levansucrase is a gene used to efficiently select a strain from which the vector portion has been dropped from the chromosome (Schafer, A. et al. Gene
145 (1994) 69-73). That is, in coryneform bacteria, when levan sucrose is expressed, levan produced by assimilating sucrose works lethally and cannot grow. Therefore, if a strain loaded with levan sucrose is left on the chromosome, the strain cannot be grown on the sucrose-containing plate, and only the strain from which the vector has been dropped can be selected on the sucrose-containing plate.

As the sacB gene or a homologous gene thereof, a gene having the following sequence can be used. Bacillus subtilis: sacB GenBank Accession Number X02730 (SEQ ID NO: 11)
Bacillus amyloliquefaciens: sacB GenBank Accession Number X52988
Zymomonas Mobilis: sacB GenBank Accession Number L33402
Bacillus stearothermophilus: surB GenBank Accession Number U34874
Lactobacillus san francisensis: frfA GenBank Accession Number AJ508391
Acetobacter xylinus: lsxA GenBank Accession Number AB034152
Glucon Acetobacter diazotrophicus: lsdA GenBank Accession Number L41732

  The transformant obtained as described above is cultured at a temperature (25 ° C.) at which the temperature-sensitive replication origin functions to obtain a strain into which the plasmid has been introduced. The plasmid-introduced strain is cultured at a high temperature (for example, 34 ° C.) at which the temperature-sensitive replication origin does not function, the temperature-sensitive plasmid is removed, and this strain is applied to a plate containing an antibiotic. Since temperature-sensitive plasmids cannot replicate at high temperatures, strains that have lost the plasmid cannot grow on plates containing antibiotics, but strains that have recombined with the mutant gene on the plasmid and the target gene on the chromosome appear. To do.

  A strain in which the recombinant DNA is incorporated into the chromosome in this way undergoes recombination with the target gene sequence originally present on the chromosome, and two fusion genes of the target gene of the chromosome and the target gene of the deletion type are recombinant DNA. The other part (vector part, temperature sensitive replication origin and drug resistance marker) is inserted between the chromosomes.

  Next, in order to leave only the deletion-type target gene on the chromosomal DNA, the target gene region is dropped from the chromosomal DNA together with the vector portion (including the temperature-sensitive replication origin and drug resistance marker). At that time, there are cases where the normal target gene is left on the chromosomal DNA and the deletion-type target gene is excised, and conversely, the deletion-type target gene is left on the chromosomal DNA and the normal target gene is excised. is there. In either case, if the cells are cultured at a temperature at which the temperature-sensitive replication origin functions, the excised DNA is retained in the cell in the form of a plasmid. Next, when culturing at a temperature (high temperature) at which the temperature-sensitive replication origin does not function, the target gene on the plasmid is removed from the cell together with the plasmid. When a plasmid loaded with sacB is used, by adding sucrose to the medium at this time, a strain in which the plasmid has dropped from the cells can be efficiently selected. By selecting a strain in which a mutation has remained in the target gene from the strain from which the plasmid has been removed, a strain in which the target gene has been replaced with a deletion type can be obtained.

As another method for imparting or enhancing L-glutamic acid-producing ability, a method for imparting resistance to an organic acid analog or a respiratory inhibitor and a method for imparting sensitivity to a cell wall synthesis inhibitor may be mentioned. For example, a method for imparting resistance to monofluoroacetic acid (Japanese Patent Laid-Open No. 50-113209), a method for imparting adenine resistance or thymine resistance (Japanese Patent Laid-Open No. 57-065198), a method of weakening urease (Japanese Patent Laid-Open No. 52-038088) ), A method for imparting resistance to malonic acid (Japanese Patent Laid-Open No. 52-038088), a method for imparting resistance to benzopyrone or naphthoquinones (Japanese Patent Laid-Open No. 56-1889), a method of imparting HOQNO resistance (Japanese Patent Laid-Open No. Sho 56-89) 140895), a method for imparting resistance to α-ketomalonic acid (Japanese Patent Laid-Open No. 57-2689), a method for imparting resistance to guanidine (Japanese Patent Laid-Open No. 56-35981), a method for imparting sensitivity to penicillin (Japanese Patent Laid-Open No. 4-88994) Etc.
Specific examples of such resistant bacteria include the following strains.
Brevibacterium flavum AJ3949 (BP-2632: see Japanese Patent Laid-Open No. 50-113209)
Corynebacterium glutamicum AJ11628 (P-5736; see JP-A-57-065198)
Brevibacterium flavum AJ11355 (FERM P-5007; see JP-A-56-1889)
Corynebacterium glutamicum AJ11368 (FERM P-P-5020; see JP 56-1889) Brevibacterium flavum AJ11217 (FERM P-4318; see JP 57-2689)
Corynebacterium glutamicum AJ11218 (FERM-P4319; see JP-A-57-2689)
Brevibacterium flavum AJ11564 (FERM P-5472; see JP 56-140895 A)
Brevibacterium flavum AJ11439 (FERM P-5136; see JP-A-56-35981)
Corynebacterium glutamicum H7684 (FERM BP-3004; see JP 04-88994 A)
Brevibacterium lactofermentum AJ11426 (FERM P-5123; see JP-A-56-048890)
Corynebacterium glutamicum AJ11440 (FERM P-5137; see JP-A-56-048890)
Brevibacterium lactofermentum AJ11796 (FERM P-6402; see JP-A-58-158192)

The coryneform bacterium of the present invention can be obtained by modifying the coryneform bacterium having L-glutamic acid producing ability as described above so that the L-glutamic acid producing ability is further improved using the yggB gene. Alternatively, after modification using the yggB gene, an operation for imparting or improving L-glutamic acid-producing ability as described above may be additionally performed.
The modification using the yggB gene includes increasing the expression level of the yggB gene as described later, and introducing a mutant yggB gene.

(I) Enhanced expression level of yggB gene
The yggB gene is known as a mechanosensitive channel and encodes a protein called mscS (FEMS Microbiol Lett. 2003 Jan 28; 218 (2): 305-9.).
By increasing the expression level of the yggB gene, the ability of coryneform bacteria to produce L-glutamic acid is improved as compared to the unmodified strain. In other words, by culturing coryneform bacteria modified to increase the expression level of the yggB gene, a larger amount of L-glutamic acid is accumulated in the medium than in the unmodified strain, or the unmodified strain Produces L-glutamic acid at a faster rate than For example, the yggB gene expression-enhanced strain preferably has a yield of L-glutamic acid that is 2% or more higher than that of the parent strain or unmodified strain, and is preferably 4% or higher. More preferably, an improvement of 6% or more is particularly preferable. The yggB gene expression-enhanced strain may have improved sterilization yield as compared to the unmodified strain. The sterilized cell yield means a product obtained by subtracting the carbon yield used for the microbial cell production from the saccharide yield.

  Confirmation that the expression level of the yggB gene has increased can be confirmed by comparing the amount of yggB m-RNA with the wild-type or unmodified strain. Examples of the method for confirming the expression level include Northern hybridization and RT-PCR (Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001)). The expression level may be increased as compared with the wild strain or the non-modified strain. For example, the expression level is 1.5 times or more, more preferably two times or more, more preferably compared to the wild strain or the non-modified strain. It is desirable that it rises 3 times or more.

Coryneform bacteria modified to increase the expression level of the yggB gene have an ability to produce L-glutamic acid as compared to an unmodified strain under at least one of the conditions including L-glutamic acid production and excess biotin. It only needs to be improved.
Here, “L-glutamic acid production conditions” means that a substance that induces L-glutamic acid production is added to a medium containing carbon sources, nitrogen sources, inorganic salts, and other organic micronutrients such as amino acids and vitamins as necessary. Or a condition that limits the amount of a substance that inhibits L-glutamic acid production in the medium, and the substance added to induce L-glutamic acid production contains saturated fatty acids such as penicillin G and Tween 40 A surfactant is exemplified, and a substance that restricts to inhibit L-glutamine production includes biotin (Amino Acid Fermentation Society Publishing Center, 1986). The addition concentration of these substances in the medium under the conditions for producing L-glutamic acid is less than 15 μg / L, preferably less than 10 μg / L, more preferably less than 5 μg / L of biotin. It does not have to be included at all. The addition concentration of penicillin in the medium is 0.1 U / ml or more, preferably 0.2 U / ml or more, more preferably 0.4 U / ml or more, and the addition concentration of the surfactant is 0.5 g / L or more, Preferably, it is 1 g / L or more, more preferably 2 g / L or more.
On the other hand, “conditions containing an excess amount of biotin” refer to conditions containing, for example, 30 μg / L or more of biotin in the medium, preferably 40 μg / L, more preferably 50 μg / L.

Examples of the yggB gene of the coryneform bacterium include a gene encoding a protein having the amino acid sequence of SEQ ID NO: 6, 62, 68, or 84. More specifically, a gene having the sequence of the nucleotide sequence 1437-3035 of SEQ ID NO: 5, a gene having the sequence of the nucleotide sequence 507-2093 of SEQ ID NO: 61, the sequence of the nucleotide sequence 403-2001 of SEQ ID NO: 67 A gene having the nucleotide sequence 501-2099 of SEQ ID NO: 83, and the like. The gene having the nucleotide sequence 1437-3035 of SEQ ID NO: 5 is the yggB gene of Corynebacterium glutamicum ATCC13032 strain, and the gene having the nucleotide sequence 507-2093 of SEQ ID NO: 61 is Corynebacterium Glutamicum (Brevibacterium flavum) This is the yggB gene of ATCC14967 strain, and the gene having the nucleotide sequence 403-2001 of SEQ ID NO: 67 is the yggB gene of Corynebacterium melacecola ATCC17965 strain. The gene having the nucleotide sequence 501-2099 of SEQ ID NO: 83 is encoded by nucleotide numbers 1336092-1337693 in the genome sequence registered as Genbank Accession No. NC_003450 of Corynebacterium glutamicum ATCC13032. It is registered as 1221 (NP_600492. Reports small-conductance ... [gi: 19552490]).
Moreover, since there may be a difference in the base sequence of the yggB gene depending on the species and strain of the coryneform bacterium, the yggB gene may be a variant of the base sequence consisting of nucleotide numbers 1437-3035 of SEQ ID NO: 5. A variant of the yggB gene can be searched by BLAST or the like with reference to the sequence of the nucleotide sequence consisting of nucleotide numbers 1437-3035 of SEQ ID NO: 5 (http://blast.genome.jp/). The variants of the yggB gene include genes that can be amplified by PCR using, for example, synthetic oligonucleotides of SEQ ID NOs: 75 and 76 using a yggB gene homolog, for example, a chromosome of a coryneform bacterium as a template. The gene of the present invention is preferably the yggB gene of a coryneform bacterium, but a gene derived from another microorganism may be used as long as it has a function in the coryneform bacterium. Alternatively, a mutant yggB gene described later may be used.

As long as the yggB gene improves L-glutamic acid-producing ability of coryneform bacteria, one or several amino acid substitutions, deletions, insertions in the amino acid sequence shown in SEQ ID NO: 6, 62, 68 or 84, Or it may have an amino acid sequence including an addition. Here, several means, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5.
The above substitution is preferably a conservative substitution, and as the conservative substitution, substitution from ala to ser or thr, substitution from arg to gln, his or lys, substitution from asn to glu, gln, lys, his or asp, asp to asn, glu or gln, cys to ser or ala, gln to asn, glu, lys, his, asp or arg, glu to gly, asn, gln, lys or asp Substitution, substitution from gly to pro, substitution from his to asn, lys, gln, arg or tyr, substitution from ile to leu, met, val or phe, substitution from 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 Substitution, substitution from thr to ser or ala, substitution from trp to phe or tyr, substitution from tyr to his, phe or trp, and substitution from val to met, ile or leu.
Furthermore, the yggB gene of the present invention is 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, based on the entire amino acid sequence of SEQ ID NO: 6, 62, 68, or 84. And a gene that improves the L-glutamic acid-producing ability of coryneform bacteria. Here, the homology should be calculated by BLAST (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) by Karlin and Altschul, FASTA (Methods Enzymol., 183, 63 (1990)) by Pearson, etc. Can do. Homology search programs (BLASTN, BLASTP, etc.) using these algorithms are available from NCBI (//www.ncbi.nlm.nih.gov).
The amino acid substitutions, deletions, insertions, additions, or inversions described above include naturally occurring mutations (mutants or variants) such as those based on individual differences or species differences in the microorganisms that retain the yggB gene. ) Is also included.

In particular, amino acids at the following positions in SEQ ID NO: 6 may be substituted or deleted. The amino acid sequence of the YggB protein conserved in coryneform bacteria is shown in SEQ ID NO: 85, and the position where amino acid substitution / deletion may occur is indicated by Xaa.
48th glutamine residue (preferably substitution with arginine residue)
275th asparagine residue (preferably substitution with serine residue)
298th glutamic acid residue (preferably substitution with alanine residue)
343th alanine residue (preferably substitution with valine residue)
396th phenylalanine residue (preferably substitution with isoleucine residue)
438th serine residue (preferably substitution with glycine residue)
445th valine residue (preferably substitution with alanine residue)
454th alanine residue (preferably substitution with valine residue)
457th proline residue (preferably substitution with serine residue)
474th serine residue (preferably substitution with asparagine residue)
517th valine residue (preferably deleted)
518th glutamic acid residue (preferably deleted)
519th alanine residue (preferably deleted)
Proline residue at position 520 (desirably deleted)

The yggB gene homologue as described above has a nucleotide sequence of SEQ ID NO: 5 such that the amino acid residue at a specific site of the encoded protein includes substitution, deletion, insertion or addition, for example, by site-directed mutagenesis. A gene having a sequence of 1437-3035, a gene having a sequence of 507-2093 of the base sequence of SEQ ID NO: 61, a gene having a sequence of 403-2001 of the base sequence of SEQ ID NO: 67, or a base of SEQ ID NO: 83 It can be obtained by modifying the gene having the 501-9999th sequence.
It can also be obtained by a conventionally known mutation treatment as follows. The mutation process includes a gene having the 1437-3035th sequence of the base sequence of SEQ ID NO: 5, a gene having the 507-2093th sequence of the base sequence of SEQ ID NO: 61, and the 403-2001th sequence of the base sequence of SEQ ID NO: 67. A method for in vitro treatment with a hydroxylamine or the like, and a microorganism having the gene, for example, an Escherichia bacterium, Examples of the method include treatment with ultraviolet light or a mutagen that is commonly used for mutagenesis, such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or ethylmethanesulfonate (EMS). In addition, amino acid substitutions, deletions, insertions, additions, or inversions as described above include naturally occurring mutations (mutants or variants) such as those based on individual differences or species differences of microorganisms that retain the yggB gene. ) Is also included. Whether these genes improve the ability to produce L-glutamic acid when introduced into coryneform bacteria is determined by, for example, introducing these genes into wild-type coryneform bacteria and producing L-glutamic acid under the above-described conditions. It can be confirmed by examining whether the performance improves.

  The yggB gene is the sequence of the 1437-3035th sequence of SEQ ID NO: 5, the 507-2093th sequence of SEQ ID NO: 61, the 403-2001 sequence of SEQ ID NO: 67, or the 501-2099th sequence of SEQ ID NO: 83. Examples thereof include DNA that hybridizes with a complementary sequence or a probe that can be prepared from these sequences under stringent conditions and improves L-glutamic acid-producing ability when introduced into coryneform bacteria. Here, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to quantify these conditions clearly, for example, DNAs with high homology, for example, DNAs having 50% or more homology hybridize and DNA with lower homology than that. A salt corresponding to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, which does not hybridize with each other, or is a condition for washing of ordinary Southern hybridization The conditions for hybridization at a concentration include conditions for washing once, more preferably 2-3 times.

As a probe, a partial sequence of a complementary sequence of the base sequence of nucleotide numbers 1437-3035 of SEQ ID NO: 5 can also be used. Such a probe is a PCR using an oligonucleotide prepared based on the nucleotide sequence of nucleotide number 1437-3035 of SEQ ID NO: 5 as a primer and a DNA fragment containing the nucleotide sequence of nucleotide number 1437-3035 of SEQ ID NO: 5 as a template. Can be produced. For example, when a DNA fragment having a length of about 300 bp is used as a probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS. In addition, the sequences of SEQ ID NOs: 75 and 76 are used for preparing probes.
The yggB gene that enhances the expression level may be a mutant yggB gene as described later.

  Methods for enhancing the expression of the yggB gene include a method for increasing the copy number of the yggB gene, a method for modifying the expression regulatory region of the yggB gene, a method for amplifying the transcriptional activation factor of the yggB gene, and the yggB gene. And the like, and the like.

  Hereinafter, a method for constructing a coryneform bacterium having L-glutamic acid-producing ability and modified so that the expression level of the yggB gene is increased will be described. These methods can be performed according to manuals such as Molecular cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001).

  The enhancement of the expression level of the gene can be achieved by increasing the copy number of the yggB gene, and the increase of the copy number can be achieved by amplifying the yggB gene with a plasmid as follows. First, the yggB gene is cloned from the chromosome of a coryneform bacterium. Chromosomal DNA is obtained from bacteria that are DNA donors, for example, the method of Saito and Miura (H. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963), Biotechnology Experiments, Nippon Biotechnology, Ed., Pp. 97-98, Bafukan, 1992). Oligonucleotides used for PCR can be synthesized based on the above known information. For example, the yggB gene can be amplified using synthetic oligonucleotides described in SEQ ID NOs: 75 and 76.

A gene fragment containing the yggB gene amplified by PCR is prepared by connecting a vector DNA capable of autonomous replication in Escherichia coli and / or coryneform bacterium cells to prepare a recombinant DNA, which is then transferred to Escherichia coli. If it is introduced, later operations will be easier. As vectors capable of autonomous replication in Escherichia coli cells, pUC19, pUC18, pHSG299,
pHSG399, pHSG398, RSF1010, pBR322, pACYC184, pMW219 and the like.

The DNA is introduced into a vector that functions in coryneform bacteria. The vector that functions in coryneform bacteria is, for example, a plasmid that can autonomously replicate in coryneform bacteria. For example, plasmids that can autonomously replicate in coryneform bacteria include, for example, the plasmid pCRY30 described in JP-A-3-210184; JP-A-2-72876 and US Pat. No. 5,185,262. Plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX described in the publication; plasmids pCRY2 and pCRY3 described in JP-A-1-191686; PHM1519 described in JP-A No. 77895; pAJ655, pAJ611 and pAJ1844 described in JP-A-58-192900; pCG1 described in JP-A-57-134500; pCG2 described in JP-A-58-35197 ; Special pCG4 and pCG11 like described in 57-183799 JP Akira, can be mentioned pVK7 described in JP-A-10-215883.

  In addition, DNA fragments capable of autonomously replicating plasmids in coryneform bacteria can be extracted from these vectors and inserted into the aforementioned Escherichia coli vector, allowing autonomous replication in both Escherichia coli and coryneform bacteria. It can be used as a so-called shuttle vector.

  To prepare a recombinant DNA by linking the yggB gene and a vector that functions in coryneform bacteria, the vector is cleaved with a restriction enzyme that matches the end of the yggB gene. This restriction enzyme site may be introduced in advance into a synthetic oligonucleotide used for yggB amplification. Ligation is usually performed using a ligase such as T4 DNA ligase.

In order to introduce the recombinant plasmid prepared as described above into a coryneform bacterium, transformation methods that have been reported so far may be used. For example, as reported for Escherichia coli K-12, a method in which recipient cells are treated with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A., J. Mol. Biol. , 53, 159 (1970)), and a method for introducing competent cells from proliferating cells and introducing DNA as reported for Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE). Gene, 1, 153 (1977)). Alternatively, DNA-receptive cells, such as those known for Bacillus subtilis, actinomycetes and yeast, are introduced into the DNA-receptor cells in a protoplast or spheroplast state that readily takes up recombinant DNA. (Chang, S. and Choen, SN, Mol. Gen. Genet., 168, 111 (1979); Bibb, MJ, Ward, JMandHopwood, OA, Nature, 274, 398 (1978); Hinnen, A., Hicks, JBand Fink, GR, Proc. Natl. Acad. Sci. USA,
75 1929 (1978)) can also be applied. In addition, transformation of coryneform bacteria can also be performed by the electric pulse method (Japanese Patent Laid-Open No. 2-207791) or the junction transfer method (Biotechnology (NY). 1991 Jan; 9 (1): 84-7). Can do.

  Increasing the copy number of the yggB gene can also be achieved by having multiple copies of the yggB gene on the chromosomal DNA of coryneform bacteria. In order to introduce a plurality of copies of the yggB gene into the chromosomal DNA of a coryneform bacterium, homologous recombination is performed using a sequence present in the chromosomal DNA as a target. As a sequence present in multiple copies on chromosomal DNA, repetitive DNA and inverted repeats present at the end of a transposable element can be used. Alternatively, as disclosed in Japanese Patent Application Laid-Open No. 2-109985, it is also possible to mount the yggB gene on a transposon, transfer it, and introduce multiple copies onto the chromosomal DNA. (JP-A-2-109985, JP-A-7-107976, Mol. Gen. Genet., 245, 397-405 (1994), Plasmid. 2000 Nov; 44 (3): 285-91). For amplification with a transposon, amplification with an artificial transposon described later can also be used.

Another possible method is to introduce the yggB gene into a replication origin that cannot replicate in the host, or a plasmid that has the ability to conjugate and transmit to a replication origin that cannot replicate in the host and amplify it on the chromosome. For example, vectors that can be used are pSUP301 (Simo et al., Bio / Technology 1, 784-791 (1983)), pK18 mob or pK19 mob (Schaefer et al., Gene
145, 69-73 (1994)), pGEM-T (Promega corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemisty 269: 32678-84; US-A 5487993) PCR (R) Blunt (Invitrogen, Groningen, Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpf et al., 1991, Journal of Bacteriology 173: 4510-4516) or pBGS8 (spratt et al., 1986, Gene, 41: 337-342) and the like. A plasmid vector containing the yggB gene is transferred into a coryneform bacterium by conjugation or transformation. The joining method is described, for example, in Schaefer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Transformation methods include, for example, Theirbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivinan (Bio / Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343- 347 (1994)).

In addition, as a means of increasing the expression of the yggB gene, the expression regulatory sequence such as the promoter of the yggB gene on the chromosomal DNA or plasmid is replaced with a strong one, and factors involved in the expression regulation of the yggB gene, such as operators and It can also be achieved by modifying the presser or linking a strong terminator downstream of the stop codon of the gene. (Hamilton et al ,; Journal of Bacterology 171: 4617-4622) For example, lac promoter, trp promoter, trc promoter, PS2 promoter and the like are known as strong promoters. Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1995, 1, 105-128). In addition, as disclosed in International Publication WO00 / 18935, it is possible to introduce a base substitution of several bases into the promoter region of the target gene, replace it with a sequence that is closer to consensus, and modify it to be stronger. . For example, it is conceivable to replace the −35 region with TTGACA and TTGCCA sequences and the −10 region with TATAAT and TATAAC sequences. Furthermore, it is known that substitution of several nucleotides in the spacer between the ribosome binding site (RBS) and the start codon, especially in the sequence immediately upstream of the start codon, has a great influence on the translation efficiency of mRNA, These can be modified. Here, the expression regulatory region of the yggB gene means a region that affects the expression level of the yggB gene, and includes, for example, an upstream region of the yggB gene. Here, as a region suitable for modification upstream of the yggB gene, a region before the start codon (for example, a region before the start codon of SEQ ID NO: 5 (before base sequence number 1436)), preferably a region of 500 bp upstream of the start codon, More desirably, a region of 300 bp upstream of the initiation codon is included.
The replacement of the expression regulatory sequence can be performed using, for example, the above-described temperature sensitive plasmid. The modification of the expression regulatory sequence may be combined with increasing the copy number of the yggB gene.

<II> Introduction of mutant yggB gene
Examples of the modification using the yggB gene include introduction of a mutant yggB gene into a coryneform bacterium. Here, “introduction of mutant yggB gene” may be introduction of mutation into yggB gene on the chromosome of coryneform bacteria, or introduction of a plasmid containing mutant yggB gene into coryneform bacteria. It may be a substitution of a yggB gene on a chromosome with a mutant yggB gene.
In the present invention, the “mutant yggB gene” is a mutation in the coding region of the yggB gene, and has a function of improving the ability to produce L-glutamic acid under conditions containing an excess amount of biotin in a coryneform bacterium. It means the yggB gene containing the mutation given to The mutant yggB gene, when introduced into coryneform bacteria, is a gene that improves L-glutamic acid-producing ability not only under conditions containing an excess amount of biotin but also under the conditions for producing L-glutamic acid as described above. Also good.
“Improved L-glutamic acid-producing ability under conditions containing an excessive amount of biotin” means that the coryneform bacterium of the present invention contains biotin at a concentration such that an unmodified strain cannot accumulate L-glutamic acid, For example, when cultured in a medium containing biotin at a concentration of 30 μg / L or more, the coryneform bacterium of the present invention accumulates a larger amount of L-glutamic acid in the medium than in the unmodified strain, or It means that L-glutamic acid is produced at a faster rate than that of the unmodified strain.

Hereinafter, a method for obtaining a mutant yggB gene of the present invention and a method for introducing a mutation into the yggB gene will be described. However, the method for obtaining a mutant yggB gene and the method for introducing a mutation into the yggB gene are not limited to the following methods.
(II-1) Method using odhA gene-deficient strain As a method for obtaining a mutant yggB gene, a strain lacking the odhA gene (sucA gene) encoding the E1o subunit of α-ketoglutarate dehydrogenase (hereinafter referred to as odhA). The present inventor has discovered that a destructive strain can be used. Construction of an odhA-disrupted strain can be performed by the method using the sacB gene as described above.

  In the present invention, α-ketoglutarate dehydrogenase (α-KDGH) activity refers to a reaction that oxidatively decarboxylates α-ketoglutarate (2-oxoglutarate) to produce succinyl-CoA (succinyl-CoA). Activity. The above reaction is catalyzed by three enzymes, α-ketoglutarate dehydrogenase (E1o EC1.2.4.2), dihydrolipoamide S-succinyltransferase, and E3 dihydrolipoamide dehydrogenase. In Escherichia coli, etc., these three enzymes form a complex. α-ketoglutarate dehydrogenase is also called oxoglutarate dehydrogenase (succinyl-transferase) or 2-oxoglutarate dehydrogenase. The α-ketoglutarate dehydrogenase activity can be measured according to the method of Shiio et al. (Isamu Shiio and Kyoko Ujigawa-Takeda, Agric. Biol. Chem., 44 (8), 1897-1904, 1980).

  The sequence of the odhA gene encoding the E1o subunit of the α-ketoglutarate dehydrogenase of coryneform bacteria has already been elucidated (Microbiology 142, 3347-3354, (1996), GenBank accession No. D84102). The base sequence of the odhA gene is shown in base numbers 443 to 4213 of SEQ ID NO: 43, and the amino acid sequence is shown in SEQ ID NO: 44. Based on the sequence of this odhA gene, for example, a primer complementary to the odhA gene as shown in SEQ ID NOs: 1 and 2 is designed, and PCR is performed using the chromosomal DNA of the ATCC13869 strain, which is a wild strain, as a template. Amplify only internal sequences. An internal fragment of the odhA gene is ligated to a plasmid to construct an odh disruption plasmid. As the plasmid used here, a temperature-sensitive plasmid for coryneform bacteria (Japanese Patent Laid-Open No. 05-007491), or pBS3, which is a suicide vector carrying levan sucrose described in Example 1, is used.

  The plasmid electric pulse method for disruption of odh (Japanese Patent Laid-Open No. 2-207791) was introduced into a strain that cannot accumulate L-glutamic acid under conditions where biotin is sufficiently present in the medium, for example, a wild strain, C. glutamicum ATCC13869, A single recombination strain that has undergone homologous recombination with the odhA gene is obtained. When a temperature sensitive plasmid is used, a recombinant strain is obtained once at a temperature at which the plasmid cannot replicate. Confirmation of the single-recombination strain can be confirmed, for example, by introducing the plasmid onto the chromosome using the oligonucleotides of SEQ ID NO: 3 and SEQ ID NO: 4.

The odhA-disrupted strain thus obtained is purified with a medium containing sugar. Natural mutations are frequently introduced into the yggB gene during the purification process. The glutamic acid-producing ability of the purified strain is confirmed by culturing in a medium containing an excessive amount of biotin. For example, 20 ml of L-glutamic acid production flask medium (glucose 30 g / l, ammonium sulfate 15 g / l, KH ₂ PO ₄ 1 g / l, MgSO ₄ .7H ₂ O 0.4 g / l, FeSO ₄ .7H ₂ O 0.01 g / l, MnSO ₄ · 4-5H ₂ O 0.01g / l, VB1 (vitamin B1) 200μg / l, Biotin 300μg / l, soybean hydrolyzate (TN total nitrogen) 0.48g / l, adjusted to pH 8.0 using KOH : Inoculate candidate strain in autoclave 115 ° C for 10 minutes), add 1g of calcium carbonate sterilized by dry heat in advance, shake culture at 31.5 ° C, complete consumption of sugar, confirm accumulation of L-glutamic acid To do. For a strain showing a yield of L-glutamic acid with respect to sugar of 50% or more, the mutation can be identified by determining the sequence of the yggB gene.

  In order to construct a strain having only the yggB mutation, the odhA gene on the chromosome destroyed by the plasmid may be restored to the wild type. In odhA-deficient strains, growth in a medium containing no sugar is remarkably slow, but if odhA reverts to the wild type, medium containing no sugar, such as CM2B (polypeptone 10 g / l, yeast extract 10 g / l, NaCl 5 g / l, Biotin 10 μg / L, agar 20 g / l, adjusted to pH 7.0 with KOH). Therefore, this odhA-disrupted strain is applied to a CM2B plate to induce a growth-improved strain. If the growth-improving strain that has emerged in this way is purified with a CM2B plate, and its antibiotic resistance sensitivity and the presence or absence of the remaining vector are examined, it can be estimated that the strain is an odhA-reverted strain. Further, by determining the base sequence of the odhA gene, it is confirmed that the odhA gene is a wild type.

  Alternatively, the mutant yggB gene region of the odhA yggB double mutant may be cloned by PCR and introduced into a wild strain. Using the chromosomal DNA of the double mutant strain as a template, PCR is performed using the synthetic DNAs shown in SEQ ID NO: 9 and SEQ ID NO: 10 to amplify the mutant yggB gene. The amplified product thus obtained was cloned into a temperature-sensitive plasmid for coryneform bacteria (Japanese Patent Laid-Open No. 05-007491), or the suspension vector pBS3 described in Example 1 and wild-type by the method using the sacB gene as described above. A single yggB mutant can be constructed by substituting the type yggB gene. Here, whether or not a mutation has been introduced into the yggB gene on the chromosome can be confirmed by determining the gene sequence of the mutant yggB gene.

(II-2) Method Using Transposable Factor Coryneform bacteria having a mutant yggB gene may be screened using a transposable factor that functions in coryneform bacteria. A transposable element includes an insertion sequence (IS element) and a transposon. The mutant yggB gene may be obtained by accidental insertion of an insertion sequence (IS factor) or a transposon into the wild-type yggB gene, or may be artificially constructed using an artificial transposon. A strain into which a transposable element has been inserted can be selected using, for example, a decrease in sensitivity to an L-glutamic acid analog as an index. As an analog of L-glutamic acid, 4-fluoroglutamic acid can be used. A transposable element insertion strain can also be selected by randomly selecting a drug resistant strain using an artificial transposon containing a drug resistant gene and confirming the yggB gene length of the resistant strain by PCR.

  As a method for introducing an artificial transposon into coryneform bacteria, the method described in JP-A-09-070291 can be used. The artificial transposon means a marker gene sandwiched between inverted repeats (IR) at both ends of a transposase structural gene, an insertion sequence (IS factor). The structural gene for transposase may be present on the same plasmid as the artificial transposon, may be present on a different plasmid, or may utilize the function of the transposon present on the host chromosome. Good. For example, the following sequences can be used as a gene encoding transposase of coryneform bacteria.

1. NCgl0179 Cgl0182; transposase
2. NCgl0235 Cgl0238; putative transposase
3. NCgl0348 Cgl0355; putative transposase
4. NCgl0688 Cgl0718; putative transposase
5. NCgl0919 Cgl0959; transposase
6. NCgl0993 Cgl1037; transposase
7. NCgl1021 Cgl1066; transposase
8. NCgl1464 Cgl1521; putative transposase
9. NCgl1496 Cgl1557; transposase
10. NCgl1662 Cgl1733; putative transposase
11. NCgl1664 Cgl1734; transposase
12. NCgl2131 Cgl2212; transposase
13. NCgl2284 Cgl2367; transposase
14. NCgl2392 Cgl2479; putative transposase
15. NCgl2418 Cgl2504; putative transposase
16. NCgl2420 Cgl2506; putative transposase
17. NCgl2460 Cgl2548; predicted transposase
18. NCgl2542 Cgl2631; predicted transposase
19. NCgl2665 Cgl2761; putative transposase
20. NCgl2748 Cgl2845; putative transposase
21. NCgl2850 Cgl2951; predicted transposase

The artificial transposon is introduced into a host coryneform bacterium mounted on an appropriate vector such as a plasmid. As a parent strain used for screening, a strain that cannot accumulate L-glutamic acid under conditions containing an excessive amount of biotin, for example, ATCC13869 strain, which is a wild strain of C. glutamicum, is desirable. Although there is no restriction | limiting in particular as a plasmid carrying an artificial transposon, Usually, what is necessary is just to use the plasmid derived from coryneform bacteria. Specifically, pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)), pAM330 (Agric. Biol. Chem., 48, 2901-2903 (1984)), and improved drug resistance thereof For example, a plasmid having a gene. Furthermore, in order to efficiently amplify the introduced artificial transposon on the chromosome, it is preferable to use a plasmid having a temperature-sensitive replication origin as described in the above (1) (see JP-A-5-7491).
As a method for introducing a plasmid carrying an artificial transposon into a coryneform bacterium, a commonly used protoplast method (Gene, 39, 281-286 (1985)), electroporation method (Bio / Technology, 7, 1067-1070 ( 1989)) or the like may be used.

When an artificial transposon is mounted on a temperature-sensitive plasmid and introduced into a coryneform bacterium, the coryneform bacterium is transformed with the constructed plasmid and cultured at 25 ° C. where the plasmid can be replicated. It is only necessary to amplify the copy-embedded transposon-carrying plasmid and introduce it into the chromosome, and then remove it by culturing at 34 ° C. This method efficiently amplifies the gene on the chromosome. In addition, there is a method of introducing into a chromosome of coryneform bacteria using a DNA fragment only of an artificial transposon or a plasmid vector that cannot replicate in coryneform bacteria (for example, a plasmid vector that replicates in Escherichia coli).
(JP-A-7-107976, Vertes, AA, Asai, Y., Inui, M., Kobayashi, M., Kurusu, Y. and Yukawa, H .: Mol. Gen. Genet., 245, 397-405 ( 1994)).

  In this manner, the strain in which the artificial transposon is inserted on the chromosome is cultured in an L-glutamic acid production medium containing an excessive amount of biotin, and a strain that accumulates L-glutamic acid is obtained. By determining the base sequence of the yggB gene on the chromosome of a strain that accumulates L-glutamic acid, a coryneform bacterium having a mutant yggB gene can be obtained.

(II-3) Method for randomly introducing mutations into yggB gene in vitro In addition, mutant yggB genes are randomly introduced in vitro, and L-glutamic acid is added without adding a surfactant or the like. It is also possible to select from clones that can be produced. As a parent strain used for screening, strains that cannot accumulate L-glutamic acid under the condition that an excess amount of biotin is contained, for example, ATCC13869 strain, ATCC13032 strain, ATCC14067 strain, C.melasecola wild strain, which are C. glutamicum wild strains, are used. The stock ATCC17965 is preferred.

  In order to screen for mutant genes, a yggB-deficient strain is first constructed. Construction of a yggB gene-disrupted strain can be performed by a method similar to the method using the sacB gene described above. For example, PCR is performed using a primer complementary to the coding region of the yggB gene, for example, the synthetic DNA shown in SEQ ID NO: 39 and the synthetic DNA shown in SEQ ID NO: 40 as a template, and the chromosomal DNA of C. glutamicum ATCC 13869 strain, and the yggB gene An N-terminal fragment of is prepared. Similarly, a C-terminal fragment is prepared using the synthetic DNAs of SEQ ID NO: 41 and SEQ ID NO: 42 as primers. SEQ ID NOs: 40 and 41 are complementary to each other. Subsequently, by performing PCR using a mixture of equal amounts of N-terminal fragment and C-terminal fragment as a template and using the synthetic DNAs of SEQ ID NO: 39 and SEQ ID NO: 42 as primers, a fragment lacking the ORF of the yggB gene can be obtained. .

  The obtained PCR fragment can be inserted into a coryneform bacterium gene disruption vector, for example, pBS4S carrying levanshuclease, to construct a plasmid for introducing a deletion mutation. The yggB-disrupting plasmid thus obtained is inserted into the chromosome of the coryneform bacterium C. glutamicum ATCC13869 strain to construct a yggB-deficient strain.

On the other hand, the mutation process of the yggB gene is performed as follows. First, the yggB gene is cloned into a plasmid that can replicate in coryneform bacteria. The resulting yggB amplification vector is dissolved in a buffer containing a mutagen, such as 500 mM phosphate buffer, 400 mM hydroxylamine, 1 mM EDTA (pH 6.0), and mutation is introduced by treating at 75 ° C. for 60 to 90 minutes. To do. The mutated plasmid is desalted with SUPREC-02 (Takara Bio) and the like, then introduced into the ATCC 13869ΔyggB strain, and a transformant is selected in a medium containing an antibiotic. As a control, the yggB amplified plasmid before the mutation treatment is also introduced into the ATCC13869ΔyggB strain. The resulting transformant is inoculated into an L-glutamic acid-producing liquid medium containing an excessive amount of biotin, and after culturing, the glutamic acid concentration is quantified. Glutamate is hardly increased in the wild-type yggB amplified plasmid-introduced strain, but there are strains that significantly accumulate glutamate in the strain into which the mutated plasmid has been introduced. A novel mutant yggB gene can be obtained by extracting a plasmid from such a strain and determining the base sequence of the yggB gene. Also, error-prone PCR, DNA shuffling,
A mutant yggB gene can also be obtained by artificially introducing a mutation into the yggB gene by genetic recombination by StEP-PCR. (Firth AE, Patrick WM; Bioinformatics. 2005 Jun 2; Statistics of protein library construction.)
As a method for introducing a mutation into the yggB gene on the chromosome as described above, in addition to the method described above, a method of irradiating a coryneform bacterium with X-rays or ultraviolet rays, or a coryneform bacterium with N-methyl-N ′ There is a method of treating with a mutation agent such as nitro-N-nitrosoguanidine. Whether or not the mutant yggB gene has been introduced by this method can be confirmed by determining the base sequence of the yggB gene on the chromosome or generating L-glutamic acid in a medium containing an excessive amount of biotin.

(II-4) Method for obtaining L-glutamic acid analog resistant strain The mutant yggB gene is obtained by culturing a coryneform bacterium having a wild-type yggB gene in a medium containing L-glutamic acid analog, and growing in the same medium. -It can also be obtained by obtaining a glutamate analog resistant strain. As the parent strain used for screening, the above-mentioned coryneform bacterium wild strain is preferable, but any strain may be used as long as it has a wild-type yggB gene. A coryneform bacterium having a plasmid carrying a wild-type yggB gene may also be used.
L-glutamic acid analogs include L-glutamic acid-γ-methyl ester, α-methylglutamic acid, β-hydroxyglutamic acid, methionine sulfoximine, glutamic acid-γ-monohydroxamate, 2-amino-4-phosphonobutyric acid, L-glutamic acid-γ-monoethyl ester, L-glutamic acid dimethyl ester, L-glutamic acid-di-t-butyl ester, monofluoroglutamic acid, L-glutamic acid diethyl ester, D-glutamic acid, 4-fluoroglutamic acid, 4-Fluoroglutamic acid analogs can be used.
L-glutamic acid analog resistant strains are obtained by the following method. Coryneform bacteria are inoculated into a minimal medium supplemented with an L-glutamic acid analog, and a strain that has formed colonies after 24 to 48 hours is obtained. The concentration of the L-glutamic acid analog contained in the medium is preferably a concentration at which coryneform bacteria in which the yggB gene is not modified cannot grow and coryneform bacteria in which a mutation is introduced into the yggB gene can grow. More specifically, when 4-fluoroglutamic acid is used, 1.25 mM, preferably 2.5 mM, more preferably 5 mM is preferred. Here, the L-glutamic acid analog resistance of the present invention means that when 4-fluoroglutamic acid is added to a minimal medium and cultured overnight, the number of viable bacteria of the parent strain (the number of bacteria having colony forming ability) is reduced to 1/100. It means that the growth is 1/10 or more even at a concentration that can be suppressed.
The obtained L-glutamic acid analog resistant strain is inoculated into an L-glutamic acid-producing liquid medium containing an excessive amount of biotin, and after culturing, the glutamic acid concentration is quantified. L-glutamic acid hardly increases in the wild type strain, but there are strains that significantly accumulate L-glutamic acid in the L-glutamic acid analog resistant strain. A novel mutant yggB gene can be obtained by amplifying the yggB gene from such a strain by PCR and determining the base sequence of the yggB gene.

<III> Mutant yggB gene of the present invention Specific examples of the mutant yggB gene are given below. However, the mutant yggB gene is not particularly limited as long as it is a mutation that can improve L-glutamic acid-producing ability under the condition that an excess amount of biotin is present in coryneform bacteria.

(III-1) C-terminal mutation This mutation is the nucleotide sequence of the region encoding the sequence of amino acid numbers 419-533 of SEQ ID NO: 6, 68, 84 or 85, or the sequence of amino acid numbers 419-529 of SEQ ID NO: 62 It is a mutation introduced in a part of. For example, this region corresponds to nucleotide numbers 2692-3035 in the nucleotide sequence of SEQ ID NO: 5. Any mutation may be introduced as long as a mutation is introduced into at least a part of the base sequence of the above region, but an insertion sequence (hereinafter referred to as IS) or a transposon inserted is preferable. The mutation may be any of those involving amino acid substitution (missense mutation), those having a frameshift mutation introduced by insertion of the IS, and those having a nonsense mutation introduced.

(III-1-1) Mutation caused by insertion of a transposable element (type 2A-1)
An example of a C-terminal mutation is a mutation in which IS is inserted after 2691st G of SEQ ID NO: 5. The nucleotide sequence of the mutant yggB gene into which this mutation has been introduced is shown in SEQ ID NO: 7, and the amino acid sequence of the mutant YggB protein encoded by the gene is shown in SEQ ID NO: 8. In SEQ ID NO: 8, the region downstream from valine at position 419 of SEQ ID NO: 6 is replaced with a sequence derived from short IS. The IS inserted in SEQ ID NO: 7 is a sequence having high homology with IS1207 (Genbank accession No. X96962) and IS719: (Genbank accession No E12759).
The mutant yggB gene is referred to as 2A-1 mutation. The 2A-1 type mutation includes a mutation that deletes or replaces the region on the C-terminal side of SEQ ID NOs: 6, 62, 68, 84, and 85.
In addition, a case in which another IS is inserted into the region, for example, a case in which an IS encoding a transposase as described above is inserted is also included in the scope of the present invention. The position where the transposase is introduced may be any position in the above region, but a position where each transposase is easily recognized or a position of a hot spot where IS is easily inserted is preferable.

(III-1-2) Mutations that substitute proline residues with other amino acids (type 66 mutation, type 22 mutation)
In addition, as an example of the C-terminal mutation, the amino acid 419-533 sequence of SEQ ID NO: 6, 68, 84 or 85, or proline present in the region of amino acid numbers 419-529 of SEQ ID NO: 62 is substituted with another amino acid. Mutations. The proline which may be substituted is present in the following position of SEQ ID NO: 6.
424th proline residue (position 424 of SEQ ID NOs: 62, 68, 84, 85)
437th proline residue (position 437 of SEQ ID NOs: 62, 68, 84, 85)
453rd proline residue (453th position of SEQ ID NOs: 62, 68, 84, 85)
The 457th proline residue (the 457th of SEQ ID NO: 62, 68, 84, 85)
462th proline residue (# 462 of SEQ ID NOs: 62, 68, 84, 85)
469th proline residue (469th position of SEQ ID NOs: 62, 68, 84, 85)
The 484th proline residue (the 484th position of SEQ ID NOs: 62, 68, 84, 85)
489th proline residue (489th position of SEQ ID NOs: 62, 68, 84, 85)
497th proline residue (position 497 of SEQ ID NOs: 62, 68, 84, 85)
515th proline residue (the 515th of SEQ ID NO: 62, 68, 84, 85)
529th proline residue (SEQ ID NO: 68, 84, 529 of 85, SEQ ID NO: 62, 525)
533th proline residue (533th in SEQ ID NO: 68, 84, 85, 529th in SEQ ID NO: 62)
The proline residue on the C-terminal side of the yggB gene is thought to play an important role in maintaining the three-dimensional structure of the YggB protein. (Protein Eng. 2002 Jan; 15 (1): 29-33, J Biol Chem. 1991 Dec 25; 266 (36): 24287-94.)
In particular, it is desirable to substitute the 424th and 437th prolines with other amino acids.
Here, the other amino acids may be any amino acids other than proline and natural amino acids, such as Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met. , Phe, Trp, Tyr, Gly, Ala, His may be substituted. In particular, the 424th proline is desirably substituted with hydrophobic amino acids Ala, Gly, Val, Leu, and Ile, and among them, branched chain amino acids Leu, Val, and Ile are desirable. (Type 66 mutation) As a mutation that substitutes 424th proline with leucine, for example, a mutation that substitutes “C” at position 1673 of SEQ ID NO: 67 with “T” can be mentioned. This mutant yggB gene is shown in SEQ ID NO: 69, and the amino acid sequence of the mutant YggB protein encoded by the gene is shown in SEQ ID NO: 70.
The 437th proline is preferably substituted with an amino acid (Thr, Ser, Tyr) having a hydroxyl group in the side chain, and among these, substitution with Ser is desirable. (Type 22 mutation) As a mutation for substituting the 437th proline with serine, for example, a mutation for substituting C at position 2745 of SEQ ID NO: 5 with T can be mentioned. This mutation may be accompanied by a mutation that substitutes C at position 3060 with T. This mutant yggB gene is shown in SEQ ID NO: 73, and the amino acid sequence of the mutant YggB protein encoded by the gene is shown in SEQ ID NO: 74.

(III-2) Mutation of transmembrane region
The YggB protein encoded by the yggB gene is presumed to have 5 transmembrane regions. In the amino acid sequences of wild-type YggB proteins of SEQ ID NOs: 6, 62, 68, 84, and 85, the transmembrane regions are amino acid numbers 1 to 23 (first transmembrane region) and 25 to 47 (second transmembrane region), respectively. ), 62 to 84 (third transmembrane region), 86 to 108 (fourth transmembrane region), and 110 to 132 (fifth transmembrane region). The DNAs encoding these correspond to the 1437-1505, 1509-1577, 1620-1688, 1692-1760, and 1764-1832 bases of SEQ ID NO: 5. The mutation of the present invention preferably has a mutation in the DNA encoding this transmembrane region, and mutagenesis is a mutation including substitution, deletion, addition, insertion or inversion of one or several amino acids. It is desirable that it does not involve a frameshift mutation or a nonsense mutation. Accordingly, in the case of amino acid sequence substitution, a missense mutation accompanied by amino acid substitution is preferable, and the above several substitutions, deletions, additions, insertions or inversions are 2 to 20, preferably 2 to 10 More preferably, it means 2 to 5, more preferably 2 to 3. In addition, amino acid insertion / deletion mutations can be introduced by introducing point mutations of 1 to several bases or base sequences without frameshift mutations, for example, 3, 6, 9, 12, 15, 18, or 21 bases. An insertion or deletion, preferably a 3, 6 or 9 base deletion or insertion, more preferably a 3 base sequence deletion or insertion is desired.

For example, the following mutations are exemplified.
(III-2-1) Mutation of the first transmembrane region (A1 mutation)
This mutation is a mutation in which one or several amino acids are inserted between the 14th leucine residue and the 15th tryptophan residue in the amino acid sequence shown in SEQ ID NOs: 6, 62, 68, 84, 85.
Specific examples include those in which 3 amino acids are introduced between the 14th leucine residue and the 15th tryptophan residue, for example, in which Cys-Ser-Leu is inserted. A mutation in which TTCATTGTG is inserted after the 1480th G of No. 5 can be mentioned. The nucleotide sequence of the mutant yggB gene into which this mutation has been introduced is shown in SEQ ID NO: 19, and the amino acid sequence of the mutant YggB protein encoded by the gene is shown in SEQ ID NO: 20.

(III-2-2) Mutation in the fourth transmembrane region (19 type mutation)
This mutation is a mutation in which the 100th alanine residue is substituted with another amino acid residue in the amino acid sequences shown in SEQ ID NOs: 6, 62, 68, 84, and 85. The other amino acid may be any amino acid residue other than alanine, and the other amino acids are arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, methionine, leucine, lysine, phenylalanine. , Proline, serine, tryptophan, tyrosine, valine, threonine, but it is desirable to be substituted with an amino acid having a hydroxyl group in the side chain (threonine, serine, tyrosine), especially threonine residues. It is desirable. An example is a mutation in which the 1734th G in SEQ ID NO: 5 is replaced with A.
The nucleotide sequence of the mutant yggB gene into which this mutation has been introduced is shown in SEQ ID NO: 21, and the amino acid sequence of the mutant YggB protein encoded by the gene is shown in SEQ ID NO: 22.

(III-2-3) Mutation in the fifth transmembrane region (L30 type mutation, type 8 mutation)
This mutation is a mutation in which the 111th alanine residue is substituted with another amino acid residue in the amino acid sequences shown in SEQ ID NOs: 6, 62, 68, 84, and 85. The other amino acid may be any amino acid residue other than alanine, and the other amino acids are arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, methionine, leucine, lysine, Phenylalanine, proline, serine, tryptophan, tyrosine, valine, threonine, but especially branched chain amino acids (valine, isoleucine, leucine), especially amino acids with valine residues and hydroxyl groups in the side chain (threonine, serine, tyrosine) ), In particular, a threonine residue. Examples of this mutation include a mutation in which the 1768th C in SEQ ID NO: 5 is replaced with T (L30 type mutation) and a mutation in which the 837th G in SEQ ID NO: 61 is replaced with A (type 8 mutation). It is done. The nucleotide sequence of the mutant yggB gene into which the L30 mutation has been introduced is shown in SEQ ID NO: 23, and the amino acid sequence of the mutant YggB protein encoded by the gene is shown in SEQ ID NO: 24. The nucleotide sequence of the mutant yggB gene into which the type 8 mutation has been introduced is shown in SEQ ID NO: 63, and the amino acid sequence of the mutant YggB protein encoded by the gene is shown in SEQ ID NO: 64.

The above-mentioned “mutant yggB gene” is a functionally equivalent gene containing the above-mentioned mutation, for example, the base of SEQ ID NO: 7, as long as it has a function of improving L-glutamic acid producing ability under conditions where biotin is present in excess. DNA comprising the nucleotide sequence of numbers 1437 to 2705, base number 1 of SEQ ID NO: 19
DNA containing the base sequence of 437 to 3044, DNA containing the base sequence of base number 1437 to 3035 of SEQ ID NO: 21, DNA containing the base sequence of base number 1437-3035 of SEQ ID NO: 23, base number 507- of SEQ ID NO: 63 Substantially homologous to a mutant gene such as DNA containing the base sequence of 2093, DNA containing the base sequence of base numbers 403-2001 of SEQ ID NO: 69, DNA containing the base sequence of base numbers 548-2146 of SEQ ID NO: 73 Such a gene may be a gene that hybridizes under stringent conditions with a polynucleotide containing a base sequence complementary thereto or a probe having a part of the base sequence. Here, the stringent condition refers to a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, normal Southern hybridization washing conditions can be mentioned, specifically, 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, more preferably The conditions include washing at a salt concentration and temperature corresponding to 68 ° C., 0.1 × SSC, 0.1% SDS, and more preferably once to 2-3 times.

  As a protein encoded by the mutant yggB gene, a functionally equivalent protein, for example, SEQ ID NO: 8, 20 as long as the activity of the protein has a function of improving L-glutamic acid-producing ability under the condition that an excess amount of biotin is present. , 22, 24, 64, 70, 74, which has an amino acid sequence that includes substitution, deletion, insertion, or addition of one or several amino acids in addition to the amino acid at the mutation point. It may be. Here, several means, for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5. The above-mentioned substitution is preferably conservative substitution (neutral mutation), and as conservative substitution, substitution from ala to ser or thr, substitution from arg to gln, his or lys, asn to glu, gln, lys, his or asp substitution, asp to asn, glu or gln substitution, cys to ser or ala substitution, gln to asn, glu, lys, his, asp or arg substitution, glu to gly, asn, gln, lys or asp substitution, gly to pro substitution, his to asn, lys, gln, arg or tyr substitution, ile to leu, met, val or phe, leu to ile, met, val or substitution from phe, substitution from lys to asn, glu, gln, his or arg, substitution from met to ile, leu, val or phe, substitution from phe to trp, tyr, met, ile or leu, from ser Substitution from thr or ala, substitution from thr to ser or ala, substitution from trp to phe or tyr, substitution from tyr to his, phe or trp, and substitution from val to met, ile or leu It is done. In addition, as described above, the Xaa amino acid of SEQ ID NO: 85 may be substituted. Furthermore, the yggB gene of the present invention is 80% or more, preferably 90% or more, more preferably 95% or more, particularly with respect to the entire amino acid sequence of SEQ ID NOs: 8, 20, 22, 24, 64, 70, 74. Preferably, it includes a homolog that encodes a protein having a homology of 97% or more and that encodes a protein having an activity equivalent to that of a protein encoded by mutant yggB in coryneform bacteria.

In particular, the following amino acids may be substituted in the amino acid sequence of SEQ ID NOs: 8, 20, 22, 24, 64, 70, 74.
Glu at position 48 (preferably substituted with Arg)
Asp at position 275 (preferably replaced with Ser)
Glu at position 298 (preferably substituted with Ala)
Ala at position 343 (preferably substituted with Val)
Phe at position 396 (preferably replaced by Ile)
Ser at position 438 (preferably replaced by Gly)
Val at position 445 (preferably substituted with Ala)
Ala at position 454 (preferably substituted with Val)
Pro at position 457 (preferably replaced with Ser)
Ser at position 474 (preferably replaced with Asp)
Val 517 (preferably deletion)
Glu at position 518 (preferably deleted)
Ala at position 519 (preferably deleted)
Pro at position 520 (preferably deletion)

<V> Method of introducing mutant yggB gene into coryneform bacterium The mutant yggB gene having the mutation as described above can be obtained, for example, by site-specific mutagenesis. More specifically, using a PCR primer having a sequence containing a mutation in the corresponding region of the yggB gene and using an overlap extension PCR method for amplifying the corresponding site, the yggB gene containing the mutation is amplified and cloned. (Urban,
A., Neukirchen, S. and Jaeger, KE, A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR. Nucleic Acids Res, 25, 2227-8. (1997)).
The coryneform bacterium of the present invention can be obtained by introducing the obtained mutant yggB gene into a coryneform bacterium. Examples of the introduction method include a method of replacing a wild-type yggB gene on a chromosome with a mutant yggB gene. The mutant yggB gene may be introduced into a strain in which the wild-type yggB gene on the chromosome is disrupted. As in the case of a single recombination strain, the mutant yggB gene may be introduced while leaving the wild-type yggB gene on the chromosome. May be present above. Replacement of the yggB gene on the chromosome can be performed using, for example, a temperature-sensitive plasmid containing the above-described sacB gene encoding levan sucrose.

  The coryneform bacterium of the present invention may have improved resistance to L-glutamic acid analogs by introducing a mutant yggB gene. In the present invention, L-glutamic acid analog means L-glutamic acid-γ-methyl ester, α-methylglutamic acid, β-hydroxyglutamic acid, methionine sulfoximine, glutamic acid-γ-monohydroxamate, 2-amino-4- Examples include phosphonobutyric acid, L-glutamic acid-γ-monoethyl ester, L-glutamic acid dimethyl ester, L-glutamic acid-di-t-butyl ester, monophloglutamic acid, L-glutamic acid diethyl ester, D-glutamic acid, 4-fluoroglutamic acid. It is done. Improved resistance to L-glutamic acid analog means that when 4-fluoroglutamic acid is added to a minimal medium and cultured overnight, the number of viable bacteria (the number of colony-forming bacteria) can be reduced to 1/100. It means that the growth is 1/10 or more even at various concentrations. Specifically, it means having resistance to 1.25 mM, preferably 2.5 mM, more preferably 5 mM or more of 4-fluoroglutamic acid.

Furthermore, a bacterium modified so as to inactivate a gene that suppresses the function of the mutant yggB gene may be used. Here, “suppressing the function of the yggB gene” means suppressing the production of glutamic acid in the mutant yggB gene-introduced strain by amplification of the gene. Examples of the gene that suppresses the function of the mutant yggB gene include the symA gene (supresser of yggB mutation). The symA gene is the Genbank Accession of Corynebacterium glutamicum ATCC13032.
It is encoded by base number 2051306..2051845 in the genome sequence registered as No. NC_003450 and registered as NCgl 1867 (NP_601149. Hypothetical prot ... [gi: 19553147]). The symA gene of C. glutamicum ATCC13869 is represented by base numbers 585 to 1121 of SEQ ID NO: 86. Inactivation of a gene can be achieved by destroying the gene or modifying it so as to reduce the expression level, and the introduction of mutations that reduce the symA gene or reduce the expression level can result in a decrease in the enzyme activity described above. A similar method can be used.

In the present invention, in addition to the modification using the yggB gene, a bacterium modified so that the activity of α-ketoglutarate dehydrogenase (hereinafter referred to as α-KGDH) may be used may be used. The phrase “α-ketoglutarate dehydrogenase activity is reduced” means that the activity of α-ketoglutarate dehydrogenase is reduced with respect to an unmodified strain such as a wild strain or a parent strain. The α-ketoglutarate dehydrogenase activity can be measured according to the method of Shiio et al. (Isamu Shiio and Kyoko Ujigawa-Takeda, Agric. Biol. Chem., 44 (8), 1897-1904, 1980). The α-KGDH activity only needs to be reduced with respect to an unmodified strain such as a wild strain or a parent strain, but is about 1/2 or less, preferably 1/4 or less, more preferably 1/10 or less with respect to the parent strain. It is desirable that it is reduced to a low level, and it may not have any activity.

Coryneform bacteria with reduced α-ketoglutarate dehydrogenase activity can be constructed using the method described above.
Among them, it is preferable to introduce a mutation into the thiamine pyrophosphate binding region (region encoded by nucleotides 2498 to 2584 of SEQ ID NO: 43 (686Gly-714Asp of SEQ ID NO: 44)).
For example, as strains with decreased α-ketoglutarate dehydrogenase activity, Brevibacterium lactofermentum ΔS strain (International Publication WO95 / 34672 pamphlet), Brevibacterium lactofermentum AJ12821 (FERM BP-4172) strain ( JP-A-06-237779). Note that either α-ketoglutarate dehydrogenase weakening or mutant yggB gene introduction may be performed first.

<3> Method for producing L-glutamic acid of the present invention The coryneform bacterium obtained as described above is cultured in a medium, L-glutamic acid is produced and accumulated in the medium, and L-glutamic acid is collected from the medium. Thus, L-glutamic acid can be produced.

  As a medium used for the culture, a normal medium containing a carbon source, a nitrogen source, inorganic salts, and other organic micronutrients such as amino acids and vitamins as necessary can be used. Either synthetic or natural media can be used. Any type of carbon source and nitrogen source may be used as long as the strain to be cultured is available.

As the carbon source, saccharides such as glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolysate, molasses can be used, other organic acids such as acetic acid and citric acid, and alcohols such as ethanol alone. Or it can use together with another carbon source. As the nitrogen source, ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate, and ammonium acetate, nitrates, and the like can be used. Organic micronutrients include amino acids, vitamins, fatty acids, nucleic acids, and peptone, casamino acids, yeast extracts, soybean protein breakdown products, etc. containing these, and auxotrophic mutations that require amino acids for growth. When using a strain, it is preferable to supplement the required nutrients. As inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like can be used.
Further, an appropriate concentration of a surfactant and penicillin may be added according to the properties of the yggB gene-modified strain to be used, and the biotin concentration may be adjusted according to the properties of the yggB gene-modified strain to be used.

  The culture is preferably carried out with aeration while controlling the fermentation temperature at 20 to 45 ° C. and the pH at 3 to 9. When the pH is lowered during the culture, for example, calcium carbonate is added or neutralized with an alkali such as ammonia gas. Under such conditions, by culturing preferably for about 10 to 120 hours, a significant amount of L-glutamic acid is accumulated in the culture solution.

  Moreover, it can also culture | cultivate, making L-glutamic acid precipitate in a culture medium using the liquid culture medium adjusted to the conditions which L-glutamic acid precipitates. Examples of conditions for precipitation of L-glutamic acid 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. Can do. (European Patent Application Publication No. 1078989)

The method for collecting L-glutamic acid from the culture solution after completion of the culture may be performed according to a known recovery method. For example, it is collected by a method of concentrating crystallization after removing cells from the culture solution or ion exchange chromatography. When cultured under conditions where L-glutamic acid is precipitated, L-glutamic acid precipitated in the culture solution can be collected by centrifugation or filtration. In this case, L-glutamic acid dissolved in the medium may be crystallized and then isolated together.

[Example]
Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.

<Construction of sacB-loaded gene disruption vector>
(A) Construction of pBS3
Construction of the sacB-loaded gene disruption vector was performed with reference to the methods of International Publication Nos. WO2005113745 and WO2005113744. The sacB gene (SEQ ID NO: 11) was obtained by PCR using Bacillus subtilis chromosomal DNA as a template and SEQ ID NOs: 13 and 14 as primers. For the PCR reaction, LA taq (TaKaRa) was used, and after 1 cycle of incubation at 94 ° C. for 5 minutes, a cycle consisting of denaturation 94 ° C. for 30 seconds, association at 49 ° C. for 30 seconds, and extension at 72 ° C. for 2 minutes was repeated 25 times. The generated PCR product was purified by a conventional method, digested with BglII and BamHI, and smoothed. This fragment was digested with AvaII of pHSG299 and then inserted into the blunted site. Using this DNA, transformation was performed using Escherichia coli JM109 competent cells (manufactured by Takara Bio Inc.), applied to LB medium containing 25 μg / ml kanamycin (hereinafter abbreviated as Km), and cultured overnight. did. Thereafter, the emerged colonies were picked up, and single colonies were separated to obtain transformants. A plasmid was extracted from the obtained transformant, and the one into which the target PCR product was inserted was named pBS3. The construction process of pBS3 is shown in FIG.

(B) Construction of pBS4S
A plasmid in which the kanamycin resistance gene was disrupted by base substitution without amino group substitution (mutation that was not cleaved by SmaI) at the SmaI site in the kanamycin resistance gene sequence present on pBS3 was obtained by crossover PCR. First, PCR is performed using pBS3 as a template and the synthetic DNAs of SEQ ID NOS: 15 and 16 as primers to obtain an N-terminal amplification product of the kanamycin resistance gene. On the other hand, in order to obtain an amplification product on the C-terminal side of the Km resistance gene, PCR was performed using pBS3 as a template and synthetic DNAs of SEQ ID NOs: 17 and 18 as templates. The PCR reaction was carried out using Pyrobest DNA Polymerase (Takara Bio Inc.), followed by one cycle of incubation at 98 ° C for 5 minutes, followed by 25 cycles of denaturation 98 ° C for 10 seconds, association at 57 ° C for 30 seconds, and extension at 72 ° C for 1 minute. By repeating, the desired PCR product can be obtained. SEQ ID NOs: 16 and 17 are partially complementary, and the SmaI site present in this sequence is destroyed by making a base substitution without an amino acid substitution. Next, in order to obtain a mutant kanamycin resistance gene fragment in which the SmaI site is destroyed, the above kanamycin resistance gene N-terminal side and C-terminal side gene products are mixed so as to be approximately equimolar, and this is used as a template. PCR was carried out using the synthetic DNAs of SEQ ID NOs: 15 and 18 as primers to obtain a Km resistance gene amplification product into which mutation was introduced. For the PCR reaction, Pyrobest DNA Polymerase (Takara Bio Inc.) was used, and after 5 cycles of incubation at 98 ° C for 5 minutes, a cycle consisting of denaturation 98 ° C for 10 seconds, association at 57 ° C for 30 seconds, and extension at 72 ° C for 1.5 minutes was repeated 25 times. As a result, the desired PCR product can be obtained.

  The PCR product was purified by a conventional method, digested with BanII, and inserted into the BanII site of pBS3. Using this DNA, transformation was performed using a competent cell of Escherichia coli JM109 (manufactured by Takara Bio Inc.), applied to an LB medium containing 25 μg / ml of kanamycin, and cultured overnight. Thereafter, the emerged colonies were picked up and separated into single colonies to obtain transformants. A plasmid was extracted from the obtained transformant, and the one into which the target PCR product was inserted was named pBS4S. The construction process of pBS4S is shown in FIG.

<Construction of odhA mutant derived from C. glutamicum ATCC13869>
The sequence of the odhA gene encoding the α-ketoglutarate dehydrogenase of coryneform bacterium has already been clarified (Microbiology 142, 3347-3354, (1996), GenBank accession No. D84102). Based on the published sequence of the odhA gene, the primers described in SEQ ID NOs: 1 and 2 were designed, and PCR was performed using the chromosomal DNA of the ATCC13869 strain as a template to amplify only the internal sequence of the odhA gene. The amplified PCR fragment was completely digested with BamHI to construct a plasmid pBS4SΔsucAint inserted into the BamHI site of pBS4S constructed in Example 1 (construction diagram is described in FIG. 3).

Introducing pBS4SΔsucAint into C. glutamicum ATCC13869 by electric pulse method (Japanese Patent Laid-Open No. 2-207791), CM-Dex agar medium containing 25 μg / ml kanamycin (glucose 5 g / l, polypeptone 10 g / l, yeast extract 10 g / l, KH ₂ PO ₄ 1g / l, MgSO ₄・ 7H ₂ O 0.4g / l, FeSO ₄・ 7H ₂ O 0.01g / l, MnSO ₄・ 4-5H ₂ O 0.01g / l, urea 3g / l, soy protein Hydrolyzed solution 1.2 g / l, agar 20 g / l, adjusted to pH 7.5 using NaOH: Autoclave 120 ° C. for 20 minutes). After culturing at 31.5 ° C., the growing strain was confirmed by PCR to be a single recombination strain in which pBS4SΔsucAint was incorporated on the chromosome by homologous recombination. Confirmation of a single recombination strain should be performed by PCR using the candidate strain's chromosome as a template and the specific sequence on pBS4S (SEQ ID NO: 3) and the sequence on the chromosome (SEQ ID NO: 4) as primers. (The pBS4S sequence does not exist on the chromosome of the non-recombinant strain, so that the fragment amplified by PCR does not appear).

The single recombination strain thus obtained was named 2A-1 strain. Wild strains ATCC13869 and 2A-1 were placed in a 20 ml flask medium (glucose 30 g / l, ammonium sulfate 15 g / l, KH ₂ PO ₄ 1 g / l, MgSO ₄ · 7H ₂ O 0.4 g / l, FeSO ₄ · 7H ₂ O 0.01 g / l, MnSO ₄・₄ ~ 5H ₂ O 0.01g / l, VB1 200μg / l, Biotin
300 μg / l, soybean hydrolyzate (TN) 0.48 g / l, adjusted to pH 8.0 with KOH: autoclave 115 ° C for 10 minutes), then added 1 g of calcium carbonate that had been sterilized by dry heat in advance And cultured with shaking at 31.5 ° C. After completely consuming sugar, the L-glutamic acid concentration in the medium was measured. The results are shown in Table 1 (OD620 is a turbidity of 101-fold dilution and is an indicator of the amount of bacterial cells, and Glu (g / L) indicates the amount of L-glutamic acid accumulated). It was confirmed that the 2A-1 strain has the ability to produce L-glutamic acid even under the condition that the parent strain ATCC 13869 does not produce any L-glutamic acid.

<Construction of 2A-1-derived odhA-reverted strain>
In the obtained 2A-1 strain, the odhA gene on the chromosome is disrupted by pBS4SΔT \ sucAint. If the chromosomal plasmid is removed from this strain, odhA reverts to the wild type. On the other hand, in the strain lacking odhA, growth in a medium containing no sugar is remarkably slow. 1), Biotin 10 μg / L, agar 20 g / l, adjusted to pH 7.0 with KOH)) Therefore, 2A-1 strain was applied to a CM2B plate to induce a growth-improved strain. The growth-improving strain 2A-1R which appeared in this way was purified with a CM2B plate, and its kanamycin sensitivity was examined.

  Since pBS4SΔsucAint contains a kanamycin resistance gene and a sacB gene that encodes levanshuclease, strains with pBS4SΔsucAint show kanamycin resistance and sucrose sensitivity, dropped strains show kanamycin sensitivity and sucrose resistance. It becomes. These results suggested that odhA reverted to the wild type in the 2A-1R strain. Furthermore, as a result of confirming the sequence of the odhA gene, it was confirmed that there was no mutation in the odhA gene. Thus, it was concluded that odhA was restored to the wild type in the 2A-1R strain. The glutamic acid-producing ability of the 2A-1R strain was confirmed by the same method as in Example 2. The results are shown in Table 2. (OD620 indicates the amount of cells diluted 101-fold, Glu (g / L) indicates the amount of accumulated L-glutamic acid) The glutamic acid accumulation of 2A-1R strain is inferior to that of 2A-1 strain, but far more than wild strain ATCC13869 It was shown to be high (Table 2). In addition, when shaking was continued even after the sugar was completely consumed, 2A-1R showed glutamic acid degradation, confirming that odhA was reverted to the wild type (FIG. 4).

<Isolation of genes involved in glutamate production of 2A-1R strain>
The 2A-1R strain can form colonies on the CM2B plate medium at a rate almost the same as that of the wild strain ATCC13869. However, the minimum medium (glucose 20 g / l, ammonium sulfate 2.64 g / L, KH ₂ PO ₄ 0.5 g / L, K ₂ HPO ₄ 0.5 g / L, MgSO ₄ · 7H ₂ O 0.25 g / L, FeSO ₄ · 7H ₂ O 0.01g / L, MnSO ₄・ 7H ₂ O 0.01g / L, CaCl ₂ 0.01g / L, CuSO ₄ 0.02mg / L, MOPS 40g / L, Protocatechuic acid 30mg / L, VB ₁・ HCl 200μg / L, Biotin 300 μg / L, agar 20 g / L, adjusted to pH 6.7 using NaOH) It was revealed that the colony formation rate on the plate was significantly lower than that of the wild strain ATCC13869. Therefore, the inventors searched for genes that can restore the growth of the 2A-1R strain in a minimal medium.

  The ATCC13869 chromosomal DNA was partially digested with Sau3AI, ligated with the shuttle vector pVK9 treated with BamHI, the ligation reaction solution was ethanol precipitated, and then electropulsed with an electrocompetent cell of E. coli DH5α (Takara Bio). Was transformed by the method. pVK9 has a blunt-ended AvaII site in pHSG299 (Takara Bio) and inserts a blunt-ended fragment excised from BamHI and KpnI in a coryneform bacterium contained in pHK4 (JP 05-007491). The shuttle vector. The transformed cells are applied to LB plate medium containing 25 μg / ml kanamycin (polypeptone 10 g / l, yeast extract 5 g / l, NaCl 5 g / l, agar 20 g / l, adjusted to pH 7.0 with NaOH). And cultured overnight at 37 ° C. All the colonies that appeared on the following day were scraped with ase and the plasmid was extracted to obtain a library of ATCC13869. This library was transformed into the 2A-1R strain obtained in Example 3 by the electric pulse method and applied to a minimal medium plate containing 25 μg / ml kanamycin, and a strain with improved colony formation rate was screened. As a result of extracting a plasmid from a strain with improved colony formation rate, it was revealed that the fragment shown in SEQ ID NO: 5 was inserted into the BamHI site of pVK9. This plasmid was named pL5k.

pL5k insertion sequence and previously published Corynebacterium glutamicum ATCC13032
As a result of comparison of the genomic sequence (Acc. No. NC — 003450), it was found that pL5k contains only the ORF having the amino acid sequence shown in SEQ ID NO: 6 as the full-length ORF. Whether ORF is a membrane protein can be predicted by the program “SOSUI” available on the Internet (2004/10/07 as of http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0E.html Linked to). As a result of analyzing this ORF by “SOSUI”, it was suggested that five transmembrane regions exist. In the amino acid sequence of SEQ ID NO: 6, the transmembrane regions correspond to the regions of amino acid numbers 1 to 23, 25 to 47, 62 to 84, 86 to 108, and 110 to 132, respectively. The DNA sequence encoding them is the nucleotide sequence of 1437-1505, 1509-1577, 1620-1688, 1692-1760, 1764-1832 of SEQ ID NO: 5, the amino acid sequence of the transmembrane region is SEQ ID NO: 25-29, Shown in Table 3

  Furthermore, as a result of examining known literatures, it became clear that this ORF is an ORF analyzed as YggB (NCgl 1221) (FEMS Microbiology letters 218 (2003) 305-309).

<Identification of mutation point of YggB gene of 2A-1R strain>
PL5K complemented the growth of the 2A-1R strain on the minimal medium, suggesting that the yggB gene of the 2A-1R strain may have some mutation. Therefore, the base sequence of the yggB gene of the 2A-1R strain was determined. As a result, it was revealed that IS was inserted into the C-terminal region of the YggB gene in the 2A-1R strain (FIG. 5). The nucleotide sequence of the mutant yggB gene of the 2A-1R strain is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8. This suggests that the glutamic acid producing ability of the 2A-1R strain may be maintained by mutation of the yggB gene. This mutation was present not only in the 2A-1R strain but also in the 2A-1 strain. When a mutation is introduced into the odhA gene, it is presumed that the mutation has occurred as a suppressor mutation for stably releasing glutamate to the outside of the cell. Here, this mutation in which IS was inserted was designated as 2A-1 mutation.

<2A-1 type yggB mutant construction and L-glutamic acid production ability evaluation>
(6-1) Introduction and evaluation of 2A-1 mutations in wild strains (single recombinant strain)
PCR was performed using the chromosomal DNA of the 2A-1 strain as a template and the synthetic DNAs shown in SEQ ID NO: 9 and SEQ ID NO: 10 as primers to amplify the yggB gene fragment having the 2A-1 type mutation. The amplification product was treated with SacI and inserted into the SacI site of pBS3 of Example 1. The plasmid in which the yggB fragment having the 2A-1 type mutation was cloned was named pBS3yggB2A.
PBS3yggB2A was introduced into C. glutamicum ATCC13869 by an electric pulse method, and applied to a CM-Dex agar medium containing 25 μg / ml kanamycin. It was confirmed by PCR that the strain grown after culturing at 31.5 ° C. was a single recombination strain in which pBS3yggB2A was incorporated on the chromosome by homologous recombination.
The obtained single recombinant strain was designated 13869-2A. In this strain, both the wild-type yggB gene and the mutant-type yggB gene are expressed.

  The glutamic acid producing ability of the obtained yggB mutation-introduced strain 13869-2A was confirmed by the method described in Example 2. The results are shown in Table 4 (OD620 indicates the amount of cells diluted 101-fold, and Glu (g / L) indicates the amount of L-glutamic acid accumulated). In the 13869-2A strain, a clear glutamic acid producing ability could be confirmed even under conditions where the ATCC 13869 strain did not produce L-glutamic acid. From this, it was shown that glutamic acid production can be induced by mutation of the YggB gene.

(6-2) Introduction and evaluation of wild type 2A-1 mutation (twice recombinant strain)
In order to construct a strain having only the mutant gene, the 13869-2A strain was cultured overnight in a CM-Dex liquid medium, and a suspension of S10 agar medium (sucrose 100 g / l, polypeptone 10 g / l, yeast extract 10 g / l, KH ₂ PO ₄ 1g / l, MgSO ₄・ 7H ₂ O 0.4g / l, FeSO ₄・ 7H ₂ O 0.01g / l, MnSO ₄・ 4-5H ₂ O 0.01g / l, urea 3g / l, soy protein The solution was applied onto a hydrolyzate 1.2 g / l, agar 20 g / l, adjusted to pH 7.5 using NaOH: autoclave 120 ° C. for 20 minutes, and cultured at 31.5 ° C. Among the emerged colonies, a strain showing kanamycin sensitivity was purified on s2B agar medium. Chromosomal DNA was prepared from these strains, PCR was performed using the synthetic DNAs shown in SEQ ID NO: 9 and SEQ ID NO: 10 as primers, mutations were confirmed, and a strain in which an IS-like sequence was inserted into the yggB gene was 13869-2A-7 It was.

  The glutamic acid-producing ability of the acquired 13869-2A-7 strain was confirmed by the method described in Example 2. The results are shown in Table 5. (OD620 represents the amount of bacterial cells diluted 101 times, and Glu (g / L) represents the amount of accumulated L-glutamic acid). Since the 13869-2A-7 strain showed the same or higher glutamate production ability as the 2A-1R strain, it was confirmed that L-glutamate production in the presence of an excess amount of biotin was caused by a mutation in the yggB gene. .

<Construction and evaluation of A1 type YggB mutant>
As a result of analyzing the above-mentioned L-glutamic acid-producing odhA mutant, five kinds of mutations were found on the yggB gene in addition to the 2A-1 mutation described above. In the following, A1 type mutation, 19 type mutation, L30 type mutation, 8 type mutation, and 66 type mutation were introduced, and the effects of the former three were confirmed by introducing mutations into the ATCC13869 chromosome. Regarding the type 8 mutation, the mutation was introduced into the chromosome of ATCC14067 to confirm the effect. Regarding the 66 type mutation, the effect was confirmed by introducing the mutation into the chromosome of C. melassecola ATCC17965.

  The A1 type mutation is a mutation in which TTCATTGTG is inserted after 1480th G of SEQ ID NO: 5, and cysteine, serine is inserted between the 14th leucine residue and 15th tryptophan residue of the amino acid sequence of SEQ ID NO: 6. , A mutation in which a leucine residue is inserted. The nucleotide sequence of the mutant yggB gene into which this mutation has been introduced is shown in SEQ ID NO: 19, and the amino acid sequence of the mutant YggB protein encoded by the gene is shown in SEQ ID NO: 20.

  A1 mutant gene can be obtained as follows. PCR is performed using the synthetic DNA shown in SEQ ID NO: 30 and the synthetic DNA shown in SEQ ID NO: 31 as primers and the chromosomal DNA of ATCC 13869 strain as a template to prepare an N-terminal fragment. Similarly, a C-terminal fragment is prepared using the synthetic DNAs of SEQ ID NO: 32 and SEQ ID NO: 33 as primers. Subsequently, a partial fragment of the A1-type YggB gene can be obtained by PCR using a mixture of equal amounts of N-terminal fragment and C-terminal fragment as a template and the synthetic DNAs of SEQ ID NO: 9 and SEQ ID NO: 34 as primers. The obtained YggB fragment is treated with SacI and inserted into the SacI site of pBS4S, whereby a mutation-introducing plasmid can be constructed. The pBS4 YggBA1 thus obtained is inserted into the chromosome of ATCC13869 strain in the same manner as described in Example 6, and then dropped. By determining the sequence of the yggB gene of the obtained kanamycin-sensitive strain and selecting a strain that has been replaced with the A1 type, a strain having an A1 type mutation can be constructed. The A1 type mutant having such mutation was designated as ATCC13869-A1.

The ATCC13869-A1 strain and the parent ATCC13869 strain were cultured in the same manner as in Example 2. After completion of the culture, the amount of L-glutamic acid contained in the culture solution was measured by a known method.
The ATCC13869-A1 strain, into which the A1 mutation was introduced on the chromosome, had significantly improved L-glutamic acid accumulation compared to the parent strain ATCC13869.

<Construction and evaluation of type 19 YggB mutant>
The type 19 mutation is a mutation in which the 1734th G in SEQ ID NO: 5 is substituted with A, and the 100th alanine in the amino acid sequence of SEQ ID NO: 6 is substituted with threonine. The nucleotide sequence of the mutant YggB gene into which this mutation has been introduced is shown in SEQ ID NO: 21, and the amino acid sequence of the mutant YggB protein encoded by the gene is shown in SEQ ID NO: 22. In the same manner as in Example 7, a yggB mutant strain into which a type 19 mutation was introduced was constructed. Specifically, PCR is performed using the synthetic DNA shown in SEQ ID NO: 30 and the synthetic DNA shown in SEQ ID NO: 35 as primers and the chromosomal DNA of ATCC 13869 strain as a template to prepare an N-terminal fragment. Similarly, a C-terminal fragment is prepared using the synthetic DNAs of SEQ ID NO: 33 and SEQ ID NO: 36 as primers. Subsequently, a partial fragment of the type 19 YggB gene can be obtained by PCR using a mixture of equal amounts of N-terminal fragment and C-terminal fragment as a template and the synthetic DNAs of SEQ ID NO: 9 and SEQ ID NO: 34 as primers. The obtained YggB fragment is treated with SacI and inserted into the SacI site of pBS4S, whereby a mutation-introducing plasmid can be constructed. PBS4 YggB19 thus obtained is inserted into the chromosome of ATCC13869 strain in the same manner as described in Example 6, and then dropped. By determining the sequence of the yggB gene of the obtained kanamycin-sensitive strain and selecting a strain that has been replaced with type 19, a strain having a type 19 mutation can be constructed. The type 19 mutant having such mutation was designated as ATCC13869-19.

  The ATCC13869-19 strain and the parent ATCC13869 strain were cultured in the same manner as in Example 2. After completion of the culture, the amount of L-glutamic acid contained in the culture solution was measured by a known method. The ATCC13869-19 strain, in which 19 mutations were introduced on the chromosome, had a significantly improved accumulation of L-glutamic acid compared to the parent strain ATCC13869.

<Construction of L30 type YggB mutant>
The L30 type mutation is a mutation in which the 1768th C in SEQ ID NO: 5 is substituted with T, and the 111th alanine in SEQ ID NO: 6 is substituted with valine. The nucleotide sequence of the mutant YggB gene into which this mutation has been introduced is shown in SEQ ID NO: 23, and the amino acid sequence of the mutant YggB protein encoded by the gene is shown in SEQ ID NO: 24.
A yggB mutant strain into which the L30 type mutation was introduced was constructed in the same manner as in Example 7. Specifically, PCR is performed using the synthetic DNA shown in SEQ ID NO: 30 and the synthetic DNA shown in SEQ ID NO: 37 as primers and the chromosomal DNA of ATCC 13869 strain as a template to prepare an N-terminal fragment. Similarly, a C-terminal fragment is prepared using the synthetic DNAs of SEQ ID NO: 34 and SEQ ID NO: 38 as primers. Subsequently, PCR is performed using a mixture of equal amounts of N-terminal fragment and C-terminal fragment as a template, and the synthetic DNAs of SEQ ID NO: 9 and SEQ ID NO: 34 as primers to obtain a partial fragment of the L30 type YggB gene. The obtained mutant YggB fragment was treated with SacI and inserted into the SacI site of pBS4S to construct a mutation-introducing plasmid. The pBS4 YggB-L thus obtained is inserted into the chromosome of ATCC13869 strain in the same manner as in Example 6, and then dropped. The sequence of the yggB gene of the obtained kanamycin sensitive strain was determined, the strain that had been replaced with the L30 type was selected, and the strain having the L30 type mutation was constructed. The L30 type mutant having such mutation was designated as ATCC13869-L.

  The ATCC13869 strain and the ATCC13869-L strain were cultured in the same manner as in Example 2, and after completion of the culture, the amount of L-glutamic acid contained in the culture solution was measured by a known method. The results are shown in Table 8. (OD620 represents the amount of bacterial cells diluted 101 times, and Glu (g / L) represents the amount of accumulated L-glutamic acid). The ATCC13869-L30 strain into which the L-30 mutation was introduced had significantly improved L-glutamic acid accumulation compared to the parent strain ATCC13869.

<Culture evaluation of mutant yggB gene-introduced strain under L-glutamic acid production conditions>
Coryneform bacteria can induce glutamate production by adding a fatty acid surfactant such as Tween 40 or by limiting biotin. Therefore, the ATCC13869 strain and the ATCC13869-19 strain were also cultured under conditions where Tween40 was added and biotin was restricted. Seed culture was carried out in 20 ml flask medium (glucose 80 g / l, ammonium sulfate 30 g / l, KH ₂ PO ₄ 1 g / l, MgSO ₄ · 7H ₂ O 0.4 g / l, FeSO ₄ · 7H ₂ O 0.01 g / l, MnSO _{4 · 4~5H 2 O 0.01g / l} , VB1 200μg / l, Biotin 60μg / l, soybean hydrolyzate (TN) 0.48g / l, adjusted to pH8.0 with KOH: autoclaved 115 ° C. 10 min) 1 g of calcium carbonate previously sterilized by dry heat was added and cultured at 31.5 ° C. with shaking. A culture solution that completely consumed sugar was used as a seed culture solution. In Tween40-added culture, 2 ml of the seed culture solution is added to a 20 ml flask medium (glucose 80 g / l, ammonium sulfate 30 g / l, KH ₂ PO ₄ 1 g / l, MgSO ₄ · 7H ₂ O 0.4 g / l, FeSO ₄ · 7H _{2 O 0.01g / l, MnSO 4 ·} 4~5H 2 O 0.01g / l, VB1 200μg / l, Biotin 60μg / l, soybean hydrolyzate (TN) 0.48g / l, adjusted to pH8.0 with KOH : Autoclave 115 ° C. for 10 minutes), 1 g of calcium carbonate previously sterilized by dry heat was added, and the mixture was cultured with shaking at 31.5 ° C. After starting the culture, when OD620 (x101) = 0.2 was reached, Tween 40 was added to a final concentration of 5 g / L and the culture was continued. In biotin-restricted culture, 1 ml of the seed culture solution is added to a 20 ml flask medium (glucose 80 g / l, ammonium sulfate 30 g / l, KH ₂ PO ₄ 1 g / l, MgSO ₄ · 7H ₂ O 0.4 g / l, FeSO ₄ · 7H ₂ O 0.01g / l, MnSO ₄ · _{4 to} 5H ₂ O 0.01g / l, VB1 200μg / l, Soybean hydrolyzate (TN) 0.48g / l, adjusted to pH 8.0 with KOH: Autoclave 115 ° C 10 1 g of calcium carbonate that had been sterilized by dry heat in advance was added and cultured at 31.5 ° C. with shaking. Under this condition, the final concentration of biotin is about 2.9 μg / L.
In both Tween 40-added culture and biotin-restricted culture, the L-glutamic acid concentration in the medium 40 hours after the start of the culture was measured. The results are shown in Table 9 (OD620 indicates the amount of bacterial cells diluted 101 times, and Glu (g / L) indicates the amount of accumulated L-glutamic acid). In the ATCC13869-19 strain, increased accumulation of L-glutamic acid was observed even under L-glutamic acid production conditions.

The ATCC13869 strain, the ATCC13869-L30, the ATCC13869-A1 strain, the ATCC13869-19 strain, and the yggB amplified strain using the plasmid were also cultured with Tween40. The cells cultured overnight on a CM-Dex plate are scraped into 1/6 plate, and 20 ml flask medium (glucose 80 g / l, ammonium sulfate)
_{30g / l, KH 2 PO 4} 1g / l, MgSO 4 · 7H 2 O 0.4g / l, FeSO 4 · 7H 2 O 0.01g / l, MnSO 4 · 4~5H 2 O 0.01g / l, VB1 200μg / l, Biotin 60μg / l, soy hydrolyzate (TN) 0.48g / l, adjusted to pH 8.0 using KOH: autoclave 115 ° C for 10 minutes), then preliminarily dry heat sterilized calcium carbonate 1g was added and cultured at 31.5 ° C with shaking. Tween 40 was added to a final concentration of 1 g / L 5 hours after the start of the culture, and the amount of bacterial cells and the yield of L-glutamic acid 24 hours after the start of the culture were analyzed. As a result, as shown in Table 10, all of ATCC13869-19, ATCC13869-L30, ATCC13869-A1 and wild-type yggB gene amplification strain (ATCC13869 / pL5k-1) yield of L-glutamic acid under L-glutamic acid production conditions Was shown to be improving.

<Construction of type 8 YggB mutant>
Type 8 mutation is a mutation in which G at position 837 in SEQ ID NO: 61 is replaced with A, and alanine at position 111 in SEQ ID NO: 62 is replaced with threonine. The nucleotide sequence of the mutant YggB gene into which this mutation has been introduced is shown in SEQ ID NO: 63, and the amino acid sequence of the mutant YggB protein encoded by the gene is shown in SEQ ID NO: 64.
A yggB mutant strain into which the type 8 mutation has been introduced is constructed in the same manner as in Example 7. Specifically, PCR is performed using the synthetic DNA shown in SEQ ID NO: 30 and the synthetic DNA shown in SEQ ID NO: 65 as primers and the chromosomal DNA of Brevibacterium flavum ATCC 14067 as a template to prepare an N-terminal fragment. Similarly, a C-terminal fragment is prepared using the synthetic DNAs of SEQ ID NO: 34 and SEQ ID NO: 66 as primers. Subsequently, PCR is performed using a mixture of equal amounts of N-terminal fragment and C-terminal fragment as a template, and the synthetic DNAs of SEQ ID NO: 9 and SEQ ID NO: 34 as primers to obtain a partial fragment of type 8 YggB gene. The obtained mutant YggB fragment is treated with SacI and inserted into the SacI site of pBS4S to construct a mutation-introducing plasmid. PBS4 YggB8 thus obtained is inserted into the chromosome of ATCC14067 strain in the same manner as described in Example 6, and then dropped. The sequence of the yggB gene of the obtained kanamycin-sensitive strain is determined, a strain that has been replaced with type 8 is selected, and a strain having a type 8 mutation is constructed. The type 8 mutant having such mutation is designated as ATCC14067yggB8.

<Construction of 66-type YggB mutant>
Type 66 mutation is a mutation in which the 1673th C in SEQ ID NO: 67 is replaced with T, and the 424th proline in SEQ ID NO: 68 is replaced with leucine. The nucleotide sequence of the mutant YggB gene into which this mutation has been introduced is shown in SEQ ID NO: 69, and the amino acid sequence of the mutant YggB protein encoded by the gene is shown in SEQ ID NO: 70.
In the same manner as in Example 7, a yggB mutant strain into which a 66 type mutation has been introduced is constructed. Specifically, PCR was performed using the synthetic DNA shown in SEQ ID NO: 30 and the synthetic DNA shown in SEQ ID NO: 71 as primers and the chromosomal DNA of C. melassecola ATCC 17965 as a template to prepare an N-terminal fragment. Similarly, a C-terminal fragment is prepared using the synthetic DNAs of SEQ ID NO: 34 and SEQ ID NO: 72 as primers. Subsequently, a partial fragment of the 66-type yggB gene can be obtained by PCR using a mixture of equal amounts of N-terminal fragment and C-terminal fragment as a template and the synthetic DNAs of SEQ ID NO: 9 and SEQ ID NO: 10 as primers. The obtained mutant YggB fragment is treated with SacI and inserted into the SacI site of pBS4S to construct a mutation-introducing plasmid. The pBS4 YggB66 thus obtained is inserted into the chromosome of the ATCC 17965 strain in the same manner as described in Example 6 and then dropped. By determining the sequence of the yggB gene of the obtained kanamycin-sensitive strain and selecting a strain that has been replaced with type 66, a strain having a type 66 mutation can be constructed. A 66-type mutant having such mutation is designated as yggB66.

<Screening of mutant yggB gene by in vitro mutation>
(13-1) Construction of a yggB-deficient strain A mutant yggB gene is randomly introduced into yggB in vitro, and a clone capable of producing glutamate without adding a surfactant is selected from them. You can also. To screen for mutant genes, a yggB-deficient strain was first constructed. PCR was performed using the synthetic DNA shown in SEQ ID NO: 39 and the synthetic DNA shown in SEQ ID NO: 40 as primers and the chromosomal DNA of ATCC 13869 strain as a template to prepare an N-terminal fragment. Similarly, a C-terminal fragment was prepared using the synthetic DNAs of SEQ ID NO: 41 and SEQ ID NO: 42 as primers. SEQ ID NOs: 40 and 41 are complementary to each other. Subsequently, PCR was performed using a mixture of equal amounts of N-terminal fragment and C-terminal fragment as a template, and the synthetic DNAs of SEQ ID NO: 39 and SEQ ID NO: 42 as primers to obtain a fragment lacking the ORF of the yggB gene.

  The obtained PCR fragment was treated with SacI and inserted into the SacI site of pBS4S to construct a plasmid for introducing a deletion mutation. The pBS4ΔYggB thus obtained was inserted into the chromosome of ATCC13869 strain by the method described in Example 6 and then dropped. PCR was performed using the obtained chromosomal DNA of the kanamycin-sensitive strain as a template and the synthetic DNAs of SEQ ID NO: 39 and SEQ ID NO: 42 as primers, and it was confirmed that the yggB gene was deleted. The obtained yggB gene-deficient strain was designated as ATCC13869ΔyggB strain.

(13-2) In vitro screening of mutant yggB gene On the other hand, the mutation treatment of the yggB gene was performed as follows. First, plasmid pL5k contains a region other than the yggB gene. Therefore, plasmid pL5kXS was obtained by treating pL5k with XhoI and SalI and self-ligating. The SalI recognition site does not exist on the nucleotide sequence of SEQ ID NO: 5, but exists at the multiple cloning site of pBS3. About 10 μg of the obtained pL5kXS was dissolved in 500 mM phosphate buffer, 400 mM hydroxylamine, 1 mM EDTA (pH 6.0), and the mutation was introduced by treating at 75 ° C. for 30 to 90 minutes. The mutated plasmid was desalted with SUPREC-02 (Takara Bio), and then introduced into the ATCC13869ΔyggB strain by the method described in Example 6, and a transformant was selected using a CM2B medium containing 25 μg / ml of Km. As a control, pL5kXS before mutation treatment was also introduced into the ATCC13869ΔyggB strain. The resulting transformant was inoculated into 2 ml of liquid medium of CM2BGU2 medium (containing 10 g / L of glucose and 15 g / L of urea in the CM2B medium described in Example 3), and after 5 minutes of incubation at 31.5 ° C., glutamic acid was used. The concentration was quantified.
Table 11 shows the results of the test tube culture evaluation of the strain transformed with pL5kXS in which ATCC13869ΔyggB was mutated in CM2BGU2 medium. Among the transformants obtained from the plasmid mixture with a mutation treatment time of 60 minutes and 90 minutes, there were 3 clones that accumulated 1 g / L or more of L-glutamic acid. In addition, glutamic acid originally contained in the medium is 0.16 g / L, ATCC13869ΔyggB / pL5kXS (no mutation treatment)
The amount of glutamic acid accumulated was 0.31 g / L.
Table 12 shows the results of test tube culture evaluation of strains transformed with pL5kXS in which ATCC13869ΔyggB was mutated in CM2BGU medium (same composition as CM2BGU2 medium except that Urea is 1.5 g / L). Among the transformants obtained from the plasmid mixture with a mutation treatment time of 90 minutes, there was one clone that accumulated 1 g / L or more of L-glutamic acid.

In Table 11, pL5kXSm-22 is a plasmid that has acquired a production ability to accumulate 1 g / L or more of L-glutamic acid by performing a mutation treatment time of 60 minutes. In Table 12, a mutation treatment time of 90 minutes is performed. A plasmid that acquired an ability to produce L-glutamic acid of 0.9 g / L or more was designated as pL5kXSm-27. The ATCC13869ΔyggB / pL5kXS strain, the ATCC13869ΔyggB / pL5kXSm-27 strain, and the ATCC13869ΔyggB / pL5kXSm-22 strain were cultured under the conditions described in Example 2 and analyzed for the amount of bacterial cells and L-glutamic acid accumulation after 4 hours. Table 13 shows the average values from three independent experiments. The ATCC13869ΔyggB / pL5kXSm-27 and ATCC13869ΔyggB / pL5kXSm-22 strains were confirmed to have significantly increased L-glutamic acid accumulation, and a mutant gene advantageous to L-glutamic acid was also found by random mutagenesis in vitro. It was shown that it can be constructed. The sequence of the yggB gene contained in pL5kXSm-22 is shown in SEQ ID NO: 73. (Type 22 mutation)
The type 22 mutation is a mutation that replaces the 437th proline with serine, for example, a mutation that replaces C at position 2745 of SEQ ID NO: 5 with T. This mutation is also accompanied by a mutation that replaces C at position 3060 with T. This mutant yggB gene is shown in SEQ ID NO: 73, and the amino acid sequence of the mutant YggB protein encoded by the gene is shown in SEQ ID NO: 74.

(13-3) Introduction of mutant gene into coryneform bacterium and confirmation of L-glutamic acid production (13-2) The novel mutant yggB gene obtained in (13-2) is introduced into coryneform bacterium. As the introduction method, the mutant gene was introduced into pBS4S by the method described in Example 6 and replaced with the wild-type yggB gene on the chromosome of ATCC13869. The strain introduced with the novel mutant gene and the parent ATCC13869 strain are cultured in the same manner as in Example 2. After completion of the culture, the amount of L-glutamic acid contained in the culture solution is measured by a known method, and it is confirmed that the mutant-introduced strain has improved L-glutamic acid accumulation. By such a method, a yggB mutant strain having improved L-glutamic acid production ability can be obtained.

<Glutamate analog sensitivity of mutant yggB gene-bearing strain>
(14-1) 4-fluoroglutamic acid sensitivity in solid medium
It was predicted that the L-glutamic acid analog sensitivity was reduced in the strains in which L-glutamic acid-producing ability was improved by the yggB mutation. Therefore, we examined changes in sensitivity to 4-fluoroglutamic acid as an analog of L-glutamic acid due to the yggB mutation. To the minimal medium described in Example 4, 4-fluoroglutamic acid adjusted to pH 6.7 with NaOH and sterilized by filtration was added to a final concentration of 7.5 mM. The ATCC13869 strain, ATCC13869-L30 strain, and ATCC13869-A1 strain are applied to a CM-Dex medium and cultured overnight, then collected and washed with a sterilized 0.85% NaCl solution to obtain the bacterial concentration shown in the upper part of FIG. Were diluted and spotted on the plate. The plate was cultured at 31.5 ° C., and the change with time is shown in FIG. ATCC13869 grows fastest under the conditions without 4-fluoroglutamic acid, whereas ATCC13869 grows under the conditions with 4-fluoroglutamic acid, while ATCC13869-L and ATCC13869-A1 grow. Was shown to be good.

(14-2) 4-Fluoroglutamic acid sensitivity in liquid medium 4-Fluoroglutamic acid adjusted to pH 6.7 with NaOH and filter sterilized in the minimum medium described in Example 4 (but not including agar) at a final concentration of 1.25 mM, 2.5 mM, 5 mM, 10 mM, and 20 mM were added. ATCC13869 strain, ATCC13869ΔyggB strain, ATCC13869-L30 strain, ATCC13869-A1 strain were applied to CM-Dex medium, cultured at 31.5 ° C overnight, then collected, washed with sterile 0.85% NaCl solution, and inoculated into liquid medium And cultured at 31.5 ° C. with shaking. In each strain, the culture was terminated when the value of OD660 in a medium to which 4-fluoroglutamic acid was not added reached 1, and the culture was appropriately diluted and applied to a CM-Dex plate. The number of colonies that appeared the next day was counted and used as the number of viable bacteria at the end of liquid culture. FIG. 7 shows the change in the relative viable cell count due to the addition of 4-fluoroglutamic acid, assuming that the viable cell count in the 4-fluoroglutamic acid-free group was 1. In the ATCC13869-A1 and ATCC13869-L30 strains, it was revealed that the sensitivity to 4-fluoroglutamic acid was decreased (resistance to 4-fluoroglutamic acid was improved).
From these results, it was shown that the yggB mutant of the present invention can also be obtained by screening using as an index the sensitivity of structural analogs of glutamic acid such as 4-fluoroglutamic acid.

<Creation and evaluation of mutant-type yggB gene-transferred strains with weakened odhA>

Using the ATCC13869-L30 strain constructed in Example 9 above, a yggB odhA double mutant was constructed, and the mutant odhA gene was evaluated.
First, a strain in which the mutation shown in Table 14 was introduced into the odhA gene of ATCC13869-L strain was constructed. In addition, in Table 14, the arrangement | sequence of the area | region corresponded to base number 2528-2562 of sequence number 43 was shown. Table 15 shows the sequence of the region corresponding to amino acid numbers 696 to 707 of SEQ ID NO: 44.
In order to construct the L30sucA8 strain into which the mutant odhA gene having the base sequence of SEQ ID NO: 45 has been introduced, the following procedure may be used. First, PCR is performed using the synthetic DNAs shown in SEQ ID NO: 53 and SEQ ID NO: 54 as primers to prepare an odhA fragment. The obtained odhA fragment was treated with BamHI and cloned into the BamHI site of plasmid pKF19m attached to Muta-Super Express Km manufactured by Takara Bio. PCR is performed using a selection primer attached to Express Km, and a plasmid containing a mutant odhA fragment is constructed by transforming sup0 E. coli strain such as MV1184 with the obtained PCR product. Next, this fragment is excised with BamHI and inserted into the BamHI site of pBS4S to construct a mutation-introducing plasmid. Using this plasmid, the ATCC13869-L strain is transformed in the same manner as described in Example 1 to obtain a strain in which the mutation introducing plasmid is inserted into the chromosome. Next, a strain showing resistance to sucrose and sensitivity to kanamycin is isolated from a strain in which a plasmid is inserted into these chromosomes. Furthermore, the L30sucA8 (odhA8) strain lacking the odhA gene can be constructed by confirming the sequence of the odhA gene and selecting a strain into which the target frameshift has been introduced.

In addition, other odhA mutant strains can be obtained using the yggB mutant strain by the following method.
For obtaining the L30sucA801 strain into which the mutant odhA gene having the base sequence of SEQ ID NO: 47 was introduced, the 5 ′ end was phosphorylated at the 5 ′ end instead of the synthetic DNA of SEQ ID NO: 55. The above method may be applied using 56 synthetic DNAs.
For obtaining the L30sucA805 strain into which the mutant odhA gene having the base sequence of SEQ ID NO: 49 was introduced, the 5 ′ end was phosphorylated instead of the synthetic DNA of SEQ ID NO: 55 in which the 5 ′ end was phosphorylated. The above method may be applied using 57 synthetic DNAs.
For obtaining the L30sucA77 strain into which the mutant odhA gene having the nucleotide sequence of SEQ ID NO: 51 was introduced, the 5 ′ end was phosphorylated at the 5 ′ end instead of the synthetic DNA at SEQ ID NO: 55. The above method may be applied using 58 synthetic DNAs.
Since the odhA gene of the L30sucA8 strain is a frameshift mutation, it does not have αKGDH activity. However, although the L30sucA801 strain, the L30sucA805 strain, and the L30sucA77 strain have a deletion in the odhA gene, they are mutations that do not cause a frameshift and have αKGDH activity, but the activity is lower than that of the unmodified strain. (Data not shown)

<Glutamic acid production culture of odhA modified strain>
The L-glutamic acid producing ability of these odhA modified strains was verified by culturing using a Sakaguchi flask. The strains shown in Table 14 were cultured overnight at 31.5 ° C. on CM-Dex agar medium. Flask medium (glucose 60 g / l, ammonium sulfate 22.5 g / l, KH ₂ PO ₄ 1 g / l, MgSO ₄ .7H ₂ O 0.4 _{g / l, FeSO 4 · 7H} 2 O 0.01g / l, MnSO 4 · 4-5H 2 O 0.01g / l, vitamin B1 200 [mu] g / l, soy protein hydrolyzate 0.48 g / l, biotin 300μg / l, KOH Adjust pH to 8.0 using 1/6 plate inoculate 20ml of 1/6 plate cells, add 1g of calcium carbonate previously sterilized by dry heat, and then perform shaking culture at 31.5 ° C and speed of 115rpm. It was. Table 16 shows the accumulation of L-glutamic acid 19 hours after the start of the culture. Among the odhA modified strains, those with L-glutamic acid accumulation almost equivalent to those of the ATCC13869-L strain were included, but the sucA801, sucA805, and sucA77 strains showed higher glutamate accumulation than the sucA8 strain. From these results, it was found that the L-glutamic acid yield was further improved by introducing a mutation into the odhA gene together with the mutation of the yggB gene.

<Construction of odhA weakened strains from ATCC14067 and ATCC14067yggB8>
An odhA-deficient strain was constructed from the yggB mutant having the type 8 mutation, and a comparative culture was performed with a strain having only the odhA-deficient strain. First, a plasmid for complete deletion of odhA was constructed. PCR was performed using the chromosomal DNA of ATCC14067 strain as a template and the synthetic DNAs of SEQ ID NO: 77 and SEQ ID NO: 78 to prepare an upstream fragment of the sucA gene. Subsequently, PCR was performed using the chromosomal DNA of the ATCC14067 strain as a template and the synthetic DNAs of SEQ ID NO: 79 and SEQ ID NO: 80 to prepare a downstream fragment of the sucA gene. Next, the upstream fragment and the downstream fragment are mixed so as to be equimolar, and PCR is performed using the synthetic DNA of SEQ ID NO: 81 and SEQ ID NO: 82 as a template to prepare a gene fragment lacking odhA did. The obtained PCR fragment was treated with BamHI and then cloned into pBS4S constructed in Example 1. The plasmid thus obtained was designated as pBSΔsucA47.
pBSΔsucA47 was inserted into the chromosome of ATCC14067 strain or ATCC14067yggB8 strain in the same manner as described in Example 6 and then dropped. Chromosomal DNA was prepared from the obtained kanamycin-sensitive strain, PCR was performed using the synthetic DNA of SEQ ID NO: 77 and SEQ ID NO: 80 as a template, and a strain lacking the odhA region was selected. The thus constructed odhA-deficient strains were designated as ATCC14067ΔodhA strain and ATCC14067ΔodhAyggB8 strain, respectively.
Table 17 shows the results of culturing the constructed ATCC14067ΔodhA strain and ATCC14067ΔodhAyggB8 strain by the method described in Example 3. It was revealed that the yggB8 mutation can improve the L-glutamic acid yield of the odhA mutant.

<Construction of symA deficient strain from 2A-1R strain>
A strain lacking the symA (suppressor of ygg mutation A) gene was constructed from the 2A-1R strain having an IS insertion mutation in yggB, which was constructed in Example 3, and subjected to comparative culture with the 2A-1R strain. The nucleic acid sequence and amino acid sequence of NCgl1867 gene of ATCC13869 strain are shown in SEQ ID NOs: 86 and 87, respectively. First, a plasmid for complete deletion of the symA gene was constructed. Using the chromosomal DNA of ATCC13869 strain as a template, PCR was performed using the synthetic DNAs of SEQ ID NO: 88 and SEQ ID NO: 89 to prepare an upstream fragment of the symA gene. Subsequently, PCR was performed using the chromosomal DNA of the ATCC 13869 strain as a template and the synthetic DNAs of SEQ ID NO: 90 and SEQ ID NO: 91 to prepare a downstream fragment of the symA gene. Next, the upstream fragment and the downstream fragment are mixed in equimolar amounts, PCR is performed using the synthetic DNAs of SEQ ID NO: 88 and SEQ ID NO: 91 as a template, and the gene fragment lacking the symA gene is obtained. Prepared. The obtained PCR fragment was treated with BglII and then cloned into pBS4S constructed in Example 1. The plasmid thus obtained was designated as pBSΔsymA.
pBSΔsymA was inserted into the chromosome of 2A-1R strain in the same manner as described in Example 6 and then dropped. Chromosomal DNA was prepared from the obtained kanamycin-sensitive strain, PCR was performed using the synthetic DNA of SEQ ID NO: 88 and SEQ ID NO: 91 using this as a template, and a strain lacking the symA region was selected. The symA deficient strain thus constructed was designated as 2A-1RΔsymA strain.
Table 18 shows the results of culturing the constructed 2A-1RΔsymA strain and the parental 2A-1R strain by the method described in Example 3. It was revealed that the ability to produce glutamate in a strain having a mutant yggB gene can be improved by deletion of the symA gene.

<Construction of wild-type yggB gene-enhanced strain>
PL5k was used as a plasmid containing the wild-type yggB gene of coryneform bacteria. pL5k is a plasmid obtained by inserting a DNA fragment obtained by partially decomposing chromosomal DNA of Corynebacterium glutamicum ATCC 13869 strain with Sau3AI into the BamHI site of pVK9. The DNA sequence inserted into the BamHI site of pVK9 is shown in SEQ ID NO: 5, and the coding region of the yggB gene is shown in nucleotide numbers 1437-3035 of SEQ ID NO: 5. PVK9 is a fragment in which the AvaII site of pHSG299 (Takara Bio) is blunt-ended, and a region capable of autonomous replication in pHK4 (JP 05-007491) is excised with BamHI and KpnI and blunt-ended. Is a shuttle vector into which is inserted. A plasmid having the same structure as pL5k was subjected to PCR using the synthetic DNAs of SEQ ID NO: 59 and SEQ ID NO: 60 as primers and the chromosomal DNA of ATCC 13869 strain as a template. It can also be constructed by inserting into

  Corynebacterium glutamicum ATCC13869 strain was transformed with pL5k by the electric pulse method (Japanese Patent Laid-Open No. 2-207791). The transformed cells are applied to CM2B plate medium containing 25 μg / ml kanamycin (polypeptone 10 g / l, yeast extract 10 g / l, NaCl 5 g / l, agar 20 g / l, adjusted to pH 7.0 with KOH) And cultured overnight at 31.5 ° C. The colonies that appeared the next day were purified with a CM2B plate medium containing 25 μg / ml kanamycin to obtain a YggB amplified strain. Plasmids were extracted from these transformants by a conventional method, and it was confirmed that the target plasmid was introduced. The YggB amplified strain thus obtained was designated as ATCC13869 / pL5k. As a control strain, strain ATCC13869 / pVK9 introduced with pVK9 was constructed in the same manner as described above.

<Evaluation of wild-type yggB gene-enhanced strain>
(2-1) Confirmation of effect in biotin-restricted culture Three clones each of the wild-type yggB gene-enhanced strain and the control strain constructed in Example 18 were selected, and 20 ml of seed culture medium (glucose 80 g / l, ammonium sulfate 30 g / l, KH ₂ PO ₄ 1g / l, MgSO ₄・ 7H ₂ O 0.4g / l, FeSO ₄・ 7H ₂ O 0.01g / l, MnSO ₄・₄ ~ 5H ₂ O 0.01g / l, VB1 200μg / l, Biotin 60μg / l, soybean hydrolyzate (TN) 0.48 g / l, adjusted to pH 8.0 using KOH: autoclave 115 ° C for 10 minutes), then added 1 g of calcium carbonate that had been sterilized by dry heat in advance, Cultured with shaking at 31.5 ° C. After complete consumption of sugar, 2 ml of the culture solution is added to 20 ml of main culture medium without glucose (glucose 80 g / l, ammonium sulfate 30 g / l, KH ₂ PO ₄ 1 g / l, MgSO ₄ · 7H ₂ O
0.4g / l, FeSO ₄・ 7H ₂ O 0.01g / l, MnSO ₄・₄ ～ 5H ₂ O 0.01g / l, VB1 200μg / l, Soybean hydrolyzate (TN) 0.48g / l, with KOH After adjusting to pH 8.0: autoclave 115 ° C. for 10 minutes, 1 g of calcium carbonate previously sterilized by dry heat was added and cultured with shaking at 31.5 ° C. After the sugar was completely consumed, the glutamic acid concentration in the medium was measured. As a result, it was shown that the accumulation of glutamic acid in the medium was improved in the yggB-enhanced strain (ATCC13869 / pL5k) compared to the vector-introduced strain. (Table 19 OD620 indicates the amount of bacterial cells, GH (g / L) indicates the amount of glutamic acid produced)

(2-2) Confirmation of Effect under Surfactant Addition Conditions Three clones of each of the wild-type YggB-enhanced strain and the control strain constructed in Example 18 were selected, and 20 ml of seed culture medium (glucose 80 g / l, ammonium sulfate 30 g / l , KH ₂ PO ₄ 1g / l, MgSO ₄・ 7H ₂ O 0.4g / l, FeSO ₄・ 7H ₂ O 0.01g / l, MnSO ₄・₄ ~ 5H ₂ O 0.01g / l, VB1 200μg / l, Biotin 60 μg / l, soybean hydrolyzate (TN) 0.48 g / l, adjusted to pH 8.0 with KOH: autoclave 115 ° C for 10 minutes), then added 1 g of calcium carbonate sterilized by dry heat in advance And cultured with shaking at 31.5 ° C. After complete consumption of sugar, 2 ml of the culture solution is added to 20 ml of the main culture medium (glucose 80 g / l, ammonium sulfate 30 g / l, KH ₂ PO ₄ 1 g / l, MgSO ₄ · 7H ₂ O 0.4 g / l, FeSO ₄・ 7H ₂ O 0.01g / l, MnSO ₄・ 4-5H ₂ O 0.01g / l, VB1 200μg / l, Biotin 60μg / l, soybean hydrolyzate (TN) 0.48g / l, pH8 using KOH. After adjusting to 0: autoclave 115 ° C. for 10 minutes), 1 g of calcium carbonate previously sterilized by dry heat was added and cultured at 31.5 ° C. with shaking. Two hours after the start of culture, Tween 40 was added to a final concentration of 5 g / L, and the culture was continued. After the sugar was completely consumed, the glutamic acid concentration in the medium was measured. As a result, it was shown that accumulation of L-glutamic acid in the medium was improved in the wild-type YggB-enhanced strain (ATCC13869 / pL5k) compared to the vector-introduced strain. (Table 20 OD620 indicates the amount of bacterial cells, and GH indicates the amount of L-glutamic acid produced.)

(2-3) Confirmation of effect in culture with penicillin G added Three clones of the wild-type yggB gene-enhanced strain and the control strain constructed in Example 18 were selected, and 20 ml of seed culture medium (glucose 80 g / l, ammonium sulfate 30 g / l). , KH ₂ PO ₄ 1g / l, MgSO ₄・ 7H ₂ O 0.4g / l, FeSO ₄・ 7H ₂ O 0.01g / l, MnSO ₄・₄ ~ 5H ₂ O 0.01g / l, VB1 200μg / l, Biotin 60 μg / l, soybean hydrolyzate (TN) 0.48 g / l, adjusted to pH 8.0 with KOH: autoclave 115 ° C for 10 minutes), then added 1 g of calcium carbonate sterilized by dry heat in advance And cultured at 31.5 ° C. with shaking. After complete consumption of sugar, 2 ml of the culture solution is added to 20 ml of the main culture medium (glucose 80 g / l, ammonium sulfate 30 g / l, KH ₂ PO ₄ 1 g / l, MgSO ₄ · 7H ₂ O 0.4 g / l, FeSO ₄・ 7H ₂ O 0.01g / l, MnSO ₄・ 4-5H ₂ O 0.01g / l, VB1 200μg / l, Biotin 60μg / l, soybean hydrolyzate (TN) 0.48g / l, pH8 using KOH. After adjusting to 0: autoclave 115 ° C. for 10 minutes), 1 g of calcium carbonate previously sterilized by dry heat was added and cultured at 31.5 ° C. with shaking. Two hours after the start of culture, penicillin G was added to a final concentration of 0.5 U / ml, and the culture was continued. After the sugar was completely consumed, the L-glutamic acid concentration in the medium was measured. As a result, it was shown that accumulation of L-glutamic acid was improved in the YggB-introduced strain (ATCC13869 / pL5k) compared to the vector-introduced strain. (Table 21)

The figure which shows the construction procedure of plasmid pBS3. The figure which shows the construction procedure of plasmid pBS4S. The figure which shows the construction procedure of plasmid pBS4sucAint. The figure which shows L-glutamic acid accumulation | storage of a yggB mutation-introduced strain. The figure which shows the mutation introduction location of 2A-1 type | mold mutation yggB gene. The figure (photograph) which shows the growth in a 4-fluoroglutamic acid containing CM-Dex plate culture medium of a mutant-type yggB gene introduction strain. The figure which shows the growth in the liquid culture medium containing a 4-fluoroglutamic acid (4-FG) of a mutant | variant yggB gene introduction | transduction strain | stump | stock.

Claims (29)

A coryneform bacterium having L-glutamic acid-producing ability, wherein the coryneform bacterium has improved L-glutamic acid-producing ability as compared with an unmodified strain by being modified using the yggB gene. The coryneform bacterium according to claim 1, wherein the yggB gene is DNA selected from any of the following (a) to (h):
(a) DNA comprising the nucleotide sequence of nucleotide numbers 1437 to 3035 of SEQ ID NO: 5,
(b) The coryneform bacterium by hybridizing under a stringent condition with a complementary base sequence of base numbers 1437 to 3035 of SEQ ID NO: 5 or a probe that can be prepared from the base sequence and introducing it into the coryneform bacterium. DNA which improves L-glutamic acid production ability of.
(c) DNA comprising the nucleotide sequence of base numbers 507 to 2093 of SEQ ID NO: 61,
(d) the coryneform bacterium by hybridizing with a complementary base sequence of base numbers 507 to 2093 of SEQ ID NO: 61 or a probe that can be prepared from the base sequence under stringent conditions and introducing it into the coryneform bacterium DNA which improves L-glutamic acid production ability of.
(e) DNA comprising the base sequence of base numbers 403 to 2001 of SEQ ID NO: 67,
(f) the coryneform bacterium by hybridizing under a stringent condition with a complementary base sequence of base numbers 403 to 2001 of SEQ ID NO: 67 or a probe that can be prepared from the base sequence and introducing it into the coryneform bacterium DNA which improves L-glutamic acid production ability of.
(g) DNA comprising the base sequence of base numbers 501 to 2099 of SEQ ID NO: 83,
(h) The coryneform bacterium by hybridizing under a stringent condition with a complementary base sequence of base numbers 501 to 2099 of SEQ ID NO: 83 or a probe that can be prepared from the base sequence and introducing it into the coryneform bacterium DNA which improves L-glutamic acid production ability of. The yggB gene is an amino acid sequence selected from SEQ ID NO: 6, 62, 68, 84, or 85, or one or several amino acids in an amino acid sequence selected from SEQ ID NO: 6, 62, 68, 84, or 85 The DNA according to claim 1, which encodes a protein having an amino acid sequence substituted, deleted, inserted or added and improves the L-glutamic acid-producing ability of the coryneform bacterium by introducing the protein into the coryneform bacterium. Coryneform bacteria. 2. The coryneform bacterium according to claim 1, which has been modified so that the expression level of the yggB gene is increased as compared with an unmodified strain. 5. The coryneform bacterium according to claim 4, wherein the coryneform bacterium has been modified so as to increase the expression level of the gene by increasing the copy number of the yggB gene or modifying the expression regulatory sequence of the gene. 2. The coryneform bacterium according to claim 1, wherein a mutant yggB gene is introduced. The mutant yggB gene has a mutation in a region encoding the sequence of amino acid numbers 419-533 of SEQ ID NO: 6, 68, 84 or 85, or the sequence of amino acid numbers 419-529 of SEQ ID NO: 62. The coryneform bacterium as described. 8. The coryneform bacterium according to claim 7, wherein the mutation is a deletion of the region. 8. The coryneform bacterium according to claim 7, wherein the mutation is an insertion sequence or a transposon insertion into the region. 8. The coryneform bacterium according to claim 7, wherein the mutation is a mutation that substitutes a proline present in the region with another amino acid. The coryneform according to claim 7, wherein the mutant yggB gene is a mutation that substitutes the proline at position 424 and / or 437 in the amino acid sequence of SEQ ID NO: 6, 62, 68, 84, or 85 with another amino acid. Type bacteria. The coryneform bacterium according to claim 6, wherein the mutant yggB gene is a gene having a mutation in a transmembrane region of a protein encoded by the yggB gene. The transmembrane region is selected from the group consisting of amino acid numbers 1-23, 25-47, 62-84, 86-108, and 110-132 in the amino acid sequence of SEQ ID NO: 6, 62, 68, 84 or 85 The coryneform bacterium according to claim 12, which is a region. The coryneform bacterium according to claim 12, wherein the mutation is introduced without accompanying frameshift. The mutant yggB gene is a gene having a mutation that substitutes alanine at position 100 and / or alanine at position 111 with another amino acid in the amino acid sequence of SEQ ID NO: 6, 62, 68, 84, or 85. 12. The coryneform bacterium according to 12. The mutant yggB gene is a gene having a mutation which inserts one or several amino acids between leucine at position 14 and tryptophan at position 15 in the amino acid sequence of SEQ ID NO: 6, 62, 68, 84 or 85. Item 13. A coryneform bacterium according to Item 12. The coryneform bacterium according to any one of claims 6 to 16, wherein L-glutamic acid analog resistance is improved by introduction of the mutant yggB gene. Furthermore, the coryneform bacterium according to any one of claims 6 to 17, which has been modified so that a gene that suppresses the function of the mutant yggB gene is inactivated. The coryneform bacterium according to claim 18, wherein the gene that suppresses the function of the mutant yggB gene is a symA gene, and the symA gene is a DNA selected from the following (i) or (j):
(i) DNA comprising the base sequence of base numbers 585 to 1121 of SEQ ID NO: 86,
(j) It hybridizes under a stringent condition with a complementary nucleotide sequence of nucleotide numbers 585 to 1121 of SEQ ID NO: 86 or a probe that can be prepared from the nucleotide sequence, and suppresses the function of the mutant yggB gene in coryneform bacteria DNA to do. Furthermore, the coryneform bacterium according to any one of claims 1 to 19, wherein the coryneform bacterium has been modified so that the α-ketoglutarate dehydrogenase activity is decreased. The coryneform bacterium according to any one of claims 1 to 20, which belongs to the genus Corynebacterium or Brevibacterium. The coryneform bacterium according to any one of claims 1 to 21 is cultured in a medium, and L-glutamic acid is produced and accumulated in the medium or in the fungus body, and L-glutamic acid is recovered from the medium or the fungus body. A process for producing L-glutamic acid characterized by A mutant yggB gene selected from the following (a) to (p):
(a) DNA encoding the amino acid sequence of SEQ ID NO: 8,
(b) An excess amount of biotin is encoded by encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 8 and introducing the protein into a coryneform bacterium. DNA which improves the L-glutamic acid-producing ability of the coryneform bacterium when cultured in a medium containing
(c) DNA encoding the amino acid sequence of SEQ ID NO: 20,
(d) an excess amount of biotin by encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 20 and introducing the protein into a coryneform bacterium. DNA which improves the L-glutamic acid-producing ability of the coryneform bacterium when cultured in a medium containing
(e) DNA encoding the amino acid sequence of SEQ ID NO: 22,
(f) An excess amount of biotin is encoded by encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 22 and introducing the protein into a coryneform bacterium. DNA which improves the L-glutamic acid-producing ability of the coryneform bacterium when cultured in a medium containing
(g) DNA encoding the amino acid sequence of SEQ ID NO: 24,
(h) In the amino acid sequence of SEQ ID NO: 24, an excess amount of biotin is encoded by encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added and introduced into a coryneform bacterium. DNA which improves the L-glutamic acid-producing ability of the coryneform bacterium when cultured in a medium containing
(i) DNA encoding the amino acid sequence of SEQ ID NO: 64,
(j) An excess amount of biotin is encoded by encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 64, and introducing the protein into a coryneform bacterium. DNA which improves the L-glutamic acid-producing ability of the coryneform bacterium when cultured in a medium containing
(k) DNA encoding the amino acid sequence of SEQ ID NO: 70,
(l) An excess amount of biotin is encoded by encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 70 and introducing the protein into a coryneform bacterium. DNA which improves the L-glutamic acid-producing ability of the coryneform bacterium when cultured in a medium containing
(m) DNA encoding the amino acid sequence of SEQ ID NO: 74,
(n) In the amino acid sequence of SEQ ID NO: 74, an excess amount of biotin is encoded by encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added and introduced into a coryneform bacterium. DNA which improves the L-glutamic acid-producing ability of the coryneform bacterium when cultured in a medium containing A method for producing a coryneform bacterium having a mutant yggB gene, wherein a coryneform bacterium lacking a gene encoding α-ketoglutarate dehydrogenase is inoculated into a medium containing an excess amount of biotin, and L- A method of obtaining a strain capable of accumulating glutamic acid. A method for producing a coryneform bacterium having a mutant yggB gene, in which a coryneform bacterium into which a yggB gene into which a mutation has been randomly introduced in vitro is introduced is inoculated into a medium containing an excess amount of biotin, and L -Obtaining a strain capable of accumulating glutamic acid. A method for producing a coryneform bacterium having a mutant yggB gene, wherein a coryneform bacterium in which a transposable element is randomly introduced on a chromosome is inoculated into a medium containing an excess amount of biotin, and L-glutamic acid is accumulated in the same medium. A method characterized by acquiring a possible stock. A method for producing a coryneform bacterium having a mutant yggB gene, comprising culturing a coryneform bacterium in a medium containing an L-glutamic acid analog to obtain a strain capable of growing in the same medium. The method for producing a coryneform bacterium according to any one of claims 24 to 27, wherein the strain having the mutant yggB gene is the coryneform bacterium according to any one of claims 6 to 16. The method for producing a coryneform bacterium according to any one of claims 24 to 28, wherein the medium containing an excess amount of biotin is a medium containing 30 µg / L or more of biotin.

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