Microorganisms for L-glutamic acid production and methods for amino acid production

Abstract

This invention relates to microorganisms that produce L-glutamic acid and a method for producing amino acids. The method involves culturing microorganisms in a culture medium that have the ability to produce one or more amino acids selected from L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline, and L-arginine, and that have been modified to increase the activity of α-ketoglutarate synthase, thereby generating and accumulating the L-amino acids in the culture medium or in the cells, and collecting the L-amino acids from the culture medium or in the cells.

Description

[0001] This application is a divisional application of the invention patent application filed on March 14, 2008, with application number 200880008072.3 (international application number PCT / JP2008 / 054735) entitled "Microorganisms for producing L-glutamic acid amino acids and methods for producing amino acids". Technical Field

[0002] This invention relates to microorganisms for producing L-glutamic acid amino acids and methods for producing L-glutamic acid amino acids. L-glutamic acid is widely used as a seasoning ingredient. Furthermore, L-glutamine, L-proline, L-ornithine, L-citrulline, and L-arginine are useful as seasonings, liver function promoters, amino acid infusions, and comprehensive amino acid preparations. Background Technology

[0003] L-glutamic acid is mainly produced by fermentation using L-glutamic acid-producing bacteria called coryneformbacteria, belonging to the genera Brevibacterium, Corynebacterium, and Microbacterium, or their mutant strains (see, for example, Non-Patent Literature 1). As a method for producing L-glutamic acid by using fermentation with other strains, the following methods are known: methods using microorganisms such as Bacillus, Streptomyces, or Penicillium (see, for example, Patent Document 1); methods using microorganisms such as Pseudomonas, Arthrobacter, Serratia, or Candida (see, for example, Patent Document 2); methods using microorganisms such as Bacillus, Pseudomonas, Serratia, or Aerobacter erogenes (now called Enterobacter erogenes) (see, for example, Patent Document 3); methods using mutant strains of Escherichia coli (see, for example, Patent Document 4), etc. In addition, methods for producing L-glutamic acid using microorganisms belonging to the genera Klebsiella, Erwinia, Pantothea, or Enterobacter have been disclosed (see, for example, Patent Documents 5-7).

[0004] Furthermore, various techniques for increasing L-glutamate production capacity by utilizing recombinant DNA technology to enhance the activity of L-glutamate biosynthesizers have been disclosed. For example, it has been reported that introducing a gene encoding citrate synthase from *Escherichia coli* or *Corynebacterium glutamicum* into *Corynebacterium* or *Bryophyte* bacteria is effective in enhancing L-glutamate production capacity in *Corynebacterium*-type bacteria (see, for example, Patent Document 8). Additionally, it has been reported that introducing a citrate synthase gene from *Corynebacterium*-type bacteria into *Enterobacteriaceae*, *Klebsiella*, *Serratia*, *Erwinia*, or *Escherichia*-family Enterobacteriaceae is effective in enhancing L-glutamate production capacity (see, for example, Patent Document 7).

[0005] Similar to L-glutamic acid, other L-glutamic acid amino acids, such as ornithine and citrulline (Non-Patent Literature 2-4), L-glutamine (Patent Literature 9), L-proline (Patent Literature 10), and L-arginine (Patent Literature 11, 12), are also produced by fermentation methods using microorganisms as described above.

[0006] Patent Document 1: U.S. Patent No. 3,220,929

[0007] Patent Document 2: U.S. Patent No. 3,563,857

[0008] Patent document 3, Japanese Patent Publication No. 32-9393

[0009] Patent document 4, Japanese Patent Application Publication No. 5-244970

[0010] Patent document 5, Japanese Patent Application Publication No. 2000-106869

[0011] Patent document 6, Japanese Patent Application Publication No. 2000-189169

[0012] Patent document 7, Japanese Patent Application Publication No. 2000-189175

[0013] Patent document No. 8, Japan Publication No. 7-121228

[0014] Patent document 9, JP2002-300887

[0015] Patent document 10 European Patent No. 1172433

[0016] Patent document 11, JP2000-287693

[0017] Patent document 12, JP2001-046082

[0018] Non-patent document 1 Akashi Kunihiko et al. "Amin Sour Yeast", published by the Society of Science and Technology, pp. 195-215, 1986

[0019] Non-patent literature 2 Lee, Y.-J. and Cho, J.-Y. 2006, Biotechnol. Lett. 28: 1849-1856

[0020] Non-patent literature 3 Choi, D Ketal. 1996. J. Ferment. Bioeng. 81: 216-219

[0021] Non-patent literature 4 Plachy, J. 1987. Kvasny Prumysl 33: 73-75 Summary of the Invention

[0022] The problem that the invention aims to solve

[0023] The objective of this invention is to provide a strain capable of efficiently producing L-glutamic acid amino acids, and a method for efficiently producing L-glutamic acid amino acids using the strain.

[0024] Problem-solving methods

[0025] As mentioned above, most technologies for improving L-glutamate production capacity involve enhancing the activity of enzymes in the TCA cycle. However, during L-glutamate fermentation via the TCA cycle, decarbonation caused by isocitrate dehydrogenase inevitably releases one molecule of CO2. Therefore, the inventors believe that to further improve production performance, this decarbonation must be reduced. Consequently, the inventors conducted in-depth research to address this issue and discovered that by increasing the activity of α-ketoglutarate synthase (an enzyme in the reduced TCA cycle) and further enhancing ferricoxin-NADP... + The production performance of L-glutamate can be improved by increasing the activity of reductases or pyruvate synthases (which have the activity of generating reduced ferricoxane or reduced flavin oxoreductin from their respective oxidized forms, which are necessary for the enzymatic activity of α-ketoglutarate synthase) or by increasing the production capacity of ferricoxane or flavin oxoreductin. This completes the present invention.

[0026] That is, the present invention is as follows.

[0027] (1) A microorganism having the ability to produce one or more L-amino acids selected from L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline and L-arginine, and said microorganism being modified to increase the activity of α-ketoglutarate synthase.

[0028] (2) The aforementioned microorganisms, wherein the activity of α-ketoglutarate synthase is increased by increasing the expression level of the gene encoding α-ketoglutarate synthase and / or increasing the translation level of the gene.

[0029] (3) The aforementioned microorganisms, wherein the activity of α-ketoglutarate synthase is increased by increasing the copy number of the gene encoding α-ketoglutarate synthase or by modifying the expression regulatory sequence of the gene.

[0030] (4) The aforementioned microorganism, wherein the gene encoding α-ketoglutarate synthase encodes a polypeptide represented by any one of (A) to (D) below, and a polypeptide represented by any one of (E) to (H):

[0031] (A) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 2;

[0032] (B) A polypeptide comprising an amino acid sequence obtained by substituting, deleting, inserting or adding one or more amino acids in SEQ ID NO: 2, wherein the polypeptide together with the polypeptide shown in any one of (E) to (H) constitutes a protein having α-ketoglutarate synthase activity.

[0033] (C) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 58;

[0034] (D) A polypeptide comprising an amino acid sequence obtained by substituting, deleting, inserting or adding one or more amino acids in SEQ ID NO: 58, and said polypeptide together with the polypeptide shown in any one of (E) to (H) constitutes a protein having α-ketoglutarate synthase activity.

[0035] (E) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 4;

[0036] (F) A polypeptide comprising an amino acid sequence obtained by substituting, deleting, inserting or adding one or more amino acids in SEQ ID NO: 4, and said polypeptide together with the polypeptide shown in any one of (A) to (D) constitutes a protein having α-ketoglutarate synthase activity.

[0037] (G) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 60;

[0038] (H) is a polypeptide containing an amino acid sequence obtained by substituting, deleting, inserting or adding one or more amino acids as in SEQ ID NO: 60, and said polypeptide together with the polypeptide shown in any one of (A) to (D) constitutes a protein having α-ketoglutarate synthase activity.

[0039] (5) The aforementioned microorganism, wherein the gene encoding α-ketoglutarate synthase comprises DNA from any one of (a) to (d) and DNA from any one of (e) to (h):

[0040] (a) DNA having the base sequence shown in SEQ ID NO: 1;

[0041] (b) DNA that hybridizes under stringent conditions to the complementary sequence of the base sequence shown in SEQ ID NO: 1 or to a probe that can be prepared from the base sequence, and the DNA encodes a polypeptide that, together with the polypeptide encoded by the DNA of any one of (e) to (h), constitutes a protein having α-ketoglutarate synthase activity.

[0042] (c) DNA having the base sequence shown in SEQ ID NO: 57;

[0043] (d) DNA that hybridizes under stringent conditions to a complementary sequence to the base sequence shown in SEQ ID NO: 57 or to a probe that can be prepared from the base sequence, and the DNA encodes a polypeptide that, together with the polypeptide encoded by any one of (e) to (h), constitutes a protein having α-ketoglutarate synthase activity.

[0044] (e) DNA having the base sequence shown in SEQ ID NO: 3;

[0045] (f) DNA that hybridizes under stringent conditions to the complementary sequence of the base sequence shown in SEQ ID NO: 3 or to a probe that can be prepared from the base sequence, and the DNA encodes a polypeptide that, together with the polypeptide encoded by the DNA of any one of (a) to (d), constitutes a protein having α-ketoglutarate synthase activity.

[0046] (g) DNA having the base sequence shown in SEQ ID NO: 59;

[0047] (h) DNA that hybridizes under stringent conditions to a complementary sequence to the base sequence shown in SEQ ID NO: 59 or to a probe that can be prepared from the base sequence, and said DNA encodes a polypeptide that, together with the polypeptide encoded by the DNA of any one of (a) to (d), constitutes a protein having α-ketoglutarate synthase activity.

[0048] (6) The aforementioned microorganism, wherein the microorganism is modified to increase ferroredoxin-NADP. + The activity of reductase.

[0049] (7) The aforementioned microorganism, wherein the microorganism is modified to increase the activity of pyruvate synthase.

[0050] (8) The aforementioned microorganism, wherein the microorganism is modified to enhance the production capacity of ferricoxin or flavin oxidoxin.

[0051] (9) The aforementioned microorganism, wherein the microorganism is modified to reduce the activity of α-ketoglutarate dehydrogenase.

[0052] (10) The aforementioned microorganism, wherein the microorganism is a bacterium belonging to a genus selected from the group consisting of Escherichia coli, Enterobacter spp., Pantotheca spp., Klebsiella spp. and Serratia spp.

[0053] (11) The aforementioned microorganism is a rod-shaped bacterium.

[0054] (12) A method for producing L-amino acids, comprising culturing the aforementioned microorganisms in a culture medium to generate and accumulate one or more L-amino acids selected from L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline, and L-arginine in the culture medium or in the microorganisms, and collecting the L-amino acids from the culture medium or the microorganisms.

[0055] (13) The aforementioned method is characterized in that the aforementioned microorganisms are cultured under aerobic conditions.

[0056] (14) The aforementioned method is characterized in that the aforementioned culture medium is a culture medium containing carbonate ions, bicarbonate ions or carbon dioxide gas, and the aforementioned microorganisms are cultured under anaerobic or microaerophilic conditions.

[0057] This invention also relates to the following aspects:

[0058] 1. A microorganism having the ability to produce one or more L-amino acids selected from L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline and L-arginine, and modified to increase the activity of α-ketoglutarate synthase.

[0059] 2. The microorganism of claim 1, wherein the activity of the α-ketoglutarate synthase is increased by increasing the expression level of the gene encoding the α-ketoglutarate synthase and / or increasing the translation level of the gene.

[0060] 3. The microorganism of item 2, wherein the activity of the α-ketoglutarate synthase is increased by increasing the copy number of the gene encoding the α-ketoglutarate synthase or by modifying the expression regulatory sequence of the gene.

[0061] 4. The microorganism described in item 2 or 3, wherein the gene encoding α-ketoglutarate synthase encodes a polypeptide represented by any one of (A) to (D) below, and a polypeptide represented by any one of (E) to (H):

[0062] (A) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 2;

[0063] (B) A polypeptide comprising an amino acid sequence obtained by substituting, deleting, inserting or adding one or more amino acids in SEQ ID NO: 2, wherein the polypeptide together with the polypeptide shown in any one of (E) to (H) constitutes a protein having α-ketoglutarate synthase activity.

[0064] (C) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 58;

[0065] (D) A polypeptide comprising an amino acid sequence obtained by substituting, deleting, inserting or adding one or more amino acids in SEQ ID NO: 58, and said polypeptide together with the polypeptide shown in any one of (E) to (H) constitutes a protein having α-ketoglutarate synthase activity.

[0066] (E) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 4;

[0067] (F) A polypeptide comprising an amino acid sequence obtained by substituting, deleting, inserting or adding one or more amino acids in SEQ ID NO: 4, and said polypeptide together with the polypeptide shown in any one of (A) to (D) constitutes a protein having α-ketoglutarate synthase activity.

[0068] (G) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 60;

[0069] (H) is a polypeptide containing an amino acid sequence obtained by substituting, deleting, inserting or adding one or more amino acids as in SEQ ID NO: 60, and said polypeptide together with the polypeptide shown in any one of (A) to (D) constitutes a protein having α-ketoglutarate synthase activity.

[0070] 5. The microorganism described in item 2 or 3, wherein the gene encoding α-ketoglutarate synthase comprises DNA from any one of (a) to (d) below, and DNA from any one of (e) to (h):

[0071] (a) DNA having the base sequence shown in SEQ ID NO: 1;

[0072] (b) DNA that hybridizes under stringent conditions to the complementary sequence of the base sequence shown in SEQ ID NO: 1 or to a probe that can be prepared from the base sequence, and the DNA encodes a polypeptide that, together with the polypeptide encoded by the DNA of any one of (e) to (h), constitutes a protein having α-ketoglutarate synthase activity.

[0073] (c) DNA having the base sequence shown in SEQ ID NO: 57;

[0074] (d) DNA that hybridizes under stringent conditions to a complementary sequence to the base sequence shown in SEQ ID NO: 57 or to a probe that can be prepared from the base sequence, and the DNA encodes a polypeptide that, together with the polypeptide encoded by any one of (e) to (h), constitutes a protein having α-ketoglutarate synthase activity.

[0075] (e) DNA having the base sequence shown in SEQ ID NO: 3;

[0076] (f) DNA that hybridizes under stringent conditions to the complementary sequence of the base sequence shown in SEQ ID NO: 3 or to a probe that can be prepared from the base sequence, and the DNA encodes a polypeptide that, together with the polypeptide encoded by the DNA of any one of (a) to (d), constitutes a protein having α-ketoglutarate synthase activity.

[0077] (g) DNA having the base sequence shown in SEQ ID NO: 59;

[0078] (h) DNA that hybridizes under stringent conditions to the complementary sequence of the base sequence shown in SEQ ID NO: 59 or to a probe that can be prepared from the base sequence, and said DNA encodes a polypeptide that, together with the polypeptide encoded by the DNA of any one of (a) to (d), constitutes a protein having α-ketoglutarate synthase activity.

[0079] 6. The microorganism of any one of claims 1-5, wherein the microorganism is modified to increase ferrugin-NADP. + The activity of reductase.

[0080] 7. The microorganism of any one of items 1-6, wherein the microorganism is modified to increase the activity of pyruvate synthase.

[0081] 8. The microorganism of any one of claims 1-7, wherein the microorganism is modified to enhance its ability to produce ferrugin or flavin ferrugin.

[0082] 9. The microorganism of any one of claims 1-8, wherein the microorganism is modified to reduce the activity of α-ketoglutarate dehydrogenase.

[0083] 10. The microorganism of any one of claims 1-9, wherein the microorganism is a bacterium belonging to a genus selected from the group consisting of: Escherichia, Enterobacter, Pantotheca, Klebsiella and Serratia.

[0084] 11. The microorganism mentioned in any one of items 1-9, wherein the microorganism is a corynebacterium.

[0085] 12. A method for producing L-amino acids, comprising culturing the microorganisms described in any one of items 1-11 in a culture medium, thereby generating and accumulating one or more L-amino acids selected from L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline, and L-arginine in the culture medium or within the microorganisms, and collecting the L-amino acids from the culture medium or the microorganisms.

[0086] 13. The method for producing L-amino acids according to item 12, characterized in that the microorganisms are cultured under aerobic conditions.

[0087] 14. The method for producing L-amino acids according to item 12, characterized in that the culture medium is a culture medium containing carbonate ions, bicarbonate ions or carbon dioxide gas, and the microorganisms are cultured under anaerobic or microaerophilic conditions.

[0088] Best mode for carrying out the invention

[0089] The present invention will now be described in detail.

[0090] <1> The microorganism of the present invention

[0091] The microorganism of the present invention is a microorganism capable of producing L-amino acids selected from L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline or L-arginine, and modified to increase the activity of α-ketoglutarate synthase.

[0092] In this invention, unless otherwise specified, "L-amino acid" refers to L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline, or L-arginine. These amino acids are called L-glutamic acid amino acids, which are L-glutamic acid or L-amino acids that can be obtained through biosynthesis using L-glutamic acid as a precursor.

[0093] "Ability to produce L-amino acids" or "L-amino acid production capacity" refers to the ability of the microorganisms of the present invention to generate and accumulate L-amino acids in cells or culture media to a degree that allows for the recovery of L-amino acids from the cells or culture medium when the microorganisms of the present invention are cultured in a culture medium. The L-amino acids produced by the microorganisms of the present invention can be one type, or two or more types of L-amino acids. Microorganisms capable of producing L-amino acids can be naturally occurring microorganisms with L-amino acid production capacity, or microorganisms obtained by modifying the microorganisms listed below using mutagenesis or recombinant DNA technology to give them L-amino acid production capacity, or microorganisms endowed with L-amino acid production capacity by introducing the genes of the present invention.

[0094] Furthermore, "increased activity of α-ketoglutarate synthase" has two meanings: increased activity of the enzyme in microorganisms that originally possess α-ketoglutarate synthase, and conferring the activity of the enzyme on microorganisms that do not possess α-ketoglutarate synthase.

[0095] <1-1> Contribution of L-amino acid production capacity

[0096] The microorganisms of the present invention can be obtained by modifying a parent strain of a microorganism capable of producing L-amino acids to increase the activity of α-ketoglutarate synthase. Furthermore, the microorganisms of the present invention can also be obtained by using a parent strain of a microorganism whose α-ketoglutarate synthase activity has been modified to enhance or confer L-amino acid production capacity.

[0097] The following examples illustrate methods for conferring L-amino acid production capacity to microorganisms and microorganisms conferring L-amino acid production capacity that can be used in this invention. However, the methods or microorganisms are not limited to those that can confer or possess L-amino acid production capacity.

[0098] As microorganisms used in this invention, examples include bacteria such as Enterobacteriaceae belonging to the genera *Escherichia*, *Enterobacter*, *Pantoea*, *Klebsiella*, *Serratia*, *Erwinia*, *Salmonella*, and *Morganella*, which are γ-proteobacteria; so-called corynebacterial bacteria belonging to the genera *Bryophyllobacterium*, *Corynebacterium*, and *Microbacterium*; and microorganisms belonging to the genera *Alicyclobacillus*, *Bacillus*, and *Saccharomyces*. γ-proteobacteria can be those classified according to the NCBI (National Center for Biotechnology Information) taxonomic database.

[0099] Microorganisms classified according to the taxonomy published at http: / / www.ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgiyymode=Undef&id=1236&lvl=3&p=mapview&p=has_linkout&p=blast_url&p=genome_blast&lin=f&keep=1&srchmode=1&unlock.

[0100] Examples of Escherichia coli include *Escherichia coli*. When using genetic engineering methods to breed *E. coli*, strain K-12 and its derivatives, such as *E. coli* MG1655 (ATCC47076) and W3110 (ATCC27325), can be used. *E. coli* strain K-12 was isolated by Stanford University in 1922. It is a lysogen of λ phage and possesses the F factor, making it a highly versatile strain capable of constructing genetic recombinants through conjugation and other methods. Furthermore, the genome sequence of *E. coli* strain K-12 has been determined, and its genetic information is freely available. *E. coli* strain K-12 and its derivatives can be obtained, for example, from the American Type Culture Collection (ATCC) (Address: PO Box 1549, Manassas, VA 20108, United States of America).

[0101] In particular, Pantotheca, Erwinia, and Enterobacteriaceae are classified as γ-Proteobacteria, and they are very closely related taxonomically (Harada H. and Ishikawa H. 1997. J. Gen. Appl. Microbiol. 43: 355-361; Kwon S.W. et al. 1997. Int. J. Syst. Bacteriol. 47: 1061-1067). In recent years, based on DNA-DNA hybridization experiments, some Enterobacteriaceae have been reclassified as Pantoea agglomerans or Pantoeadispersa, etc. (Gavini, F. et al. 1989. Int. J. Syst. Bacteriol. 39: 337-345). In addition, some bacteria belonging to the genus Erwinia have been reclassified as Pantoea anananas and Pantoeastewartii (see Mergaert, J. et al. 1993. Int. J. Syst. Bacteriol. 43: 162-173).

[0102] Enterobacteriaceae include, for example, *Enterobacter agglomerans* and *Enterobacter aerogenes*. Specifically, the strains exemplified in European Patent Application Publication 952221 can be used. *Enterobacter agglomerans* strain ATCC12287 is a representative strain of the *Enterobacteria* genus.

[0103] Representative strains of the Pantoea genus include Pantoea ananatis, Pantoea stearothermia, Pantoea clumpida, and Pantoea citrea. Specifically, the following strains can be listed.

[0104] Pantoeaananatis AJ13355 (FERMBP-6614) (European Patent Application Publication No. 0952221)

[0105] Pantoeaananatis AJ13356 (FERMBP-6615) (European Patent Application Publication No. 0952221)

[0106] Moreover, these strains were described as Enterobacter clumps in European Patent Application Publication No. 0952221, but now, as mentioned above, they have been reclassified as Pantoea ananatis based on 16S rRNA base sequence analysis.

[0107] Examples of Erwinia species include *Erwinia amylovora* and *Erwinia carotovora*; examples of Klebsiella species include *Klebsiella planticola*. Specifically, the following strains can be listed.

[0108] Erwinia amyloliquefaciens ATCC15580 strain

[0109] Erwinia carotene ATCC15713 strain

[0110] Plant-grown Klebsiella pneumoniae strain AJ13399 (FERMBP-6600) (European Patent Application Publication No. 955368)

[0111] Plant-grown Klebsiella pneumoniae strain AJ13410 (FERMBP-6617) (European Patent Application Publication No. 955368)

[0112] The term "corynebacterium" as used in this invention includes microorganisms defined in Bergey's Manual of Determinative Bacteriology, 8th edition, page 599 (1974). These include aerobic, Gram-positive, non-acid-resistant, non-spore-forming bacilli that were formerly classified under the genus *Brevibacterium* but are now uniformly classified under the genus *Corynebacterium* (Liebl, W. et al. 1991, Int. J. Syst. Bacteriol. 41: 255-260), as well as *Brevibacterium* and *Microbacterium* bacteria that are very closely related to *Corynebacterium*.

[0113] Corynebacterium-type bacteria suitable for use in the production of L-glutamic acid amino acids can be listed as, for example, the strains shown below.

[0114] Corynebacterium acetoacidophilum

[0115] Corynebacterium acetoglutamicum

[0116] Corynebacterium alkanolyticum

[0117] Corynebacterium callunae

[0118] Corynebacterium glutamicum

[0119] Corynebacterium lilium

[0120] Corynebacterium melassecola

[0121] Corynebacterium thermoaminogenes (Corynebacterium efficiens)

[0122] Corynebacterium herculis

[0123] Brevibacterium divaricatum (Corynebacterium glutamicum)

[0124] Brevibacterium flavum (Corynebacterium glutamicum)

[0125] Brevibacterium immariophilum

[0126] Brevibacterium lactofermentum (Corynebacterium glutamicum)

[0127] Brevibacterium roseum

[0128] Brevibacterium saccharolyticum

[0129] Brevibacterium thiogenitalis

[0130] Brevibacteriumammoniagenes (Corynebacteriumammoniagenes)

[0131] Brevibacterium album

[0132] Brevibacteriumcerinum

[0133] Microbacterium amammoniaphilum

[0134] Specifically, strains such as those listed below can be cited.

[0135] Corynebacterium thermophilum AJ12340 (FERMBP-1539)

[0136] Corynebacterium glutamicum ATCC13032

[0137] Corynebacterium glutamicum (Breastbacterium flavum) ATCC13826, ATCC14067

[0138] Lactobacillus fermentum (Corynebacterium glutamicum) ATCC13665, ATCC13869

[0139] Ammonia-producing short bacilli (Ammonia-producing Corynebacterium) ATCC6871

[0140] L-glutamic acid producing bacteria

[0141] As a method to endow or enhance the ability to produce L-glutamate, one example is to increase the expression of genes encoding enzymes related to L-glutamate biosynthesis by modification. Examples of enzymes related to L-glutamate biosynthesis include, for example: glutamate dehydrogenase (GDH, (gdhA)), glutamine synthase (glnA), glutamate synthase (gltAB), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglycerate mutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose diphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), glucose phosphate isomerase (pgi), 6-phosphogluconic acid dehydratase (edd), 2-keto-3-deoxy-6-phosphogluconic acid aldolase (eda), transhydrogenases, etc. In addition, the gene name is in parentheses after the enzyme name (the same applies below).

[0142] The following describes a method for modifying microorganisms to increase the expression of the genes encoding the enzymes related to L-glutamate biosynthesis.

[0143] The first method is to increase the copy number of the target gene. For example, the copy number of the gene can be increased by cloning the target gene into a suitable vector and transforming the host bacteria with the resulting vector. When the base sequence of the target gene has been elucidated in bacteria such as Escherichia and Corynebacterium, primers can be synthesized based on its base sequence, and the gene can be obtained using genomic DNA as a template via PCR (polymerase chain reaction) (see White, T.J. et al. 1989. Trends Genet. 5: 185-189). As glutamate dehydrogenases, the *E. coli* gdhA gene (Vallea, F. et al. 1984. Gene 27: 193-199) and *Corynebacterium glutamicum* gdh gene (Bormann, E.R. et al. 1992. Mol. Microbiol. 6: 317-326) are known; as phosphoenolpyruvate carboxylases, the *E. coli* ppc gene (Fujita, N. et al. 1984. J. Biochem. (Tokyo) 95: 909-916) and *Corynebacterium glutamicum* ppc gene (Eikmanns, B.J. et al. 1989. Mol. Gen. Genet. 218: 330-339) are known.

[0144] As vectors for transformation, plasmids capable of autonomous replication in the microorganisms used can be listed. For example, plasmids capable of autonomous replication in microorganisms belonging to the Enterobacteriaceae family include pUC19, pUC18, pBR322, RSF1010, pHSG299, pHSG298, pHSG399, pHSG398, pSTV28, pSTV29 (pHSG and pSTV are available from TAKARABio), pMW119, pMW118, pMW219, and pMW218 (pMW is available from NIPPONGENE). In addition, plasmids used for Corynebacterium-type bacteria include pAM330 (Japanese Patent Application Publication No. 58-67699), pHM1519 (Japanese Patent Application Publication No. 58-77895), pSFK6 (refer to Japanese Patent Application Publication No. 2000-262288), pVK7 (US Patent Application Publication No. 2003-0175912), pAJ655, pAJ611, pAJ1844 (Japanese Patent Application Publication No. 58-192900), pCG1 (Japanese Patent Application Publication No. 57-134500), pCG2 (Japanese Patent Application Publication No. 58-35197), pCG4, pCG11 (Japanese Patent Application Publication No. 57-183799), and pHK4 (Japanese Patent Application Publication No. 5-7491). Furthermore, DNA fragments capable of autonomously replicating plasmids in corynebacteria can be extracted from these vectors and inserted into the E. coli vector, thus enabling its use as a shuttle vector capable of autonomous replication in both E. coli and corynebacteria. Moreover, phage DNA can be used instead of plasmids as the vector.

[0145] Examples of transformation methods include: treating recipient bacterial cells with calcium chloride to increase DNA permeability, a method reported for Escherichia coli K-12 (Mandel, M. and Higa, AJ Mol. Biol. 1970. 53: 159-162); or preparing competent cells from cells in the proliferation phase and introducing DNA, a method reported for Bacillus subtilis (Duncan, CH et al. 1997. Gene1: 153-167). Alternatively, a method can be used to prepare DNA recipient bacteria cells into protoplasts or protoplast spheres that are easily introduced with recombinant DNA, and then introduce the recombinant DNA into the DNA recipient bacteria. This method is known to have been used for Bacillus subtilis, actinomycetes, and yeast (Chang, S. and Choen, SN., 1979. Mol. Gen. Genet. 168: 111-115; Bibb, M.J. et al., 1978. Nature 274: 398-400; Hinnen, A. et al., 1978. Proc. Natl. Acad. Sci. USA 75: 1929-1933). Furthermore, microbial transformation can also be performed using the electroporation method (Japanese Patent Application Publication No. 2-207791).

[0146] Increasing gene copy number can also be achieved by introducing multiple copies of the target gene into the genomic DNA of microorganisms. To introduce multiple copies of the target gene into the genomic DNA of microorganisms, sequences existing in multiple copies on the genomic DNA can be used as targets, and homologous recombination can be performed (Miller I, JH Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Repetitive DNA or inverted repeat sequences located at the ends of transposon elements can be used as sequences existing in multiple copies on the genomic DNA. Alternatively, as disclosed in Japanese Patent Application Publication No. 2-109985, the target gene can be loaded into a transposon, causing it to transfer and thus introducing multiple gene copies into the genomic DNA. Furthermore, the target gene can be integrated into the host genome using the Mu phage method (Japanese Patent Application Publication No. 2-109985). The fact that the target gene has been transferred to the genome can be confirmed by performing Southren hybridization using a portion of the gene as a probe.

[0147] As for the copy number, it can be any number as long as it can enhance the activity of the target gene product, but ideally it is more than two copies.

[0148] The second method involves replacing the expression regulatory sequences of the target gene, such as the promoter, on the genomic DNA or plasmid with expression regulatory sequences of appropriate strength to enhance the expression of the target gene. For example, the thr promoter, lac promoter, trp promoter, trc promoter, pL promoter, and tac promoter are commonly known promoters. Examples of highly expressed promoters in Corynebacterium-type bacteria include the promoter of the elongation factor Tu (EF-Tu) gene tuf (SEQ ID NO: 77), and promoters of genes encoding cochaperonin GroES-chaperonin GroEL, thioredoxin reductase, phosphoglycerate mutase, and glyceraldehyde-3-phosphate dehydrogenase (WO2006 / 028063 and EP1697525). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein and Doi’s paper (Goldstein, MA and Doi R.H. 1995. Biotechnol. Annu. Rev., 1: 105-128).

[0149] Furthermore, as described in International Publication WO00 / 18935, a promoter of suitable strength can be modified by introducing several base substitutions into the promoter region of a gene. Substitution of expression regulatory sequences can be performed, for example, as with gene substitution using temperature-sensitive plasmids. Examples of temperature-sensitive origin of replication vectors that can be used in *E. coli* or *Pantoea ananatis* include, for instance, the temperature-sensitive plasmid pMAN997 or its derivatives as described in International Publication WO99 / 03988. Furthermore, substitution of expression regulatory sequences can also be performed using linear DNA methods: a method called "Red-driven integration" utilizing the Red recombinase of λ phage (Datsenko, K.A. and Wanner, BL, 2000. Proc. Natl. Acad. Sci. USA. 97: 6640-6645), and a method combining Red-driven integration with a λ phage-derived excision system (Cho, E. Hetal. 2002. J. Bacteriol. 184: 5200-5203) (see WO2005 / 010175), etc. Moreover, modification of expression regulatory sequences can be combined with methods described above for increasing gene copy number.

[0150] Furthermore, it is known that substitution of several nucleotides in the spacer sequence between the ribosome binding site (RBS) and the start codon, especially in the sequence immediately upstream of the start codon, has a significant impact on mRNA translation efficiency, and these sequences can be modified to increase translation yield.

[0151] Examples of microorganisms that have been modified in a manner such as those described above to increase the expression of genes related to L-glutamate production include those described in International Publication No. WO99 / 07853 and European Patent No. 1352966.

[0152] When introducing a target gene into the aforementioned plasmid or genome, the promoter used to express these genes can be any promoter capable of functioning in the microorganism used, the promoter of the gene itself, or a modified promoter. Gene expression can also be regulated by appropriately selecting a promoter that functions strongly in the microorganism used, or by bringing the -35 and -10 regions of the promoter closer to a shared sequence. Microorganisms whose expression of genes such as glutamate dehydrogenase is enhanced by modification as described above are described in WO00 / 18935, European Patent Application Publication 1010755, and other specifications.

[0153] The methods for enhancing gene expression described above can also be applied to the gene encoding α-ketoglutarate synthase, which will be discussed later.

[0154] Modifications to confer L-glutamate production capacity can also be achieved by reducing or eliminating the activity of enzymes that catalyze reactions that branch off from the L-glutamate biosynthetic pathway and generate compounds other than L-glutamate. Examples of enzymes that catalyze reactions that branch off from the L-glutamate biosynthetic pathway and generate compounds other than L-glutamate include α-ketoglutarate dehydrogenase, isocitrate lyase, acetate kinase, acetylhydroxylase, acetyllactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, and 1-pyrrolino-5-carboxylic acid dehydrogenase. Among these, reducing or eliminating the activity of α-ketoglutarate dehydrogenase is particularly preferred.

[0155] To reduce or eliminate the activity of enzymes such as those described above, conventional mutagenesis methods or genetic engineering techniques can be used to introduce mutations into the genes of these enzymes that reduce or eliminate their activity in cells. Mutagenesis methods include, for example, irradiation with X-rays or ultraviolet light, or treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine. The site where the mutation is introduced into the gene can be the coding region of the enzyme protein or an expression regulatory region such as a promoter. Furthermore, in terms of genetic engineering techniques, methods such as gene recombination, transduction, and cell fusion can be used.

[0156] The reduction or absence of target enzyme activity in cells, and the degree of reduction, can be confirmed by measuring the enzyme activity of cell extracts or purified fractions of candidate strains and comparing them with wild-type strains. For example, α-ketoglutarate dehydrogenase activity can be measured according to the method of Reed and Mukherjee (Reed, L.J. and Mukherjee, BB 1969. Methods in Enzymology 13: 55-61).

[0157] Methods for depleting or reducing α-ketoglutarate dehydrogenase activity in Escherichia coli are described in Japanese Patent Application Publication Nos. 5-244970 and 7-203980, among others. Furthermore, methods for depleting or reducing α-ketoglutarate dehydrogenase activity in Corynebacterium species are described in International Publication No. 95 / 34672. Moreover, methods for depleting or reducing α-ketoglutarate dehydrogenase activity in Enterobacteriaceae are disclosed in Japanese Patent Application Publication No. 2001-333769.

[0158] For example, to reduce the activity of α-ketoglutarate dehydrogenase, the sucA(odhA) gene encoding the E1o subunit of this enzyme can be modified. Examples of strains that reduce α-ketoglutarate dehydrogenase activity include the following.

[0159] Lactobacillus fermentum strain ΔS (International Publication No. 95 / 34672)

[0160] Lactobacillus fermentum AJ12821 (FERMBP-4172; see French Patent Publication No. 9401748).

[0161] Flavobacterium flammatum AJ12822 (FERMBP-4173; see French Patent Publication No. 9401748)

[0162] Corynebacterium glutamicum AJ12823 (FERMBP-4174; see French Patent Publication No. 9401748).

[0163] Corynebacterium glutamicum ATCC13869OAGN, OA2-2, OAGN2-2 (refer to International Publication No. 2006 / 028298)

[0164] Escherichia coli AJ12624 (FERMBP-3853)

[0165] Escherichia coli AJ12628 (FERMBP-3854)

[0166] Escherichia coli AJ12949 (FERMBP-4881)

[0167] Lactobacillus fermentum strain ΔS (refer to International Publication No. 95 / 34672)

[0168] Pantoeaananatis AJ13601 (FERMBP-7207 European Patent Publication 1078989)

[0169] Pantoeaananatis AJ13356 (FERMBP-6615, US Patent No. 6,331,419)

[0170] PantoeaananatisSC17sucA(FERMBP-8646WO2005 / 085419)

[0171] Plant-growing Klebsiella pneumoniae AJ13410 (FERMBP-6617, US Patent No. 6,197,559)

[0172] As an example of an L-glutamic acid-producing bacterium of Pantoeaananatis, strain Pantoeaananatis AJ13355 can be cited. Pantoeaananatis AJ13355 is a strain isolated from soil in Iwata City, Shizuoka Prefecture, capable of propagating in a medium containing L-glutamic acid and a carbon source at low pH. Pantoeaananatis AJ13355 was deposited on February 19, 1998, at the Patent Biological Collection Center of the National Institute of Advanced Industrial Science and Technology (address: 1-1-1-Chuo-6, Higashi, Tsukuba City, Ibaraki Prefecture, Japan 305-8566, Japan), accession number FERMP-16644; and on January 11, 1999, it was transferred to international deposit under the Budapest Treaty, accession number FERMP-6614. Furthermore, this strain was initially identified as *Enterobacter agglomerans* during isolation and was preserved as *Enterobacter agglomerans* AJ13355. However, in recent years, based on 16S rRNA base sequence analysis and other factors, it has been reclassified as *Pantoea ananatis*.

[0173] Furthermore, L-glutamate-producing bacteria of *Pantoea ananatis* can be listed as those belonging to the *Pantoea* genus that exhibit deficient or reduced α-ketoglutarate dehydrogenase (αKGDH) activity. Examples of such strains include AJ13356 (US Patent No. 6,331,419), derived from strain AJ13355 by deleting the αKGDH-E1 subunit gene (sucA), and SC17sucA (US Patent No. 6,596,517), a sucA gene-deficient strain derived from strain SC17, selected as a mucin-low production mutant strain from AJ13355. AJ13356 was deposited on February 19, 1998, at the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (now the Patent Biological Collection Center of the National Institute of Advanced Industrial Science and Technology, 〒305-8566, 1-1 Higashi-1-1 Chuo-6, Tsukuba City, Ibaraki Prefecture, Japan), with accession number FERMP-16645; and on January 11, 1999, it was transferred to international deposit under the Budapest Treaty, with accession number FERMP-6616. AJ13355, AJ13356, and strain AJ13601 (described later) are deposited as *Enterobacter clumps* at the aforementioned institutions, but are referred to as *Pantoea ananatis* in this specification. Furthermore, strain SC17sucA, internally designated as strain AJ417, was deposited on February 26, 2004, at the Patent Biological Collection Center of the National Institute of Advanced Industrial Science and Technology, with accession number FERMP-08646.

[0174] Furthermore, examples of L-glutamate-producing bacteria in *Pantoea ananatis* include strains SC17sucA / RSFCPG+pSTVCB, AJ13601, NP106, and NA1. Strain SC17sucA / RSFCPG+pSTVCB was obtained by introducing plasmid RSFCPG, containing the *E. coli*-derived citrate synthase gene (gltA), phosphoenolpyruvate carboxylase gene (ppsA), and glutamate dehydrogenase gene (gdhA), and plasmid pSTVCB, containing the *Brevibacterium lactis*-derived citrate synthase gene (gltA), into strain SC17sucA. Strain AJ13601 was selected from this SC17sucA / RSFCPG+pSTVCB strain as a strain exhibiting resistance to high concentrations of L-glutamate at low pH. Furthermore, strain NP106 was obtained by detaching plasmid RSFCPG+pSTVCB from strain AJ13601 as described in the examples. Strain AJ13601 was deposited on August 18, 1999, at the Patent Biology Collection Center of the National Institute of Advanced Industrial Science and Technology (address: 1-1-1-Chuo-6, Higashi, Tsukuba City, Ibaraki Prefecture, Japan 305-8566, Japan), accession number FERMP-17516; and was transferred to international deposit under the Budapest Treaty on July 6, 2000, accession number FERMP-7207.

[0175] Furthermore, as a method to confer L-glutamate production capacity to Corynebacterium-type bacteria, the following methods can also be used: amplifying the yggB gene (NCgl1221; NP_600492[gi:19552490]) or introducing a mutant yggB gene with a mutation introduced into the coding region (WO2006 / 070944).

[0176] L-glutamate production capacity can also be conferred by amplifying the yhfK gene, which is the L-glutamate secretion gene (WO2005 / 085419).

[0177] Other methods for conferring or enhancing L-glutamate production capacity include: methods for conferring resistance to organic acid analogs, respiratory chain inhibitors, etc., and methods for conferring sensitivity to cell wall synthesis inhibitors. Examples include: methods for conferring resistance to monofluoroacetic acid (Japanese Patent Application Laid-Open No. 50-113209), methods for conferring resistance to adenine or thymine (Japanese Patent Application Laid-Open No. 57-065198), methods for weakening urease (Japanese Patent Application Laid-Open No. 52-038088), methods for conferring resistance to malonic acid (Japanese Patent Application Laid-Open No. 52-038088), methods for conferring resistance to benzopyranone or naphthoquinones (Japanese Patent Application Laid-Open No. 56-1889), methods for conferring resistance to HOQNO (Japanese Patent Application Laid-Open No. 56-140895), methods for conferring resistance to α-ketomalonic acid (Japanese Patent Application Laid-Open No. 57-2689), methods for conferring resistance to guanidine (Japanese Patent Application Laid-Open No. 56-35981), and methods for conferring sensitivity to penicillin (Japanese Patent Application Laid-Open No. 4-88994), etc.

[0178] The following strains can be listed as specific examples of such resistant bacteria:

[0179] Brevibacterium flavum AJ3949 (FERMBP-2632: refer to Japanese Patent Application Laid-Open No. 50-113209)

[0180] Corynebacterium glutamicum AJ11628 (FERMP-5736; see Japanese Patent Application Publication No. 57-065198)

[0181] Fermentobacterium flavum AJ11355 (FERMP-5007; see Japanese Patent Application Publication No. 56-1889)

[0182] Corynebacterium glutamicum AJ11368 (FERMP-5020; see Japanese Patent Application Publication No. 56-1889)

[0183] Fermentobacterium flavum AJ11217 (FERMP-4318; see Japanese Patent Application Publication No. 57-2689)

[0184] Corynebacterium glutamicum AJ11218 (FERMP-4319; see Japanese Patent Application Publication No. 57-2689)

[0185] Fermentobacterium flavum AJ11564 (FERMP-5472; see Japanese Patent Application Publication No. 56-140895)

[0186] Fermentobacterium flavum AJ11439 (FERMP-5136; see Japanese Patent Application Publication No. 56-35981)

[0187] Corynebacterium glutamicum H7684 (FERMBP-3004; see Japanese Patent Application Publication No. 04-88994)

[0188] Lactobacillus fermentum AJ11426 (FERMP-5123; see Japanese Patent Application Publication No. 56-048890)

[0189] Corynebacterium glutamicum AJ11440 (FERMP-5137; see Japanese Patent Application Publication No. 56-048890)

[0190] Lactobacillus fermentum AJ11796 (FERMP-6402; see Japanese Patent Application Publication No. 58-158192)

[0191] L-glutamine producing bacteria

[0192] Preferred examples of microorganisms capable of producing L-glutamine include bacteria with enhanced glutamate dehydrogenase activity, bacteria with enhanced glutamine synthase (glnA) activity, and bacteria with disrupted glutaminase genes (European Patent Application Publications Nos. 1229121 and 1424398). Enhanced glutamine synthase activity can also be achieved through disruption of the glutamine adenylate transferase gene (glnE) or the PII regulatory protein gene (glnB) (EP1229121). Furthermore, as preferred examples of L-glutamine-producing bacteria, bacteria belonging to the genus *Escherichia* possessing a mutant glutamine synthase described below, in which the tyrosine residue at position 397 of the mutant glutamine synthase is replaced by another amino acid residue (US Patent Application Publication No. 2003-0148474).

[0193] Methods for conferring or enhancing L-glutamine production capacity include: methods for conferring resistance to 6-diazo-5-oxo-leucine (Japanese Patent Application Laid-Open No. 3-232497), methods for conferring resistance to purine analogs and methionine sulfoxide (Japanese Patent Application Laid-Open No. 61-202694), and methods for conferring resistance to α-ketomaleic acid (Japanese Patent Application Laid-Open No. 56-151495), etc. Specific examples of Corynebacterium-type bacteria with L-glutamine production capacity include the following microorganisms:

[0194] Flavobacterium flammatum AJ11573 (FERMP-5492, TEDA 56-161495)

[0195] Flavobacterium flammatum AJ11576 (FERMBP-10381, TEDA 56-161495)

[0196] Fermentobacterium flavum AJ12212 (FERMP-8123, ITEMS 61-202694)

[0197] L-proline producing bacteria

[0198] Examples of microorganisms capable of producing L-proline include bacteria carrying γ-glutamyl kinase that relieves L-proline feedback inhibition and bacteria with weakened L-proline degradation systems. A method for modifying bacteria using DNA encoding γ-glutamyl kinase that relieves L-proline feedback inhibition is disclosed in the work of Dandekar and Uratsu (J. Bacteriol. 170, 12: 5943-5945 (1988)). Furthermore, methods for obtaining bacteria with weakened L-proline degradation systems include, for example, introducing mutations into the proline dehydrogenase gene that reduce enzyme activity. Specific examples of bacteria capable of producing L-proline include Escherichia coli strains NRLRB-12403 and NRLRB-12404 (British Patent 2075056), Escherichia coli strain VKPMB-8012 (US Patent Publication 2002-0058315), and strains carrying the following plasmid mutants: plasmid mutants disclosed in German Patent No. 3127361, or plasmid mutants disclosed in the literature of Bloom F.R. et al. (The 15th Miami Winter Symposium, 1983, p. 34).

[0199] In addition, preferred microorganisms with L-proline production capacity include Escherichia coli strain 702 (VKPMB-8011), which is resistant to 3,4-dehydroxyproline and azacyclobutane-2-carboxylic acid; strain 702ilvA (VKPMB-8012), which is an ilvA-deficient strain of strain 702; and Escherichia coli that enhances the activity of proteins encoded by the b2682, b2683, b1242 or b3434 genes (Japanese Patent Application Laid-Open No. 2002-300874), etc.

[0200] L-arginine producing bacteria

[0201] As microorganisms capable of producing L-arginine, examples include *E. coli* mutant strains resistant to α-methylmethionine, p-fluorophenylalanine, D-arginine, arginine isohydroxamic acid, S-(2-aminoethyl)-cysteine, α-methylserine, β-2-thiophene alanine, or sulfaguanidine (see Japanese Patent Application Publication No. 56-106598). Furthermore, *E. coli* strain 237 (Russian Patent Application No. 2000117677), an L-arginine-producing strain with feedback inhibition resistance to L-arginine and carrying highly active N-acetylglutamate synthase, is also a suitable L-arginine-producing strain. This strain was deposited on April 10, 2000, at the Russian National Collection of Industrial Microorganisms (VKPM), GNII Genetika, accession number VKPMB-7925, and transferred to international deposit under the Budapest Treaty on May 18, 2001. Alternatively, *Escherichia coli* strain 382, ​​a derivative of strain 237, is an L-arginine-producing bacterium with improved ability to assimilate acetic acid (Japanese Patent Application Publication No. 2002-017342). *Escherichia coli* strain 382 was deposited on April 10, 2000, at the Russian National Collection of Industrial Microorganisms (VKPM), accession number VKPMB-7926.

[0202] Furthermore, as microorganisms endowed with the ability to produce L-arginine, microorganisms that increase the expression levels of genes encoding enzymes related to L-arginine biosynthesis can be used. For example, enzymes selected from the L-arginine biosynthesis system include one or more of N-acetylglutamate synthase (argA), N-acetylglutamyl-phosphoreductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), acetyl-ornithine transaminase (argD), acetyl-ornithine deacetylase (argE), ornithine carbamoyltransferase (argF), arginine succinate synthase (argG), arginine succinate lyase (argH), and carbamoyl phosphate synthase (carAB). For N-acetylglutamate synthase (argA), a mutant gene is more preferred, in which the amino acid sequence corresponding to positions 15 to 19 of the wild type is substituted, and the feedback inhibition caused by L-arginine is relieved (European Application Publication No. 1170361).

[0203] For L-arginine-producing bacteria belonging to the Corynebacterium type, there are no special restrictions on the type of Corynebacterium that can produce L-arginine, as long as they possess this ability. This includes, for example: wild-type Corynebacterium strains; Corynebacterium strains resistant to sulfonamides, 2-thiazolinone, or α-amino-β-hydroxyvalerate; and, in addition to 2-thiazolinone resistance, possessing L-histidine, L-proline, L-threonine, L-isoleucine, L-methionine, or L-tryptophan. Nutritionally deficient corynebacteria (Japanese Patent Application Publication No. 54-44096); corynebacteria resistant to ketomalonic acid, fluoromalonic acid, or monofluoroacetic acid (Japanese Patent Application Publication No. 57-18989); corynebacteria resistant to argininol (Japanese Patent Application Publication No. 62-24075); or corynebacteria resistant to X-guanidine (where X is a derivative of a fatty acid or aliphatic chain) (Japanese Patent Application Publication No. 2-186995), etc.

[0204] In addition, *Corynebacterium* bacteria capable of producing L-arginine can be selected as the following strains: mutant strains resistant to 5-azouracil, 6-azouracil, 2-thiouracil, 5-fluorouracil, 5-bromouracil, 5-azocytosine, and 6-azocytosine; mutant strains resistant to arginine isohydroxamic acid or 2-thiouracil; mutant strains resistant to arginine isohydroxamic acid and 6-azouracil (Japanese Patent Application Laid-Open No. 49-126819); mutant strains resistant to histidine or tryptophan analogs (Japanese Patent Application Laid-Open No. 52-114092); and strains resistant to at least one of methionine, histidine, threonine, proline, isoleucine, lysine, adenine, guanine, or uracil (or uracil precursor). Mutant strains exhibiting nutritional auxotrophicity (see Japanese Patent Application Publication No. 52-99289); mutant strains resistant to arginine isohydroxamic acid (Japanese Patent Application Publication No. 51-6754); mutant strains exhibiting succinic acid auxotrophicity or resistance to nucleic acid base analogs (Japanese Patent Application Publication No. 58-9692); mutant strains with impaired arginine degradation ability, resistance to arginine antagonists and canavalialine, and lysine auxotrophicity (Japanese Patent Application Publication No. 52-8729); mutant strains resistant to arginine, arginine isohydroxamic acid, high arginine, D-arginine, and canavalialine, or arginine isohydroxamic acid and 6-azouridine (Japanese Patent Application Publication No. 53-143288); and mutant strains resistant to canavalialine (Japanese Patent Application Publication No. 53-3586), etc.

[0205] The following strains can be cited as specific examples of rod-shaped bacteria capable of producing L-arginine.

[0206] Yellow short bacillus AJ11169 (FERMBP-6892)

[0207] Lactobacillus fermentum AJ12092 (FERMBP-6906)

[0208] Yellow short bacillus AJ11336 (FERMBP-6893)

[0209] Yellow short bacillus AJ11345 (FERMBP-6894)

[0210] Lactobacillus fermentum AJ12430 (FERMBP-2228)

[0211] Furthermore, strains deficient in the arginine repressor ArgR (US Patent Application Publication No. 2002-0045223) and strains with increased intracellular glutamine synthase activity (US Patent Application Publication No. 2005-0014236) can also be used.

[0212] The biosynthetic pathways of L-citrulline and L-ornithine are the same as those of L-arginine. The production capacity of these amino acids can be conferred by increasing the enzyme activity of the following enzymes: N-acetylglutamate synthase (argA), N-acetylglutamyl phosphoryl reductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), acetylornithine transaminase (argD), and acetylornithine deacetylase (argE) (International Publication No. 2006-35831).

[0213] Furthermore, in the L-amino acid producing bacteria used in this invention, in addition to the genes encoding enzymes of the natural biosynthetic system, genes related to sugar uptake, sugar metabolism (glycolysis system), and energy metabolism can also be amplified.

[0214] Genes related to sugar metabolism include those encoding enzymes in the glycolysis system or sugar uptake genes, such as glucose-6-phosphate isomerase gene (pgi; International Publication No. 01 / 02542), phosphoenolpyruvate synthase gene (pps; European Patent Application Publication No. 877090), phosphoglucose mutase gene (pgm; International Publication No. 03 / 04598), fructose-2-phosphate aldolase gene (pfkBfbp; International Publication No. 03 / 04664), and pyruvate kinase gene (pykF; International Publication No. 03). The following gene sequences are listed in the patent application: pamphlet 008609, transaldolase gene (talB; international publication pamphlet 03 / 008611), fumarate gene (fum; international publication pamphlet 01 / 02545), phosphoenolpyruvate synthase gene (pps; European patent application pamphlet 877090), non-PTS sucrose uptake gene (csc; European patent application pamphlet 149911), and sucrose assimilation gene (scrAB operon; international publication pamphlet 90 / 04636).

[0215] Examples of genes involved in energy metabolism include the transhydrogenase gene (pntAB; U.S. Patent No. 5,830,716) and the cytochrome botype oxidase gene (cyoB; European Patent Application Publication No. 1070376).

[0216] In addition, when glycerol is used as a carbon source, in order to improve glycerol assimilation, the expression of the glpR gene (EP1715056) can be weakened, or the expression of glycerol metabolism genes such as glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, tpiA, gldA, dhaK, dhaL, dhaM, dhaR, fsa, and talC (EP1715055A) can be enhanced.

[0217] <1-2> Enhanced α-ketoglutarate synthase activity

[0218] The microorganisms of the present invention are microorganisms capable of producing L-glutamic acid amino acids and modified to increase the activity of α-ketoglutarate synthase. Modification to increase α-ketoglutarate synthase activity means modification that increases the activity of α-ketoglutarate synthase compared to the parent strain (e.g., wild-type or unmodified strain). Furthermore, as mentioned above, in microorganisms that originally lacked α-ketoglutarate synthase activity, the modified microorganisms exhibit increased α-ketoglutarate synthase activity compared to unmodified strains.

[0219] In this invention, "α-ketoglutarate synthase" refers to an enzyme (EC 1.2.7.3) that, as described above, catalyzes the reaction of succinyl-CoA and CO2 to produce α-ketoglutarate in the presence of an electron donor, such as ferrugin. α-ketoglutarate synthase is sometimes also named α-ketoglutarate oxide reductase, α-ketoglutarate ferrugin oxide reductase, 2-oxoglutarate synthase, 2-oxoglutarate oxide reductase, or 2-oxoglutarate ferrugin oxide reductase. Ferroreductin or flavin oxidoreductin can be used as the electron donor.

[0220] Furthermore, the parental strains modified to enhance α-ketoglutarate synthase can be strains that naturally possess the gene encoding α-ketoglutarate synthase, or strains that do not naturally possess the α-ketoglutarate synthase gene but are endowed with the activity of the enzyme by introducing the α-ketoglutarate synthase gene, thereby increasing their L-glutamate production capacity.

[0221] Modification can be performed first to enhance the enzyme activity of α-ketoglutarate synthase, and then L-glutamate production capacity can be conferred. Alternatively, L-glutamate production capacity can be conferred first, and then modification can be performed to enhance the enzyme activity of α-ketoglutarate synthase. Moreover, the activity of α-ketoglutarate synthase can be enhanced by methods such as enhancing gene expression as described above. That is, the expression of endogenous α-ketoglutarate synthase genes can be enhanced by modifying expression regulatory regions, such as promoter modification, or the expression of exogenous α-ketoglutarate synthase genes can be enhanced by introducing plasmids containing α-ketoglutarate synthase genes.

[0222] The enhanced activity of α-ketoglutarate synthase has been confirmed by preparing crude enzyme solutions from unmodified and modified microorganisms and comparing their α-ketoglutarate synthase activities. The activity of α-ketoglutarate synthase can be determined, for example, by the method of Yun et al. (Yun, N. Retal. 2001. Biochem. Biophy. Res. Commum. 282: 589-594). For example, it can be determined by spectroscopic methods using oxidized methyl viologen (as an electron acceptor), CoA, and crude enzyme solution, to measure the increase in the amount of reduced methyl viologen due to the decarbonylation of α-ketoglutarate upon addition. One unit of enzyme activity (U) is expressed as the reduction of 1 μmol of methyl viologen per minute. Enhancing the activity of α-ketoglutarate synthase is sufficient if it is improved compared to the wild-type strain. However, when the parent strain possesses α-ketoglutarate synthase activity, it is ideal that the enzyme activity increases by at least 1.5 times, more preferably at least 2 times, and even more preferably at least 3 times compared to the parent strain. Furthermore, when the parent strain does not possess α-ketoglutarate synthase activity, it is sufficient to produce α-ketoglutarate synthase by introducing the α-ketoglutarate synthase gene. However, it is preferable that the modification results in a measurable enzyme activity, ideally at least 0.001 U / mg, more preferably at least 0.005 U / mg, and even more preferably at least 0.01 U / mg relative to bacterial protein.

[0223] Genes encoding α-ketoglutarate synthase can be derived from bacteria exhibiting a reducing TCA cycle, such as *Chlorobium*, *Desulfobacter*, *Aquifex*, *Hydrogenobacter*, *Thermoproteus*, and *Pyrobaculum*, or their homologs. Specifically, the α-ketoglutarate synthase genes of *Chlorobium tetrapidum* and *Hydrogenobacter thermophilus* can be cited. Furthermore, genes presumed to be α-ketoglutarate synthase genes also exist in *Blastopirellula marina* (Schlesner, H. et al. 2004. Int. J. Syst. Evol. Microbiol. 54: 1567-1580), a marine bacterium belonging to the order Planctomycetes. Furthermore, genes with high homology to the α-ketoglutarate synthase gene have also been found in the genomes of sulfur-oxidizing bacteria such as *Sulfurimonas denitrificans* (Brinkhoff, T. et al. 1999. Int. J. Syst. Bacteriol. 49: 875-879) and the methanogenic bacterium *Methanococcus maripaludis* (Jones, W. J. et al. Arch. Microbiol. 1983. 135: 91-97), and the α-ketoglutarate synthase gene can also be selected from them.

[0224] α-Ketoglutarate synthase is known to function as a complex of multiple peptides. The genome sequence of *Chlorobium tepidum* (Genbank accession number NC_002932) has been determined (Eisen, JA et al. 2002. Proc. Natl. Acad. Sci. USA 99: 9509-9514). The α-ketoglutarate synthase gene can be represented by the base sequences shown in SEQ ID NO: 1 and 3. SEQ ID NO: 1 shows the base sequence of the α-ketoglutarate synthase α-subunit gene located at base numbers 170164–172047 (complementary strand) in the *Chlorobium tepidum* genome sequence, and SEQ ID NO: 3 shows the base sequence of the β-subunit gene located at base numbers 169132–170160 (complementary strand) in the *Chlorobium tepidum* genome sequence. SEQ ID NO: 2 is the amino acid sequence of the α subunit of α-ketoglutarate synthase (Genbank accession number NP_661069), and SEQ ID NO: 4 is the amino acid sequence of the β subunit (Genbank accession number NP_661068). The genome sequence of Blastopirellulamarina (Genbank accession number AANZ00000000) has been determined (Fuchsman, CA, and Rocap, G. Appl. Environ. Microbiol. 2006. 72: 6841-6844), and the base sequences shown in SEQ ID NO: 57 and 59 can be used as examples of the α-ketoglutarate synthase gene. SEQ ID NO: 57 is the base sequence of the α-subunit gene of α-ketoglutarate synthase located at base numbers 3180–5045 (complementary strand) in the Blastopirellulamarina genome sequence; SEQ ID NO: 59 is the base sequence of the β-subunit gene located at base numbers 2089–3108 (complementary strand) in the Blastopirellulamarina genome sequence; SEQ ID NO: 58 is the amino acid sequence of the α-subunit of α-ketoglutarate synthase; and SEQ ID NO: 60 is the amino acid sequence of the β-subunit.

[0225] The α-ketoglutarate synthase gene of *Hydrogenobacterium thermophilum* (GenBank accession number AB046568) (Yun, NRetal. 2001. Biochem. Biophy. Res. Commum. 282: 589-594) has been cloned, and its α subunit (GenBank accession number BAB21494) and β subunit (GenBank accession number BAB21495) have been identified. Furthermore, examples include the α-ketoglutarate synthase gene composed of four genes (HP0588, HP0589, HP0590, and HP0591) located at positions 620219–623070 in the genome sequence of Helicobacter pylori (GenBank accession number NC_00091), and the α-ketoglutarate synthase gene composed of two genes (SSO2815 and SSO2816) located at positions 2575303–2578105 in the genome sequence of Sulfolobus sofataricus (GenBank accession number NC_002754). Furthermore, the α-ketoglutarate synthase gene can be cloned from bacteria such as *Aureobacterium*, *Desulfurobacterium*, *Liquidus*, *Hydrogenobacterium*, *Thermoplasia*, *Thermoplasia*, *Sulfurimonas*, and *Methanococcus* based on homology with the genes in the examples above.

[0226] The α-subunit gene and β-subunit gene of α-ketoglutarate synthase are preferably from the same biological source, but any gene capable of forming a protein with α-ketoglutarate synthase activity is acceptable; the genes can also be from different biological sources. Preferred combinations are: the α-subunit or a conserved variant thereof composed of the amino acid sequence of SEQ ID NO: 2 and the β-subunit or a conserved variant thereof composed of the amino acid sequence of SEQ ID NO: 4; and the α-subunit or a conserved variant thereof composed of the amino acid sequence of SEQ ID NO: 58 and the β-subunit or a conserved variant thereof composed of the amino acid sequence of SEQ ID NO: 60, but are not limited thereto. "Conserved variant" will be described later.

[0227] The microorganisms of the present invention can also be microorganisms modified to increase the activity of α-ketoglutarate synthase, wherein the modification increases the activity of regenerating electron donors from the oxidized form to the reduced form (an activity essential for the activity of α-ketoglutarate synthase) compared to the parental strain, such as a wild-type strain or an unmodified strain. The activity of regenerating oxidized electron donors to the reduced form includes, for example, ferricredoxin-NADP. +The activity can be either reductase activity or pyruvate synthase activity. Additionally, the parent strain can be a microorganism that has been modified to increase the expression of the α-ketoglutarate synthase gene, in addition to enhancing the regenerative activity of the electron donor. Furthermore, the parent strain can be a microorganism that naturally possesses a gene encoding the regenerative activity of the electron donor, or a microorganism that does not naturally possess the regenerative activity of the electron donor but has been endowed with this activity by introducing a gene encoding it, thereby increasing its L-glutamate production capacity.

[0228] "Feroxanone-NADP" + "Reductase" refers to an enzyme that reversibly catalyzes the following reaction (EC 1.18.1.2).

[0229] Reduced ferricyanide + NADP + →Oxidized ferricredoxin + NADPH + H +

[0230] This reaction is reversible and can produce reduced ferricoxin in the presence of NADPH and oxidized ferricoxin. Ferricoxin can be replaced with flavin ferricoxin, named flavin ferricoxin-NADP. + The enzyme reductase also has the same function. Frioxonin-NADP has been identified. + Reductases are ubiquitous in organisms from microorganisms to higher organisms (see Carrillo, N. and Ceccarelli, EA 2003. Eur. J. Biochem. 270: 1900-1915; Ceccarelli, EA et al. 2004. Biochim. Biophys. Acta. 1698: 155-165), and have also been named ferritin-NADP. + Oxidase reductase, NADPH-ferroredoxin oxidase reductase.

[0231] ferricyanide-NADP + The confirmation of the enhanced reductase activity was achieved by preparing crude enzyme solutions from unmodified and modified microorganisms and comparing their ferroredoxin-NADP content. + This is achieved through reductase activity. (Feroxin-NADP) + The activity of reductase can be determined, for example, by Blaschkowski et al. (Blaschkowski, HP et al. 1982. Eur. J. Biochem. 123: 563-569). For instance, it can be determined by spectroscopic methods using ferredoxin as a substrate to measure the amount of NADPH reduced. One unit of enzyme activity (U) is expressed as the amount of 1 μmol of NADPH oxidized per minute. When the parental strain possesses ferredoxin-NADP...+ When it comes to reductase activity, if the activity of the parent strain is already sufficiently high, there is no need to enhance it. However, ideally, the enzyme activity should increase by more than 1.5 times, more preferably more than 2 times, and more preferably more than 3 times compared to the parent strain.

[0232] The protein encoding ferricoxin-NADP has been found in many biological species. + Reductase genes can be used, as long as they are active in the target L-amino acid producing strain. In *E. coli*, they are used as flavin-NADP... + The reductase has been identified by the fpr gene (Bianchi, V. et al. 1993. J. Bacteriol. 175: 1590-1595). Furthermore, the NADPH-pseudomonas putida reductase gene and the putida redoxin gene are known to exist as operons in *Pseudomonas putida* (Koga, H. et al. 1989. J. Biochem. (Tokyo) 106: 831-836).

[0233] Flavoredoxin-NADP in Escherichia coli + Reductases can be exemplified by the fpr gene, located at bases 4111749–4112495 (complementary strand) in the genome sequence of *Escherichia coli* strain K-12 (Genbank accession number U00096), with the base sequence shown in SEQ ID NO: 5. The amino acid sequence of Fpr is shown in SEQ ID NO: 6 (Genbank accession number AAC76906). Furthermore, ferritin-NADP was found at bases 2526234–2527211 in the genome sequence of *Corynebacterium glutamicum* (Genbank accession number BA00036). + Reductase gene (Genbank accession number: BAB99777).

[0234] "Pyruvate synthase" refers to an enzyme (EC1.2.7.1) that reversibly catalyzes the following reaction to produce pyruvate from acetyl-CoA and CO2.

[0235] Reduced ferricyanide + acetyl-CoA + CO2 → oxidized ferricyanide + pyruvate

[0236] This reaction is reversible, producing reduced ferricrene in the presence of pyruvate and oxidized ferricrene. This enzyme is also known as pyruvate oxide reductase, pyruvate ferricrene (flavin ferricrene) reductase, or pyruvate ferricrene oxide reductase. By combining this enzyme activity with α-ketoglutarate synthase activity, the reduced ferricrene consumed by α-ketoglutarate synthase activity can be regenerated through the reverse reaction activity of pyruvate synthase.

[0237] The enhanced pyruvate synthase activity can be confirmed by preparing crude enzyme solutions from microorganisms before and after enhancement and comparing their pyruvate synthase activities. Pyruvate synthase activity can be determined, for example, by the method of Yoon et al. (Yoon, K. Setal. 1997. Arch. Microbiol. 167: 275-279). The determination principle is the same as that for the above-described α-ketoglutarate synthase activity determination; for example, it can be determined by using pyruvate as a substrate and employing spectroscopic methods to determine the amount of methyl viologen reduced in the pyruvate decarbonation reaction. One unit of enzyme activity (U) is expressed as the amount of methyl viologen reduced per minute (1 μmol). When the parent strain possesses pyruvate synthase activity, ideally, the enzyme activity should increase by at least 1.5 times, more preferably at least 2 times, and even more preferably at least 3 times compared to the parent strain. Furthermore, when the parental strain does not possess pyruvate synthase activity, pyruvate synthase can be generated simply by introducing a pyruvate synthase gene. However, it is preferable that the enzyme activity is enhanced to a measurable level, preferably 0.001 U / mg or more, more preferably 0.005 U / mg or more, and even more preferably 0.01 U / mg or more.

[0238] Genes encoding pyruvate synthase can utilize genes from bacteria with a reducing TCA cycle, such as the pyruvate synthase gene of *Sulphurella vulgaris* and the pyruvate synthase gene of *Hydrogenobacterium thermophilum*.

[0239] Specifically, as a pyruvate synthase gene of *A. aeruginosa*, an example can be found is the gene with base numbers 1534432–1537989 in the *A. aeruginosa* genome sequence (Genbank accession number NC_002932), having the base sequence shown in SEQ ID NO: 7. SEQ ID NO: 8 shows the amino acid sequence encoded by this gene (Genbank accession number AAC76906). Furthermore, it is known that pyruvate synthase in *Hydrogenobacter thermophilus* is formed by a complex of four subunits: δ subunit (Genbank accession number BAA95604), α subunit (Genbank accession number BAA95605), β subunit (Genbank accession number BAA95606), and γ subunit (Genbank accession number BAA95607) (Ikeda, T. et al. 2006. Biochem. Biophys. Res. Commun. 340: 76-82). Furthermore, examples include a pyruvate synthase gene composed of four genes (HP1108, HP1109, HP1110, and HP1111) located at positions 1170138–1173296 in the *Helicobacter pylori* genome sequence (GenBank accession number NC000915), and a pyruvate synthase gene composed of four genes (SSO1208, SSO7412, SSO1207, and SSO1206) located at positions 1047593–1044711 in the *Sulphurobacterium sulfideum* genome sequence (GenBank accession number NC002754). Moreover, pyruvate synthase genes can also be cloned from bacteria of the genera *Aureobacterium*, *Desulfurobacterium*, *Liquigenes*, *Hydrogenobacterium*, *Thermoplasia*, and *Thermoplasia* based on homology with the genes described above.

[0240] For α-ketoglutarate synthase activity, the presence of ferricoxin or flavinoxorubicin as an electron donor is necessary. Therefore, microorganisms modified to increase α-ketoglutarate synthase activity, such modifications enhance the production of ferricoxin or flavinoxorubicin. Furthermore, in addition to modifications that enhance α-ketoglutarate synthase activity, flavinoxorubicin-NADP... + In addition to enhancing the activity of reductase or pyruvate synthase, modifications can also be made to increase the production capacity of ferroredoxin or flavin oxidoredoxin.

[0241] In this invention, "ferredoxin" refers to a protein containing non-porphyrin iron (Fe) and sulfur atoms, and bound to an iron-sulfur cluster called 4Fe-4S, 3Fe-4S, or 2Fe-2S, which functions as a single-electron transporter. "Flavin-redoxin" refers to a protein containing an FMN (flavin mononucleotide) as a cofactor, which functions as a single or double-electron transporter. Ferroredoxin and flavin-redoxin are described in the literature of McLean et al. (McLean, KJ et al. 2005. Biochem. Soc. Trans. 33: 796-801).

[0242] Moreover, the parent strains used for modification can be strains that naturally and intrinsically possess genes encoding ferricodoxin or flavinoxin, or strains that do not naturally possess ferricodoxin or flavinoxin genes but have been endowed with activity by introducing ferricodoxin or flavinoxin genes, thereby increasing their L-glutamic acid production capacity.

[0243] The increased production capacity of ferricoxin or flavinoxin compared to the parental strain (e.g., wild-type or unmodified strain) can be confirmed by comparing the amount of ferricoxin or flavinoxin mRNA with that of the wild-type or unmodified strain. Methods for confirming expression levels include Southern hybridization and RT-PCR (Sambrook, J. et al. 1989. Molecular Cloning A Laboratory Manual / Second Edition, Cold Spring Harbor Laboratory Press, New York). Regarding expression levels, any increase compared to the wild-type or unmodified strain is acceptable; ideally, it should be at least 1.5-fold, more preferably 2-fold, and more preferably 3-fold.

[0244] Furthermore, the fact that the production capacity of ferrous reductin or flavin reductin is increased compared to the parental strain (e.g., wild-type or unmodified strain) can be confirmed by SDS-PAGE, two-dimensional electrophoresis, or Western blotting using antibodies (Sambrook, J. et al. 1989. Molecular Cloning A Laboratory Manual / Second Edition, Cold Spring Harbor Laboratory Press, New York). Regarding the production amount, any increase compared to the wild-type or unmodified strain is acceptable; ideally, it should be at least 1.5 times, more preferably 2 times, and more preferably 3 times higher than the wild-type or unmodified strain.

[0245] The activities of ferricoxin and flavin ferricoxin can be determined by adding them to appropriate redox reaction systems. For example, Boyer et al. disclosed the following method: via ferricoxin-NADP... + The reduction of ferricoxane by reductase can quantify the reduction of cytochrome C induced by the generated reduced ferricoxane (Boyer, MEetal. 2006. Biotechnol. Bioeng. 94: 128-138). Furthermore, the activity of flavin-NADP can be measured using flavin-NADP... + The reductase was determined using the same method.

[0246] Genes encoding ferricodoxins or flavinodoxins are widely distributed. Any gene can be used as long as the encoded ferricodoxin or flavinodoxin can be utilized by α-ketoglutarate synthase and the electron donor regeneration system. For example, in *E. coli*, the *fdx* gene is present as the gene encoding a ferricodoxin with a 2Fe-2S cluster (Ta, DT, and Vickery, LE 1992. J. Biol. Chem. 267: 11120-11125), suggesting that the *yfhL* gene encodes a ferricodoxin with a 4Fe-4S cluster. Furthermore, *fldA* (Osborne, C. et al. 1991. J. Bacteriol. 173: 1729-1737) and *fldB* (Gaudu, P., and Weiss, B. 2000. J. Bacteriol. 182: 1788-1793) are known flavinodoxin genes. In the genome sequence of *Corynebacterium glutamicum* (Genbank accession number BA00036), the feroxin gene *fdx* (Genbank accession number BAB97942) was found at positions 562643–562963, and *fer* (Genbank accession number BAB98495) was found at positions 1148953–1149270. Furthermore, numerous feroxin genes exist in *Agropyron globulinum*, among which feroxin I and feroxin II have been identified as 4Fe-4S type feroxin genes acting as electron acceptors for pyruvate synthase (Yoon, K. Setal. 2001. J. Biol. Chem. 276: 44027-44036). Ferroxin genes or flavin-oxidoreductin genes derived from bacteria with a reducing TCA cycle, such as the feroxin gene of *Hydrogenobacter thermophilus*, can also be used.

[0247] Specifically, the ferredoxin genes of *E. coli* include, for example, the fdx gene located at positions 2654770–2655105 (complementary strand) in the genome sequence of *E. coli* strain K-12 (Genbank accession number U00096), and the yfhL gene located at positions 2697685–2697945, as shown in SEQ ID NO: 11. The amino acid sequences of Fdx and YfhL (Genbank accession numbers AAC75578 and AAC75615, respectively) are shown in SEQ ID NO: 10 and SEQ ID NO: 12. Genes encoding flavin redoxins in *Escherichia coli* include, for example, the fldA gene shown in SEQ ID NO: 13, located at base positions 710688–710158 (complementary strand) in the genome sequence of *Escherichia coli* strain K-12 (Genbank accession number U00096), and the fldB gene shown in SEQ ID NO: 15, located at base positions 3037877–3038398. SEQ ID NO: 14 and SEQ ID NO: 16 show the amino acid sequences encoded by the fldA and fldB genes (Genbank accession numbers AAC73778 and AAC75933, respectively).

[0248] Examples of ferricoxin genes from *Aureobacterium tumefaciens* include, for instance, the ferricoxin I gene shown in SEQ ID NO: 17, located at base positions 1184078–1184266 of the *Aureobacterium tumefaciens* genome sequence (Genbank accession number NC_002932), and the ferricoxin II gene shown in SEQ ID NO: 19, located at base positions 1184476–1184664. The amino acid sequences encoding ferricoxin I and ferricoxin II (Genbank accession numbers AAM72491 and AAM72490, respectively) are shown in SEQ ID NO: 18 and SEQ ID NO: 20. Additionally, examples include the ferricoxin gene of *Hydrogenobacter thermophilus* (Genbank accession number BAE02673) and the ferricoxin gene of *Lepiota sulfidea* shown at base positions 2345414–2345728 of the *Lepiota sulfidea* genome sequence. Furthermore, genes can be cloned from bacteria such as *Aureobacterium*, *Desulfurobacterium*, *Liquidus*, *Hydrogenobacterium*, *Thermoplasticum*, and *Corynebacterium* based on homology with the genes in the examples above. They can also be cloned from γ-proteobacteria such as *Enterobacter*, *Klebsiella*, *Serratia*, *Erwinia*, and *Yersinia*; rod-shaped bacteria such as *Corynebacterium glutamicum* and *Brevibacterium lactis*; *Pseudomonas aeruginosa* and other *Pseudomonas* bacteria; and *Mycobacterium* bacteria such as *Mycobacterium tuberculosis*.

[0249] These encode α-ketoglutarate synthase and ferroredoxin-NADP. + The genes for reductase, pyruvate synthase, ferrous reductin, and flavin reductin (hereinafter collectively referred to as the genes of the present invention) can be genes encoding conserved variants of amino acid sequences obtained by substitution, deletion, insertion, or addition of one or more amino acids at one or more positions, as long as the activity or function of the encoded protein is not impaired. The term "one or more" varies depending on the position of the amino acid residues in the protein's three-dimensional structure and the type of amino acid; preferably 1 to 20, more preferably 1 to 10, and particularly preferably 1 to 5.

[0250] The aforementioned substitutions are preferably conservative substitutions, which are neutral mutations that do not change function. Regarding conservative mutations, when the substitution site is an aromatic amino acid, it is a substitution mutation between Phe, Trp, and Tyr; when the substitution site is a hydrophobic amino acid, it is a substitution mutation between Leu, Ile, and Val; when the substitution site is a polar amino acid, it is a substitution mutation between Gln and Asn; when the substitution site is a basic amino acid, it is a substitution mutation between Lys, Arg, and His; when the substitution site is an acidic amino acid, it is a substitution mutation between Asp and Glu; and when the substitution site is an amino acid with a hydroxyl group, it is a substitution mutation between Ser and Thr.

[0251] More specifically, examples include: replacing Ala with Ser or Thr; replacing Arg with Gln, His, or Lys; replacing Asn with Glu, Gln, Lys, His, or Asp; replacing Asp with Asn, Glu, or Gln; replacing Cys with Ser or Ala; replacing Gln with Asn, Glu, Lys, His, Asp, or Arg; replacing Glu with Gly, Asn, Gln, Lys, or Asp; replacing Gly with Pro; replacing His with Asn, Lys, Gln, Arg, or Tyr; and replacing L with... Ile can be replaced by eu, Met, Val, or Phe; Leu can be replaced by Ile, Met, Val, or Phe; Lys can be replaced by Asn, Glu, Gln, His, or Arg; Met can be replaced by Ile, Leu, Val, or Phe; Phe can be replaced by Trp, Tyr, Met, Ile, or Leu; Ser can be replaced by Thr or Ala; Thr can be replaced by Ser or Ala; Trp can be replaced by Phe or Tyr; Tyr can be replaced by His, Phe, or Trp; and Val can be replaced by Met, Ile, or Leu. Furthermore, such amino acid substitutions, deletions, insertions, additions, or inversions also include those resulting from naturally occurring mutations (mutants or variants) due to individual or interspecies differences in the microorganisms carrying the gene of this invention.

[0252] Furthermore, codons that are easy to use in the host in which the genes of the present invention are introduced can be substituted. Similarly, as long as the genes of the present invention are functional, the N-terminal and / or C-terminal sides of the encoded protein can be extended or truncated. For example, the length of the extension is 50 or less, preferably 20 or less, more preferably 10 or less, and particularly preferably 5 or less amino acid residues.

[0253] Genes encoding conserved variants as described above can be obtained, for example, by modifying the base sequence using site-directed mutagenesis, such that amino acid residues at specific sites of the encoded protein contain substitutions, deletions, insertions, or additions. Alternatively, they can be obtained through conventionally known mutagenesis treatments. Examples of mutagenesis treatments include: in vitro treatment of the gene of the present invention with hydroxylamine or the like, and treatment of microorganisms carrying the gene (e.g., Escherichia coli) with ultraviolet light irradiation or with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS) used in conventional mutagenesis treatments. Furthermore, the amino acid substitutions, deletions, insertions, additions, or inversions described above also include those resulting from naturally occurring mutations (mutants or variants) due to individual or interspecies differences in the microorganisms carrying the gene of the present invention. Whether these genes encode functional α-ketoglutarate synthase or ferroredoxin-NADP is another matter. + Reductase, pyruvate synthase, ferroredoxin, or flavin reductase can be identified, for example, by introducing these genes into microorganisms and measuring the activity of each gene product.

[0254] The gene of the present invention can be capable of hybridizing under stringent conditions with DNA having the above-described base sequence or a probe that can be prepared from DNA having these base sequences, and encodes α-ketoglutarate synthase and ferroredoxin-NADP. + DNA containing reductase, pyruvate synthase, feroxin, or flavin oxidoxin.

[0255] Here, "strict conditions" refer to conditions under which so-called specific hybrids are formed but non-specific hybrids are not. It is difficult to explicitly quantify these conditions. For example, conditions under which DNAs with high homology (e.g., DNAs with 70% or more, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more homology) hybridize, while DNAs with lower homology do not hybridize; or conditions under washing conditions equivalent to those used in conventional Southern hybridization, i.e., washing once at 60°C with 1×SSC and 0.1% SDS, preferably 0.1×SSC and 0.1% SDS, more preferably 68°C with 0.1×SSC and 0.1% SDS, preferably 2 to 3 times.

[0256] Furthermore, in this specification, "homology" sometimes refers to "identity".

[0257] The probe can also be a portion of the gene sequence of the present invention. Such probes can be prepared using methods well known to those skilled in the art, using oligonucleotides prepared based on the base sequences of each gene as primers and DNA fragments containing each gene as templates for PCR reactions. Moreover, when using DNA fragments of approximately 300 bp in length as probes, the washing conditions after hybridization under the above conditions can be, for example, 50°C, 2×SSC, and 0.1% SDS.

[0258] The above description of conserved variants also applies to the enzymes and genes described in the attribution of L-amino acid production capacity.

[0259] The modifications described above for enhancing the expression of the gene of the present invention can be performed as described in the method for enhancing the expression of the target gene in the section on conferring L-amino acid production capacity. The gene of the present invention can be obtained by PCR using the genomic DNA of a microorganism carrying the gene as a template.

[0260] For example, the α-ketoglutarate synthase gene of *Sulphurella vulgaris* can be obtained by PCR using primers prepared based on the base sequences of SEQ ID NO: 1 and 3, such as the primers shown in SEQ ID NO: 21 and 22, with *Sulphurella vulgaris* genomic DNA as a template.

[0261] Flavoredoxin-NADP of Escherichia coli + The reductase gene can be obtained by PCR using primers prepared based on the base sequence of SEQ ID NO: 5, such as the primers shown in SEQ ID NO: 32 and 33, with E. coli genomic DNA as a template.

[0262] The pyruvate synthase gene of *S. green sulfur* can be obtained by PCR using primers prepared based on the base sequence of SEQ ID NO: 7, such as the primers shown in SEQ ID NO: 23 and 24, with *S. green sulfur* genomic DNA as a template.

[0263] The ferric reductin gene fdx of Escherichia coli can be obtained by PCR using primers prepared based on the base sequence of SEQ ID NO: 9, such as the primers shown in SEQ ID NO: 38 and 39, with E. coli genomic DNA as a template; the ferric reductin gene yfhL can be obtained by PCR using primers prepared based on the base sequence of SEQ ID NO: 11, such as the primers shown in SEQ ID NO: 40 and 41, with E. coli genomic DNA as a template.

[0264] The flavin-redoxin gene fldA of *E. coli* can be obtained by PCR using primers prepared based on the base sequence of SEQ ID NO: 13, such as primers shown in SEQ ID NO: 34 and 35, with *E. coli* genomic DNA as a template; the flavin-redoxin gene fldB can be obtained by PCR using primers prepared based on the base sequence of SEQ ID NO: 15, such as primers shown in SEQ ID NO: 36 and 37, with *E. coli* genomic DNA as a template.

[0265] In addition, the ferricodoxin I gene of *Sulphurella vulgaris* can be obtained by PCR using primers prepared based on the base sequence of SEQ ID NO: 17, such as the primers shown in SEQ ID NO: 25 and 26, with *Sulphurella vulgaris* genomic DNA as a template.

[0266] For genes of the present invention derived from other microorganisms, they can also be obtained from the genomic DNA or genomic DNA library of the microorganism by PCR using oligonucleotides prepared based on the sequence information of the aforementioned genes or the known gene or protein sequence information of the microorganism as primers, or by hybridization using oligonucleotides prepared based on the sequence information as probes. Furthermore, genomic DNA can be prepared from microorganisms serving as DNA donors using, for example, the methods of Saito and Miura (see Saito, H. and Miura, KI 1963. Biochem. Biophys. Acta, 72, 619-629; Biotechnology Experiments, edited by the Japanese Society of Biotechnology, pp. 97-98, Peifukan, 1992).

[0267] The enhancement of gene expression can be achieved by using the methods described above, such as transformation or homologous recombination to increase the copy number of the gene, or by modifying the expression regulatory sequence of the gene. Furthermore, the enhancement of gene expression can be achieved by amplifying activators that can increase gene expression and / or deleting or attenuating regulators that can decrease gene expression.

[0268] Furthermore, when increasing the copy number of a gene, it is sufficient to enhance the activity of the product of the target gene; there are no special restrictions on the copy number. When the microorganism already possesses the target gene, the copy number is preferably 2 or more. Additionally, when the microorganism does not originally possess the gene of this invention, the copy number of the introduced gene can be 1 or 2 or more.

[0269] When a gene is composed of multiple subunits, such as α-ketoglutarate synthase, the expression of the gene encoding each subunit can be enhanced individually, or the expression of these genes can be enhanced simultaneously in a polycistronic manner. Furthermore, when introducing genes into microorganisms using vectors, the genes encoding each subunit can be simultaneously carried on a single vector molecule, or separately carried on different vector molecules. Additionally, when inserting genes into the genome, the genes encoding each subunit can be simultaneously inserted into the same site on the genome, or separately inserted into different locations.

[0270] Furthermore, preferably, the microorganisms of the present invention, in addition to enhancing α-ketoglutarate synthase activity, also reduce α-ketoglutarate dehydrogenase activity. Such microorganisms can be obtained, for example, by transforming microorganisms whose gene encoding α-ketoglutarate dehydrogenase has been disrupted using a recombinant vector containing the gene of the present invention, respectively.

[0271] In this invention, α-ketoglutarate dehydrogenase (hereinafter also referred to as "α-KGDH") activity refers to the activity of catalyzing the oxidation and decarbonation of α-ketoglutarate (2-oxoglutarate) to generate succinyl-CoA. This reaction is catalyzed by three enzymes: α-KGDH (E1o: α-ketoglutarate dehydrogenase, EC: 1.2.4.2), dihydrolipoamide S-succinyltransferase (E2o: dihydrolipoamide-S-succinyltransferase; EC: 2.3.1.61), and dihydrolipoamide dehydrogenase (E3: dihydrolipoamide dehydrogenase; EC: 1.8.1.4). That is, each of these three subunits catalyzes a separate reaction, and the activity of catalyzing the combined reaction of these three reactions is called α-KGDH activity. The activity of α-KGDH can be confirmed by the method of Shiio and Ujigawa-Takeda (Isamu Shiio and Ujigawa-Takeda, Agric. Biol. Chem., 44(8), 1897-1904, 1980).

[0272] E1o: 2-oxoglutarate + [dihydrolipoyllysine-residuesuccinyltransferase]lipoyllysine → [dihydrolipoyllysine-residuesuccinyltransferase]S-succinyldihydrolipoyllysine + CO2

[0273] E2o: CoA+ enzyme N6-(S-succinyl dihydrolipoyl)lysine → succinyl-CoA+ enzyme N6-(dihydrolipoyl)lysine

[0274] E3: Protein N6-(dihydrolipoyl)lysine + NAD + =Protein N6-(Lipoic acid)lysine + NADH + H +

[0275] Moreover, α-KGDH is also known as oxoglutarate dehydrogenase or 2-oxoglutarate dehydrogenase.

[0276] In Enterobacteriaceae bacteria such as *Pantoea ananatis*, subunit proteins possessing these three enzymatic activities form a complex. Furthermore, each subunit is encoded by a separate sucA, sucB, and lpd gene, with the sucA and sucB genes located downstream of the succinate dehydrogenase iron-sulfur protein gene (sdhB) (US Patent No. 6,331,419). Moreover, the aforementioned patent describes these genes as belonging to the clumping Enterobacter aJ13355 strain, which was later reclassified as *Pantoea ananatis*.

[0277] The gene encoding α-KGDH in Enterobacteriaceae includes the base sequences of the sucA and sucB genes of *Pantoea ananatis*, a portion of the upstream sdhB gene, and a fragment of the downstream sucC gene, as shown in SEQ ID NO: 46. Furthermore, the amino acid sequences encoded by a portion of their sdhB gene, sucA, sucB, and sucC genes are shown in SEQ ID NO: 47–50, respectively. Additionally, sucA and sucB, encoding α-KGDH in *Escherichia coli*, are disclosed in GenBank NP_415254 and NP_415255, respectively.

[0278] Furthermore, in Corynebacterium-type bacteria, the E1o subunit is encoded by the odhA gene (also known as the sucA gene, accessed via GenBank accession number NCgl1084, NC_003450), and the E3 subunit is encoded by the lpd gene (GenBank accession number Y16642). On the other hand, it is speculated that the E2o subunit, together with the E1o subunit, is encoded by the odhA gene as a bifunctional protein (see Usuda, Y. et al., Microbiology 1996, 142: 3347-3354), or by a gene different from the odhA gene, accessed via GenBank accession number NCgl2126, NC_003450. Therefore, in this invention, the odhA gene is the gene encoding the E1o subunit, but it can also simultaneously encode the E2o subunit.

[0279] The base sequence of the *Odhya microbesii* *odhA* gene and the amino acid sequence of the E1o subunit (Genbank accession number NC_003450, NCgl1084, WO2006 / 028298) are shown in SEQ ID NO: 51 and 52. Furthermore, the base sequence of the aforementioned Genbank accession number NC_003450, NCgl2126, and the amino acid sequence of the E2o subunit encoded by that sequence are shown in SEQ ID NO: 53 and 54.

[0280] Furthermore, when cultured under anaerobic or microaerophilic conditions, the microorganisms of the present invention can also be modified to not produce organic acids or ethanol under anaerobic or microaerophilic conditions, except for enhanced α-ketoglutarate synthase activity. Examples of organic acids include lactic acid, formic acid, and acetic acid. A method for modifying the microorganisms to prevent the production of organic acids or ethanol is to disrupt the gene encoding lactate dehydrogenase (Verumi, GNetal. 2002. J. Industrial Microbiol. Biotechnol. 28: 325-332; JP 2005-95169).

[0281] <2> The method for producing L-glutamic acid of the present invention

[0282] The method of the present invention is a method for producing the following L-amino acids: Microorganisms having the ability to produce one or more L-amino acids selected from L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline, and L-arginine, and modified to increase the activity of α-ketoglutarate synthase, are cultured in a culture medium. The L-amino acids are generated and accumulated in the culture medium or within the cells of the microorganisms, and the L-amino acids are collected from the culture medium or the cells. The method of the present invention can be carried out under aerobic conditions, or under anaerobic or microaerobic conditions. Aerobic conditions are typically achieved by introducing oxygen-containing gas into the culture medium or by stirring the culture medium. Furthermore, anaerobic or microaerobic conditions are typically achieved by not aerating or stirring, or by reducing the aeration rate or stirring frequency.

[0283] When culturing microorganisms in a culture medium, the bacterial cells obtained by slant culture in solid culture medium such as agar can be directly inoculated into the liquid culture medium. However, it is preferable to pre-culture the microorganisms in liquid culture medium (seed culture) and then inoculate the obtained bacterial cells into the main culture medium (fermentation medium).

[0284] The culture medium used for cultivation can be a conventional microbial culture medium containing carbon sources, nitrogen sources, inorganic salts, and organic micronutrients such as amino acids and vitamins as needed. For example, a common culture medium can be used by adding natural nutrient sources such as meat extract, yeast extract, and peptone to a component containing inorganic salts such as ammonium sulfate, potassium phosphate, and magnesium sulfate.

[0285] There are no special restrictions on the carbon source, as long as it can be assimilated by microorganisms to produce L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline, or L-arginine. Commonly used carbohydrates include galactose, lactose, glucose, fructose, sucrose, disaccharides, starch, cellulose, and fatty acids. Alcoholic sugars such as glycerol, mannitol, xylitol, ribitol, and ethanol can also be used. Among these, glucose, fructose, sucrose, glycerol, and ethanol are preferred, with glucose and glycerol being particularly preferred. Crude glycerol preferably comes from biodiesel fuel production. The carbon source can be one type or a mixture of two or more.

[0286] In addition, starch saccharification solutions, molasses, crude glycerol, etc., containing the aforementioned sugars can also be used. There are no particular restrictions on the concentration of the aforementioned carbon sources; however, it is advantageous to use the highest possible concentration without inhibiting the formation of L-amino acids, typically within the range of 5–30% (w / v), preferably 10–20% (w / v) for fermentation. Furthermore, carbon sources can be added as needed to compensate for the reduction of carbon sources during fermentation.

[0287] Furthermore, there are no specific restrictions on the nitrogen source, as long as it can be assimilated by microorganisms to produce L-amino acids. Specifically, examples include ammonium salts, nitrates, urea, soybean hydrolysate, casein breakdown products, peptone, yeast extract, meat extract, corn steep liquor, and various organic or inorganic nitrogen-containing compounds. As inorganic salts, various phosphates, sulfates, and metal salts such as magnesium, potassium, manganese, iron, and zinc can be used. In addition, vitamins such as biotin, pantothenic acid, inositol, and niacin, as well as growth-promoting factors such as nucleotides and amino acids, can be added as needed. Furthermore, to suppress foaming during cultivation, it is ideal to add an appropriate amount of commercially available antifoaming agent to the culture medium.

[0288] The pH can be adjusted by adding ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide, etc. The preferred pH in the main culture is usually pH 5-10, preferably pH 6-9.5. Therefore, alkaline substances, carbonates, urea, etc. should be used as needed to adjust the pH of the culture medium within the above range during culture.

[0289] The culture medium used in this invention preferably contains the aforementioned carbon source, as well as carbonate ions, bicarbonate ions, or carbon dioxide gas, and is capable of culturing under aerobic or microaerobic / anaerobic conditions. Carbonate ions or bicarbonate ions can be supplied by carbonates or bicarbonates such as magnesium carbonate, sodium carbonate, sodium bicarbonate, potassium carbonate, and potassium bicarbonate, which can also be used as neutralizing agents, and may be supplied by carbon dioxide gas as needed. Furthermore, carbonate ions and bicarbonate ions are added at a concentration of 0.001–5 M, preferably 0.1–3 M, more preferably 1–2 M. When carbon dioxide gas is present, it contains 50 mg–25 g, preferably 100 mg–15 g, more preferably 150 mg–10 g of carbon dioxide gas per 1 L of solution.

[0290] Generally, when culturing microorganisms, it is preferable to culture them under aerobic conditions where oxygen is supplied through aeration and stirring. However, in the method of the present invention, culturing can be carried out under aerobic conditions with aeration and stirring, or under anaerobic / microaerobic conditions without aeration and oxygen supply. Various dissolved oxygen concentration conditions can be set by, for example, by reducing the aeration rate or stirring, sealing the container under aeration conditions, introducing an inert gas containing carbon dioxide, etc.

[0291] The incubation temperature is typically 25℃ to 40℃, preferably 30℃ to 37℃. The incubation time is preferably 1 hour to 168 hours, more preferably 3 hours to 72 hours.

[0292] The cultivation period can be divided into cultivation for microbial proliferation and cultivation for L-amino acid production, and different culture media or conditions can be used for each. For example, after microbial proliferation is achieved under aeration or stirring, L-amino acids can be produced under anaerobic or microaerobic conditions.

[0293] In addition, a liquid culture medium prepared to precipitate L-glutamic acid can be used, allowing L-glutamic acid to precipitate from the medium during culturing. Examples of conditions for L-glutamic acid precipitation include, for example, pH 5.0 to 4.0, preferably pH 4.5 to 4.0, more preferably pH 4.3 to 4.0, and particularly preferably pH 4.0.

[0294] After cultivation, L-amino acids can be collected from the culture medium using known recovery methods. For example, methods such as removing bacterial cells from the culture medium followed by concentration and crystallization, or ion-exchange chromatography, can be used. When L-glutamic acid precipitates during cultivation, the precipitated L-glutamic acid can be collected by centrifugation or filtration. In this case, it can be separated along with the L-glutamic acid after crystallization of the dissolved L-glutamic acid in the culture medium.

[0295] Example

[0296] The present invention will be described in more detail below through embodiments, but the present invention is not limited to these embodiments.

[0297] [Example 1] <Construction of expression plasmids for α-ketoglutarate synthase gene, pyruvate synthase gene, and ferricoxin gene derived from *Sulphurella multocida*>

[0298] *Sulphurella vulgaris* is a mesophilic autotrophic bacterium with an optimum growth temperature of 48°C. The genome sequence of the TLS strain has been elucidated by Eisen et al. (Eisen, JA et al. 2002. Proc. Natl. Acad. Sci. USA 99:9509-9514). The α-ketoglutarate synthase gene, pyruvate synthase gene, and ferricredoxin gene were isolated from this strain, and plasmids expressing all three genes were constructed.

[0299] <1-1> Construction of an expression plasmid for the α-ketoglutarate synthase gene derived from *Sulphurella multocida*

[0300] Using genomic DNA from *Thiobacillus aeruginosa* strain TLS (ATCC49652) as a template, PCR was performed using the oligonucleotides shown in SEQ ID NO: 21 and 22 to amplify a gene fragment containing the α and β subunits of α-ketoglutarate synthase. The resulting gene fragment was cut with BamHI and inserted into the BamHI site of pSTV28 (manufactured by TAKARABio) to construct an α-ketoglutarate synthase gene expression plasmid, named pSTV-KGS. In this plasmid, the α-ketoglutarate synthase gene is located downstream of the pSV28-derived lac promoter and can be expressed by this promoter.

[0301] <1-2> Construction of expression plasmids for α-ketoglutarate synthase gene and pyruvate synthase gene derived from green sulfur bacteria

[0302] Using genomic DNA from *Thiobacillus aeruginosa* strain TLS (ATCC49652) as a template, PCR was performed using the oligonucleotides shown in SEQ ID NO: 23 and 24 to amplify the pyruvate synthase gene fragment. This gene fragment was then cut with XbaI and ligated to a fragment obtained by treating pSTV-KGS with XbaI to construct plasmids for expressing the α-ketoglutarate synthase gene and pyruvate synthase gene, named pSTV-KGS-PS.

[0303] <1-3> Construction of expression plasmids for α-ketoglutarate synthase gene, pyruvate synthase gene, and ferricoxin gene derived from *Sulphurella multocida*

[0304] Using genomic DNA from *Thiobacillus aeruginosa* strain TLS (ATCC49652) as a template, PCR was performed using the oligonucleotides shown in SEQ ID NO: 25 and 26 to amplify the ferredoxin I gene fragment. This gene fragment was digested with SmaI and ligated to a fragment obtained by treating pSTV-KGS-PS with SmaI, constructing a plasmid for expressing the α-ketoglutarate synthase gene, pyruvate synthase gene, and ferredoxin I gene, named pSTV-FdI-KGS-PS. In this plasmid, the α-ketoglutarate synthase gene, pyruvate synthase gene, and ferredoxin I are located downstream of the lac promoter of pSTV28 and can be expressed by this promoter.

[0305] [Example 2] <Construction of Escherichia coli α-ketoglutarate dehydrogenase-deficient strain>

[0306] <2-1> Construction of plasmids for disrupting the sucA gene in Escherichia coli

[0307] A strain of *Escherichia coli* MG1655 with a disrupted sucA gene encoding the E1 subunit of α-ketoglutarate dehydrogenase was prepared. Primers were synthesized based on the sucA gene sequence located at bases 757929–760730 in the genomic sequence (Genbank accession number U00096). Using genomic DNA of *E. coli* MG1655 as a template, the N-terminal and C-terminal fragments of the sucA gene were amplified by PCR. The sucA gene sequence of *E. coli* is shown in SEQ ID NO: 55, and the amino acid sequence of the E1 subunit encoded by this gene is shown in SEQ ID NO: 56. Oligonucleotides of SEQ ID NO: 42 and 43 were used as primers for PCR amplification of the N-terminal fragment, and oligonucleotides of SEQ ID NO: 44 and 45 were used as primers for PCR amplification of the C-terminal fragment. The oligonucleotide of SEQ ID NO: 42 is designed with a HindIII site, and the oligonucleotide of SEQ ID NO: 45 is designed with an XbaI site.

[0308] The amplified DNA fragments after PCR were purified using the QIAquick PCR Purification Kit (Quiagen). Using the purified N-terminal and C-terminal DNA fragments, and primers for SEQ ID NO: 42 and 45, a depleted sucA fragment was obtained via crossover PCR (Link, A.J. et al. 1997. J. Bacteriol. 179: 6228-6237). The purified DNA fragment was digested with HindIII and XbaI (TAKARABio) and cloned into the temperature-sensitive plasmid pMAN997 (Matsui, H. et al. 2001. Biosci. Biotechnol. Biochem. 65: 570-578; WO99 / 03988), which was also digested with HindIII and XbaI. This constructed a plasmid for sucA destruction and named pMANΔsucA.

[0309] <2-2> Construction of an α-ketoglutarate dehydrogenase-deficient strain derived from Escherichia coli MG1655

[0310] Escherichia coli MG1655 was transformed with plasmid pMANΔsucA. Colonies were selected on LB agar plates (containing 25 μg / mL ampicillin) at 30°C. The selected clones were incubated overnight at 30°C. The culture was then diluted 1000-fold and inoculated onto LB agar plates, where colonies were selected at 42°C. The selected clones were plated on LB agar plates and incubated at 30°C. One-eighth of the plate was then suspended in 2 mL of LB medium and incubated with shaking at 42°C for 4–5 hours. A 10,000-fold diluted bacterial culture was inoculated onto LB agar plates. By inoculating hundreds of colonies onto both LB and LB agar plates and confirming growth, ampicillin-sensitive strains were selected. Colony PCR was performed on several ampicillin-sensitive strains, confirming the deletion of the sucA gene. This yields the sucA-deleted strain MG1655ΔsucA, derived from Escherichia coli strain MG1655.

[0311] [Example 3] <Construction of Escherichia coli lactate dehydrogenase-deficient strain>

[0312] Lactate dehydrogenase is an enzyme that uses NADH as a coenzyme to produce lactate from pyruvate. To inhibit lactate production in *E. coli* cultures under anaerobic conditions, a strain encoding the ldhA gene for lactate dehydrogenase was constructed. This gene deletion was achieved using a method called "Red-driven integration" developed by Datsenko and Wanner (Datsenko, KA, and Wanner, BL2000. Proc. Natl. Acad. Sci. USA. 97: 6640-6645) combined with a λ phage-derived excision system (Cho, E Hetal. 2002. J. Bacteriol. 184: 5200-5203). Using this method, a synthetic oligonucleotide obtained by designing a portion of the target gene to its 5' side and a portion of an antibiotic resistance gene to its 3' side was used as primers to obtain PCR products. These PCR products enabled the one-step construction of a gene-damaged strain. By further combining the use of excision systems derived from λ phage, it is possible to remove antibiotic resistance genes from introduced gene-damaging strains.

[0313] <3-1> Construction of a strain with the ldhA gene missing, encoding lactate dehydrogenase

[0314] According to WO2005 / 010175, PCR was performed using the following synthetic oligonucleotides as primers and plasmid pMW118-attL-Cm-attR as a template. These synthetic oligonucleotides have a sequence corresponding to a portion of the ldhA gene at their 5' end and sequences corresponding to the respective ends of attL and attR of λ phage at their 3' end. The sequences of the synthetic oligonucleotides used as primers are shown in SEQ ID NO: 27, 28. The amplified PCR product was purified using agarose gel electroporation and introduced into *E. coli* strain MG1655ΔsucA, which carries the temperature-sensitive replication plasmid pKD46. Then, ampicillin-sensitive strains with plasmid pKD46 detached were obtained, and the ldhA gene deletion was confirmed by PCR. Furthermore, to remove the att-cat gene introduced into the ldhA gene, transformation was performed using the helper plasmid pMW-intxis-ts, and ampicillin-resistant strains were selected. In addition, the plasmid pMW-intxis-ts has an integrase (Int) gene and an excisionase (Xis) gene derived from λ phage, and its replication is temperature-sensitive.

[0315] Then, ldhA-damaged strains detached from att-cat and pMW-intxis-ts were obtained using ampicillin and chloramphenicol sensitivity tests. Genomic DNA was prepared from the obtained ldhA-deficient candidate strains, and PCR was performed using the oligonucleotides shown in SEQ ID NO: 29 and 30. The results were analyzed by electrophoresis. For one ldhA-deficient strain, a band approximately 1.0 kb smaller than the band observed during PCR using genomic DNA prepared from MG1655ΔsucA strain as a template was identified. This ldhA-deficient strain was named MG1655ΔsucAΔldhA strain.

[0316] [Example 4] <Determination of α-ketoglutarate synthase activity in strains expressing the α-ketoglutarate synthase gene from *S. aeruginosa*>

[0317] To confirm the active expression of the α-ketoglutarate synthase gene from *S. aeruginosa* in *Escherichia coli*, an expression vector for the α-ketoglutarate synthase gene from *S. aeruginosa* was constructed, introduced into MG1655ΔsucA, and its activity was measured.

[0318] <4-1> Construction of an expression plasmid for the α-ketoglutarate synthase gene derived from green sulfur bacteria

[0319] Using genomic DNA from *Thiobacillus aeruginosa* strain TLS (ATCC49652) as a template, the α-ketoglutarate synthase gene fragment was amplified by PCR using the oligonucleotides shown in SEQ ID NO: 21 and 22. The resulting gene fragment was cut with BamHI and inserted into the BamHI site of pUC18 (manufactured by TAKARABio) to construct an α-ketoglutarate synthase gene expression plasmid, named pUC-KGS. In this plasmid, the α-ketoglutarate synthase gene is located downstream of the lac promoter of pUC18 and can be expressed by this promoter. Furthermore, the copy number of pUC18 is higher than that of pSTV28, making enzyme activity detection easier.

[0320] <4-2> Preparation of crude enzyme solution from α-ketoglutarate synthase gene expression strains derived from *Sulphurella multocida*

[0321] The pUC-KGS and pUC18 vectors used as a control were introduced into MG1655ΔsucA via electroporation, and transformants were obtained using ampicillin resistance as an indicator. After confirming that the plasmids had been introduced, the α-ketoglutarate synthase gene expression strain derived from *Thiopyridamole* was named MG1655ΔsucA / pUC-KGS, and the control strain was named MG1655ΔsucA / pUC18.

[0322] The above-mentioned strain was inoculated into LB medium containing 1 mM IPTG and cultured overnight at 37°C. The bacterial cells, equivalent to 10 ml, were recovered by centrifugation and resuspended in 300 μl of 50 mM HEPES buffer (pH 8.0). The suspension was then disrupted using an ultrasonic homogenizer, and the supernatant obtained after centrifugation at 15000 rpm for 15 minutes was used as the crude enzyme solution.

[0323] <4-3> Determination of α-ketoglutarate synthase activity in crude enzyme solutions of strains expressing the α-ketoglutarate synthase gene from *S. aeruginosa*

[0324] The reaction was initiated by adding 50 μl of crude enzyme solution to 1 ml of the reaction solution shown below. First, the reaction solution containing all components except the substrate α-ketoglutarate was added to the cuvette, and the cuvette was sealed with a rubber stopper and aluminum cap. Argon gas was blown into the cuvette for 5 minutes using a syringe to reduce the oxygen concentration. The cuvette was placed on a spectrophotometer (Hitachi U-3210 Spectrophotometer), and α-ketoglutarate solution was added using a syringe to start the reaction. The reaction was carried out at 37°C for 30 minutes, and the absorbance at 578 nm was measured periodically to track changes in the amount of reduced methylviologen.

[0325] [Reaction solution]

[0326] MgCl2 1mM

[0327] Dithiothreitol 1mM

[0328] Methyl viologen 5mM

[0329] CoA 0.25mM

[0330] α-Ketoglutaric acid 10mM (add just before the assay begins)

[0331] HEPES (pH 8.0) 50mM

[0332] The results are shown in Table 1. In the table, KGS represents α-ketoglutarate synthase. In the above reaction system, reduced methyl viologen increased due to the decarbonation of α-ketoglutarate catalyzed by α-ketoglutarate synthase. The absorbance coefficient of reduced methyl viologen at 578 nm was ε578 = 9.8 / mM / cm. 1 U of enzyme activity is expressed as the amount of methyl viologen reduced per minute (1 μmol / m). No α-ketoglutarate synthase activity was detected in the control strain MG1655ΔsucA / pUC18, but an activity of 0.1 U / mg was confirmed in the α-ketoglutarate synthase gene expression strain MG1655ΔsucA / pUC18-KGS derived from *Thiopyridae*.

[0333] Table 1

[0334] strain KGS activity (U / mg) MG1655ΔsucA / pUC18 0.00 MG1655ΔsucA / pUC-KGS 0.10

[0335] [Example 5] <Effects of α-ketoglutarate synthase gene, pyruvate synthase gene, and ferricoxin gene expression strains derived from *S. aeruginosa* on L-glutamate production capacity under oxygen-limited conditions with glucose as the carbon source>

[0336] To investigate the effect of α-ketoglutarate synthase expression on L-glutamate production under oxygen-limited conditions, the α-ketoglutarate synthase gene, pyruvate synthase gene, and ferricodin gene from *S. aeruginosa* were expressed using plasmid pSTV-KGS-PS-FdI and introduced into MG1655ΔsucAΔldhA, followed by culture.

[0337] The pSTV-FdI-KGS-PS vector and the control vector pSTV28 were introduced into MG1655ΔsucAΔldhA via electroporation, and transformants were obtained using chloramphenicol resistance as an indicator. After confirming the plasmid introduction, the strain expressing the α-ketoglutarate synthase gene, pyruvate synthase gene, and ferricoxin gene from *Thiopyrum aeruginosa* was named MG1655ΔsucAΔldhA / pSTV-FdI-KGS-PS, and the control strain was named MG1655ΔsucAΔldhA / pSTV28.

[0338] The L-glutamic acid production capacity was studied by culturing the strain prepared above.

[0339] MG1655ΔsucAΔldhA / pSTV-FdI-KGS-PS and the control strain MG1655ΔsucAΔldhA / pSTV28 were inoculated into LB medium and cultured overnight at 37°C. One-sixth plate volume of the bacterial culture was then inoculated into 50 ml of glucose medium (the following composition) in a 500 ml Erlenmeyer flask and cultured at 37°C for 31 hours. To implement oxygen restriction, the culture was carried out with a stirring speed of 100 rpm. After culture, the accumulated L-glutamate in the medium was determined using a Biotech Analyzer (Asahi Kasei Corporation).

[0340] [Composition of glucose culture medium]

[0341] Glucose 40g / L

[0342] MgSO4·7H2O 1.0 g / L

[0343] (NH4)2SO4 20g / L

[0344] KH2PO4 1.0 g / L

[0345] Yeast extract 2.0g / L

[0346] FeSO4·7H2O 0.01g / L

[0347] MnSO4·5H2O 0.01g / L

[0348] Thiamine hydrochloride 0.01 g / L

[0349] Chloramphenicol 25mg / L

[0350] Calcium carbonate 50g / L

[0351] Sterilization conditions: pH 7.0 (adjusted with KOH), 120℃, 20 minutes.

[0352] The results are shown in Table 2. Compared with the control MG1655ΔsucAΔldhA / pSTV28, the L-glutamate yield relative to consumed glucose was increased by 0.6% in the MG1655ΔsucAΔldhA / pSTV-FdI-KGS-PS vector containing the expression vectors for α-ketoglutarate synthase, pyruvate synthase, and ferricoxin genes derived from *Thalassiopeia aurea*.

[0353] Table 2

[0354]

[0355] [Example 6] <Effects of α-ketoglutarate synthase gene, pyruvate synthase gene, and ferricoxin gene expression strains derived from *S. aeruginosa* on L-glutamate production capacity using glycerol as a carbon source>

[0356] The L-glutamic acid production capacity using glycerol as a carbon source was investigated using α-ketoglutarate synthase gene, pyruvate synthase gene, and ferricredoxin gene expression strain MG1655ΔsucAΔldhA / pSTV-FdI-KGS-PS and control strain MG1655ΔsucAΔldhA / pSTV28 derived from *Sulphurella multocida*.

[0357] MG1655ΔsucAΔldhA / pSTV-FdI-KGS-PS and the control strain MG1655ΔsucAΔldhA / pSTV28 were inoculated into LB medium and cultured overnight at 37°C. Half a plate of cells was then inoculated into 20 ml of glycerol medium (the following composition) in a 500 ml Sakaguchi flask and cultured aerobically at 37°C and 120 rpm for 29 hours. After culture, the accumulated L-glutamate in the medium was determined using a Biotech Analyzer (Asahi Kasei Corporation).

[0358] [Composition of glycerol culture medium]

[0359] 50g / L of glycerin

[0360] MgSO4·7H2O 1.0 g / L

[0361] (NH4)2SO4 20g / L

[0362] KH2PO4 1.0 g / L

[0363] Yeast extract 2.0g / L

[0364] FeSO4·7H2O 0.01g / L

[0365] MnSO4·5H2O 0.01g / L

[0366] Thiamine hydrochloride (Thiamine HCl) 0.01 g / L

[0367] Chloramphenicol 25mg / L

[0368] Calcium carbonate 30g / L

[0369] Sterilization conditions: pH 7.0 (adjusted with KOH), 120℃, 20 minutes.

[0370] The results are shown in Table 3. Compared with the control MG1655ΔsucAΔldhA / pSTV28, the L-glutamate yield relative to consumed glycerol was increased by 6% in MG1655ΔsucAΔldhA / pSTV-FdI-KGS-PS, which introduced the expression vectors for α-ketoglutarate synthase, pyruvate synthase, and ferricredoxin from *Thalassiopeia aurea*.

[0371] Table 3

[0372]

[0373] [Example 7] <α-ketoglutarate synthase gene from *Sulphurella multocida*, flavin-NADP from *Escherichia coli*> + Construction of reductase gene and expression plasmids for ferricoxin / flavin reductin derived from E. coli >

[0374] As a coenzyme regeneration system essential for α-ketoglutarate synthase activity, flavin-NADP derived from E. coli is used. + A plasmid was constructed to simultaneously express the reductase gene and the flavin-NADP gene from *E. coli*. + The reductase gene used was the fpr gene; the flavin oxidoreductin gene of E. coli used the fldA and fldB genes; and the ferreductin gene used the fdx and yfhL genes.

[0375] <7-1> Construction of an expression plasmid for the α-ketoglutarate synthase gene derived from green sulfur bacteria

[0376] The pSTV-KGS constructed in Example 1 was cut with BamHI, and the resulting α-ketoglutarate synthase gene fragment was inserted into the BamHI site of pMWPthr to construct the α-ketoglutarate synthase gene expression vector pMWPthr-KGS. The plasmid pMWPthr has a promoter region (Pthr) of the threonine operon (thrABC) located between the HindIII and XbaI sites in the vector pMW118 (manufactured by NIPPONGENE), as shown in SEQ ID NO: 31, in the genome sequence of E. coli strain K-12 (Genbank accession number U00096). When the gene is cloned downstream of this promoter, the plasmid can express the gene.

[0377] <7-2> E. coli-derived flavin-NADP + Construction of reductase gene amplification vector

[0378] Using genomic DNA of Escherichia coli strain MG1655 as a template, flavin-NADP was amplified by PCR using the oligonucleotides shown in SEQ ID NO: 32 and 33. + A reductase gene fragment was obtained. This gene fragment was cut with SmaI and inserted into the SmaI site of pMWPthr to construct flavin-NADP. + The plasmid used for amplifying the reductase gene was named pMWPthr-fpr.

[0379] <7-3> Construction of an amplification vector for the flavin redoxin (fldA) gene derived from Escherichia coli

[0380] Using genomic DNA from *Escherichia coli* strain MG1655 as a template, the flavin redoxin (fldA) gene fragment was amplified by PCR using the oligonucleotides shown in SEQ ID NO: 34 and 35. This gene fragment was then cut with EcoRI and inserted into the EcoRI site of pMWPthr to construct the plasmid pMWPthr-fldA for flavin redoxin (fldA) gene amplification.

[0381] <7-4> Construction of a plasmid for amplifying the flavin redoxin (fldB) gene from *E. coli*

[0382] Using genomic DNA from *Escherichia coli* strain MG1655 as a template, the flavin redoxin (fldB) gene fragment was amplified by PCR using the oligonucleotides shown in SEQ ID NO: 36 and 37. This gene fragment was then cut with EcoRI and inserted into the EcoRI site of pMWPthr to construct the plasmid pMWPthr-fldB for flavin redoxin (fldB) gene amplification.

[0383] <7-5> Construction of a plasmid for amplifying the ferroredoxin (fdx) gene from E. coli

[0384] Using genomic DNA from Escherichia coli strain MG1655 as a template, the ferricodoxin (fdx) gene fragment was amplified by PCR using the oligonucleotides shown in SEQ ID NO: 38 and 39. This gene fragment was then cut with EcoRI and inserted into the EcoRI site of pMWPthr to construct the plasmid pMWPthr-fdx for ferricodoxin (fdx) gene amplification.

[0385] <7-6> Construction of a plasmid for amplifying the ferroredoxin (yfhL) gene from *E. coli*

[0386] Using genomic DNA from *Escherichia coli* strain MG1655 as a template, the ferritin (yfhL) gene fragment was amplified by PCR using the oligonucleotides shown in SEQ ID NO: 40 and 41. This gene fragment was then cut with EcoRI and inserted into the EcoRI site of pMWPthr, thereby constructing the plasmid pMWPthr-yfhL for ferritin (yfhL) gene amplification.

[0387] <7-7> Used for expressing the α-ketoglutarate synthase gene from *Sulphurella multocida* and for amplifying ferrugin-NADP from *E. coli*. + Construction of plasmids for reductase gene and flavin reductase (fldA) gene

[0388] pMWPthr-fldA was digested with EcoRI, and the resulting fldA gene fragment was ligated with the fragment obtained from treating pMWPthr-KGS with EcoRI to obtain pMWPthr-KGS-fldA. Then, pMWPthr-fpr was digested with SmaI, and the resulting fpr gene fragment was ligated with the fragment obtained from treating pMWPthr-KGS-fldA with SmaI, thus constructing a gene for expressing α-ketoglutarate synthase and enhancing flavin-NADP from *E. coli*. + The plasmid pMWPthr-KGS-fpr-fldA expresses the reductase gene and the flavin oxidoreductin (fldA) gene.

[0389] <7-8> Used to express the α-ketoglutarate synthase gene from *Sulphurella multocida* and amplify flavin-NADP from *Escherichia coli*. + Construction of plasmids for reductase gene and flavin reductase (fldB) gene

[0390] pMWPthr-fldB was digested with EcoRI, and the resulting fldB gene fragment was ligated with the fragment obtained by treating pMWPthr-KGS with EcoRI to obtain pMWPthr-KGS-fldB. Then, pMWPthr-fpr was digested with SmaI, and the resulting fpr gene fragment was ligated with the fragment obtained by treating pMWPthr-KGS-fldB with SmaI to construct a gene for enhancing α-ketoglutarate synthase and flavin-NADP. + The plasmid pMWPthr-KGS-fpr-fldB expresses the reductase gene and the flavin oxidoreductin (fldB) gene.

[0391] <7-9> Used to express the α-ketoglutarate synthase gene from *Sulphurella multocida* and amplify flavin-NADP from *Escherichia coli*. +Construction of plasmids for reductase gene and ferricoxin (fdx) gene

[0392] pMWPthr-fdx was digested with EcoRI, and the resulting fdx gene fragment was ligated with the fragment obtained by treating pMWPthr-KGS with EcoRI to obtain pMWPthr-KGS-fdx. Then, pMWPthr-fpr was digested with SmaI, and the resulting fpr gene fragment was ligated with the fragment obtained by treating pMWPthr-KGS-fdx with SmaI to construct a gene for enhancing α-ketoglutarate synthase and flavin-NADP. + The vector pMWPthr-KGS-fpr-fdx for expressing the reductase gene and ferroredoxin (fdx) gene.

[0393] <7-10> Used to express the α-ketoglutarate synthase gene from *Sulphurella multocida* and amplify flavin-NADP from *Escherichia coli*. + Construction of plasmids for reductase gene and ferricoxin (yfhL) gene

[0394] pMWPthr-yfhL was digested with EcoRI, and the resulting yfhL gene fragment was ligated with the fragment obtained by treating pMWPthr-KGS with EcoRI to obtain pMWPthr-KGS-yfhL. Then, pMWPthr-fpr was digested with SmaI, and the resulting fpr gene fragment was ligated with the fragment obtained by treating pMWPthr-KGS-yfhL with SmaI to construct a gene for enhancing α-ketoglutarate synthase and flavin-NADP. + The plasmid pMWPthr-KGS-fpr-yfhL expresses the reductase gene and the ferroredoxin (yfhL) gene.

[0395] Among the plasmids mentioned above, the α-ketoglutarate synthase gene from *Sulphurella multocida* is transcribed via Pthr, while other genes are transcribed via read-through from Pthr.

[0396] [Example 8] <α-ketoglutarate synthase gene from *Sulphurella multocida*, flavin-NADP from *Escherichia coli*> + The effects of enhanced expression of reductase genes and flavin-oxidoreductin / ferreductin genes from *E. coli* on L-glutamate production capacity using glycerol as a carbon source >

[0397] To investigate flavin-NADP derived from E. coli +The effect of enhanced α-ketoglutarate synthase activity from *S. aeruginosa* caused by amplification of reductase genes and *E. coli*-derived flavin-redoxin or ferroredoxin genes on L-glutamate production under oxygen-limited conditions, along with the aforementioned methods for enhancing *S. aeruginosa*-derived α-ketoglutarate synthase and *E. coli*-derived flavin-redoxin-NADP... + The reductase gene and the vectors expressing the flavin oxoreductin or ferroreductin gene from E. coli were introduced into MG1655ΔsucAΔldhA and cultured.

[0398] <8-1> The gene for amplifying α-ketoglutarate synthase from *Sulphurella multocida* and flavin-NADP from *Escherichia coli* will be used. + Plasmids containing reductase genes and flavin-oxidoreductin-ferroreductin genes from *E. coli* were introduced into strain MG1655ΔsucAΔldhA.

[0399] The following vectors were introduced via electroporation into MG1655ΔsucAΔldhA: pMWPthr-KGS, for expressing the α-ketoglutarate synthase gene from *Thiobacillus aeruginosa*, and pMWPthr-KGS, for enhancing the α-ketoglutarate synthase gene and flavin-NADP from *E. coli*. + The reductase gene and the expression plasmids pMWPthr-KGS-fpr-fldA, pMWPthr-KGS-fpr-fldB, pMWPthr-KGS-fpr-fdx, or pMWPthr-KGS-fpr-yfhL of flavin oxidoreductin-NADP from *E. coli* were used as controls. + The reductase gene was amplified using the plasmid pMWPthr-fpr. Transformants were obtained using kanamycin resistance as an indicator. After confirming the plasmid introduction, the introduced strains were named MG1655ΔsucAΔldhA / pMWPthr-KGS, MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fldA, MG1655ΔsucAΔldhA / pMWPthrKGS-fpr-fldB, MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fdx, MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-yfhL, MG1655ΔsucAΔldhA / pMWPthr, and MG1655ΔsucAΔldhA / pMWPthr-fpr.

[0400] <8-2> α-Ketoglutarate synthase gene from *Sulphurella multocida* and flavin-NADP from *Escherichia coli*. +Effects of enhanced expression of reductase gene and flavin-oxidoreductin-ferroreductin gene from *E. coli* on L-glutamate production capacity using glycerol as a carbon source.

[0401] The α-ketoglutarate synthase gene expression strain MG1655ΔsucAΔldhA / pMWPthr-KGS derived from *Thiobacillus aeruginosa*, prepared in <8-1>, was used. The α-ketoglutarate synthase gene from *Thiobacillus aeruginosa* and flavin-NADP from *Escherichia coli* were also used. + The reductase gene, the enhanced expression strains of flavin-oxidoreductin-ferroreductin from *E. coli* MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fldA, MG1655ΔsucAΔldhA / pMWPthrKGS-fpr-fldB, MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fdx, MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-yfhL, the control strain MG1655ΔsucAΔldhA / pMWPthr, and flavin-oxidoreductin-NADP from *E. coli* + The L-glutamate production capacity under aerobic conditions was investigated using the reductase gene amplification strain MG1655ΔsucAΔldhA / pMWPthr-fpr with glycerol as the carbon source.

[0402] Half the volume of each strain of bacteria, equivalent to one plate of a plate obtained from an overnight pre-culture at 37°C using LB medium, was inoculated into 20 mL of medium (a glycerol medium prepared by replacing chloramphenicol with 40 mg / L kanamycin on the basis of the glycerol medium described in Example 6) in a 500 mL volumetric flask and cultured at 37°C and 120 rpm for 29 hours. After culture, the L-glutamate accumulated in the medium was determined using a Biotech Analyzer (manufactured by Asahi Kasei Corporation).

[0403] The results are shown in Table 4. Compared with the control MG1655ΔsucAΔldhA / pMWPthr and E. coli-derived flavin-NADP... + Compared to the amplified reductase gene strain MG1655ΔsucAΔldhA / pMWPthr-fpr, the α-ketoglutarate synthase gene expression strain MG1655ΔsucAΔldhA / pMWPthr-KGS from *Thiopyridae* showed a 3% increase in L-glutamate yield relative to glycerol consumption; while the α-ketoglutarate synthase gene from *Thiopyridae* and the flavin-NADP gene from *E. coli* showed a 3% increase in L-glutamate yield relative to glycerol consumption. +Increased yields of 8–9% were observed in the reductase gene and the flavin oxoreductin / ferreductin gene expression-enhanced strains MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fldA, MG1655ΔsucAΔldhA / pMWPthrKGS-fpr-fldB, MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fdx, and MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-yfhL.

[0404] Table 4

[0405]

[0406] [Example 9] <α-ketoglutarate synthase gene from *Sulphurella multocida*, flavin-NADP from *Escherichia coli*> + The effects of introducing plasmids containing reductase genes and flavin-oxidoreductin-ferroreductin genes from *E. coli* on L-glutamate production capacity under oxygen-limited conditions with glucose as the carbon source >

[0407] For the control MG1655ΔsucAΔldhA / pMWPthr constructed in Example 8, the α-ketoglutarate synthase gene from *Thiobacillus aeruginosa* and the flavin-NADP from *Escherichia coli* were used. + The reductase gene and the flavin-oxidoreductin-ferroreductin gene expression-enhanced strains MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fldA, MG1655ΔsucAΔldhA / pMWPthrKGS-fpr-fldB, MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fdx, and MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-yfhL were pre-cultured overnight at 37°C using LB medium. The bacterial cells, equivalent to 1 / 6 of the plate volume, were inoculated into 50 mL of medium (based on the glucose medium described in Example 5, with chloramphenicol replaced by 40 mg / L kanamycin) in a 500 mL Erlenmeyer flask and cultured at 37°C for 38 hours. For oxygen restriction, the culture was carried out with a stirring speed of 100 rpm. After the culture was completed, the amount of L-glutamic acid accumulated in the culture medium was determined using Biotech Analyzer (Asahi Kasei Corporation).

[0408] The results are shown in Table 5. Compared with the control MG1655ΔsucAΔldhA / pMWPthr, the α-ketoglutarate synthase gene from *Thiobacillus aeruginosa* and the flavin-NADP gene from *Escherichia coli* showed significant differences.+ The yield of L-glutamic acid was increased in the reductase gene and in the flavin oxoreductin / ferreductin gene expression-enhanced strains MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fldA, MG1655ΔsucAΔldhA / pMWPthrKGS-fpr-fldB, MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fdx, and MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-yfhL.

[0409] Table 5

[0410]

[0411] [Example 10] <α-ketoglutarate synthase gene from *Sulphurella multocida*, flavin-NADP from *Escherichia coli*> + The effects of enhanced expression of reductase genes and flavin reductin from *E. coli* on L-glutamate production capacity using glucose as a carbon source >

[0412] The α-ketoglutarate synthase gene from *Sulphurella multocida* prepared in Example 8 and the flavin-NADP from *Escherichia coli* were used. + The L-glutamate production capacity under aerobic conditions with glucose as the carbon source was investigated by comparing the expression-enhanced strain MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fldA with the control strain MG1655ΔsucAΔldhA / pMWPthr and the reductase gene and the flavin oxidoreductin gene derived from E. coli.

[0413] Each strain was inoculated with 1 / 4 the volume of cells from a plate obtained by overnight pre-culture at 37°C using LB medium into 20 mL of glucose + methionine medium in a 500 mL volumetric flask (the glucose medium described in Example 5 was prepared by replacing chloramphenicol with 40 mg / L kanamycin and adding 0.2 g / L DL-methionine). The medium was incubated at 37°C and 120 rpm for 25 hours. After incubation, the accumulated L-glutamate in the medium was determined using a Biotech Analyzer (manufactured by Asahi Kasei Corporation).

[0414] The results are shown in Table 6. Compared with the control MG1655ΔsucAΔldhA / pMWPthr, the α-ketoglutarate synthase gene from *Thiobacillus aeruginosa* and the flavin-NADP gene from *Escherichia coli* showed significant differences. +In the recombinant strain MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fldA with enhanced expression of the reductase gene and the flavodoxin gene derived from Escherichia coli, the yield was confirmed to have increased by approximately 6%.

[0415] Table 6

[0416]

[0417] 〔Example 11〕<Effect of the recombinant strain with enhanced expression of the α-ketoglutarate synthase gene derived from Chlorobium limicola, the flavodoxin-NADP reductase gene derived from Escherichia coli, and the flavodoxin gene derived from Escherichia coli on the L-glutamic acid production ability when using glycerol as a carbon source> +

[0418] To study the effect of enhanced α-ketoglutarate synthase activity in Pantoea ananatis on L-glutamic acid production, the plasmid pMWPthr-KGS-fpr-fldA carrying the α-ketoglutarate synthase gene derived from Chlorobium limicola, the flavodoxin-NADP reductase gene derived from Escherichia coli, and the flavodoxin gene derived from Escherichia coli prepared in Example 7 and the control plasmid pMWPthr were introduced into the L-glutamic acid-producing bacterium Pantoea ananatis, and the bacteria were cultured. +

[0419] <11-1>Construction of the L-glutamic acid-producing bacterium Pantoea ananatis

[0420] The plasmid RSFCPG (refer to the specification of European Patent Application Publication No. 1233068) carrying the citrate synthase gene (gltA), phosphoenolpyruvate carboxylase gene (ppc), and glutamate dehydrogenase gene (gdhA) derived from Escherichia coli was treated with BglII and KpnI, made blunt-ended, and then ligated to remove the gltA gene. The resulting plasmid was used to transform Escherichia coli JM109 strain. The plasmid was extracted from the obtained transformant and named pRSF-ppc-gdhA. The plasmid pRSF-ppc-gdhA was introduced into the L-glutamic acid-producing bacterium Pantoea ananatis NP106 strain to construct NP106 / pRSF-ppc-gdhA.

[0421] ​​The NP106 strain was obtained as follows: The Pantoeaananatis AJ13601 strain from the previous example was cultured overnight in LBGM9 liquid medium at 34°C with shaking. The culture was then diluted to 100–200 colonies per plate and plated onto LBGM9 plates containing 12.5 mg / L tetracycline. Colonies that appeared were copied onto LBGM9 plates containing 12.5 mg / L tetracycline and 25 mg / L chloramphenicol. Chloramphenicol-sensitive strains were selected, thus obtaining the pSTVCB-exfoliated strain, which was named G106S. Furthermore, the G106S strain was cultured overnight in LBGM9 liquid medium at 34°C with shaking. The culture was then diluted to 100–200 colonies per plate and plated onto drug-free LBGM9 plates. The resulting colonies were cloned onto LBGM9 plates containing 12.5 mg / L tetracycline and LBGM9 plates without the drug. Tetracycline-sensitive strains were selected, thus obtaining strains with detached RSFCPG plasmids, which were named NP106. The NP106 strain obtained in this way does not carry the RSFCPG and pSTVCB plasmids carried by strain AJ13601.

[0422] <11-2> Incorporate α-ketoglutarate synthase gene from *Sulphurella multocida* and flavin-NADP from *Escherichia coli*. + The reductase gene and flavin reductase (fldA) gene were expressed by introducing a plasmid into the NP106 / pRSF-ppc-gdhA strain.

[0423] Electroporation was used to introduce, respectively, α-ketoglutarate synthase gene enhancer and E. coli-derived flavin-NADP into NP106 / pRSF-ppc-gdhA. + The reductase gene and the flavin-oxidoreductin-ferroreductin gene from *E. coli*, expressed by the plasmid pMWPthr-KGS-fpr-fldA, along with the control plasmid pMWPthr, were used to obtain transformants, with resistance to both kanamycin and tetracycline as indicators. After confirming the introduction of the plasmids, the introduced strains were named NP106 / pRSF-ppc-gdhA / pMWPthr-KGS-fpr-fldA and NP106 / pRSF-ppc-gdhA / pMWPthr, respectively.

[0424] <11-3> Using glycerol as a carbon source, α-ketoglutarate synthase gene from *S. green sulfur* and flavin-NADP from *E. coli* were cultured under aerobic conditions. + The reductase gene and flavin reductin (fldA) gene amplification plasmid were introduced into the strain.

[0425] For the two strains constructed in <11-1>, a pre-culture was performed overnight at 37°C using LBMG medium (LB medium supplemented with 0.5 g / L glucose, 2 mM MgSO4, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, and 6 g / L Na2HPO4). One-quarter plate volume of bacterial cells was inoculated into 20 ml of pantothenic acid glycerol medium (the following composition) in a 500 ml Sakaguchi flask and incubated at 34°C for 48 hours at 120 rpm. After incubation, the accumulated L-glutamate in the medium was determined using a Biotech Analyzer (Asahi Kasei Corporation).

[0426] [Composition of glycerol culture medium for pan-based bacteria]

[0427] Glycerin 40g / L

[0428] MgSO4·7H2O 1.0 g / L

[0429] (NH4)2SO4 20g / L

[0430] KH2PO4 2.0g / L

[0431] Yeast extract 2.0g / L

[0432] FeSO4·7H2O 0.02g / L

[0433] MnSO4·5H2O 0.02g / L

[0434] Thiamine hydrochloride 0.01 g / L

[0435] L-Lysine 0.2g / L

[0436] DL-Diaminopimelic acid 0.2 g / L

[0437] L-methionine 0.2 g / L

[0438] Kanamycin 40 mg / L

[0439] Tetracycline 2.5 mg / L

[0440] Calcium carbonate 20g / L

[0441] Sterilization conditions: pH 7.0 (adjusted with KOH), 120℃, 20 minutes.

[0442] The results are shown in Table 7. Compared with the control NP106 / pRSF-ppc-gdhA / pMWPthr, the α-ketoglutarate synthase gene from *Thiobacillus aeruginosa* and the flavin-NADP gene from *Escherichia coli* showed significant differences. +In the reductase gene and the flavodoxin gene expression-enhanced strain NP106 / pRSF-ppc-gdhA / pMWPthr-KGS-fpr-fldA derived from Escherichia coli, it was confirmed that the L-glutamic acid yield increased by about 3%.

[0443] Table 7

[0444]

[0445] 〔Example 12〕<Effect of the α-ketoglutarate synthase gene expression-enhanced strain derived from Chlorobium on the L-glutamic acid production ability under aerobic conditions when using glucose as a carbon source>

[0446] Using the strain MG1655ΔsucAΔldhA / pMWPthr-KGS in which only the α-ketoglutarate synthase gene derived from Chlorobium was enhanced and the control strain MG1655ΔsucAΔldhA / pMWPthr prepared in Example 8, the L-glutamic acid production ability under aerobic conditions when using glucose as a carbon source was investigated.

[0447] The cells of each strain equivalent to 1 / 4 plate amount of the plate obtained by pre-culturing overnight at 37°C in LB medium were inoculated into 20 mL of glucose + methionine medium (in the glucose medium described in Example 5, the glucose concentration was 35.0 g / L, 40 mg / L of kanamycin was added instead of chloramphenicol, and 0.2 g / L of DL-methionine was added) in a 500 mL Sakaguchi flask, and cultured at 37°C and a stirring speed of 120 rpm for 23 hours. After the culture was completed, the L-glutamic acid accumulated in the medium was measured using a Biotech Analyzer (manufactured by Asahi Kasei Corporation).

[0448] The results are shown in Table 8. Compared with the control MG1655ΔsucAΔldhA / pMWPthr, in the strain MG1655ΔsucAΔldhA / pMWPthr-KGS in which only the expression of the α-ketoglutarate synthase gene derived from Chlorobium was enhanced, it was confirmed that the yield increased by about 6%.

[0449] Table 8

[0450]

[0451] 〔Example 13〕<Effect of the α-ketoglutarate synthase gene-enhanced strain derived from Chlorobium in Pantoea ananatis on the L-glutamic acid production ability under aerobic culture conditions when using glycerol as a carbon source>

[0452] To investigate the effect of enhanced α-ketoglutarate synthase activity in Pantoeaananatis on L-glutamate production, the α-ketoglutarate synthase gene expression plasmid pMWPthr-KGS from *Sulphurella vulgaris* prepared in Example 7 and the control plasmid pMWPthr were introduced into *Pantoeaananatis* L-glutamate producing bacteria and cultured.

[0453] The α-ketoglutarate synthase gene expression enhancement plasmid pMWPthr-KGS and the control plasmid pMWPthr, prepared in Example 7, were introduced into NP106 / pRSF-ppc-gdhA prepared in Example 11 via electroporation. Transformants were obtained using resistance to both kanamycin and tetracycline as indicators. After confirming successful plasmid introduction, the introduced strains were named NP106 / pRSF-ppc-gdhA / pMWPthr-KGS and NP106 / pRSF-ppc-gdhA / pMWPthr, respectively.

[0454] NP106 / pRSF-ppc-gdhA / pMWPthr-KGS and NP106 / pRSF-ppc-gdhA / pMWPthr were cultured overnight at 37°C using LBMG. One-quarter plate volume of the bacterial cells was inoculated into 20 mL of pantothenic acid glycerol medium (the following composition) in a 500 mL Sakaguchi flask and incubated at 34°C for 66 hours at 100 rpm. After incubation, the accumulated L-glutamate in the medium was determined using a Biotech Analyzer (Asahi Kasei Corporation).

[0455] [Composition of glycerol culture medium for pan-based bacteria]

[0456] 30g / L of glycerin

[0457] MgSO4·7H2O 0.5 g / L

[0458] (NH4)2SO4 20g / L

[0459] KH2PO4 2.0g / L

[0460] Yeast extract 2.0g / L

[0461] FeSO4·7H2O 0.02g / L

[0462] MnSO4·5H2O 0.02g / L

[0463] Calcium pantothenate 18 mg / L

[0464] Thiamine hydrochloride 0.01 g / L

[0465] L-Lysine 0.2g / L

[0466] DL-Diaminopimelic acid 0.2 g / L

[0467] L-Methionine 0.2 g / L

[0468] Kanamycin 40 mg / L

[0469] Tetracycline 12.5 mg / L

[0470] Calcium carbonate 20 g / L

[0471] pH 7.0 (adjusted with KOH) Sterilization conditions: 115 °C, 10 minutes

[0472] The results are shown in Table 9. Compared with the control NP106 / pRSF-ppc-gdhA / pMWPthr, in the enhanced strain of α-ketoglutarate synthase gene expression derived from Chlorobium limicola, NP106 / pRSF-ppc-gdhA / pMWPthr-KGS, it was confirmed that the L-glutamic acid yield increased by about 8%.

[0473] Table 9

[0474]

[0475] 〔Example 14〕<Effect of an enhanced strain of α-ketoglutarate synthase gene derived from Chlorobium limicola in Pantoea ananatis on L-glutamic acid production ability under conditions of limited oxygen concentration when using glycerol as a carbon source>

[0476] To study the effect of only enhanced α-ketoglutarate synthase activity in Pantoea ananatis on L-glutamic acid production in microaerobic culture using glycerol as a carbon source, microaerobic culture of NP106 / pRSF-ppc-gdhA / pMWPthr-KGS and NP106 / pRSF-ppc-gdhA / pMWPthr prepared in Example 13 was carried out using glycerol as a carbon source.

[0477] NP106 / pRSF-ppc-gdhA / pMWPthr-KGS and NP106 / pRSF-ppc-gdhA / pMWPthr were pre-cultured overnight at 37 °C in LBMG medium. Bacterial cells equivalent to 1 / 4 of a plate amount were inoculated into 20 mL of the following Pantoea glycerol medium in a 500 mL Sakaguchi flask and cultured at 100 rpm at 34 °C for 90 hours. After the culture was completed, the L-glutamic acid accumulated in the medium was measured using a Biotech Analyzer (Asahi Kasei Corporation).

[0478] 〔Composition of Pantoea glycerol medium〕

[0479] 30g / L of glycerin

[0480] MgSO4·7H2O 0.5 g / L

[0481] (NH4)2SO4 20g / L

[0482] KH2PO4 2.0g / L

[0483] Yeast extract 2.0g / L

[0484] FeSO4·7H2O 0.02g / L

[0485] MnSO4·5H2O 0.02g / L

[0486] Thiamine hydrochloride 0.01 g / L

[0487] Kanamycin 40 mg / L

[0488] Tetracycline 12.5 mg / L

[0489] Calcium carbonate 20g / L

[0490] Sterilization conditions: pH 7.0 (adjusted with KOH), 115℃, 10 minutes.

[0491] The results are shown in Table 10. Compared with the control NP106 / pRSF-ppc-gdhA / pMWPthr, the L-glutamate yield was confirmed to be increased by about 30% in the α-ketoglutarate synthase gene-enhanced strain NP106 / pRSF-ppc-gdhA / pMWPthr-KGS derived from *Sulphurella multocida*.

[0492] Table 10

[0493]

[0494] [Example 15] <Effect of α-ketoglutarate synthase gene expression strain from *Blastopirellulamarina* on L-glutamate production capacity using glycerol as a carbon source>

[0495] To investigate the effect of α-ketoglutarate synthase activity expression from Blastopirellulamarina on L-glutamate production using glycerol as the carbon source, a plasmid for expressing the α-ketoglutarate synthase gene from Blastopirellulamarina was constructed and introduced into Escherichia coli MG1655ΔsucAΔldhA for culture.

[0496] <15-1> Construction of plasmid for expression of α-ketoglutarate synthase gene derived from Blastopirellulamarina

[0497] Using genomic DNA from Blastopirellulamarina DSM3645 (ATCC49069) as a template, PCR was performed using the oligonucleotides shown in SEQ ID NO: 61 and 62 to amplify gene fragments containing the α and β subunits of α-ketoglutarate synthase. The resulting gene fragments were cut with KpnI and EcoRI and inserted into fragments obtained by cutting pMWPthr with KpnI and EcoRI, thus constructing a plasmid for expressing the α-ketoglutarate synthase gene from Blastopirellulamarina, named pMWPthr-BlaKGS.

[0498] <15-2> Culture of α-ketoglutarate synthase gene expression strains derived from *Blastopirellulamarina* under aerobic conditions with glycerol as a carbon source.

[0499] Transformants were obtained by electroporation of the α-ketoglutarate synthase gene expression strain pMWPthr-BlaKGS and the control strain pMWPthr, respectively, with kanamycin resistance as an indicator. After confirming plasmid introduction, the α-ketoglutarate synthase gene expression strain derived from Blastopirellulamarina was named MG1655ΔsucAΔldhA / pMWPthr-BlaKGS, and the control strain was named MG1655ΔsucAΔldhA / pMWPthr.

[0500] The MG1655ΔsucAΔldhA / pMWPthr-BlaKGS strain and the control strain MG1655ΔsucAΔldhA / pMWPthr were inoculated into LB medium and pre-cultured overnight at 37°C. Half the volume of the bacterial culture was then inoculated into 20 mL of the following glycerol medium in a 500 mL volumetric flask and aerobically cultured at 37°C and 120 rpm for 22 hours. After culture, the accumulated L-glutamate in the medium was determined using a Biotech Analyzer (Asahi Kasei Corporation).

[0501] [Composition of glycerol culture medium]

[0502] 30g / L of glycerin

[0503] MgSO4·7H2O 1.0 g / L

[0504] (NH4)2SO4 20g / L

[0505] KH2PO4 1.0 g / L

[0506] Yeast extract 2.0 g / L

[0507] FeSO4·7H2O 0.01 g / L

[0508] MnSO4·5H2O 0.01 g / L

[0509] Thiamine hydrochloride 0.01 g / L

[0510] Kanamycin 40 mg / L

[0511] Calcium carbonate 30 g / L

[0512] pH 7.0 (adjusted with KOH) Sterilization conditions: 120 °C, 20 minutes

[0513] The results are shown in Table 11. Compared with the control MG1655ΔsucAΔldhA / pMWPthr, in the expression vector MG1655ΔsucAΔldhA / pMWPthr-BlaKGS into which only the α-ketoglutarate synthase gene derived from Blastopirellula marina was introduced, the L-glutamate yield relative to glycerol consumption increased by about 8%.

[0514] Table 11

[0515]

[0516] 〔Example 16〕<Effect of the α-ketoglutarate synthase gene expression-enhanced strain derived from Blastopirellula marina on the L-glutamate production ability under aerobic conditions in a glucose carbon source>

[0517] Using the α-ketoglutarate synthase gene expression-enhanced strain MG1655ΔsucAΔldhA / pMWPthr-BlaKGS derived from Blastopirellula marina prepared in Example 15 and the control strain MG1655ΔsucAΔldhA / pMWPthr, the L-glutamate production ability when using glucose as the carbon source was investigated.

[0518] Half the volume of each strain of bacteria, equivalent to one plate obtained from an overnight LB culture at 37°C, was inoculated into 20 mL of glucose + methionine medium (the glucose medium described in Example 5, with a glucose concentration of 30.0 g / L, 40 mg / L kanamycin instead of chloramphenicol, and 0.2 g / L DL-methionine) in a 500 mL Sakaguchi flask and cultured at 37°C and 120 rpm for 24 hours. After culture, the L-glutamate accumulated in the medium was determined using a Biotech Analyzer (manufactured by Asahi Kasei Corporation).

[0519] The results are shown in Table 12. Compared with the control MG1655ΔsucAΔldhA / pMWPthr, the yield was confirmed to be about 6% higher in the strain MG1655ΔsucAΔldhA / pMWPthr-KGS, which showed enhanced expression of the α-ketoglutarate synthase gene from Blastopirellulamarina.

[0520] Table 12

[0521]

[0522] [Example 17] <Construction of strain 2256ΔldhΔsucA from wild-type lactobacillus>

[0523] A strain with a gene deletion of sucA (odhA) encoding the E1o subunit of α-ketoglutarate dehydrogenase was constructed from *Corynebacterium glutamicum* strain 2256Δldh (refer to WO2005 / 113744). The 2256Δldh strain was an ldhA-deficient strain obtained from *Corynebacterium glutamicum* strain 2256 (ATCC13869).

[0524] Based on the publicly available base sequence (SEQ ID NO: 51) of the E1o subunit gene (hereinafter referred to as the sucA gene) of *Corynebacterium glutamicum* ATCC13032 (GenBank accession number NC_003450), DNA was designed and synthesized as primers. Crossover PCR was used to obtain the α-ketoglutarate dehydrogenase gene fragment from *Lactobacillus fermentum* strain 2256, which lacks the ORF of this gene. Specifically, using the genomic DNA of *Lactobacillus fermentum* strain 2256 as a template and the synthesized DNA of SEQ ID NO: 63 and 64 as primers, PCR was performed using conventional methods to obtain the N-terminal amplification product of the sucA gene. On the other hand, to obtain the C-terminal amplification product of the sucA gene, PCR was performed using the genomic DNA of *Lactobacillus fermentum* strain 2256 as a template and the synthesized DNA of SEQ ID NO: 65 and 66 as primers, using conventional methods. SEQ ID NO: 64 and 65 are complementary.

[0525] Then, the N- and C-terminal gene products of sucA were mixed in essentially equimolar amounts as a template. Using the synthetic DNA of SEQ ID NO: 63 and 66 as primers, PCR was performed using conventional methods to obtain an amplified sucA gene product with most of its internal sequence missing. The generated PCR product was purified using conventional methods, digested with BamHI, and then inserted into the SalI site of pBS3 as described in International Publication WO2005 / 113744. This DNA was used to transform competent E. coli JM109 cells (manufactured by Takara Bio Inc.), and plated on LB medium containing 100 μM IPTG, 40 μg / mL X-Gal, and 25 μg / mL Km, and cultured overnight. Then, white colonies were picked and isolated as single colonies to obtain transformants. Plasmids were extracted from the obtained transformants, and the plasmid containing the target PCR product was named pΔsucA56-1.

[0526] Because pΔldh56-1 lacks a region enabling autonomous intracellular replication in Corynebacterium-type bacteria, strains that integrate the plasmid into their genomic DNA via homologous recombination will appear as transformants when Corynebacterium-type bacteria are transformed using this plasmid, although at a very low frequency. High concentrations of plasmid pΔldh56-1 were used to transform *Brugia lactis* strain 2256 using the electroporation method. The transformed strains were plated on CM-Dex medium containing 25 μg / mL kanamycin and incubated at 31.5°C for approximately 30 hours. In the strains grown on this medium, homologous recombination occurred between the ldh gene fragment of the plasmid and the same gene in the genome of *Brugia lactis* strain 2256, resulting in the insertion of the kanamycin resistance gene and the sacB gene derived from the plasmid into the genome.

[0527] [CM-Dex medium]

[0528] glucose 5g / L

[0529] Multipeptone 10g / L

[0530] Yeast extract 10g / L

[0531] KH2PO4 1g / L

[0532] MgSO4·7H2O 0.4 g / L

[0533] FeSO4·7H2O 0.01g / L

[0534] MnSO4·7H2O 0.01g / L

[0535] urea 3g / L

[0536] Soybean hydrolysate 1.2 g / L

[0537] Biotin 10 μg / L

[0538] Sterilization conditions: pH 7.5 (adjusted with NaOH), 120℃, 20 minutes.

[0539] Then, these first-round recombinants were cultured overnight at 31.5°C in kanamycin-free CM-Dex liquid medium. After appropriate dilution, they were spread onto kanamycin-free Dex-S10 medium containing 10% sucrose (a medium prepared by replacing the glucose in CM-Dex medium with 10 g / L sucrose) and cultured at 31.5°C for approximately 30 hours. Approximately 60 strains were obtained, and it was considered that these 60 strains became sucrose-insensitive by shedding the sacB gene through a second round of homologous recombination.

[0540] Among the strains obtained in this way were strains with the sucA gene replaced by a defective strain derived from pΔsucA56-1, and strains with the sucA gene restored to wild-type. Whether the sucA gene is mutant or wild-type can be easily confirmed by directly performing PCR on bacterial cells cultured in Dex-S10 agar medium to detect the sucA gene. Such strains were used as sucA-deficient strains in the following experiments: when analyzed using primers (SEQ ID NO: 63 and SEQ ID NO: 66) for PCR amplification of the sucA gene, the size of the PCR product was smaller than that obtained using the genomic DNA of strain 2256 as a template. As a result of the analysis of sucrose-insensitive strains using the above method, a strain possessing only the defective sucA gene was selected and named strain 2256ΔldhΔsucA.

[0541] [Example 18] <Effect of an α-ketoglutarate synthase gene expression strain derived from *Lactobacillus aeruginosa* on L-glutamate production capacity under oxygen-limited conditions with glucose as the carbon source>

[0542] To investigate the effect of α-ketoglutarate synthase expression from *S. green sulfur* on L-glutamate production in *Brevibacterium lactis* using glucose as the carbon source, a plasmid for expressing the α-ketoglutarate synthase gene from *S. green sulfur* was constructed and introduced into *Brevibacterium lactis* strain 2256ΔldhΔsucA for culture.

[0543] <18-1> Construction of a plasmid for expressing the α-ketoglutarate synthase gene from *Sulphurella multocida*

[0544] DNA was designed and synthesized as primers based on the base sequences near the tuf and KGS genes of Corynebacterium glutamicum ATCC13032 (GenBank Database accession number NC_003450), and crossover PCR was performed to obtain the KGS gene fragment with the upstream of the gene replaced by the tuf promoter.

[0545] Specifically, using pMWPthr-KGS-fpr-fldA as a template (pMWPthr-KGS-fpr-fldA is the gene constructed in Example 7 for expressing the α-ketoglutarate synthase gene from *Sulphurella multocida* and enhancing the ferricopentaprotein-NADP from *Escherichia coli*), + Using the primers shown in SEQ ID NO: 67 and 68, the KGS fragment was amplified by PCR. This KGS fragment was then treated with PstI and XbaI and inserted into the PstI and XbaI sites of pVK9 to construct the plasmid pVKKGS carrying KGS. A portion of the KGS ORF can be excised from this plasmid by treating it with PstI and AatII.

[0546] Furthermore, using the plasmid pMWPthr-KGS-fpr-fldA constructed in Example 7 as a template, the fragment (A) containing the N-terminus of KGS was amplified by PCR using primers shown in SEQ ID NO: 69 and 70. On the other hand, using the DNA of plasmid pVKPtuf as a template, the tuf promoter fragment (B) was amplified by PCR using primers shown in SEQ ID NO: 71 and 72. SEQ ID NO: 69 and 72 are complementary to each other.

[0547] Then, using fragments (A) and (B) as templates, and primers shown in SEQ ID NO: 73 and 74, a fragment containing the KGSN terminal side of the natural promoter replaced by the tuf promoter was constructed via cross-PCR. The generated PCR product was purified using conventional methods and treated with PstI and AatII, and then inserted into the PstI and AatII sites of pVKKGS, thus constructing the plasmid pVKPtuf-KGS for amplifying KGS with the natural promoter replaced by the tuf gene promoter.

[0548] Furthermore, pVK9 is a shuttle vector obtained as follows: the AvaII site of pHSG299 (TAKARABio) is blunt-terminated, and a fragment containing a region in pHK4 (TEKP 05-007491) capable of autonomous replication within Corynebacterium-type bacteria is cut out using BamHI and KpnI and blunt-terminated. In addition, the comparative control pVKPtuf is a plasmid constructed by inserting a tuf promoter fragment into the PstI site of pVK: the tuf promoter fragment was obtained by PCR amplification using the genome of *Bacillus flavus* strain MJ-233 (FERMBP-1497) as a template, using primers shown in SEQ ID NO: 75 and 76. The tuf promoter sequence of *Bacillus flavus* strain MJ-233 is shown in SEQ ID NO: 77. The tuf promoter fragment can also be obtained similarly using the genome of *Corynebacterium glutamicum* strain ATCC13032 as a template.

[0549] <18-2> The α-ketoglutarate synthase gene from *Sulphurella multocida* was introduced into strain 2256ΔldhAΔsucA.

[0550] Transformants were obtained by electroporation of pVKPtuf-KGS and the control vector pVKPtuf into 2256ΔldhAΔsucA, with kanamycin resistance as an indicator. After confirming plasmid introduction, the α-ketoglutarate synthase gene expression strain from *Thiopyridamole* was named 2256ΔldhAΔsucA / pVKPtuf-KGS, and the control strain was named 2256ΔldhAΔsucA / pVKPtuf.

[0551] <18-3> Culture of a strain of α-ketoglutarate synthase gene expression derived from *Sulphurella multocida* under anaerobic conditions with glucose as the carbon source.

[0552] The L-glutamate production capacity under anaerobic culture conditions using the α-ketoglutarate synthase gene expression strain 2256ΔldhAΔsucA / pVKPtuf-KGS (constructed in <18-2>) and the control strain 2256ΔldhAΔsucA / pVKPtuf, with glucose as the carbon source, was investigated. Cells obtained from overnight pre-culture in CM-Dex agar at 31.5°C were inoculated into 3 mL of seed culture medium and cultured aerobically in test tubes at 31.5°C with shaking for approximately 16 hours.

[0553] [Seed Culture Medium]

[0554] glucose 10g / L

[0555] (NH4)2SO4 2.5g / L

[0556] KH2PO4 0.5g / L

[0557] MgSO4·7H2O 0.25g / L

[0558] urea 2g / L

[0559] FeSO4·7H2O 0.01g / L

[0560] MnSO4·7H2O 0.01g / L

[0561] Biotin 50 μg / L

[0562] VB1·HCl 100μg / L

[0563] Protocatechuic acid 15mg / L

[0564] CuSO4 0.02 mg / L

[0565] CaCl2 10 mg / L

[0566] Sterilization conditions: pH 7.0 (adjusted with KOH), 115℃, 10 minutes.

[0567] Add 3 mL of main culture medium to 3 mL of seed culture broth. To prevent aeration, seal the test tube with a silicone stopper and then incubate with shaking at 31.5 °C. After 66 hours, stop the culture and determine the accumulated L-glutamate in the culture medium using a Biotech Analyzer (manufactured by Asahi Kasei Corporation).

[0568] [Main Culture Medium]

[0569] Glucose 60g / L

[0570] (NH4)2SO4 30g / L

[0571] KH2PO4 4g / L

[0572] urea 6g / L

[0573] FeSO4·7H2O 0.02g / L

[0574] MnSO4·7H2O 0.02g / L

[0575] Biotin 400 μg / L

[0576] VB1·HCl 400μg / L

[0577] MgCO4 50g / L

[0578] Sterilization conditions: pH 6.8 (adjusted with NaOH), 115℃, 10 minutes.

[0579] The results are shown in Table 13. Compared with the control 2256ΔldhAΔsucA / pVKPtuf, the L-glutamate yield relative to glucose consumption was increased by 0.6% in 2256ΔldhAΔsucA / pVKPtuf-KGS, which was infused with an expression vector containing only the α-ketoglutarate synthase gene from *Thiopyrrolizum aureum*.

[0580] Table 13

[0581] strain OD620 Residual glucose (g / L) L-glutamic acid yield (%) 2256ΔldhΔsucA / pVKPtuf 7.6 0.48 3.1 2256ΔldhΔsucA / pVKPtuf-KGS 6.6 6.02 3.7

[0582] [Sequence List Description]

[0583] SEQ ID NO: 1: Base sequence of the α-ketoglutarate synthase α-subunit gene of *Sulphurella multocida*

[0584] SEQ ID NO: 2: Amino acid sequence of the α-subunit of α-ketoglutarate synthase from *Sulphurella multocida*

[0585] SEQ ID NO: 3: Base sequence of the α-ketoglutarate synthase β-subunit gene of *Sulphurella multocida*

[0586] SEQ ID NO: 4: Amino acid sequence of the β-subunit of α-ketoglutarate synthase from *Sulphurella multocida*

[0587] SEQ ID NO: 5: Base sequence of the fpr gene in Escherichia coli

[0588] SEQ ID NO: 6: Amino acid sequence encoded by the fpr gene of Escherichia coli

[0589] SEQ ID NO: 7: Base sequence of the pyruvate synthase gene of *Sulphurella vulgaris*

[0590] SEQ ID NO: 8: Amino acid sequence of pyruvate synthase from *Sulphurella multocida*

[0591] SEQ ID NO: 9: Base sequence of the fdx gene in Escherichia coli

[0592] SEQ ID NO: 10: Amino acid sequence encoded by the fdx gene of Escherichia coli

[0593] SEQ ID NO: 11: Base sequence of the yfhL gene in Escherichia coli

[0594] SEQ ID NO: 12: Amino acid sequence encoded by the yfhL gene in Escherichia coli

[0595] SEQ ID NO: 13: Base sequence of the fldA gene in Escherichia coli

[0596] SEQ ID NO: 14: Amino acid sequence encoded by the fldA gene of Escherichia coli

[0597] SEQ ID NO: 15: Base sequence of the fldB gene in Escherichia coli

[0598] SEQ ID NO: 16: Amino acid sequence encoded by the fldB gene of Escherichia coli

[0599] SEQ ID NO: 17: Base sequence of the ferricoxin I gene of *Sulphurella vulgaris*

[0600] SEQ ID NO: 18: Amino acid sequence encoded by the ferricoxin I gene of *Sulphurella vulgaris*

[0601] SEQ ID NO: 19: Base sequence of the ferroredoxin II gene of *Sulphurella multocida*

[0602] SEQ ID NO: 20: Amino acid sequence encoded by the ferricoxin II gene of *Sulphurella vulgaris*

[0603] SEQ ID NO: 21: Primer 1 for amplifying the α-ketoglutarate synthase gene of *Sulphurella vulgaris*

[0604] SEQ ID NO: 22: Primer 2 for amplifying the α-ketoglutarate synthase gene of *Sulphurella vulgaris*

[0605] SEQ ID NO: 23: Primer 1 for amplifying the pyruvate synthase gene of *Sulphurella vulgaris*

[0606] SEQ ID NO: 24: Primer 2 for amplifying the pyruvate synthase gene of *Sulphurella vulgaris*

[0607] SEQ ID NO: 25: Primer 1 for amplifying the ferricoxin I gene of *Sulphurella vulgaris*

[0608] SEQ ID NO: 26: Primer 2 for amplifying the ferricoxin I gene of *Sulphurella vulgaris*

[0609] SEQ ID NO: 27: Primer 1 for ldhA in defective Escherichia coli

[0610] SEQ ID NO: 28: Primer 2 for ldhA in defective Escherichia coli

[0611] SEQ ID NO: 29: Primer 1 used to confirm ldhA deficiency in Escherichia coli

[0612] SEQ ID NO: 30: Primer 2 used to confirm ldhA deficiency in Escherichia coli

[0613] SEQ ID NO: 31: Threonine operon promoter sequence of Escherichia coli

[0614] SEQ ID NO: 32: Flavin-NADP of Escherichia coli + Primer 1 for reductase gene amplification

[0615] SEQ ID NO: 33: Flavin-NADP of Escherichia coli + Primer 2 for reductase gene amplification

[0616] SEQ ID NO: 34: Primer 1 for amplification of the fldA gene in Escherichia coli

[0617] SEQ ID NO: 35: Primer 2 for amplification of the fldA gene in Escherichia coli

[0618] SEQ ID NO: 36: Primer 1 for amplification of the fldB gene in Escherichia coli

[0619] SEQ ID NO: 37: Primer 2 for amplification of the fldB gene in Escherichia coli

[0620] SEQ ID NO: 38: Primer 1 for amplification of the fdx gene in Escherichia coli

[0621] SEQ ID NO: 39: Primer 2 for amplification of the fdx gene in Escherichia coli

[0622] SEQ ID NO: 40: Primer 1 for amplification of the yhfL gene in Escherichia coli

[0623] SEQ ID NO: 41: Primer 2 for amplification of the yfhL gene in Escherichia coli

[0624] SEQ ID NO: 42: PCR primers for amplification of the N-terminal fragment of the sucA gene in Escherichia coli

[0625] SEQ ID NO: 43: PCR primers for amplification of the N-terminal fragment of the sucA gene in Escherichia coli

[0626] SEQ ID NO: 44: PCR primers for amplification of the C-terminal fragment of the sucA gene in Escherichia coli

[0627] SEQ ID NO: 45: PCR primers for amplification of the C-terminal fragment of the sucA gene in Escherichia coli

[0628] SEQ ID NO: 46: Base sequence of the α-KGDH subunit gene and neighboring genes of Pantoea ananatis

[0629] sdhB: 2~121

[0630] sucA: 322~3129

[0631] sucB: 3145~4368

[0632] sucC: 4437~4556

[0633] SEQ ID NO: 47: Partial amino acid sequence of succinate dehydrogenase iron-sulfur protein of Pantoea ananatis.

[0634] SEQ ID NO: 48: Amino acid sequence of the α-KGDHE1o subunit of Pantoea ananatis

[0635] SEQ ID NO: 49: Amino acid sequence of the α-KGDHE2o subunit of Pantoea ananatis

[0636] SEQ ID NO: 50: Part of the β subunit of succinyl-CoA synthase in Pantoea ananatis

[0637] SEQ ID NO: 51: Base sequence of the sucA (odhA) gene in *Brucea lactis*.

[0638] SEQ ID NO: 52: Amino acid sequence of the E1o subunit encoded by sucA (odhA) of *Lactobacillus fermentum*.

[0639] SEQ ID NO: 53: Base sequence of the gene encoding the E2o subunit of *Brevibacterium lactis* (Genbank accession number NC_003450, NCgl2126).

[0640] SEQ ID NO: 54: Amino acid sequence of the E2o subunit encoded by NCgl2126 of *Lactobacillus fermentum*.

[0641] SEQ ID NO: 55: Base sequence of the sucA gene in Escherichia coli

[0642] SEQ ID NO: 56: Amino acid sequence of the α-KGDHE1 subunit encoded by the sucA gene in Escherichia coli.

[0643] SEQ ID NO: 57: Base sequence of the α-ketoglutarate synthase α-subunit gene of *Blastopirellulamarina*

[0644] SEQ ID NO: 58: Amino acid sequence of the α-ketoglutarate synthase α-subunit of Blastopirellulamarina

[0645] SEQ ID NO: 59: Base sequence of the α-ketoglutarate synthase β-subunit gene of *Blastopirellulamarina*

[0646] SEQ ID NO: 60: Amino acid sequence of the α-ketoglutarate synthase β-subunit of Blastopirellulamarina

[0647] SEQ ID NO: 61: Primer 1 for amplifying the α-ketoglutarate synthase gene of Blastopirellulamarina

[0648] SEQ ID NO: 62: Primer 2 for amplifying the α-ketoglutarate synthase gene of Blastopirellulamarina

[0649] SEQ ID NO: 63: Primer 1 for amplifying the N-terminal fragment of the sucA gene in *Lactobacillus fermentum*.

[0650] SEQ ID NO: 64: Primer 2 for amplifying the N-terminal fragment of the sucA gene in *Lactobacillus fermentum*.

[0651] SEQ ID NO: 65: Primer 1 for amplifying the C-terminal fragment of the sucA gene in *Brucea lactis*.

[0652] SEQ ID NO: 66: Primer 2 for amplifying the C-terminal fragment of the sucA gene in *Brucea lactis*

[0653] SEQ ID NO: 67: Primers 1 for full-length amplification of the α-ketoglutarate synthase gene of *Sulphurella vulgaris*

[0654] SEQ ID NO: 68: Primers 2 for full-length amplification of the α-ketoglutarate synthase gene of *Sulphurella vulgaris*

[0655] SEQ ID NO: 69: Primer 1 for amplifying the N-terminal fragment of the α-ketoglutarate synthase gene from *S. aeruginosa*

[0656] SEQ ID NO: 70: Primer 2 for amplifying the N-terminal fragment of the α-ketoglutarate synthase gene from *S. aeruginosa*.

[0657] SEQ ID NO: 71: Primer 1 for amplifying the tuf promoter of *Bacillus flavus*

[0658] SEQ ID NO: 72: Primer 2 for amplifying the tuf promoter of *Bacillus flavus*

[0659] SEQ ID NO: 73: Ptuf-KGS fragment amplification primer 1

[0660] SEQ ID NO: 74: Ptuf-KGS fragment amplification primer 2

[0661] SEQ ID NO: 75: Primer 3 for amplifying the tuf promoter of *Bacillus flavus*.

[0662] SEQ ID NO: 76: Primer 4 for amplifying the tuf promoter of *Bacillus flavus*.

[0663] SEQ ID NO: 77: Base sequence of the tuf gene promoter of *Bacillus flavus*

[0664] Industrial applicability

[0665] By using the microorganisms of this invention, the fermentation production of L-glutamic acid amino acids can be carried out efficiently. Furthermore, the method of this invention reduces carbon dioxide emissions by inhibiting decarbonation and utilizing carbon dioxide fixation reactions, making it an environmentally friendly approach.

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Examples

Embodiment 1

[0297] [Example 1]

[0298] Chlorothiobacter is a medium-high-temperature autotrophic bacterium with an optimum growth temperature of 48°C. The genome sequence of the TLS strain has been elucidated by Eisen et al. ). The α-ketoglutarate synthase gene, pyruvate synthase gene and ferredoxin gene were isolated from the strain, and a plasmid expressing these three genes simultaneously was constructed.

[0299] Construction of α-ketoglutarate synthase gene expression plasmid derived from green sulfur bacteria

[0300] Using the genomic DNA of Sulfur chlorosis TLS strain (ATCC49652) as a template, the oligonucleotides shown in SEQ ID NO: 21 and 22 were used for PCR to amplify the α- and β-subunits of α-ketoglutarate synthase base gene segment. The resulting gene fragment was cut with BamHI and inserted into the BamHI site of pSTV28 (manufactured by TAKARA Bio) to construct a plasmid for expressing the α-ketoglutarate synthase gene, which was named pSTV-KGS. In this plasmid, th...

Embodiment 2

[0305] [Example 2]

[0306] Construction of Escherichia coli sucA gene disruption plasmid

[0307] A strain in which the sucA gene encoding the E1 subunit of α-ketoglutarate dehydrogenase was disrupted was prepared from Escherichia coli MG1655 strain. Primers were synthesized based on the base sequence of the sucA gene located at base number 757929-760730 in the genome sequence (Genbank accession number. U00096), and the N of the sucA gene was amplified by PCR using the genomic DNA of Escherichia coli MG1655 strain as a template. terminal and C-terminal fragments. The base sequence of the sucA gene of Escherichia coli is shown in SEQ ID NO: 55, and the amino acid sequence of the E1 subunit encoded by the gene is shown in SEQ ID NO: 56. The oligonucleotides of SEQ ID NO: 42 and 43 were used as PCR primers for N-terminal fragment amplification, and the oligonucleotides of SEQ ID NO: 44 and 45 were used as PCR primers for C-terminal fragment amplification. The oligonucleotid...

Embodiment 3

[0311] [Example 3]

[0312]Lactate dehydrogenase is an enzyme that produces lactate from pyruvate using NADH as a coenzyme. In order to inhibit the production of lactic acid in Escherichia coli cultured under oxygen-limited conditions, a deficient strain of the ldhA gene encoding lactate dehydrogenase was constructed. Deletion of this gene utilizes a method called "Red-driven integration" developed by Datsenko and Wanner (Datsenko, K.A. and Wanner, B.L. 2000.Proc.Natl.Acad.Sci.USA.97:6640-6645) Performed with a lambda phage-derived excision system (Cho, E.H. et al. 2002. J. Bacteriol. 184:5200-5203). By this method, a synthetic oligonucleotide in which a part of the gene of interest is designed on the 5' side and a part of the antibiotic resistance gene is designed on the 3' side is used as a primer to obtain a PCR product, and the use of such a PCR product can One-step construction of gene disruption strains. The antibiotic resistance gene introduced into the gene-disrupt...

[0001] This application is a divisional application of the invention patent application filed on March 14, 2008, with application number 200880008072.3 (international application number PCT / JP2008 / 054735) entitled "Microorganisms for producing L-glutamic acid amino acids and methods for producing amino acids". Technical Field

[0002] This invention relates to microorganisms for producing L-glutamic acid amino acids and methods for producing L-glutamic acid amino acids. L-glutamic acid is widely used as a seasoning ingredient. Furthermore, L-glutamine, L-proline, L-ornithine, L-citrulline, and L-arginine are useful as seasonings, liver function promoters, amino acid infusions, and comprehensive amino acid preparations. Background Technology

[0003] L-glutamic acid is mainly produced by fermentation using L-glutamic acid-producing bacteria called coryneformbacteria, belonging to the genera Brevibacterium, Corynebacterium, and Microbacterium, or their mutant strains (see, for example, Non-Patent Literature 1). As a method for producing L-glutamic acid by using fermentation with other strains, the following methods are known: methods using microorganisms such as Bacillus, Streptomyces, or Penicillium (see, for example, Patent Document 1); methods using microorganisms such as Pseudomonas, Arthrobacter, Serratia, or Candida (see, for example, Patent Document 2); methods using microorganisms such as Bacillus, Pseudomonas, Serratia, or Aerobacter erogenes (now called Enterobacter erogenes) (see, for example, Patent Document 3); methods using mutant strains of Escherichia coli (see, for example, Patent Document 4), etc. In addition, methods for producing L-glutamic acid using microorganisms belonging to the genera Klebsiella, Erwinia, Pantothea, or Enterobacter have been disclosed (see, for example, Patent Documents 5-7).

[0004] Furthermore, various techniques for increasing L-glutamate production capacity by utilizing recombinant DNA technology to enhance the activity of L-glutamate biosynthesizers have been disclosed. For example, it has been reported that introducing a gene encoding citrate synthase from *Escherichia coli* or *Corynebacterium glutamicum* into *Corynebacterium* or *Bryophyte* bacteria is effective in enhancing L-glutamate production capacity in *Corynebacterium*-type bacteria (see, for example, Patent Document 8). Additionally, it has been reported that introducing a citrate synthase gene from *Corynebacterium*-type bacteria into *Enterobacteriaceae*, *Klebsiella*, *Serratia*, *Erwinia*, or *Escherichia*-family Enterobacteriaceae is effective in enhancing L-glutamate production capacity (see, for example, Patent Document 7).

[0005] Similar to L-glutamic acid, other L-glutamic acid amino acids, such as ornithine and citrulline (Non-Patent Literature 2-4), L-glutamine (Patent Literature 9), L-proline (Patent Literature 10), and L-arginine (Patent Literature 11, 12), are also produced by fermentation methods using microorganisms as described above.

[0006] Patent Document 1: U.S. Patent No. 3,220,929

[0007] Patent Document 2: U.S. Patent No. 3,563,857

[0008] Patent document 3, Japanese Patent Publication No. 32-9393

[0009] Patent document 4, Japanese Patent Application Publication No. 5-244970

[0010] Patent document 5, Japanese Patent Application Publication No. 2000-106869

[0011] Patent document 6, Japanese Patent Application Publication No. 2000-189169

[0012] Patent document 7, Japanese Patent Application Publication No. 2000-189175

[0013] Patent document No. 8, Japan Publication No. 7-121228

[0014] Patent document 9, JP2002-300887

[0015] Patent document 10 European Patent No. 1172433

[0016] Patent document 11, JP2000-287693

[0017] Patent document 12, JP2001-046082

[0018] Non-patent document 1 Akashi Kunihiko et al. "Amin Sour Yeast", published by the Society of Science and Technology, pp. 195-215, 1986

[0019] Non-patent literature 2 Lee, Y.-J. and Cho, J.-Y. 2006, Biotechnol. Lett. 28: 1849-1856

[0020] Non-patent literature 3 Choi, D Ketal. 1996. J. Ferment. Bioeng. 81: 216-219

[0021] Non-patent literature 4 Plachy, J. 1987. Kvasny Prumysl 33: 73-75 Summary of the Invention

[0022] The problem that the invention aims to solve

[0023] The objective of this invention is to provide a strain capable of efficiently producing L-glutamic acid amino acids, and a method for efficiently producing L-glutamic acid amino acids using the strain.

[0024] Problem-solving methods

[0025] As mentioned above, most technologies for improving L-glutamate production capacity involve enhancing the activity of enzymes in the TCA cycle. However, during L-glutamate fermentation via the TCA cycle, decarbonation caused by isocitrate dehydrogenase inevitably releases one molecule of CO2. Therefore, the inventors believe that to further improve production performance, this decarbonation must be reduced. Consequently, the inventors conducted in-depth research to address this issue and discovered that by increasing the activity of α-ketoglutarate synthase (an enzyme in the reduced TCA cycle) and further enhancing ferricoxin-NADP... + The production performance of L-glutamate can be improved by increasing the activity of reductases or pyruvate synthases (which have the activity of generating reduced ferricoxane or reduced flavin oxoreductin from their respective oxidized forms, which are necessary for the enzymatic activity of α-ketoglutarate synthase) or by increasing the production capacity of ferricoxane or flavin oxoreductin. This completes the present invention.

[0026] That is, the present invention is as follows.

[0027] (1) A microorganism having the ability to produce one or more L-amino acids selected from L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline and L-arginine, and said microorganism being modified to increase the activity of α-ketoglutarate synthase.

[0028] (2) The aforementioned microorganisms, wherein the activity of α-ketoglutarate synthase is increased by increasing the expression level of the gene encoding α-ketoglutarate synthase and / or increasing the translation level of the gene.

[0029] (3) The aforementioned microorganisms, wherein the activity of α-ketoglutarate synthase is increased by increasing the copy number of the gene encoding α-ketoglutarate synthase or by modifying the expression regulatory sequence of the gene.

[0030] (4) The aforementioned microorganism, wherein the gene encoding α-ketoglutarate synthase encodes a polypeptide represented by any one of (A) to (D) below, and a polypeptide represented by any one of (E) to (H):

[0031] (A) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 2;

[0032] (B) A polypeptide comprising an amino acid sequence obtained by substituting, deleting, inserting or adding one or more amino acids in SEQ ID NO: 2, wherein the polypeptide together with the polypeptide shown in any one of (E) to (H) constitutes a protein having α-ketoglutarate synthase activity.

[0033] (C) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 58;

[0034] (D) A polypeptide comprising an amino acid sequence obtained by substituting, deleting, inserting or adding one or more amino acids in SEQ ID NO: 58, and said polypeptide together with the polypeptide shown in any one of (E) to (H) constitutes a protein having α-ketoglutarate synthase activity.

[0035] (E) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 4;

[0036] (F) A polypeptide comprising an amino acid sequence obtained by substituting, deleting, inserting or adding one or more amino acids in SEQ ID NO: 4, and said polypeptide together with the polypeptide shown in any one of (A) to (D) constitutes a protein having α-ketoglutarate synthase activity.

[0037] (G) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 60;

[0038] (H) is a polypeptide containing an amino acid sequence obtained by substituting, deleting, inserting or adding one or more amino acids as in SEQ ID NO: 60, and said polypeptide together with the polypeptide shown in any one of (A) to (D) constitutes a protein having α-ketoglutarate synthase activity.

[0039] (5) The aforementioned microorganism, wherein the gene encoding α-ketoglutarate synthase comprises DNA from any one of (a) to (d) and DNA from any one of (e) to (h):

[0040] (a) DNA having the base sequence shown in SEQ ID NO: 1;

[0041] (b) DNA that hybridizes under stringent conditions to the complementary sequence of the base sequence shown in SEQ ID NO: 1 or to a probe that can be prepared from the base sequence, and the DNA encodes a polypeptide that, together with the polypeptide encoded by the DNA of any one of (e) to (h), constitutes a protein having α-ketoglutarate synthase activity.

[0042] (c) DNA having the base sequence shown in SEQ ID NO: 57;

[0043] (d) DNA that hybridizes under stringent conditions to a complementary sequence to the base sequence shown in SEQ ID NO: 57 or to a probe that can be prepared from the base sequence, and the DNA encodes a polypeptide that, together with the polypeptide encoded by any one of (e) to (h), constitutes a protein having α-ketoglutarate synthase activity.

[0044] (e) DNA having the base sequence shown in SEQ ID NO: 3;

[0045] (f) DNA that hybridizes under stringent conditions to the complementary sequence of the base sequence shown in SEQ ID NO: 3 or to a probe that can be prepared from the base sequence, and the DNA encodes a polypeptide that, together with the polypeptide encoded by the DNA of any one of (a) to (d), constitutes a protein having α-ketoglutarate synthase activity.

[0046] (g) DNA having the base sequence shown in SEQ ID NO: 59;

[0047] (h) DNA that hybridizes under stringent conditions to a complementary sequence to the base sequence shown in SEQ ID NO: 59 or to a probe that can be prepared from the base sequence, and said DNA encodes a polypeptide that, together with the polypeptide encoded by the DNA of any one of (a) to (d), constitutes a protein having α-ketoglutarate synthase activity.

[0048] (6) The aforementioned microorganism, wherein the microorganism is modified to increase ferroredoxin-NADP. + The activity of reductase.

[0049] (7) The aforementioned microorganism, wherein the microorganism is modified to increase the activity of pyruvate synthase.

[0050] (8) The aforementioned microorganism, wherein the microorganism is modified to enhance the production capacity of ferricoxin or flavin oxidoxin.

[0051] (9) The aforementioned microorganism, wherein the microorganism is modified to reduce the activity of α-ketoglutarate dehydrogenase.

[0052] (10) The aforementioned microorganism, wherein the microorganism is a bacterium belonging to a genus selected from the group consisting of Escherichia coli, Enterobacter spp., Pantotheca spp., Klebsiella spp. and Serratia spp.

[0053] (11) The aforementioned microorganism is a rod-shaped bacterium.

[0054] (12) A method for producing L-amino acids, comprising culturing the aforementioned microorganisms in a culture medium to generate and accumulate one or more L-amino acids selected from L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline, and L-arginine in the culture medium or in the microorganisms, and collecting the L-amino acids from the culture medium or the microorganisms.

[0055] (13) The aforementioned method is characterized in that the aforementioned microorganisms are cultured under aerobic conditions.

[0056] (14) The aforementioned method is characterized in that the aforementioned culture medium is a culture medium containing carbonate ions, bicarbonate ions or carbon dioxide gas, and the aforementioned microorganisms are cultured under anaerobic or microaerophilic conditions.

[0057] This invention also relates to the following aspects:

[0058] 1. A microorganism having the ability to produce one or more L-amino acids selected from L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline and L-arginine, and modified to increase the activity of α-ketoglutarate synthase.

[0059] 2. The microorganism of claim 1, wherein the activity of the α-ketoglutarate synthase is increased by increasing the expression level of the gene encoding the α-ketoglutarate synthase and / or increasing the translation level of the gene.

[0060] 3. The microorganism of item 2, wherein the activity of the α-ketoglutarate synthase is increased by increasing the copy number of the gene encoding the α-ketoglutarate synthase or by modifying the expression regulatory sequence of the gene.

[0061] 4. The microorganism described in item 2 or 3, wherein the gene encoding α-ketoglutarate synthase encodes a polypeptide represented by any one of (A) to (D) below, and a polypeptide represented by any one of (E) to (H):

[0062] (A) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 2;

[0063] (B) A polypeptide comprising an amino acid sequence obtained by substituting, deleting, inserting or adding one or more amino acids in SEQ ID NO: 2, wherein the polypeptide together with the polypeptide shown in any one of (E) to (H) constitutes a protein having α-ketoglutarate synthase activity.

[0064] (C) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 58;

[0065] (D) A polypeptide comprising an amino acid sequence obtained by substituting, deleting, inserting or adding one or more amino acids in SEQ ID NO: 58, and said polypeptide together with the polypeptide shown in any one of (E) to (H) constitutes a protein having α-ketoglutarate synthase activity.

[0066] (E) A polypeptide containing the amino acid sequence shown in SEQ ID NO: 4;

[0067] (F) A polypeptide comprising an amino acid sequence obtained by substituting, deleting, inserting or adding one or more amino acids in SEQ ID NO: 4, and said polypeptide together with the polypeptide shown in any one of (A) to (D) constitutes a protein having α-ketoglutarate synthase activity.

[0068] (G) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 60;

[0069] (H) is a polypeptide containing an amino acid sequence obtained by substituting, deleting, inserting or adding one or more amino acids as in SEQ ID NO: 60, and said polypeptide together with the polypeptide shown in any one of (A) to (D) constitutes a protein having α-ketoglutarate synthase activity.

[0070] 5. The microorganism described in item 2 or 3, wherein the gene encoding α-ketoglutarate synthase comprises DNA from any one of (a) to (d) below, and DNA from any one of (e) to (h):

[0071] (a) DNA having the base sequence shown in SEQ ID NO: 1;

[0072] (b) DNA that hybridizes under stringent conditions to the complementary sequence of the base sequence shown in SEQ ID NO: 1 or to a probe that can be prepared from the base sequence, and the DNA encodes a polypeptide that, together with the polypeptide encoded by the DNA of any one of (e) to (h), constitutes a protein having α-ketoglutarate synthase activity.

[0073] (c) DNA having the base sequence shown in SEQ ID NO: 57;

[0074] (d) DNA that hybridizes under stringent conditions to a complementary sequence to the base sequence shown in SEQ ID NO: 57 or to a probe that can be prepared from the base sequence, and the DNA encodes a polypeptide that, together with the polypeptide encoded by any one of (e) to (h), constitutes a protein having α-ketoglutarate synthase activity.

[0075] (e) DNA having the base sequence shown in SEQ ID NO: 3;

[0076] (f) DNA that hybridizes under stringent conditions to the complementary sequence of the base sequence shown in SEQ ID NO: 3 or to a probe that can be prepared from the base sequence, and the DNA encodes a polypeptide that, together with the polypeptide encoded by the DNA of any one of (a) to (d), constitutes a protein having α-ketoglutarate synthase activity.

[0077] (g) DNA having the base sequence shown in SEQ ID NO: 59;

[0078] (h) DNA that hybridizes under stringent conditions to the complementary sequence of the base sequence shown in SEQ ID NO: 59 or to a probe that can be prepared from the base sequence, and said DNA encodes a polypeptide that, together with the polypeptide encoded by the DNA of any one of (a) to (d), constitutes a protein having α-ketoglutarate synthase activity.

[0079] 6. The microorganism of any one of claims 1-5, wherein the microorganism is modified to increase ferrugin-NADP. + The activity of reductase.

[0080] 7. The microorganism of any one of items 1-6, wherein the microorganism is modified to increase the activity of pyruvate synthase.

[0081] 8. The microorganism of any one of claims 1-7, wherein the microorganism is modified to enhance its ability to produce ferrugin or flavin ferrugin.

[0082] 9. The microorganism of any one of claims 1-8, wherein the microorganism is modified to reduce the activity of α-ketoglutarate dehydrogenase.

[0083] 10. The microorganism of any one of claims 1-9, wherein the microorganism is a bacterium belonging to a genus selected from the group consisting of: Escherichia, Enterobacter, Pantotheca, Klebsiella and Serratia.

[0084] 11. The microorganism mentioned in any one of items 1-9, wherein the microorganism is a corynebacterium.

[0085] 12. A method for producing L-amino acids, comprising culturing the microorganisms described in any one of items 1-11 in a culture medium, thereby generating and accumulating one or more L-amino acids selected from L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline, and L-arginine in the culture medium or within the microorganisms, and collecting the L-amino acids from the culture medium or the microorganisms.

[0086] 13. The method for producing L-amino acids according to item 12, characterized in that the microorganisms are cultured under aerobic conditions.

[0087] 14. The method for producing L-amino acids according to item 12, characterized in that the culture medium is a culture medium containing carbonate ions, bicarbonate ions or carbon dioxide gas, and the microorganisms are cultured under anaerobic or microaerophilic conditions.

[0088] Best mode for carrying out the invention

[0089] The present invention will now be described in detail.

[0090] <1> The microorganism of the present invention

[0091] The microorganism of the present invention is a microorganism capable of producing L-amino acids selected from L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline or L-arginine, and modified to increase the activity of α-ketoglutarate synthase.

[0092] In this invention, unless otherwise specified, "L-amino acid" refers to L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline, or L-arginine. These amino acids are called L-glutamic acid amino acids, which are L-glutamic acid or L-amino acids that can be obtained through biosynthesis using L-glutamic acid as a precursor.

[0093] "Ability to produce L-amino acids" or "L-amino acid production capacity" refers to the ability of the microorganisms of the present invention to generate and accumulate L-amino acids in cells or culture media to a degree that allows for the recovery of L-amino acids from the cells or culture medium when the microorganisms of the present invention are cultured in a culture medium. The L-amino acids produced by the microorganisms of the present invention can be one type, or two or more types of L-amino acids. Microorganisms capable of producing L-amino acids can be naturally occurring microorganisms with L-amino acid production capacity, or microorganisms obtained by modifying the microorganisms listed below using mutagenesis or recombinant DNA technology to give them L-amino acid production capacity, or microorganisms endowed with L-amino acid production capacity by introducing the genes of the present invention.

[0094] Furthermore, "increased activity of α-ketoglutarate synthase" has two meanings: increased activity of the enzyme in microorganisms that originally possess α-ketoglutarate synthase, and conferring the activity of the enzyme on microorganisms that do not possess α-ketoglutarate synthase.

[0095] <1-1> Contribution of L-amino acid production capacity

[0096] The microorganisms of the present invention can be obtained by modifying a parent strain of a microorganism capable of producing L-amino acids to increase the activity of α-ketoglutarate synthase. Furthermore, the microorganisms of the present invention can also be obtained by using a parent strain of a microorganism whose α-ketoglutarate synthase activity has been modified to enhance or confer L-amino acid production capacity.

[0097] The following examples illustrate methods for conferring L-amino acid production capacity to microorganisms and microorganisms conferring L-amino acid production capacity that can be used in this invention. However, the methods or microorganisms are not limited to those that can confer or possess L-amino acid production capacity.

[0098] As microorganisms used in this invention, examples include bacteria such as Enterobacteriaceae belonging to the genera *Escherichia*, *Enterobacter*, *Pantoea*, *Klebsiella*, *Serratia*, *Erwinia*, *Salmonella*, and *Morganella*, which are γ-proteobacteria; so-called corynebacterial bacteria belonging to the genera *Bryophyllobacterium*, *Corynebacterium*, and *Microbacterium*; and microorganisms belonging to the genera *Alicyclobacillus*, *Bacillus*, and *Saccharomyces*. γ-proteobacteria can be those classified according to the NCBI (National Center for Biotechnology Information) taxonomic database.

[0099] Microorganisms classified according to the taxonomy published at http: / / www.ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgiyymode=Undef&id=1236&lvl=3&p=mapview&p=has_linkout&p=blast_url&p=genome_blast&lin=f&keep=1&srchmode=1&unlock.

[0100] Examples of Escherichia coli include *Escherichia coli*. When using genetic engineering methods to breed *E. coli*, strain K-12 and its derivatives, such as *E. coli* MG1655 (ATCC47076) and W3110 (ATCC27325), can be used. *E. coli* strain K-12 was isolated by Stanford University in 1922. It is a lysogen of λ phage and possesses the F factor, making it a highly versatile strain capable of constructing genetic recombinants through conjugation and other methods. Furthermore, the genome sequence of *E. coli* strain K-12 has been determined, and its genetic information is freely available. *E. coli* strain K-12 and its derivatives can be obtained, for example, from the American Type Culture Collection (ATCC) (Address: PO Box 1549, Manassas, VA 20108, United States of America).

[0101] In particular, Pantotheca, Erwinia, and Enterobacteriaceae are classified as γ-Proteobacteria, and they are very closely related taxonomically (Harada H. and Ishikawa H. 1997. J. Gen. Appl. Microbiol. 43: 355-361; Kwon S.W. et al. 1997. Int. J. Syst. Bacteriol. 47: 1061-1067). In recent years, based on DNA-DNA hybridization experiments, some Enterobacteriaceae have been reclassified as Pantoea agglomerans or Pantoeadispersa, etc. (Gavini, F. et al. 1989. Int. J. Syst. Bacteriol. 39: 337-345). In addition, some bacteria belonging to the genus Erwinia have been reclassified as Pantoea anananas and Pantoeastewartii (see Mergaert, J. et al. 1993. Int. J. Syst. Bacteriol. 43: 162-173).

[0102] Enterobacteriaceae include, for example, *Enterobacter agglomerans* and *Enterobacter aerogenes*. Specifically, the strains exemplified in European Patent Application Publication 952221 can be used. *Enterobacter agglomerans* strain ATCC12287 is a representative strain of the *Enterobacteria* genus.

[0103] Representative strains of the Pantoea genus include Pantoea ananatis, Pantoea stearothermia, Pantoea clumpida, and Pantoea citrea. Specifically, the following strains can be listed.

[0104] Pantoeaananatis AJ13355 (FERMBP-6614) (European Patent Application Publication No. 0952221)

[0105] Pantoeaananatis AJ13356 (FERMBP-6615) (European Patent Application Publication No. 0952221)

[0106] Moreover, these strains were described as Enterobacter clumps in European Patent Application Publication No. 0952221, but now, as mentioned above, they have been reclassified as Pantoea ananatis based on 16S rRNA base sequence analysis.

[0107] Examples of Erwinia species include *Erwinia amylovora* and *Erwinia carotovora*; examples of Klebsiella species include *Klebsiella planticola*. Specifically, the following strains can be listed.

[0108] Erwinia amyloliquefaciens ATCC15580 strain

[0109] Erwinia carotene ATCC15713 strain

[0110] Plant-grown Klebsiella pneumoniae strain AJ13399 (FERMBP-6600) (European Patent Application Publication No. 955368)

[0111] Plant-grown Klebsiella pneumoniae strain AJ13410 (FERMBP-6617) (European Patent Application Publication No. 955368)

[0112] The term "corynebacterium" as used in this invention includes microorganisms defined in Bergey's Manual of Determinative Bacteriology, 8th edition, page 599 (1974). These include aerobic, Gram-positive, non-acid-resistant, non-spore-forming bacilli that were formerly classified under the genus *Brevibacterium* but are now uniformly classified under the genus *Corynebacterium* (Liebl, W. et al. 1991, Int. J. Syst. Bacteriol. 41: 255-260), as well as *Brevibacterium* and *Microbacterium* bacteria that are very closely related to *Corynebacterium*.

[0113] Corynebacterium-type bacteria suitable for use in the production of L-glutamic acid amino acids can be listed as, for example, the strains shown below.

[0114] Corynebacterium acetoacidophilum

[0115] Corynebacterium acetoglutamicum

[0116] Corynebacterium alkanolyticum

[0117] Corynebacterium callunae

[0118] Corynebacterium glutamicum

[0119] Corynebacterium lilium

[0120] Corynebacterium melassecola

[0121] Corynebacterium thermoaminogenes (Corynebacterium efficiens)

[0122] Corynebacterium herculis

[0123] Brevibacterium divaricatum (Corynebacterium glutamicum)

[0124] Brevibacterium flavum (Corynebacterium glutamicum)

[0125] Brevibacterium immariophilum

[0126] Brevibacterium lactofermentum (Corynebacterium glutamicum)

[0127] Brevibacterium roseum

[0128] Brevibacterium saccharolyticum

[0129] Brevibacterium thiogenitalis

[0130] Brevibacteriumammoniagenes (Corynebacteriumammoniagenes)

[0131] Brevibacterium album

[0132] Brevibacteriumcerinum

[0133] Microbacterium amammoniaphilum

[0134] Specifically, strains such as those listed below can be cited.

[0135] Corynebacterium thermophilum AJ12340 (FERMBP-1539)

[0136] Corynebacterium glutamicum ATCC13032

[0137] Corynebacterium glutamicum (Breastbacterium flavum) ATCC13826, ATCC14067

[0138] Lactobacillus fermentum (Corynebacterium glutamicum) ATCC13665, ATCC13869

[0139] Ammonia-producing short bacilli (Ammonia-producing Corynebacterium) ATCC6871

[0140] L-glutamic acid producing bacteria

[0141] As a method to endow or enhance the ability to produce L-glutamate, one example is to increase the expression of genes encoding enzymes related to L-glutamate biosynthesis by modification. Examples of enzymes related to L-glutamate biosynthesis include, for example: glutamate dehydrogenase (GDH, (gdhA)), glutamine synthase (glnA), glutamate synthase (gltAB), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglycerate mutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose diphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), glucose phosphate isomerase (pgi), 6-phosphogluconic acid dehydratase (edd), 2-keto-3-deoxy-6-phosphogluconic acid aldolase (eda), transhydrogenases, etc. In addition, the gene name is in parentheses after the enzyme name (the same applies below).

[0142] The following describes a method for modifying microorganisms to increase the expression of the genes encoding the enzymes related to L-glutamate biosynthesis.

[0143] The first method is to increase the copy number of the target gene. For example, the copy number of the gene can be increased by cloning the target gene into a suitable vector and transforming the host bacteria with the resulting vector. When the base sequence of the target gene has been elucidated in bacteria such as Escherichia and Corynebacterium, primers can be synthesized based on its base sequence, and the gene can be obtained using genomic DNA as a template via PCR (polymerase chain reaction) (see White, T.J. et al. 1989. Trends Genet. 5: 185-189). As glutamate dehydrogenases, the *E. coli* gdhA gene (Vallea, F. et al. 1984. Gene 27: 193-199) and *Corynebacterium glutamicum* gdh gene (Bormann, E.R. et al. 1992. Mol. Microbiol. 6: 317-326) are known; as phosphoenolpyruvate carboxylases, the *E. coli* ppc gene (Fujita, N. et al. 1984. J. Biochem. (Tokyo) 95: 909-916) and *Corynebacterium glutamicum* ppc gene (Eikmanns, B.J. et al. 1989. Mol. Gen. Genet. 218: 330-339) are known.

[0144] As vectors for transformation, plasmids capable of autonomous replication in the microorganisms used can be listed. For example, plasmids capable of autonomous replication in microorganisms belonging to the Enterobacteriaceae family include pUC19, pUC18, pBR322, RSF1010, pHSG299, pHSG298, pHSG399, pHSG398, pSTV28, pSTV29 (pHSG and pSTV are available from TAKARABio), pMW119, pMW118, pMW219, and pMW218 (pMW is available from NIPPONGENE). In addition, plasmids used for Corynebacterium-type bacteria include pAM330 (Japanese Patent Application Publication No. 58-67699), pHM1519 (Japanese Patent Application Publication No. 58-77895), pSFK6 (refer to Japanese Patent Application Publication No. 2000-262288), pVK7 (US Patent Application Publication No. 2003-0175912), pAJ655, pAJ611, pAJ1844 (Japanese Patent Application Publication No. 58-192900), pCG1 (Japanese Patent Application Publication No. 57-134500), pCG2 (Japanese Patent Application Publication No. 58-35197), pCG4, pCG11 (Japanese Patent Application Publication No. 57-183799), and pHK4 (Japanese Patent Application Publication No. 5-7491). Furthermore, DNA fragments capable of autonomously replicating plasmids in corynebacteria can be extracted from these vectors and inserted into the E. coli vector, thus enabling its use as a shuttle vector capable of autonomous replication in both E. coli and corynebacteria. Moreover, phage DNA can be used instead of plasmids as the vector.

[0145] Examples of transformation methods include: treating recipient bacterial cells with calcium chloride to increase DNA permeability, a method reported for Escherichia coli K-12 (Mandel, M. and Higa, AJ Mol. Biol. 1970. 53: 159-162); or preparing competent cells from cells in the proliferation phase and introducing DNA, a method reported for Bacillus subtilis (Duncan, CH et al. 1997. Gene1: 153-167). Alternatively, a method can be used to prepare DNA recipient bacteria cells into protoplasts or protoplast spheres that are easily introduced with recombinant DNA, and then introduce the recombinant DNA into the DNA recipient bacteria. This method is known to have been used for Bacillus subtilis, actinomycetes, and yeast (Chang, S. and Choen, SN., 1979. Mol. Gen. Genet. 168: 111-115; Bibb, M.J. et al., 1978. Nature 274: 398-400; Hinnen, A. et al., 1978. Proc. Natl. Acad. Sci. USA 75: 1929-1933). Furthermore, microbial transformation can also be performed using the electroporation method (Japanese Patent Application Publication No. 2-207791).

[0146] Increasing gene copy number can also be achieved by introducing multiple copies of the target gene into the genomic DNA of microorganisms. To introduce multiple copies of the target gene into the genomic DNA of microorganisms, sequences existing in multiple copies on the genomic DNA can be used as targets, and homologous recombination can be performed (Miller I, JH Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Repetitive DNA or inverted repeat sequences located at the ends of transposon elements can be used as sequences existing in multiple copies on the genomic DNA. Alternatively, as disclosed in Japanese Patent Application Publication No. 2-109985, the target gene can be loaded into a transposon, causing it to transfer and thus introducing multiple gene copies into the genomic DNA. Furthermore, the target gene can be integrated into the host genome using the Mu phage method (Japanese Patent Application Publication No. 2-109985). The fact that the target gene has been transferred to the genome can be confirmed by performing Southren hybridization using a portion of the gene as a probe.

[0147] As for the copy number, it can be any number as long as it can enhance the activity of the target gene product, but ideally it is more than two copies.

[0148] The second method involves replacing the expression regulatory sequences of the target gene, such as the promoter, on the genomic DNA or plasmid with expression regulatory sequences of appropriate strength to enhance the expression of the target gene. For example, the thr promoter, lac promoter, trp promoter, trc promoter, pL promoter, and tac promoter are commonly known promoters. Examples of highly expressed promoters in Corynebacterium-type bacteria include the promoter of the elongation factor Tu (EF-Tu) gene tuf (SEQ ID NO: 77), and promoters of genes encoding cochaperonin GroES-chaperonin GroEL, thioredoxin reductase, phosphoglycerate mutase, and glyceraldehyde-3-phosphate dehydrogenase (WO2006 / 028063 and EP1697525). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein and Doi’s paper (Goldstein, MA and Doi R.H. 1995. Biotechnol. Annu. Rev., 1: 105-128).

[0149] Furthermore, as described in International Publication WO00 / 18935, a promoter of suitable strength can be modified by introducing several base substitutions into the promoter region of a gene. Substitution of expression regulatory sequences can be performed, for example, as with gene substitution using temperature-sensitive plasmids. Examples of temperature-sensitive origin of replication vectors that can be used in *E. coli* or *Pantoea ananatis* include, for instance, the temperature-sensitive plasmid pMAN997 or its derivatives as described in International Publication WO99 / 03988. Furthermore, substitution of expression regulatory sequences can also be performed using linear DNA methods: a method called "Red-driven integration" utilizing the Red recombinase of λ phage (Datsenko, K.A. and Wanner, BL, 2000. Proc. Natl. Acad. Sci. USA. 97: 6640-6645), and a method combining Red-driven integration with a λ phage-derived excision system (Cho, E. Hetal. 2002. J. Bacteriol. 184: 5200-5203) (see WO2005 / 010175), etc. Moreover, modification of expression regulatory sequences can be combined with methods described above for increasing gene copy number.

[0150] Furthermore, it is known that substitution of several nucleotides in the spacer sequence between the ribosome binding site (RBS) and the start codon, especially in the sequence immediately upstream of the start codon, has a significant impact on mRNA translation efficiency, and these sequences can be modified to increase translation yield.

[0151] Examples of microorganisms that have been modified in a manner such as those described above to increase the expression of genes related to L-glutamate production include those described in International Publication No. WO99 / 07853 and European Patent No. 1352966.

[0152] When introducing a target gene into the aforementioned plasmid or genome, the promoter used to express these genes can be any promoter capable of functioning in the microorganism used, the promoter of the gene itself, or a modified promoter. Gene expression can also be regulated by appropriately selecting a promoter that functions strongly in the microorganism used, or by bringing the -35 and -10 regions of the promoter closer to a shared sequence. Microorganisms whose expression of genes such as glutamate dehydrogenase is enhanced by modification as described above are described in WO00 / 18935, European Patent Application Publication 1010755, and other specifications.

[0153] The methods for enhancing gene expression described above can also be applied to the gene encoding α-ketoglutarate synthase, which will be discussed later.

[0154] Modifications to confer L-glutamate production capacity can also be achieved by reducing or eliminating the activity of enzymes that catalyze reactions that branch off from the L-glutamate biosynthetic pathway and generate compounds other than L-glutamate. Examples of enzymes that catalyze reactions that branch off from the L-glutamate biosynthetic pathway and generate compounds other than L-glutamate include α-ketoglutarate dehydrogenase, isocitrate lyase, acetate kinase, acetylhydroxylase, acetyllactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, and 1-pyrrolino-5-carboxylic acid dehydrogenase. Among these, reducing or eliminating the activity of α-ketoglutarate dehydrogenase is particularly preferred.

[0155] To reduce or eliminate the activity of enzymes such as those described above, conventional mutagenesis methods or genetic engineering techniques can be used to introduce mutations into the genes of these enzymes that reduce or eliminate their activity in cells. Mutagenesis methods include, for example, irradiation with X-rays or ultraviolet light, or treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine. The site where the mutation is introduced into the gene can be the coding region of the enzyme protein or an expression regulatory region such as a promoter. Furthermore, in terms of genetic engineering techniques, methods such as gene recombination, transduction, and cell fusion can be used.

[0156] The reduction or absence of target enzyme activity in cells, and the degree of reduction, can be confirmed by measuring the enzyme activity of cell extracts or purified fractions of candidate strains and comparing them with wild-type strains. For example, α-ketoglutarate dehydrogenase activity can be measured according to the method of Reed and Mukherjee (Reed, L.J. and Mukherjee, BB 1969. Methods in Enzymology 13: 55-61).

[0157] Methods for depleting or reducing α-ketoglutarate dehydrogenase activity in Escherichia coli are described in Japanese Patent Application Publication Nos. 5-244970 and 7-203980, among others. Furthermore, methods for depleting or reducing α-ketoglutarate dehydrogenase activity in Corynebacterium species are described in International Publication No. 95 / 34672. Moreover, methods for depleting or reducing α-ketoglutarate dehydrogenase activity in Enterobacteriaceae are disclosed in Japanese Patent Application Publication No. 2001-333769.

[0158] For example, to reduce the activity of α-ketoglutarate dehydrogenase, the sucA(odhA) gene encoding the E1o subunit of this enzyme can be modified. Examples of strains that reduce α-ketoglutarate dehydrogenase activity include the following.

[0159] Lactobacillus fermentum strain ΔS (International Publication No. 95 / 34672)

[0160] Lactobacillus fermentum AJ12821 (FERMBP-4172; see French Patent Publication No. 9401748).

[0161] Flavobacterium flammatum AJ12822 (FERMBP-4173; see French Patent Publication No. 9401748)

[0162] Corynebacterium glutamicum AJ12823 (FERMBP-4174; see French Patent Publication No. 9401748).

[0163] Corynebacterium glutamicum ATCC13869OAGN, OA2-2, OAGN2-2 (refer to International Publication No. 2006 / 028298)

[0164] Escherichia coli AJ12624 (FERMBP-3853)

[0165] Escherichia coli AJ12628 (FERMBP-3854)

[0166] Escherichia coli AJ12949 (FERMBP-4881)

[0167] Lactobacillus fermentum strain ΔS (refer to International Publication No. 95 / 34672)

[0168] Pantoeaananatis AJ13601 (FERMBP-7207 European Patent Publication 1078989)

[0169] Pantoeaananatis AJ13356 (FERMBP-6615, US Patent No. 6,331,419)

[0170] PantoeaananatisSC17sucA(FERMBP-8646WO2005 / 085419)

[0171] Plant-growing Klebsiella pneumoniae AJ13410 (FERMBP-6617, US Patent No. 6,197,559)

[0172] As an example of an L-glutamic acid-producing bacterium of Pantoeaananatis, strain Pantoeaananatis AJ13355 can be cited. Pantoeaananatis AJ13355 is a strain isolated from soil in Iwata City, Shizuoka Prefecture, capable of propagating in a medium containing L-glutamic acid and a carbon source at low pH. Pantoeaananatis AJ13355 was deposited on February 19, 1998, at the Patent Biological Collection Center of the National Institute of Advanced Industrial Science and Technology (address: 1-1-1-Chuo-6, Higashi, Tsukuba City, Ibaraki Prefecture, Japan 305-8566, Japan), accession number FERMP-16644; and on January 11, 1999, it was transferred to international deposit under the Budapest Treaty, accession number FERMP-6614. Furthermore, this strain was initially identified as *Enterobacter agglomerans* during isolation and was preserved as *Enterobacter agglomerans* AJ13355. However, in recent years, based on 16S rRNA base sequence analysis and other factors, it has been reclassified as *Pantoea ananatis*.

[0173] Furthermore, L-glutamate-producing bacteria of *Pantoea ananatis* can be listed as those belonging to the *Pantoea* genus that exhibit deficient or reduced α-ketoglutarate dehydrogenase (αKGDH) activity. Examples of such strains include AJ13356 (US Patent No. 6,331,419), derived from strain AJ13355 by deleting the αKGDH-E1 subunit gene (sucA), and SC17sucA (US Patent No. 6,596,517), a sucA gene-deficient strain derived from strain SC17, selected as a mucin-low production mutant strain from AJ13355. AJ13356 was deposited on February 19, 1998, at the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (now the Patent Biological Collection Center of the National Institute of Advanced Industrial Science and Technology, 〒305-8566, 1-1 Higashi-1-1 Chuo-6, Tsukuba City, Ibaraki Prefecture, Japan), with accession number FERMP-16645; and on January 11, 1999, it was transferred to international deposit under the Budapest Treaty, with accession number FERMP-6616. AJ13355, AJ13356, and strain AJ13601 (described later) are deposited as *Enterobacter clumps* at the aforementioned institutions, but are referred to as *Pantoea ananatis* in this specification. Furthermore, strain SC17sucA, internally designated as strain AJ417, was deposited on February 26, 2004, at the Patent Biological Collection Center of the National Institute of Advanced Industrial Science and Technology, with accession number FERMP-08646.

[0174] Furthermore, examples of L-glutamate-producing bacteria in *Pantoea ananatis* include strains SC17sucA / RSFCPG+pSTVCB, AJ13601, NP106, and NA1. Strain SC17sucA / RSFCPG+pSTVCB was obtained by introducing plasmid RSFCPG, containing the *E. coli*-derived citrate synthase gene (gltA), phosphoenolpyruvate carboxylase gene (ppsA), and glutamate dehydrogenase gene (gdhA), and plasmid pSTVCB, containing the *Brevibacterium lactis*-derived citrate synthase gene (gltA), into strain SC17sucA. Strain AJ13601 was selected from this SC17sucA / RSFCPG+pSTVCB strain as a strain exhibiting resistance to high concentrations of L-glutamate at low pH. Furthermore, strain NP106 was obtained by detaching plasmid RSFCPG+pSTVCB from strain AJ13601 as described in the examples. Strain AJ13601 was deposited on August 18, 1999, at the Patent Biology Collection Center of the National Institute of Advanced Industrial Science and Technology (address: 1-1-1-Chuo-6, Higashi, Tsukuba City, Ibaraki Prefecture, Japan 305-8566, Japan), accession number FERMP-17516; and was transferred to international deposit under the Budapest Treaty on July 6, 2000, accession number FERMP-7207.

[0175] Furthermore, as a method to confer L-glutamate production capacity to Corynebacterium-type bacteria, the following methods can also be used: amplifying the yggB gene (NCgl1221; NP_600492[gi:19552490]) or introducing a mutant yggB gene with a mutation introduced into the coding region (WO2006 / 070944).

[0176] L-glutamate production capacity can also be conferred by amplifying the yhfK gene, which is the L-glutamate secretion gene (WO2005 / 085419).

[0177] Other methods for conferring or enhancing L-glutamate production capacity include: methods for conferring resistance to organic acid analogs, respiratory chain inhibitors, etc., and methods for conferring sensitivity to cell wall synthesis inhibitors. Examples include: methods for conferring resistance to monofluoroacetic acid (Japanese Patent Application Laid-Open No. 50-113209), methods for conferring resistance to adenine or thymine (Japanese Patent Application Laid-Open No. 57-065198), methods for weakening urease (Japanese Patent Application Laid-Open No. 52-038088), methods for conferring resistance to malonic acid (Japanese Patent Application Laid-Open No. 52-038088), methods for conferring resistance to benzopyranone or naphthoquinones (Japanese Patent Application Laid-Open No. 56-1889), methods for conferring resistance to HOQNO (Japanese Patent Application Laid-Open No. 56-140895), methods for conferring resistance to α-ketomalonic acid (Japanese Patent Application Laid-Open No. 57-2689), methods for conferring resistance to guanidine (Japanese Patent Application Laid-Open No. 56-35981), and methods for conferring sensitivity to penicillin (Japanese Patent Application Laid-Open No. 4-88994), etc.

[0178] The following strains can be listed as specific examples of such resistant bacteria:

[0179] Brevibacterium flavum AJ3949 (FERMBP-2632: refer to Japanese Patent Application Laid-Open No. 50-113209)

[0180] Corynebacterium glutamicum AJ11628 (FERMP-5736; see Japanese Patent Application Publication No. 57-065198)

[0181] Fermentobacterium flavum AJ11355 (FERMP-5007; see Japanese Patent Application Publication No. 56-1889)

[0182] Corynebacterium glutamicum AJ11368 (FERMP-5020; see Japanese Patent Application Publication No. 56-1889)

[0183] Fermentobacterium flavum AJ11217 (FERMP-4318; see Japanese Patent Application Publication No. 57-2689)

[0184] Corynebacterium glutamicum AJ11218 (FERMP-4319; see Japanese Patent Application Publication No. 57-2689)

[0185] Fermentobacterium flavum AJ11564 (FERMP-5472; see Japanese Patent Application Publication No. 56-140895)

[0186] Fermentobacterium flavum AJ11439 (FERMP-5136; see Japanese Patent Application Publication No. 56-35981)

[0187] Corynebacterium glutamicum H7684 (FERMBP-3004; see Japanese Patent Application Publication No. 04-88994)

[0188] Lactobacillus fermentum AJ11426 (FERMP-5123; see Japanese Patent Application Publication No. 56-048890)

[0189] Corynebacterium glutamicum AJ11440 (FERMP-5137; see Japanese Patent Application Publication No. 56-048890)

[0190] Lactobacillus fermentum AJ11796 (FERMP-6402; see Japanese Patent Application Publication No. 58-158192)

[0191] L-glutamine producing bacteria

[0192] Preferred examples of microorganisms capable of producing L-glutamine include bacteria with enhanced glutamate dehydrogenase activity, bacteria with enhanced glutamine synthase (glnA) activity, and bacteria with disrupted glutaminase genes (European Patent Application Publications Nos. 1229121 and 1424398). Enhanced glutamine synthase activity can also be achieved through disruption of the glutamine adenylate transferase gene (glnE) or the PII regulatory protein gene (glnB) (EP1229121). Furthermore, as preferred examples of L-glutamine-producing bacteria, bacteria belonging to the genus *Escherichia* possessing a mutant glutamine synthase described below, in which the tyrosine residue at position 397 of the mutant glutamine synthase is replaced by another amino acid residue (US Patent Application Publication No. 2003-0148474).

[0193] Methods for conferring or enhancing L-glutamine production capacity include: methods for conferring resistance to 6-diazo-5-oxo-leucine (Japanese Patent Application Laid-Open No. 3-232497), methods for conferring resistance to purine analogs and methionine sulfoxide (Japanese Patent Application Laid-Open No. 61-202694), and methods for conferring resistance to α-ketomaleic acid (Japanese Patent Application Laid-Open No. 56-151495), etc. Specific examples of Corynebacterium-type bacteria with L-glutamine production capacity include the following microorganisms:

[0194] Flavobacterium flammatum AJ11573 (FERMP-5492, TEDA 56-161495)

[0195] Flavobacterium flammatum AJ11576 (FERMBP-10381, TEDA 56-161495)

[0196] Fermentobacterium flavum AJ12212 (FERMP-8123, ITEMS 61-202694)

[0197] L-proline producing bacteria

[0198] Examples of microorganisms capable of producing L-proline include bacteria carrying γ-glutamyl kinase that relieves L-proline feedback inhibition and bacteria with weakened L-proline degradation systems. A method for modifying bacteria using DNA encoding γ-glutamyl kinase that relieves L-proline feedback inhibition is disclosed in the work of Dandekar and Uratsu (J. Bacteriol. 170, 12: 5943-5945 (1988)). Furthermore, methods for obtaining bacteria with weakened L-proline degradation systems include, for example, introducing mutations into the proline dehydrogenase gene that reduce enzyme activity. Specific examples of bacteria capable of producing L-proline include Escherichia coli strains NRLRB-12403 and NRLRB-12404 (British Patent 2075056), Escherichia coli strain VKPMB-8012 (US Patent Publication 2002-0058315), and strains carrying the following plasmid mutants: plasmid mutants disclosed in German Patent No. 3127361, or plasmid mutants disclosed in the literature of Bloom F.R. et al. (The 15th Miami Winter Symposium, 1983, p. 34).

[0199] In addition, preferred microorganisms with L-proline production capacity include Escherichia coli strain 702 (VKPMB-8011), which is resistant to 3,4-dehydroxyproline and azacyclobutane-2-carboxylic acid; strain 702ilvA (VKPMB-8012), which is an ilvA-deficient strain of strain 702; and Escherichia coli that enhances the activity of proteins encoded by the b2682, b2683, b1242 or b3434 genes (Japanese Patent Application Laid-Open No. 2002-300874), etc.

[0200] L-arginine producing bacteria

[0201] As microorganisms capable of producing L-arginine, examples include *E. coli* mutant strains resistant to α-methylmethionine, p-fluorophenylalanine, D-arginine, arginine isohydroxamic acid, S-(2-aminoethyl)-cysteine, α-methylserine, β-2-thiophene alanine, or sulfaguanidine (see Japanese Patent Application Publication No. 56-106598). Furthermore, *E. coli* strain 237 (Russian Patent Application No. 2000117677), an L-arginine-producing strain with feedback inhibition resistance to L-arginine and carrying highly active N-acetylglutamate synthase, is also a suitable L-arginine-producing strain. This strain was deposited on April 10, 2000, at the Russian National Collection of Industrial Microorganisms (VKPM), GNII Genetika, accession number VKPMB-7925, and transferred to international deposit under the Budapest Treaty on May 18, 2001. Alternatively, *Escherichia coli* strain 382, ​​a derivative of strain 237, is an L-arginine-producing bacterium with improved ability to assimilate acetic acid (Japanese Patent Application Publication No. 2002-017342). *Escherichia coli* strain 382 was deposited on April 10, 2000, at the Russian National Collection of Industrial Microorganisms (VKPM), accession number VKPMB-7926.

[0202] Furthermore, as microorganisms endowed with the ability to produce L-arginine, microorganisms that increase the expression levels of genes encoding enzymes related to L-arginine biosynthesis can be used. For example, enzymes selected from the L-arginine biosynthesis system include one or more of N-acetylglutamate synthase (argA), N-acetylglutamyl-phosphoreductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), acetyl-ornithine transaminase (argD), acetyl-ornithine deacetylase (argE), ornithine carbamoyltransferase (argF), arginine succinate synthase (argG), arginine succinate lyase (argH), and carbamoyl phosphate synthase (carAB). For N-acetylglutamate synthase (argA), a mutant gene is more preferred, in which the amino acid sequence corresponding to positions 15 to 19 of the wild type is substituted, and the feedback inhibition caused by L-arginine is relieved (European Application Publication No. 1170361).

[0203] For L-arginine-producing bacteria belonging to the Corynebacterium type, there are no special restrictions on the type of Corynebacterium that can produce L-arginine, as long as they possess this ability. This includes, for example: wild-type Corynebacterium strains; Corynebacterium strains resistant to sulfonamides, 2-thiazolinone, or α-amino-β-hydroxyvalerate; and, in addition to 2-thiazolinone resistance, possessing L-histidine, L-proline, L-threonine, L-isoleucine, L-methionine, or L-tryptophan. Nutritionally deficient corynebacteria (Japanese Patent Application Publication No. 54-44096); corynebacteria resistant to ketomalonic acid, fluoromalonic acid, or monofluoroacetic acid (Japanese Patent Application Publication No. 57-18989); corynebacteria resistant to argininol (Japanese Patent Application Publication No. 62-24075); or corynebacteria resistant to X-guanidine (where X is a derivative of a fatty acid or aliphatic chain) (Japanese Patent Application Publication No. 2-186995), etc.

[0204] In addition, *Corynebacterium* bacteria capable of producing L-arginine can be selected as the following strains: mutant strains resistant to 5-azouracil, 6-azouracil, 2-thiouracil, 5-fluorouracil, 5-bromouracil, 5-azocytosine, and 6-azocytosine; mutant strains resistant to arginine isohydroxamic acid or 2-thiouracil; mutant strains resistant to arginine isohydroxamic acid and 6-azouracil (Japanese Patent Application Laid-Open No. 49-126819); mutant strains resistant to histidine or tryptophan analogs (Japanese Patent Application Laid-Open No. 52-114092); and strains resistant to at least one of methionine, histidine, threonine, proline, isoleucine, lysine, adenine, guanine, or uracil (or uracil precursor). Mutant strains exhibiting nutritional auxotrophicity (see Japanese Patent Application Publication No. 52-99289); mutant strains resistant to arginine isohydroxamic acid (Japanese Patent Application Publication No. 51-6754); mutant strains exhibiting succinic acid auxotrophicity or resistance to nucleic acid base analogs (Japanese Patent Application Publication No. 58-9692); mutant strains with impaired arginine degradation ability, resistance to arginine antagonists and canavalialine, and lysine auxotrophicity (Japanese Patent Application Publication No. 52-8729); mutant strains resistant to arginine, arginine isohydroxamic acid, high arginine, D-arginine, and canavalialine, or arginine isohydroxamic acid and 6-azouridine (Japanese Patent Application Publication No. 53-143288); and mutant strains resistant to canavalialine (Japanese Patent Application Publication No. 53-3586), etc.

[0205] The following strains can be cited as specific examples of rod-shaped bacteria capable of producing L-arginine.

[0206] Yellow short bacillus AJ11169 (FERMBP-6892)

[0207] Lactobacillus fermentum AJ12092 (FERMBP-6906)

[0208] Yellow short bacillus AJ11336 (FERMBP-6893)

[0209] Yellow short bacillus AJ11345 (FERMBP-6894)

[0210] Lactobacillus fermentum AJ12430 (FERMBP-2228)

[0211] Furthermore, strains deficient in the arginine repressor ArgR (US Patent Application Publication No. 2002-0045223) and strains with increased intracellular glutamine synthase activity (US Patent Application Publication No. 2005-0014236) can also be used.

[0212] The biosynthetic pathways of L-citrulline and L-ornithine are the same as those of L-arginine. The production capacity of these amino acids can be conferred by increasing the enzyme activity of the following enzymes: N-acetylglutamate synthase (argA), N-acetylglutamyl phosphoryl reductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), acetylornithine transaminase (argD), and acetylornithine deacetylase (argE) (International Publication No. 2006-35831).

[0213] Furthermore, in the L-amino acid producing bacteria used in this invention, in addition to the genes encoding enzymes of the natural biosynthetic system, genes related to sugar uptake, sugar metabolism (glycolysis system), and energy metabolism can also be amplified.

[0214] Genes related to sugar metabolism include those encoding enzymes in the glycolysis system or sugar uptake genes, such as glucose-6-phosphate isomerase gene (pgi; International Publication No. 01 / 02542), phosphoenolpyruvate synthase gene (pps; European Patent Application Publication No. 877090), phosphoglucose mutase gene (pgm; International Publication No. 03 / 04598), fructose-2-phosphate aldolase gene (pfkBfbp; International Publication No. 03 / 04664), and pyruvate kinase gene (pykF; International Publication No. 03). The following gene sequences are listed in the patent application: pamphlet 008609, transaldolase gene (talB; international publication pamphlet 03 / 008611), fumarate gene (fum; international publication pamphlet 01 / 02545), phosphoenolpyruvate synthase gene (pps; European patent application pamphlet 877090), non-PTS sucrose uptake gene (csc; European patent application pamphlet 149911), and sucrose assimilation gene (scrAB operon; international publication pamphlet 90 / 04636).

[0215] Examples of genes involved in energy metabolism include the transhydrogenase gene (pntAB; U.S. Patent No. 5,830,716) and the cytochrome botype oxidase gene (cyoB; European Patent Application Publication No. 1070376).

[0216] In addition, when glycerol is used as a carbon source, in order to improve glycerol assimilation, the expression of the glpR gene (EP1715056) can be weakened, or the expression of glycerol metabolism genes such as glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, tpiA, gldA, dhaK, dhaL, dhaM, dhaR, fsa, and talC (EP1715055A) can be enhanced.

[0217] <1-2> Enhanced α-ketoglutarate synthase activity

[0218] The microorganisms of the present invention are microorganisms capable of producing L-glutamic acid amino acids and modified to increase the activity of α-ketoglutarate synthase. Modification to increase α-ketoglutarate synthase activity means modification that increases the activity of α-ketoglutarate synthase compared to the parent strain (e.g., wild-type or unmodified strain). Furthermore, as mentioned above, in microorganisms that originally lacked α-ketoglutarate synthase activity, the modified microorganisms exhibit increased α-ketoglutarate synthase activity compared to unmodified strains.

[0219] In this invention, "α-ketoglutarate synthase" refers to an enzyme (EC 1.2.7.3) that, as described above, catalyzes the reaction of succinyl-CoA and CO2 to produce α-ketoglutarate in the presence of an electron donor, such as ferrugin. α-ketoglutarate synthase is sometimes also named α-ketoglutarate oxide reductase, α-ketoglutarate ferrugin oxide reductase, 2-oxoglutarate synthase, 2-oxoglutarate oxide reductase, or 2-oxoglutarate ferrugin oxide reductase. Ferroreductin or flavin oxidoreductin can be used as the electron donor.

[0220] Furthermore, the parental strains modified to enhance α-ketoglutarate synthase can be strains that naturally possess the gene encoding α-ketoglutarate synthase, or strains that do not naturally possess the α-ketoglutarate synthase gene but are endowed with the activity of the enzyme by introducing the α-ketoglutarate synthase gene, thereby increasing their L-glutamate production capacity.

[0221] Modification can be performed first to enhance the enzyme activity of α-ketoglutarate synthase, and then L-glutamate production capacity can be conferred. Alternatively, L-glutamate production capacity can be conferred first, and then modification can be performed to enhance the enzyme activity of α-ketoglutarate synthase. Moreover, the activity of α-ketoglutarate synthase can be enhanced by methods such as enhancing gene expression as described above. That is, the expression of endogenous α-ketoglutarate synthase genes can be enhanced by modifying expression regulatory regions, such as promoter modification, or the expression of exogenous α-ketoglutarate synthase genes can be enhanced by introducing plasmids containing α-ketoglutarate synthase genes.

[0222] The enhanced activity of α-ketoglutarate synthase has been confirmed by preparing crude enzyme solutions from unmodified and modified microorganisms and comparing their α-ketoglutarate synthase activities. The activity of α-ketoglutarate synthase can be determined, for example, by the method of Yun et al. (Yun, N. Retal. 2001. Biochem. Biophy. Res. Commum. 282: 589-594). For example, it can be determined by spectroscopic methods using oxidized methyl viologen (as an electron acceptor), CoA, and crude enzyme solution, to measure the increase in the amount of reduced methyl viologen due to the decarbonylation of α-ketoglutarate upon addition. One unit of enzyme activity (U) is expressed as the reduction of 1 μmol of methyl viologen per minute. Enhancing the activity of α-ketoglutarate synthase is sufficient if it is improved compared to the wild-type strain. However, when the parent strain possesses α-ketoglutarate synthase activity, it is ideal that the enzyme activity increases by at least 1.5 times, more preferably at least 2 times, and even more preferably at least 3 times compared to the parent strain. Furthermore, when the parent strain does not possess α-ketoglutarate synthase activity, it is sufficient to produce α-ketoglutarate synthase by introducing the α-ketoglutarate synthase gene. However, it is preferable that the modification results in a measurable enzyme activity, ideally at least 0.001 U / mg, more preferably at least 0.005 U / mg, and even more preferably at least 0.01 U / mg relative to bacterial protein.

[0223] Genes encoding α-ketoglutarate synthase can be derived from bacteria exhibiting a reducing TCA cycle, such as *Chlorobium*, *Desulfobacter*, *Aquifex*, *Hydrogenobacter*, *Thermoproteus*, and *Pyrobaculum*, or their homologs. Specifically, the α-ketoglutarate synthase genes of *Chlorobium tetrapidum* and *Hydrogenobacter thermophilus* can be cited. Furthermore, genes presumed to be α-ketoglutarate synthase genes also exist in *Blastopirellula marina* (Schlesner, H. et al. 2004. Int. J. Syst. Evol. Microbiol. 54: 1567-1580), a marine bacterium belonging to the order Planctomycetes. Furthermore, genes with high homology to the α-ketoglutarate synthase gene have also been found in the genomes of sulfur-oxidizing bacteria such as *Sulfurimonas denitrificans* (Brinkhoff, T. et al. 1999. Int. J. Syst. Bacteriol. 49: 875-879) and the methanogenic bacterium *Methanococcus maripaludis* (Jones, W. J. et al. Arch. Microbiol. 1983. 135: 91-97), and the α-ketoglutarate synthase gene can also be selected from them.

[0224] α-Ketoglutarate synthase is known to function as a complex of multiple peptides. The genome sequence of *Chlorobium tepidum* (Genbank accession number NC_002932) has been determined (Eisen, JA et al. 2002. Proc. Natl. Acad. Sci. USA 99: 9509-9514). The α-ketoglutarate synthase gene can be represented by the base sequences shown in SEQ ID NO: 1 and 3. SEQ ID NO: 1 shows the base sequence of the α-ketoglutarate synthase α-subunit gene located at base numbers 170164–172047 (complementary strand) in the *Chlorobium tepidum* genome sequence, and SEQ ID NO: 3 shows the base sequence of the β-subunit gene located at base numbers 169132–170160 (complementary strand) in the *Chlorobium tepidum* genome sequence. SEQ ID NO: 2 is the amino acid sequence of the α subunit of α-ketoglutarate synthase (Genbank accession number NP_661069), and SEQ ID NO: 4 is the amino acid sequence of the β subunit (Genbank accession number NP_661068). The genome sequence of Blastopirellulamarina (Genbank accession number AANZ00000000) has been determined (Fuchsman, CA, and Rocap, G. Appl. Environ. Microbiol. 2006. 72: 6841-6844), and the base sequences shown in SEQ ID NO: 57 and 59 can be used as examples of the α-ketoglutarate synthase gene. SEQ ID NO: 57 is the base sequence of the α-subunit gene of α-ketoglutarate synthase located at base numbers 3180–5045 (complementary strand) in the Blastopirellulamarina genome sequence; SEQ ID NO: 59 is the base sequence of the β-subunit gene located at base numbers 2089–3108 (complementary strand) in the Blastopirellulamarina genome sequence; SEQ ID NO: 58 is the amino acid sequence of the α-subunit of α-ketoglutarate synthase; and SEQ ID NO: 60 is the amino acid sequence of the β-subunit.

[0225] The α-ketoglutarate synthase gene of *Hydrogenobacterium thermophilum* (GenBank accession number AB046568) (Yun, NRetal. 2001. Biochem. Biophy. Res. Commum. 282: 589-594) has been cloned, and its α subunit (GenBank accession number BAB21494) and β subunit (GenBank accession number BAB21495) have been identified. Furthermore, examples include the α-ketoglutarate synthase gene composed of four genes (HP0588, HP0589, HP0590, and HP0591) located at positions 620219–623070 in the genome sequence of Helicobacter pylori (GenBank accession number NC_00091), and the α-ketoglutarate synthase gene composed of two genes (SSO2815 and SSO2816) located at positions 2575303–2578105 in the genome sequence of Sulfolobus sofataricus (GenBank accession number NC_002754). Furthermore, the α-ketoglutarate synthase gene can be cloned from bacteria such as *Aureobacterium*, *Desulfurobacterium*, *Liquidus*, *Hydrogenobacterium*, *Thermoplasia*, *Thermoplasia*, *Sulfurimonas*, and *Methanococcus* based on homology with the genes in the examples above.

[0226] The α-subunit gene and β-subunit gene of α-ketoglutarate synthase are preferably from the same biological source, but any gene capable of forming a protein with α-ketoglutarate synthase activity is acceptable; the genes can also be from different biological sources. Preferred combinations are: the α-subunit or a conserved variant thereof composed of the amino acid sequence of SEQ ID NO: 2 and the β-subunit or a conserved variant thereof composed of the amino acid sequence of SEQ ID NO: 4; and the α-subunit or a conserved variant thereof composed of the amino acid sequence of SEQ ID NO: 58 and the β-subunit or a conserved variant thereof composed of the amino acid sequence of SEQ ID NO: 60, but are not limited thereto. "Conserved variant" will be described later.

[0227] The microorganisms of the present invention can also be microorganisms modified to increase the activity of α-ketoglutarate synthase, wherein the modification increases the activity of regenerating electron donors from the oxidized form to the reduced form (an activity essential for the activity of α-ketoglutarate synthase) compared to the parental strain, such as a wild-type strain or an unmodified strain. The activity of regenerating oxidized electron donors to the reduced form includes, for example, ferricredoxin-NADP. +The activity can be either reductase activity or pyruvate synthase activity. Additionally, the parent strain can be a microorganism that has been modified to increase the expression of the α-ketoglutarate synthase gene, in addition to enhancing the regenerative activity of the electron donor. Furthermore, the parent strain can be a microorganism that naturally possesses a gene encoding the regenerative activity of the electron donor, or a microorganism that does not naturally possess the regenerative activity of the electron donor but has been endowed with this activity by introducing a gene encoding it, thereby increasing its L-glutamate production capacity.

[0228] "Feroxanone-NADP" + "Reductase" refers to an enzyme that reversibly catalyzes the following reaction (EC 1.18.1.2).

[0229] Reduced ferricyanide + NADP + →Oxidized ferricredoxin + NADPH + H +

[0230] This reaction is reversible and can produce reduced ferricoxin in the presence of NADPH and oxidized ferricoxin. Ferricoxin can be replaced with flavin ferricoxin, named flavin ferricoxin-NADP. + The enzyme reductase also has the same function. Frioxonin-NADP has been identified. + Reductases are ubiquitous in organisms from microorganisms to higher organisms (see Carrillo, N. and Ceccarelli, EA 2003. Eur. J. Biochem. 270: 1900-1915; Ceccarelli, EA et al. 2004. Biochim. Biophys. Acta. 1698: 155-165), and have also been named ferritin-NADP. + Oxidase reductase, NADPH-ferroredoxin oxidase reductase.

[0231] ferricyanide-NADP + The confirmation of the enhanced reductase activity was achieved by preparing crude enzyme solutions from unmodified and modified microorganisms and comparing their ferroredoxin-NADP content. + This is achieved through reductase activity. (Feroxin-NADP) + The activity of reductase can be determined, for example, by Blaschkowski et al. (Blaschkowski, HP et al. 1982. Eur. J. Biochem. 123: 563-569). For instance, it can be determined by spectroscopic methods using ferredoxin as a substrate to measure the amount of NADPH reduced. One unit of enzyme activity (U) is expressed as the amount of 1 μmol of NADPH oxidized per minute. When the parental strain possesses ferredoxin-NADP...+ When it comes to reductase activity, if the activity of the parent strain is already sufficiently high, there is no need to enhance it. However, ideally, the enzyme activity should increase by more than 1.5 times, more preferably more than 2 times, and more preferably more than 3 times compared to the parent strain.

[0232] The protein encoding ferricoxin-NADP has been found in many biological species. + Reductase genes can be used, as long as they are active in the target L-amino acid producing strain. In *E. coli*, they are used as flavin-NADP... + The reductase has been identified by the fpr gene (Bianchi, V. et al. 1993. J. Bacteriol. 175: 1590-1595). Furthermore, the NADPH-pseudomonas putida reductase gene and the putida redoxin gene are known to exist as operons in *Pseudomonas putida* (Koga, H. et al. 1989. J. Biochem. (Tokyo) 106: 831-836).

[0233] Flavoredoxin-NADP in Escherichia coli + Reductases can be exemplified by the fpr gene, located at bases 4111749–4112495 (complementary strand) in the genome sequence of *Escherichia coli* strain K-12 (Genbank accession number U00096), with the base sequence shown in SEQ ID NO: 5. The amino acid sequence of Fpr is shown in SEQ ID NO: 6 (Genbank accession number AAC76906). Furthermore, ferritin-NADP was found at bases 2526234–2527211 in the genome sequence of *Corynebacterium glutamicum* (Genbank accession number BA00036). + Reductase gene (Genbank accession number: BAB99777).

[0234] "Pyruvate synthase" refers to an enzyme (EC1.2.7.1) that reversibly catalyzes the following reaction to produce pyruvate from acetyl-CoA and CO2.

[0235] Reduced ferricyanide + acetyl-CoA + CO2 → oxidized ferricyanide + pyruvate

[0236] This reaction is reversible, producing reduced ferricrene in the presence of pyruvate and oxidized ferricrene. This enzyme is also known as pyruvate oxide reductase, pyruvate ferricrene (flavin ferricrene) reductase, or pyruvate ferricrene oxide reductase. By combining this enzyme activity with α-ketoglutarate synthase activity, the reduced ferricrene consumed by α-ketoglutarate synthase activity can be regenerated through the reverse reaction activity of pyruvate synthase.

[0237] The enhanced pyruvate synthase activity can be confirmed by preparing crude enzyme solutions from microorganisms before and after enhancement and comparing their pyruvate synthase activities. Pyruvate synthase activity can be determined, for example, by the method of Yoon et al. (Yoon, K. Setal. 1997. Arch. Microbiol. 167: 275-279). The determination principle is the same as that for the above-described α-ketoglutarate synthase activity determination; for example, it can be determined by using pyruvate as a substrate and employing spectroscopic methods to determine the amount of methyl viologen reduced in the pyruvate decarbonation reaction. One unit of enzyme activity (U) is expressed as the amount of methyl viologen reduced per minute (1 μmol). When the parent strain possesses pyruvate synthase activity, ideally, the enzyme activity should increase by at least 1.5 times, more preferably at least 2 times, and even more preferably at least 3 times compared to the parent strain. Furthermore, when the parental strain does not possess pyruvate synthase activity, pyruvate synthase can be generated simply by introducing a pyruvate synthase gene. However, it is preferable that the enzyme activity is enhanced to a measurable level, preferably 0.001 U / mg or more, more preferably 0.005 U / mg or more, and even more preferably 0.01 U / mg or more.

[0238] Genes encoding pyruvate synthase can utilize genes from bacteria with a reducing TCA cycle, such as the pyruvate synthase gene of *Sulphurella vulgaris* and the pyruvate synthase gene of *Hydrogenobacterium thermophilum*.

[0239] Specifically, as a pyruvate synthase gene of *A. aeruginosa*, an example can be found is the gene with base numbers 1534432–1537989 in the *A. aeruginosa* genome sequence (Genbank accession number NC_002932), having the base sequence shown in SEQ ID NO: 7. SEQ ID NO: 8 shows the amino acid sequence encoded by this gene (Genbank accession number AAC76906). Furthermore, it is known that pyruvate synthase in *Hydrogenobacter thermophilus* is formed by a complex of four subunits: δ subunit (Genbank accession number BAA95604), α subunit (Genbank accession number BAA95605), β subunit (Genbank accession number BAA95606), and γ subunit (Genbank accession number BAA95607) (Ikeda, T. et al. 2006. Biochem. Biophys. Res. Commun. 340: 76-82). Furthermore, examples include a pyruvate synthase gene composed of four genes (HP1108, HP1109, HP1110, and HP1111) located at positions 1170138–1173296 in the *Helicobacter pylori* genome sequence (GenBank accession number NC000915), and a pyruvate synthase gene composed of four genes (SSO1208, SSO7412, SSO1207, and SSO1206) located at positions 1047593–1044711 in the *Sulphurobacterium sulfideum* genome sequence (GenBank accession number NC002754). Moreover, pyruvate synthase genes can also be cloned from bacteria of the genera *Aureobacterium*, *Desulfurobacterium*, *Liquigenes*, *Hydrogenobacterium*, *Thermoplasia*, and *Thermoplasia* based on homology with the genes described above.

[0240] For α-ketoglutarate synthase activity, the presence of ferricoxin or flavinoxorubicin as an electron donor is necessary. Therefore, microorganisms modified to increase α-ketoglutarate synthase activity, such modifications enhance the production of ferricoxin or flavinoxorubicin. Furthermore, in addition to modifications that enhance α-ketoglutarate synthase activity, flavinoxorubicin-NADP... + In addition to enhancing the activity of reductase or pyruvate synthase, modifications can also be made to increase the production capacity of ferroredoxin or flavin oxidoredoxin.

[0241] In this invention, "ferredoxin" refers to a protein containing non-porphyrin iron (Fe) and sulfur atoms, and bound to an iron-sulfur cluster called 4Fe-4S, 3Fe-4S, or 2Fe-2S, which functions as a single-electron transporter. "Flavin-redoxin" refers to a protein containing an FMN (flavin mononucleotide) as a cofactor, which functions as a single or double-electron transporter. Ferroredoxin and flavin-redoxin are described in the literature of McLean et al. (McLean, KJ et al. 2005. Biochem. Soc. Trans. 33: 796-801).

[0242] Moreover, the parent strains used for modification can be strains that naturally and intrinsically possess genes encoding ferricodoxin or flavinoxin, or strains that do not naturally possess ferricodoxin or flavinoxin genes but have been endowed with activity by introducing ferricodoxin or flavinoxin genes, thereby increasing their L-glutamic acid production capacity.

[0243] The increased production capacity of ferricoxin or flavinoxin compared to the parental strain (e.g., wild-type or unmodified strain) can be confirmed by comparing the amount of ferricoxin or flavinoxin mRNA with that of the wild-type or unmodified strain. Methods for confirming expression levels include Southern hybridization and RT-PCR (Sambrook, J. et al. 1989. Molecular Cloning A Laboratory Manual / Second Edition, Cold Spring Harbor Laboratory Press, New York). Regarding expression levels, any increase compared to the wild-type or unmodified strain is acceptable; ideally, it should be at least 1.5-fold, more preferably 2-fold, and more preferably 3-fold.

[0244] Furthermore, the fact that the production capacity of ferrous reductin or flavin reductin is increased compared to the parental strain (e.g., wild-type or unmodified strain) can be confirmed by SDS-PAGE, two-dimensional electrophoresis, or Western blotting using antibodies (Sambrook, J. et al. 1989. Molecular Cloning A Laboratory Manual / Second Edition, Cold Spring Harbor Laboratory Press, New York). Regarding the production amount, any increase compared to the wild-type or unmodified strain is acceptable; ideally, it should be at least 1.5 times, more preferably 2 times, and more preferably 3 times higher than the wild-type or unmodified strain.

[0245] The activities of ferricoxin and flavin ferricoxin can be determined by adding them to appropriate redox reaction systems. For example, Boyer et al. disclosed the following method: via ferricoxin-NADP... + The reduction of ferricoxane by reductase can quantify the reduction of cytochrome C induced by the generated reduced ferricoxane (Boyer, MEetal. 2006. Biotechnol. Bioeng. 94: 128-138). Furthermore, the activity of flavin-NADP can be measured using flavin-NADP... + The reductase was determined using the same method.

[0246] Genes encoding ferricodoxins or flavinodoxins are widely distributed. Any gene can be used as long as the encoded ferricodoxin or flavinodoxin can be utilized by α-ketoglutarate synthase and the electron donor regeneration system. For example, in *E. coli*, the *fdx* gene is present as the gene encoding a ferricodoxin with a 2Fe-2S cluster (Ta, DT, and Vickery, LE 1992. J. Biol. Chem. 267: 11120-11125), suggesting that the *yfhL* gene encodes a ferricodoxin with a 4Fe-4S cluster. Furthermore, *fldA* (Osborne, C. et al. 1991. J. Bacteriol. 173: 1729-1737) and *fldB* (Gaudu, P., and Weiss, B. 2000. J. Bacteriol. 182: 1788-1793) are known flavinodoxin genes. In the genome sequence of *Corynebacterium glutamicum* (Genbank accession number BA00036), the feroxin gene *fdx* (Genbank accession number BAB97942) was found at positions 562643–562963, and *fer* (Genbank accession number BAB98495) was found at positions 1148953–1149270. Furthermore, numerous feroxin genes exist in *Agropyron globulinum*, among which feroxin I and feroxin II have been identified as 4Fe-4S type feroxin genes acting as electron acceptors for pyruvate synthase (Yoon, K. Setal. 2001. J. Biol. Chem. 276: 44027-44036). Ferroxin genes or flavin-oxidoreductin genes derived from bacteria with a reducing TCA cycle, such as the feroxin gene of *Hydrogenobacter thermophilus*, can also be used.

[0247] Specifically, the ferredoxin genes of *E. coli* include, for example, the fdx gene located at positions 2654770–2655105 (complementary strand) in the genome sequence of *E. coli* strain K-12 (Genbank accession number U00096), and the yfhL gene located at positions 2697685–2697945, as shown in SEQ ID NO: 11. The amino acid sequences of Fdx and YfhL (Genbank accession numbers AAC75578 and AAC75615, respectively) are shown in SEQ ID NO: 10 and SEQ ID NO: 12. Genes encoding flavin redoxins in *Escherichia coli* include, for example, the fldA gene shown in SEQ ID NO: 13, located at base positions 710688–710158 (complementary strand) in the genome sequence of *Escherichia coli* strain K-12 (Genbank accession number U00096), and the fldB gene shown in SEQ ID NO: 15, located at base positions 3037877–3038398. SEQ ID NO: 14 and SEQ ID NO: 16 show the amino acid sequences encoded by the fldA and fldB genes (Genbank accession numbers AAC73778 and AAC75933, respectively).

[0248] Examples of ferricoxin genes from *Aureobacterium tumefaciens* include, for instance, the ferricoxin I gene shown in SEQ ID NO: 17, located at base positions 1184078–1184266 of the *Aureobacterium tumefaciens* genome sequence (Genbank accession number NC_002932), and the ferricoxin II gene shown in SEQ ID NO: 19, located at base positions 1184476–1184664. The amino acid sequences encoding ferricoxin I and ferricoxin II (Genbank accession numbers AAM72491 and AAM72490, respectively) are shown in SEQ ID NO: 18 and SEQ ID NO: 20. Additionally, examples include the ferricoxin gene of *Hydrogenobacter thermophilus* (Genbank accession number BAE02673) and the ferricoxin gene of *Lepiota sulfidea* shown at base positions 2345414–2345728 of the *Lepiota sulfidea* genome sequence. Furthermore, genes can be cloned from bacteria such as *Aureobacterium*, *Desulfurobacterium*, *Liquidus*, *Hydrogenobacterium*, *Thermoplasticum*, and *Corynebacterium* based on homology with the genes in the examples above. They can also be cloned from γ-proteobacteria such as *Enterobacter*, *Klebsiella*, *Serratia*, *Erwinia*, and *Yersinia*; rod-shaped bacteria such as *Corynebacterium glutamicum* and *Brevibacterium lactis*; *Pseudomonas aeruginosa* and other *Pseudomonas* bacteria; and *Mycobacterium* bacteria such as *Mycobacterium tuberculosis*.

[0249] These encode α-ketoglutarate synthase and ferroredoxin-NADP. + The genes for reductase, pyruvate synthase, ferrous reductin, and flavin reductin (hereinafter collectively referred to as the genes of the present invention) can be genes encoding conserved variants of amino acid sequences obtained by substitution, deletion, insertion, or addition of one or more amino acids at one or more positions, as long as the activity or function of the encoded protein is not impaired. The term "one or more" varies depending on the position of the amino acid residues in the protein's three-dimensional structure and the type of amino acid; preferably 1 to 20, more preferably 1 to 10, and particularly preferably 1 to 5.

[0250] The aforementioned substitutions are preferably conservative substitutions, which are neutral mutations that do not change function. Regarding conservative mutations, when the substitution site is an aromatic amino acid, it is a substitution mutation between Phe, Trp, and Tyr; when the substitution site is a hydrophobic amino acid, it is a substitution mutation between Leu, Ile, and Val; when the substitution site is a polar amino acid, it is a substitution mutation between Gln and Asn; when the substitution site is a basic amino acid, it is a substitution mutation between Lys, Arg, and His; when the substitution site is an acidic amino acid, it is a substitution mutation between Asp and Glu; and when the substitution site is an amino acid with a hydroxyl group, it is a substitution mutation between Ser and Thr.

[0251] More specifically, examples include: replacing Ala with Ser or Thr; replacing Arg with Gln, His, or Lys; replacing Asn with Glu, Gln, Lys, His, or Asp; replacing Asp with Asn, Glu, or Gln; replacing Cys with Ser or Ala; replacing Gln with Asn, Glu, Lys, His, Asp, or Arg; replacing Glu with Gly, Asn, Gln, Lys, or Asp; replacing Gly with Pro; replacing His with Asn, Lys, Gln, Arg, or Tyr; and replacing L with... Ile can be replaced by eu, Met, Val, or Phe; Leu can be replaced by Ile, Met, Val, or Phe; Lys can be replaced by Asn, Glu, Gln, His, or Arg; Met can be replaced by Ile, Leu, Val, or Phe; Phe can be replaced by Trp, Tyr, Met, Ile, or Leu; Ser can be replaced by Thr or Ala; Thr can be replaced by Ser or Ala; Trp can be replaced by Phe or Tyr; Tyr can be replaced by His, Phe, or Trp; and Val can be replaced by Met, Ile, or Leu. Furthermore, such amino acid substitutions, deletions, insertions, additions, or inversions also include those resulting from naturally occurring mutations (mutants or variants) due to individual or interspecies differences in the microorganisms carrying the gene of this invention.

[0252] Furthermore, codons that are easy to use in the host in which the genes of the present invention are introduced can be substituted. Similarly, as long as the genes of the present invention are functional, the N-terminal and / or C-terminal sides of the encoded protein can be extended or truncated. For example, the length of the extension is 50 or less, preferably 20 or less, more preferably 10 or less, and particularly preferably 5 or less amino acid residues.

[0253] Genes encoding conserved variants as described above can be obtained, for example, by modifying the base sequence using site-directed mutagenesis, such that amino acid residues at specific sites of the encoded protein contain substitutions, deletions, insertions, or additions. Alternatively, they can be obtained through conventionally known mutagenesis treatments. Examples of mutagenesis treatments include: in vitro treatment of the gene of the present invention with hydroxylamine or the like, and treatment of microorganisms carrying the gene (e.g., Escherichia coli) with ultraviolet light irradiation or with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS) used in conventional mutagenesis treatments. Furthermore, the amino acid substitutions, deletions, insertions, additions, or inversions described above also include those resulting from naturally occurring mutations (mutants or variants) due to individual or interspecies differences in the microorganisms carrying the gene of the present invention. Whether these genes encode functional α-ketoglutarate synthase or ferroredoxin-NADP is another matter. + Reductase, pyruvate synthase, ferroredoxin, or flavin reductase can be identified, for example, by introducing these genes into microorganisms and measuring the activity of each gene product.

[0254] The gene of the present invention can be capable of hybridizing under stringent conditions with DNA having the above-described base sequence or a probe that can be prepared from DNA having these base sequences, and encodes α-ketoglutarate synthase and ferroredoxin-NADP. + DNA containing reductase, pyruvate synthase, feroxin, or flavin oxidoxin.

[0255] Here, "strict conditions" refer to conditions under which so-called specific hybrids are formed but non-specific hybrids are not. It is difficult to explicitly quantify these conditions. For example, conditions under which DNAs with high homology (e.g., DNAs with 70% or more, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more homology) hybridize, while DNAs with lower homology do not hybridize; or conditions under washing conditions equivalent to those used in conventional Southern hybridization, i.e., washing once at 60°C with 1×SSC and 0.1% SDS, preferably 0.1×SSC and 0.1% SDS, more preferably 68°C with 0.1×SSC and 0.1% SDS, preferably 2 to 3 times.

[0256] Furthermore, in this specification, "homology" sometimes refers to "identity".

[0257] The probe can also be a portion of the gene sequence of the present invention. Such probes can be prepared using methods well known to those skilled in the art, using oligonucleotides prepared based on the base sequences of each gene as primers and DNA fragments containing each gene as templates for PCR reactions. Moreover, when using DNA fragments of approximately 300 bp in length as probes, the washing conditions after hybridization under the above conditions can be, for example, 50°C, 2×SSC, and 0.1% SDS.

[0258] The above description of conserved variants also applies to the enzymes and genes described in the attribution of L-amino acid production capacity.

[0259] The modifications described above for enhancing the expression of the gene of the present invention can be performed as described in the method for enhancing the expression of the target gene in the section on conferring L-amino acid production capacity. The gene of the present invention can be obtained by PCR using the genomic DNA of a microorganism carrying the gene as a template.

[0260] For example, the α-ketoglutarate synthase gene of *Sulphurella vulgaris* can be obtained by PCR using primers prepared based on the base sequences of SEQ ID NO: 1 and 3, such as the primers shown in SEQ ID NO: 21 and 22, with *Sulphurella vulgaris* genomic DNA as a template.

[0261] Flavoredoxin-NADP of Escherichia coli + The reductase gene can be obtained by PCR using primers prepared based on the base sequence of SEQ ID NO: 5, such as the primers shown in SEQ ID NO: 32 and 33, with E. coli genomic DNA as a template.

[0262] The pyruvate synthase gene of *S. green sulfur* can be obtained by PCR using primers prepared based on the base sequence of SEQ ID NO: 7, such as the primers shown in SEQ ID NO: 23 and 24, with *S. green sulfur* genomic DNA as a template.

[0263] The ferric reductin gene fdx of Escherichia coli can be obtained by PCR using primers prepared based on the base sequence of SEQ ID NO: 9, such as the primers shown in SEQ ID NO: 38 and 39, with E. coli genomic DNA as a template; the ferric reductin gene yfhL can be obtained by PCR using primers prepared based on the base sequence of SEQ ID NO: 11, such as the primers shown in SEQ ID NO: 40 and 41, with E. coli genomic DNA as a template.

[0264] The flavin-redoxin gene fldA of *E. coli* can be obtained by PCR using primers prepared based on the base sequence of SEQ ID NO: 13, such as primers shown in SEQ ID NO: 34 and 35, with *E. coli* genomic DNA as a template; the flavin-redoxin gene fldB can be obtained by PCR using primers prepared based on the base sequence of SEQ ID NO: 15, such as primers shown in SEQ ID NO: 36 and 37, with *E. coli* genomic DNA as a template.

[0265] In addition, the ferricodoxin I gene of *Sulphurella vulgaris* can be obtained by PCR using primers prepared based on the base sequence of SEQ ID NO: 17, such as the primers shown in SEQ ID NO: 25 and 26, with *Sulphurella vulgaris* genomic DNA as a template.

[0266] For genes of the present invention derived from other microorganisms, they can also be obtained from the genomic DNA or genomic DNA library of the microorganism by PCR using oligonucleotides prepared based on the sequence information of the aforementioned genes or the known gene or protein sequence information of the microorganism as primers, or by hybridization using oligonucleotides prepared based on the sequence information as probes. Furthermore, genomic DNA can be prepared from microorganisms serving as DNA donors using, for example, the methods of Saito and Miura (see Saito, H. and Miura, KI 1963. Biochem. Biophys. Acta, 72, 619-629; Biotechnology Experiments, edited by the Japanese Society of Biotechnology, pp. 97-98, Peifukan, 1992).

[0267] The enhancement of gene expression can be achieved by using the methods described above, such as transformation or homologous recombination to increase the copy number of the gene, or by modifying the expression regulatory sequence of the gene. Furthermore, the enhancement of gene expression can be achieved by amplifying activators that can increase gene expression and / or deleting or attenuating regulators that can decrease gene expression.

[0268] Furthermore, when increasing the copy number of a gene, it is sufficient to enhance the activity of the product of the target gene; there are no special restrictions on the copy number. When the microorganism already possesses the target gene, the copy number is preferably 2 or more. Additionally, when the microorganism does not originally possess the gene of this invention, the copy number of the introduced gene can be 1 or 2 or more.

[0269] When a gene is composed of multiple subunits, such as α-ketoglutarate synthase, the expression of the gene encoding each subunit can be enhanced individually, or the expression of these genes can be enhanced simultaneously in a polycistronic manner. Furthermore, when introducing genes into microorganisms using vectors, the genes encoding each subunit can be simultaneously carried on a single vector molecule, or separately carried on different vector molecules. Additionally, when inserting genes into the genome, the genes encoding each subunit can be simultaneously inserted into the same site on the genome, or separately inserted into different locations.

[0270] Furthermore, preferably, the microorganisms of the present invention, in addition to enhancing α-ketoglutarate synthase activity, also reduce α-ketoglutarate dehydrogenase activity. Such microorganisms can be obtained, for example, by transforming microorganisms whose gene encoding α-ketoglutarate dehydrogenase has been disrupted using a recombinant vector containing the gene of the present invention, respectively.

[0271] In this invention, α-ketoglutarate dehydrogenase (hereinafter also referred to as "α-KGDH") activity refers to the activity of catalyzing the oxidation and decarbonation of α-ketoglutarate (2-oxoglutarate) to generate succinyl-CoA. This reaction is catalyzed by three enzymes: α-KGDH (E1o: α-ketoglutarate dehydrogenase, EC: 1.2.4.2), dihydrolipoamide S-succinyltransferase (E2o: dihydrolipoamide-S-succinyltransferase; EC: 2.3.1.61), and dihydrolipoamide dehydrogenase (E3: dihydrolipoamide dehydrogenase; EC: 1.8.1.4). That is, each of these three subunits catalyzes a separate reaction, and the activity of catalyzing the combined reaction of these three reactions is called α-KGDH activity. The activity of α-KGDH can be confirmed by the method of Shiio and Ujigawa-Takeda (Isamu Shiio and Ujigawa-Takeda, Agric. Biol. Chem., 44(8), 1897-1904, 1980).

[0272] E1o: 2-oxoglutarate + [dihydrolipoyllysine-residuesuccinyltransferase]lipoyllysine → [dihydrolipoyllysine-residuesuccinyltransferase]S-succinyldihydrolipoyllysine + CO2

[0273] E2o: CoA+ enzyme N6-(S-succinyl dihydrolipoyl)lysine → succinyl-CoA+ enzyme N6-(dihydrolipoyl)lysine

[0274] E3: Protein N6-(dihydrolipoyl)lysine + NAD + =Protein N6-(Lipoic acid)lysine + NADH + H +

[0275] Moreover, α-KGDH is also known as oxoglutarate dehydrogenase or 2-oxoglutarate dehydrogenase.

[0276] In Enterobacteriaceae bacteria such as *Pantoea ananatis*, subunit proteins possessing these three enzymatic activities form a complex. Furthermore, each subunit is encoded by a separate sucA, sucB, and lpd gene, with the sucA and sucB genes located downstream of the succinate dehydrogenase iron-sulfur protein gene (sdhB) (US Patent No. 6,331,419). Moreover, the aforementioned patent describes these genes as belonging to the clumping Enterobacter aJ13355 strain, which was later reclassified as *Pantoea ananatis*.

[0277] The gene encoding α-KGDH in Enterobacteriaceae includes the base sequences of the sucA and sucB genes of *Pantoea ananatis*, a portion of the upstream sdhB gene, and a fragment of the downstream sucC gene, as shown in SEQ ID NO: 46. Furthermore, the amino acid sequences encoded by a portion of their sdhB gene, sucA, sucB, and sucC genes are shown in SEQ ID NO: 47–50, respectively. Additionally, sucA and sucB, encoding α-KGDH in *Escherichia coli*, are disclosed in GenBank NP_415254 and NP_415255, respectively.

[0278] Furthermore, in Corynebacterium-type bacteria, the E1o subunit is encoded by the odhA gene (also known as the sucA gene, accessed via GenBank accession number NCgl1084, NC_003450), and the E3 subunit is encoded by the lpd gene (GenBank accession number Y16642). On the other hand, it is speculated that the E2o subunit, together with the E1o subunit, is encoded by the odhA gene as a bifunctional protein (see Usuda, Y. et al., Microbiology 1996, 142: 3347-3354), or by a gene different from the odhA gene, accessed via GenBank accession number NCgl2126, NC_003450. Therefore, in this invention, the odhA gene is the gene encoding the E1o subunit, but it can also simultaneously encode the E2o subunit.

[0279] The base sequence of the *Odhya microbesii* *odhA* gene and the amino acid sequence of the E1o subunit (Genbank accession number NC_003450, NCgl1084, WO2006 / 028298) are shown in SEQ ID NO: 51 and 52. Furthermore, the base sequence of the aforementioned Genbank accession number NC_003450, NCgl2126, and the amino acid sequence of the E2o subunit encoded by that sequence are shown in SEQ ID NO: 53 and 54.

[0280] Furthermore, when cultured under anaerobic or microaerophilic conditions, the microorganisms of the present invention can also be modified to not produce organic acids or ethanol under anaerobic or microaerophilic conditions, except for enhanced α-ketoglutarate synthase activity. Examples of organic acids include lactic acid, formic acid, and acetic acid. A method for modifying the microorganisms to prevent the production of organic acids or ethanol is to disrupt the gene encoding lactate dehydrogenase (Verumi, GNetal. 2002. J. Industrial Microbiol. Biotechnol. 28: 325-332; JP 2005-95169).

[0281] <2> The method for producing L-glutamic acid of the present invention

[0282] The method of the present invention is a method for producing the following L-amino acids: Microorganisms having the ability to produce one or more L-amino acids selected from L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline, and L-arginine, and modified to increase the activity of α-ketoglutarate synthase, are cultured in a culture medium. The L-amino acids are generated and accumulated in the culture medium or within the cells of the microorganisms, and the L-amino acids are collected from the culture medium or the cells. The method of the present invention can be carried out under aerobic conditions, or under anaerobic or microaerobic conditions. Aerobic conditions are typically achieved by introducing oxygen-containing gas into the culture medium or by stirring the culture medium. Furthermore, anaerobic or microaerobic conditions are typically achieved by not aerating or stirring, or by reducing the aeration rate or stirring frequency.

[0283] When culturing microorganisms in a culture medium, the bacterial cells obtained by slant culture in solid culture medium such as agar can be directly inoculated into the liquid culture medium. However, it is preferable to pre-culture the microorganisms in liquid culture medium (seed culture) and then inoculate the obtained bacterial cells into the main culture medium (fermentation medium).

[0284] The culture medium used for cultivation can be a conventional microbial culture medium containing carbon sources, nitrogen sources, inorganic salts, and organic micronutrients such as amino acids and vitamins as needed. For example, a common culture medium can be used by adding natural nutrient sources such as meat extract, yeast extract, and peptone to a component containing inorganic salts such as ammonium sulfate, potassium phosphate, and magnesium sulfate.

[0285] There are no special restrictions on the carbon source, as long as it can be assimilated by microorganisms to produce L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline, or L-arginine. Commonly used carbohydrates include galactose, lactose, glucose, fructose, sucrose, disaccharides, starch, cellulose, and fatty acids. Alcoholic sugars such as glycerol, mannitol, xylitol, ribitol, and ethanol can also be used. Among these, glucose, fructose, sucrose, glycerol, and ethanol are preferred, with glucose and glycerol being particularly preferred. Crude glycerol preferably comes from biodiesel fuel production. The carbon source can be one type or a mixture of two or more.

[0286] In addition, starch saccharification solutions, molasses, crude glycerol, etc., containing the aforementioned sugars can also be used. There are no particular restrictions on the concentration of the aforementioned carbon sources; however, it is advantageous to use the highest possible concentration without inhibiting the formation of L-amino acids, typically within the range of 5–30% (w / v), preferably 10–20% (w / v) for fermentation. Furthermore, carbon sources can be added as needed to compensate for the reduction of carbon sources during fermentation.

[0287] Furthermore, there are no specific restrictions on the nitrogen source, as long as it can be assimilated by microorganisms to produce L-amino acids. Specifically, examples include ammonium salts, nitrates, urea, soybean hydrolysate, casein breakdown products, peptone, yeast extract, meat extract, corn steep liquor, and various organic or inorganic nitrogen-containing compounds. As inorganic salts, various phosphates, sulfates, and metal salts such as magnesium, potassium, manganese, iron, and zinc can be used. In addition, vitamins such as biotin, pantothenic acid, inositol, and niacin, as well as growth-promoting factors such as nucleotides and amino acids, can be added as needed. Furthermore, to suppress foaming during cultivation, it is ideal to add an appropriate amount of commercially available antifoaming agent to the culture medium.

[0288] The pH can be adjusted by adding ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide, etc. The preferred pH in the main culture is usually pH 5-10, preferably pH 6-9.5. Therefore, alkaline substances, carbonates, urea, etc. should be used as needed to adjust the pH of the culture medium within the above range during culture.

[0289] The culture medium used in this invention preferably contains the aforementioned carbon source, as well as carbonate ions, bicarbonate ions, or carbon dioxide gas, and is capable of culturing under aerobic or microaerobic / anaerobic conditions. Carbonate ions or bicarbonate ions can be supplied by carbonates or bicarbonates such as magnesium carbonate, sodium carbonate, sodium bicarbonate, potassium carbonate, and potassium bicarbonate, which can also be used as neutralizing agents, and may be supplied by carbon dioxide gas as needed. Furthermore, carbonate ions and bicarbonate ions are added at a concentration of 0.001–5 M, preferably 0.1–3 M, more preferably 1–2 M. When carbon dioxide gas is present, it contains 50 mg–25 g, preferably 100 mg–15 g, more preferably 150 mg–10 g of carbon dioxide gas per 1 L of solution.

[0290] Generally, when culturing microorganisms, it is preferable to culture them under aerobic conditions where oxygen is supplied through aeration and stirring. However, in the method of the present invention, culturing can be carried out under aerobic conditions with aeration and stirring, or under anaerobic / microaerobic conditions without aeration and oxygen supply. Various dissolved oxygen concentration conditions can be set by, for example, by reducing the aeration rate or stirring, sealing the container under aeration conditions, introducing an inert gas containing carbon dioxide, etc.

[0291] The incubation temperature is typically 25℃ to 40℃, preferably 30℃ to 37℃. The incubation time is preferably 1 hour to 168 hours, more preferably 3 hours to 72 hours.

[0292] The cultivation period can be divided into cultivation for microbial proliferation and cultivation for L-amino acid production, and different culture media or conditions can be used for each. For example, after microbial proliferation is achieved under aeration or stirring, L-amino acids can be produced under anaerobic or microaerobic conditions.

[0293] In addition, a liquid culture medium prepared to precipitate L-glutamic acid can be used, allowing L-glutamic acid to precipitate from the medium during culturing. Examples of conditions for L-glutamic acid precipitation include, for example, pH 5.0 to 4.0, preferably pH 4.5 to 4.0, more preferably pH 4.3 to 4.0, and particularly preferably pH 4.0.

[0294] After cultivation, L-amino acids can be collected from the culture medium using known recovery methods. For example, methods such as removing bacterial cells from the culture medium followed by concentration and crystallization, or ion-exchange chromatography, can be used. When L-glutamic acid precipitates during cultivation, the precipitated L-glutamic acid can be collected by centrifugation or filtration. In this case, it can be separated along with the L-glutamic acid after crystallization of the dissolved L-glutamic acid in the culture medium.

[0295] Example

[0296] The present invention will be described in more detail below through embodiments, but the present invention is not limited to these embodiments.

[0297] [Example 1] <Construction of expression plasmids for α-ketoglutarate synthase gene, pyruvate synthase gene, and ferricoxin gene derived from *Sulphurella multocida*>

[0298] *Sulphurella vulgaris* is a mesophilic autotrophic bacterium with an optimum growth temperature of 48°C. The genome sequence of the TLS strain has been elucidated by Eisen et al. (Eisen, JA et al. 2002. Proc. Natl. Acad. Sci. USA 99:9509-9514). The α-ketoglutarate synthase gene, pyruvate synthase gene, and ferricredoxin gene were isolated from this strain, and plasmids expressing all three genes were constructed.

[0299] <1-1> Construction of an expression plasmid for the α-ketoglutarate synthase gene derived from *Sulphurella multocida*

[0300] Using genomic DNA from *Thiobacillus aeruginosa* strain TLS (ATCC49652) as a template, PCR was performed using the oligonucleotides shown in SEQ ID NO: 21 and 22 to amplify a gene fragment containing the α and β subunits of α-ketoglutarate synthase. The resulting gene fragment was cut with BamHI and inserted into the BamHI site of pSTV28 (manufactured by TAKARABio) to construct an α-ketoglutarate synthase gene expression plasmid, named pSTV-KGS. In this plasmid, the α-ketoglutarate synthase gene is located downstream of the pSV28-derived lac promoter and can be expressed by this promoter.

[0301] <1-2> Construction of expression plasmids for α-ketoglutarate synthase gene and pyruvate synthase gene derived from green sulfur bacteria

[0302] Using genomic DNA from *Thiobacillus aeruginosa* strain TLS (ATCC49652) as a template, PCR was performed using the oligonucleotides shown in SEQ ID NO: 23 and 24 to amplify the pyruvate synthase gene fragment. This gene fragment was then cut with XbaI and ligated to a fragment obtained by treating pSTV-KGS with XbaI to construct plasmids for expressing the α-ketoglutarate synthase gene and pyruvate synthase gene, named pSTV-KGS-PS.

[0303] <1-3> Construction of expression plasmids for α-ketoglutarate synthase gene, pyruvate synthase gene, and ferricoxin gene derived from *Sulphurella multocida*

[0304] Using genomic DNA from *Thiobacillus aeruginosa* strain TLS (ATCC49652) as a template, PCR was performed using the oligonucleotides shown in SEQ ID NO: 25 and 26 to amplify the ferredoxin I gene fragment. This gene fragment was digested with SmaI and ligated to a fragment obtained by treating pSTV-KGS-PS with SmaI, constructing a plasmid for expressing the α-ketoglutarate synthase gene, pyruvate synthase gene, and ferredoxin I gene, named pSTV-FdI-KGS-PS. In this plasmid, the α-ketoglutarate synthase gene, pyruvate synthase gene, and ferredoxin I are located downstream of the lac promoter of pSTV28 and can be expressed by this promoter.

[0305] [Example 2] <Construction of Escherichia coli α-ketoglutarate dehydrogenase-deficient strain>

[0306] <2-1> Construction of plasmids for disrupting the sucA gene in Escherichia coli

[0307] A strain of *Escherichia coli* MG1655 with a disrupted sucA gene encoding the E1 subunit of α-ketoglutarate dehydrogenase was prepared. Primers were synthesized based on the sucA gene sequence located at bases 757929–760730 in the genomic sequence (Genbank accession number U00096). Using genomic DNA of *E. coli* MG1655 as a template, the N-terminal and C-terminal fragments of the sucA gene were amplified by PCR. The sucA gene sequence of *E. coli* is shown in SEQ ID NO: 55, and the amino acid sequence of the E1 subunit encoded by this gene is shown in SEQ ID NO: 56. Oligonucleotides of SEQ ID NO: 42 and 43 were used as primers for PCR amplification of the N-terminal fragment, and oligonucleotides of SEQ ID NO: 44 and 45 were used as primers for PCR amplification of the C-terminal fragment. The oligonucleotide of SEQ ID NO: 42 is designed with a HindIII site, and the oligonucleotide of SEQ ID NO: 45 is designed with an XbaI site.

[0308] The amplified DNA fragments after PCR were purified using the QIAquick PCR Purification Kit (Quiagen). Using the purified N-terminal and C-terminal DNA fragments, and primers for SEQ ID NO: 42 and 45, a depleted sucA fragment was obtained via crossover PCR (Link, A.J. et al. 1997. J. Bacteriol. 179: 6228-6237). The purified DNA fragment was digested with HindIII and XbaI (TAKARABio) and cloned into the temperature-sensitive plasmid pMAN997 (Matsui, H. et al. 2001. Biosci. Biotechnol. Biochem. 65: 570-578; WO99 / 03988), which was also digested with HindIII and XbaI. This constructed a plasmid for sucA destruction and named pMANΔsucA.

[0309] <2-2> Construction of an α-ketoglutarate dehydrogenase-deficient strain derived from Escherichia coli MG1655

[0310] Escherichia coli MG1655 was transformed with plasmid pMANΔsucA. Colonies were selected on LB agar plates (containing 25 μg / mL ampicillin) at 30°C. The selected clones were incubated overnight at 30°C. The culture was then diluted 1000-fold and inoculated onto LB agar plates, where colonies were selected at 42°C. The selected clones were plated on LB agar plates and incubated at 30°C. One-eighth of the plate was then suspended in 2 mL of LB medium and incubated with shaking at 42°C for 4–5 hours. A 10,000-fold diluted bacterial culture was inoculated onto LB agar plates. By inoculating hundreds of colonies onto both LB and LB agar plates and confirming growth, ampicillin-sensitive strains were selected. Colony PCR was performed on several ampicillin-sensitive strains, confirming the deletion of the sucA gene. This yields the sucA-deleted strain MG1655ΔsucA, derived from Escherichia coli strain MG1655.

[0311] [Example 3] <Construction of Escherichia coli lactate dehydrogenase-deficient strain>

[0312] Lactate dehydrogenase is an enzyme that uses NADH as a coenzyme to produce lactate from pyruvate. To inhibit lactate production in *E. coli* cultures under anaerobic conditions, a strain encoding the ldhA gene for lactate dehydrogenase was constructed. This gene deletion was achieved using a method called "Red-driven integration" developed by Datsenko and Wanner (Datsenko, KA, and Wanner, BL2000. Proc. Natl. Acad. Sci. USA. 97: 6640-6645) combined with a λ phage-derived excision system (Cho, E Hetal. 2002. J. Bacteriol. 184: 5200-5203). Using this method, a synthetic oligonucleotide obtained by designing a portion of the target gene to its 5' side and a portion of an antibiotic resistance gene to its 3' side was used as primers to obtain PCR products. These PCR products enabled the one-step construction of a gene-damaged strain. By further combining the use of excision systems derived from λ phage, it is possible to remove antibiotic resistance genes from introduced gene-damaging strains.

[0313] <3-1> Construction of a strain with the ldhA gene missing, encoding lactate dehydrogenase

[0314] According to WO2005 / 010175, PCR was performed using the following synthetic oligonucleotides as primers and plasmid pMW118-attL-Cm-attR as a template. These synthetic oligonucleotides have a sequence corresponding to a portion of the ldhA gene at their 5' end and sequences corresponding to the respective ends of attL and attR of λ phage at their 3' end. The sequences of the synthetic oligonucleotides used as primers are shown in SEQ ID NO: 27, 28. The amplified PCR product was purified using agarose gel electroporation and introduced into *E. coli* strain MG1655ΔsucA, which carries the temperature-sensitive replication plasmid pKD46. Then, ampicillin-sensitive strains with plasmid pKD46 detached were obtained, and the ldhA gene deletion was confirmed by PCR. Furthermore, to remove the att-cat gene introduced into the ldhA gene, transformation was performed using the helper plasmid pMW-intxis-ts, and ampicillin-resistant strains were selected. In addition, the plasmid pMW-intxis-ts has an integrase (Int) gene and an excisionase (Xis) gene derived from λ phage, and its replication is temperature-sensitive.

[0315] Then, ldhA-damaged strains detached from att-cat and pMW-intxis-ts were obtained using ampicillin and chloramphenicol sensitivity tests. Genomic DNA was prepared from the obtained ldhA-deficient candidate strains, and PCR was performed using the oligonucleotides shown in SEQ ID NO: 29 and 30. The results were analyzed by electrophoresis. For one ldhA-deficient strain, a band approximately 1.0 kb smaller than the band observed during PCR using genomic DNA prepared from MG1655ΔsucA strain as a template was identified. This ldhA-deficient strain was named MG1655ΔsucAΔldhA strain.

[0316] [Example 4] <Determination of α-ketoglutarate synthase activity in strains expressing the α-ketoglutarate synthase gene from *S. aeruginosa*>

[0317] To confirm the active expression of the α-ketoglutarate synthase gene from *S. aeruginosa* in *Escherichia coli*, an expression vector for the α-ketoglutarate synthase gene from *S. aeruginosa* was constructed, introduced into MG1655ΔsucA, and its activity was measured.

[0318] <4-1> Construction of an expression plasmid for the α-ketoglutarate synthase gene derived from green sulfur bacteria

[0319] Using genomic DNA from *Thiobacillus aeruginosa* strain TLS (ATCC49652) as a template, the α-ketoglutarate synthase gene fragment was amplified by PCR using the oligonucleotides shown in SEQ ID NO: 21 and 22. The resulting gene fragment was cut with BamHI and inserted into the BamHI site of pUC18 (manufactured by TAKARABio) to construct an α-ketoglutarate synthase gene expression plasmid, named pUC-KGS. In this plasmid, the α-ketoglutarate synthase gene is located downstream of the lac promoter of pUC18 and can be expressed by this promoter. Furthermore, the copy number of pUC18 is higher than that of pSTV28, making enzyme activity detection easier.

[0320] <4-2> Preparation of crude enzyme solution from α-ketoglutarate synthase gene expression strains derived from *Sulphurella multocida*

[0321] The pUC-KGS and pUC18 vectors used as a control were introduced into MG1655ΔsucA via electroporation, and transformants were obtained using ampicillin resistance as an indicator. After confirming that the plasmids had been introduced, the α-ketoglutarate synthase gene expression strain derived from *Thiopyridamole* was named MG1655ΔsucA / pUC-KGS, and the control strain was named MG1655ΔsucA / pUC18.

[0322] The above-mentioned strain was inoculated into LB medium containing 1 mM IPTG and cultured overnight at 37°C. The bacterial cells, equivalent to 10 ml, were recovered by centrifugation and resuspended in 300 μl of 50 mM HEPES buffer (pH 8.0). The suspension was then disrupted using an ultrasonic homogenizer, and the supernatant obtained after centrifugation at 15000 rpm for 15 minutes was used as the crude enzyme solution.

[0323] <4-3> Determination of α-ketoglutarate synthase activity in crude enzyme solutions of strains expressing the α-ketoglutarate synthase gene from *S. aeruginosa*

[0324] The reaction was initiated by adding 50 μl of crude enzyme solution to 1 ml of the reaction solution shown below. First, the reaction solution containing all components except the substrate α-ketoglutarate was added to the cuvette, and the cuvette was sealed with a rubber stopper and aluminum cap. Argon gas was blown into the cuvette for 5 minutes using a syringe to reduce the oxygen concentration. The cuvette was placed on a spectrophotometer (Hitachi U-3210 Spectrophotometer), and α-ketoglutarate solution was added using a syringe to start the reaction. The reaction was carried out at 37°C for 30 minutes, and the absorbance at 578 nm was measured periodically to track changes in the amount of reduced methylviologen.

[0325] [Reaction solution]

[0326] MgCl2 1mM

[0327] Dithiothreitol 1mM

[0328] Methyl viologen 5mM

[0329] CoA 0.25mM

[0330] α-Ketoglutaric acid 10mM (add just before the assay begins)

[0331] HEPES (pH 8.0) 50mM

[0332] The results are shown in Table 1. In the table, KGS represents α-ketoglutarate synthase. In the above reaction system, reduced methyl viologen increased due to the decarbonation of α-ketoglutarate catalyzed by α-ketoglutarate synthase. The absorbance coefficient of reduced methyl viologen at 578 nm was ε578 = 9.8 / mM / cm. 1 U of enzyme activity is expressed as the amount of methyl viologen reduced per minute (1 μmol / m). No α-ketoglutarate synthase activity was detected in the control strain MG1655ΔsucA / pUC18, but an activity of 0.1 U / mg was confirmed in the α-ketoglutarate synthase gene expression strain MG1655ΔsucA / pUC18-KGS derived from *Thiopyridae*.

[0333] Table 1

[0334] strain KGS activity (U / mg) MG1655ΔsucA / pUC18 0.00 MG1655ΔsucA / pUC-KGS 0.10

[0335] [Example 5] <Effects of α-ketoglutarate synthase gene, pyruvate synthase gene, and ferricoxin gene expression strains derived from *S. aeruginosa* on L-glutamate production capacity under oxygen-limited conditions with glucose as the carbon source>

[0336] To investigate the effect of α-ketoglutarate synthase expression on L-glutamate production under oxygen-limited conditions, the α-ketoglutarate synthase gene, pyruvate synthase gene, and ferricodin gene from *S. aeruginosa* were expressed using plasmid pSTV-KGS-PS-FdI and introduced into MG1655ΔsucAΔldhA, followed by culture.

[0337] The pSTV-FdI-KGS-PS vector and the control vector pSTV28 were introduced into MG1655ΔsucAΔldhA via electroporation, and transformants were obtained using chloramphenicol resistance as an indicator. After confirming the plasmid introduction, the strain expressing the α-ketoglutarate synthase gene, pyruvate synthase gene, and ferricoxin gene from *Thiopyrum aeruginosa* was named MG1655ΔsucAΔldhA / pSTV-FdI-KGS-PS, and the control strain was named MG1655ΔsucAΔldhA / pSTV28.

[0338] The L-glutamic acid production capacity was studied by culturing the strain prepared above.

[0339] MG1655ΔsucAΔldhA / pSTV-FdI-KGS-PS and the control strain MG1655ΔsucAΔldhA / pSTV28 were inoculated into LB medium and cultured overnight at 37°C. One-sixth plate volume of the bacterial culture was then inoculated into 50 ml of glucose medium (the following composition) in a 500 ml Erlenmeyer flask and cultured at 37°C for 31 hours. To implement oxygen restriction, the culture was carried out with a stirring speed of 100 rpm. After culture, the accumulated L-glutamate in the medium was determined using a Biotech Analyzer (Asahi Kasei Corporation).

[0340] [Composition of glucose culture medium]

[0341] Glucose 40g / L

[0342] MgSO4·7H2O 1.0 g / L

[0343] (NH4)2SO4 20g / L

[0344] KH2PO4 1.0 g / L

[0345] Yeast extract 2.0g / L

[0346] FeSO4·7H2O 0.01g / L

[0347] MnSO4·5H2O 0.01g / L

[0348] Thiamine hydrochloride 0.01 g / L

[0349] Chloramphenicol 25mg / L

[0350] Calcium carbonate 50g / L

[0351] Sterilization conditions: pH 7.0 (adjusted with KOH), 120℃, 20 minutes.

[0352] The results are shown in Table 2. Compared with the control MG1655ΔsucAΔldhA / pSTV28, the L-glutamate yield relative to consumed glucose was increased by 0.6% in the MG1655ΔsucAΔldhA / pSTV-FdI-KGS-PS vector containing the expression vectors for α-ketoglutarate synthase, pyruvate synthase, and ferricoxin genes derived from *Thalassiopeia aurea*.

[0353] Table 2

[0354]

[0355] [Example 6] <Effects of α-ketoglutarate synthase gene, pyruvate synthase gene, and ferricoxin gene expression strains derived from *S. aeruginosa* on L-glutamate production capacity using glycerol as a carbon source>

[0356] The L-glutamic acid production capacity using glycerol as a carbon source was investigated using α-ketoglutarate synthase gene, pyruvate synthase gene, and ferricredoxin gene expression strain MG1655ΔsucAΔldhA / pSTV-FdI-KGS-PS and control strain MG1655ΔsucAΔldhA / pSTV28 derived from *Sulphurella multocida*.

[0357] MG1655ΔsucAΔldhA / pSTV-FdI-KGS-PS and the control strain MG1655ΔsucAΔldhA / pSTV28 were inoculated into LB medium and cultured overnight at 37°C. Half a plate of cells was then inoculated into 20 ml of glycerol medium (the following composition) in a 500 ml Sakaguchi flask and cultured aerobically at 37°C and 120 rpm for 29 hours. After culture, the accumulated L-glutamate in the medium was determined using a Biotech Analyzer (Asahi Kasei Corporation).

[0358] [Composition of glycerol culture medium]

[0359] 50g / L of glycerin

[0360] MgSO4·7H2O 1.0 g / L

[0361] (NH4)2SO4 20g / L

[0362] KH2PO4 1.0 g / L

[0363] Yeast extract 2.0g / L

[0364] FeSO4·7H2O 0.01g / L

[0365] MnSO4·5H2O 0.01g / L

[0366] Thiamine hydrochloride (Thiamine HCl) 0.01 g / L

[0367] Chloramphenicol 25mg / L

[0368] Calcium carbonate 30g / L

[0369] Sterilization conditions: pH 7.0 (adjusted with KOH), 120℃, 20 minutes.

[0370] The results are shown in Table 3. Compared with the control MG1655ΔsucAΔldhA / pSTV28, the L-glutamate yield relative to consumed glycerol was increased by 6% in MG1655ΔsucAΔldhA / pSTV-FdI-KGS-PS, which introduced the expression vectors for α-ketoglutarate synthase, pyruvate synthase, and ferricredoxin from *Thalassiopeia aurea*.

[0371] Table 3

[0372]

[0373] [Example 7] <α-ketoglutarate synthase gene from *Sulphurella multocida*, flavin-NADP from *Escherichia coli*> + Construction of reductase gene and expression plasmids for ferricoxin / flavin reductin derived from E. coli >

[0374] As a coenzyme regeneration system essential for α-ketoglutarate synthase activity, flavin-NADP derived from E. coli is used. + A plasmid was constructed to simultaneously express the reductase gene and the flavin-NADP gene from *E. coli*. + The reductase gene used was the fpr gene; the flavin oxidoreductin gene of E. coli used the fldA and fldB genes; and the ferreductin gene used the fdx and yfhL genes.

[0375] <7-1> Construction of an expression plasmid for the α-ketoglutarate synthase gene derived from green sulfur bacteria

[0376] The pSTV-KGS constructed in Example 1 was cut with BamHI, and the resulting α-ketoglutarate synthase gene fragment was inserted into the BamHI site of pMWPthr to construct the α-ketoglutarate synthase gene expression vector pMWPthr-KGS. The plasmid pMWPthr has a promoter region (Pthr) of the threonine operon (thrABC) located between the HindIII and XbaI sites in the vector pMW118 (manufactured by NIPPONGENE), as shown in SEQ ID NO: 31, in the genome sequence of E. coli strain K-12 (Genbank accession number U00096). When the gene is cloned downstream of this promoter, the plasmid can express the gene.

[0377] <7-2> E. coli-derived flavin-NADP + Construction of reductase gene amplification vector

[0378] Using genomic DNA of Escherichia coli strain MG1655 as a template, flavin-NADP was amplified by PCR using the oligonucleotides shown in SEQ ID NO: 32 and 33. + A reductase gene fragment was obtained. This gene fragment was cut with SmaI and inserted into the SmaI site of pMWPthr to construct flavin-NADP. + The plasmid used for amplifying the reductase gene was named pMWPthr-fpr.

[0379] <7-3> Construction of an amplification vector for the flavin redoxin (fldA) gene derived from Escherichia coli

[0380] Using genomic DNA from *Escherichia coli* strain MG1655 as a template, the flavin redoxin (fldA) gene fragment was amplified by PCR using the oligonucleotides shown in SEQ ID NO: 34 and 35. This gene fragment was then cut with EcoRI and inserted into the EcoRI site of pMWPthr to construct the plasmid pMWPthr-fldA for flavin redoxin (fldA) gene amplification.

[0381] <7-4> Construction of a plasmid for amplifying the flavin redoxin (fldB) gene from *E. coli*

[0382] Using genomic DNA from *Escherichia coli* strain MG1655 as a template, the flavin redoxin (fldB) gene fragment was amplified by PCR using the oligonucleotides shown in SEQ ID NO: 36 and 37. This gene fragment was then cut with EcoRI and inserted into the EcoRI site of pMWPthr to construct the plasmid pMWPthr-fldB for flavin redoxin (fldB) gene amplification.

[0383] <7-5> Construction of a plasmid for amplifying the ferroredoxin (fdx) gene from E. coli

[0384] Using genomic DNA from Escherichia coli strain MG1655 as a template, the ferricodoxin (fdx) gene fragment was amplified by PCR using the oligonucleotides shown in SEQ ID NO: 38 and 39. This gene fragment was then cut with EcoRI and inserted into the EcoRI site of pMWPthr to construct the plasmid pMWPthr-fdx for ferricodoxin (fdx) gene amplification.

[0385] <7-6> Construction of a plasmid for amplifying the ferroredoxin (yfhL) gene from *E. coli*

[0386] Using genomic DNA from *Escherichia coli* strain MG1655 as a template, the ferritin (yfhL) gene fragment was amplified by PCR using the oligonucleotides shown in SEQ ID NO: 40 and 41. This gene fragment was then cut with EcoRI and inserted into the EcoRI site of pMWPthr, thereby constructing the plasmid pMWPthr-yfhL for ferritin (yfhL) gene amplification.

[0387] <7-7> Used for expressing the α-ketoglutarate synthase gene from *Sulphurella multocida* and for amplifying ferrugin-NADP from *E. coli*. + Construction of plasmids for reductase gene and flavin reductase (fldA) gene

[0388] pMWPthr-fldA was digested with EcoRI, and the resulting fldA gene fragment was ligated with the fragment obtained from treating pMWPthr-KGS with EcoRI to obtain pMWPthr-KGS-fldA. Then, pMWPthr-fpr was digested with SmaI, and the resulting fpr gene fragment was ligated with the fragment obtained from treating pMWPthr-KGS-fldA with SmaI, thus constructing a gene for expressing α-ketoglutarate synthase and enhancing flavin-NADP from *E. coli*. + The plasmid pMWPthr-KGS-fpr-fldA expresses the reductase gene and the flavin oxidoreductin (fldA) gene.

[0389] <7-8> Used to express the α-ketoglutarate synthase gene from *Sulphurella multocida* and amplify flavin-NADP from *Escherichia coli*. + Construction of plasmids for reductase gene and flavin reductase (fldB) gene

[0390] pMWPthr-fldB was digested with EcoRI, and the resulting fldB gene fragment was ligated with the fragment obtained by treating pMWPthr-KGS with EcoRI to obtain pMWPthr-KGS-fldB. Then, pMWPthr-fpr was digested with SmaI, and the resulting fpr gene fragment was ligated with the fragment obtained by treating pMWPthr-KGS-fldB with SmaI to construct a gene for enhancing α-ketoglutarate synthase and flavin-NADP. + The plasmid pMWPthr-KGS-fpr-fldB expresses the reductase gene and the flavin oxidoreductin (fldB) gene.

[0391] <7-9> Used to express the α-ketoglutarate synthase gene from *Sulphurella multocida* and amplify flavin-NADP from *Escherichia coli*. +Construction of plasmids for reductase gene and ferricoxin (fdx) gene

[0392] pMWPthr-fdx was digested with EcoRI, and the resulting fdx gene fragment was ligated with the fragment obtained by treating pMWPthr-KGS with EcoRI to obtain pMWPthr-KGS-fdx. Then, pMWPthr-fpr was digested with SmaI, and the resulting fpr gene fragment was ligated with the fragment obtained by treating pMWPthr-KGS-fdx with SmaI to construct a gene for enhancing α-ketoglutarate synthase and flavin-NADP. + The vector pMWPthr-KGS-fpr-fdx for expressing the reductase gene and ferroredoxin (fdx) gene.

[0393] <7-10> Used to express the α-ketoglutarate synthase gene from *Sulphurella multocida* and amplify flavin-NADP from *Escherichia coli*. + Construction of plasmids for reductase gene and ferricoxin (yfhL) gene

[0394] pMWPthr-yfhL was digested with EcoRI, and the resulting yfhL gene fragment was ligated with the fragment obtained by treating pMWPthr-KGS with EcoRI to obtain pMWPthr-KGS-yfhL. Then, pMWPthr-fpr was digested with SmaI, and the resulting fpr gene fragment was ligated with the fragment obtained by treating pMWPthr-KGS-yfhL with SmaI to construct a gene for enhancing α-ketoglutarate synthase and flavin-NADP. + The plasmid pMWPthr-KGS-fpr-yfhL expresses the reductase gene and the ferroredoxin (yfhL) gene.

[0395] Among the plasmids mentioned above, the α-ketoglutarate synthase gene from *Sulphurella multocida* is transcribed via Pthr, while other genes are transcribed via read-through from Pthr.

[0396] [Example 8] <α-ketoglutarate synthase gene from *Sulphurella multocida*, flavin-NADP from *Escherichia coli*> + The effects of enhanced expression of reductase genes and flavin-oxidoreductin / ferreductin genes from *E. coli* on L-glutamate production capacity using glycerol as a carbon source >

[0397] To investigate flavin-NADP derived from E. coli +The effect of enhanced α-ketoglutarate synthase activity from *S. aeruginosa* caused by amplification of reductase genes and *E. coli*-derived flavin-redoxin or ferroredoxin genes on L-glutamate production under oxygen-limited conditions, along with the aforementioned methods for enhancing *S. aeruginosa*-derived α-ketoglutarate synthase and *E. coli*-derived flavin-redoxin-NADP... + The reductase gene and the vectors expressing the flavin oxoreductin or ferroreductin gene from E. coli were introduced into MG1655ΔsucAΔldhA and cultured.

[0398] <8-1> The gene for amplifying α-ketoglutarate synthase from *Sulphurella multocida* and flavin-NADP from *Escherichia coli* will be used. + Plasmids containing reductase genes and flavin-oxidoreductin-ferroreductin genes from *E. coli* were introduced into strain MG1655ΔsucAΔldhA.

[0399] The following vectors were introduced via electroporation into MG1655ΔsucAΔldhA: pMWPthr-KGS, for expressing the α-ketoglutarate synthase gene from *Thiobacillus aeruginosa*, and pMWPthr-KGS, for enhancing the α-ketoglutarate synthase gene and flavin-NADP from *E. coli*. + The reductase gene and the expression plasmids pMWPthr-KGS-fpr-fldA, pMWPthr-KGS-fpr-fldB, pMWPthr-KGS-fpr-fdx, or pMWPthr-KGS-fpr-yfhL of flavin oxidoreductin-NADP from *E. coli* were used as controls. + The reductase gene was amplified using the plasmid pMWPthr-fpr. Transformants were obtained using kanamycin resistance as an indicator. After confirming the plasmid introduction, the introduced strains were named MG1655ΔsucAΔldhA / pMWPthr-KGS, MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fldA, MG1655ΔsucAΔldhA / pMWPthrKGS-fpr-fldB, MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fdx, MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-yfhL, MG1655ΔsucAΔldhA / pMWPthr, and MG1655ΔsucAΔldhA / pMWPthr-fpr.

[0400] <8-2> α-Ketoglutarate synthase gene from *Sulphurella multocida* and flavin-NADP from *Escherichia coli*. +Effects of enhanced expression of reductase gene and flavin-oxidoreductin-ferroreductin gene from *E. coli* on L-glutamate production capacity using glycerol as a carbon source.

[0401] The α-ketoglutarate synthase gene expression strain MG1655ΔsucAΔldhA / pMWPthr-KGS derived from *Thiobacillus aeruginosa*, prepared in <8-1>, was used. The α-ketoglutarate synthase gene from *Thiobacillus aeruginosa* and flavin-NADP from *Escherichia coli* were also used. + The reductase gene, the enhanced expression strains of flavin-oxidoreductin-ferroreductin from *E. coli* MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fldA, MG1655ΔsucAΔldhA / pMWPthrKGS-fpr-fldB, MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fdx, MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-yfhL, the control strain MG1655ΔsucAΔldhA / pMWPthr, and flavin-oxidoreductin-NADP from *E. coli* + The L-glutamate production capacity under aerobic conditions was investigated using the reductase gene amplification strain MG1655ΔsucAΔldhA / pMWPthr-fpr with glycerol as the carbon source.

[0402] Half the volume of each strain of bacteria, equivalent to one plate of a plate obtained from an overnight pre-culture at 37°C using LB medium, was inoculated into 20 mL of medium (a glycerol medium prepared by replacing chloramphenicol with 40 mg / L kanamycin on the basis of the glycerol medium described in Example 6) in a 500 mL volumetric flask and cultured at 37°C and 120 rpm for 29 hours. After culture, the L-glutamate accumulated in the medium was determined using a Biotech Analyzer (manufactured by Asahi Kasei Corporation).

[0403] The results are shown in Table 4. Compared with the control MG1655ΔsucAΔldhA / pMWPthr and E. coli-derived flavin-NADP... + Compared to the amplified reductase gene strain MG1655ΔsucAΔldhA / pMWPthr-fpr, the α-ketoglutarate synthase gene expression strain MG1655ΔsucAΔldhA / pMWPthr-KGS from *Thiopyridae* showed a 3% increase in L-glutamate yield relative to glycerol consumption; while the α-ketoglutarate synthase gene from *Thiopyridae* and the flavin-NADP gene from *E. coli* showed a 3% increase in L-glutamate yield relative to glycerol consumption. +Increased yields of 8–9% were observed in the reductase gene and the flavin oxoreductin / ferreductin gene expression-enhanced strains MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fldA, MG1655ΔsucAΔldhA / pMWPthrKGS-fpr-fldB, MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fdx, and MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-yfhL.

[0404] Table 4

[0405]

[0406] [Example 9] <α-ketoglutarate synthase gene from *Sulphurella multocida*, flavin-NADP from *Escherichia coli*> + The effects of introducing plasmids containing reductase genes and flavin-oxidoreductin-ferroreductin genes from *E. coli* on L-glutamate production capacity under oxygen-limited conditions with glucose as the carbon source >

[0407] For the control MG1655ΔsucAΔldhA / pMWPthr constructed in Example 8, the α-ketoglutarate synthase gene from *Thiobacillus aeruginosa* and the flavin-NADP from *Escherichia coli* were used. + The reductase gene and the flavin-oxidoreductin-ferroreductin gene expression-enhanced strains MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fldA, MG1655ΔsucAΔldhA / pMWPthrKGS-fpr-fldB, MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fdx, and MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-yfhL were pre-cultured overnight at 37°C using LB medium. The bacterial cells, equivalent to 1 / 6 of the plate volume, were inoculated into 50 mL of medium (based on the glucose medium described in Example 5, with chloramphenicol replaced by 40 mg / L kanamycin) in a 500 mL Erlenmeyer flask and cultured at 37°C for 38 hours. For oxygen restriction, the culture was carried out with a stirring speed of 100 rpm. After the culture was completed, the amount of L-glutamic acid accumulated in the culture medium was determined using Biotech Analyzer (Asahi Kasei Corporation).

[0408] The results are shown in Table 5. Compared with the control MG1655ΔsucAΔldhA / pMWPthr, the α-ketoglutarate synthase gene from *Thiobacillus aeruginosa* and the flavin-NADP gene from *Escherichia coli* showed significant differences.+ The yield of L-glutamic acid was increased in the reductase gene and in the flavin oxoreductin / ferreductin gene expression-enhanced strains MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fldA, MG1655ΔsucAΔldhA / pMWPthrKGS-fpr-fldB, MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fdx, and MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-yfhL.

[0409] Table 5

[0410]

[0411] [Example 10] <α-ketoglutarate synthase gene from *Sulphurella multocida*, flavin-NADP from *Escherichia coli*> + The effects of enhanced expression of reductase genes and flavin reductin from *E. coli* on L-glutamate production capacity using glucose as a carbon source >

[0412] The α-ketoglutarate synthase gene from *Sulphurella multocida* prepared in Example 8 and the flavin-NADP from *Escherichia coli* were used. + The L-glutamate production capacity under aerobic conditions with glucose as the carbon source was investigated by comparing the expression-enhanced strain MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fldA with the control strain MG1655ΔsucAΔldhA / pMWPthr and the reductase gene and the flavin oxidoreductin gene derived from E. coli.

[0413] Each strain was inoculated with 1 / 4 the volume of cells from a plate obtained by overnight pre-culture at 37°C using LB medium into 20 mL of glucose + methionine medium in a 500 mL volumetric flask (the glucose medium described in Example 5 was prepared by replacing chloramphenicol with 40 mg / L kanamycin and adding 0.2 g / L DL-methionine). The medium was incubated at 37°C and 120 rpm for 25 hours. After incubation, the accumulated L-glutamate in the medium was determined using a Biotech Analyzer (manufactured by Asahi Kasei Corporation).

[0414] The results are shown in Table 6. Compared with the control MG1655ΔsucAΔldhA / pMWPthr, the α-ketoglutarate synthase gene from *Thiobacillus aeruginosa* and the flavin-NADP gene from *Escherichia coli* showed significant differences. +In the recombinant strain MG1655ΔsucAΔldhA / pMWPthr-KGS-fpr-fldA with enhanced expression of the reductase gene and the flavodoxin gene derived from Escherichia coli, the yield was confirmed to have increased by approximately 6%.

[0415] Table 6

[0416]

[0417] 〔Example 11〕<Effect of the recombinant strain with enhanced expression of the α-ketoglutarate synthase gene derived from Chlorobium limicola, the flavodoxin-NADP reductase gene derived from Escherichia coli, and the flavodoxin gene derived from Escherichia coli on the L-glutamic acid production ability when using glycerol as a carbon source> +

[0418] To study the effect of enhanced α-ketoglutarate synthase activity in Pantoea ananatis on L-glutamic acid production, the plasmid pMWPthr-KGS-fpr-fldA carrying the α-ketoglutarate synthase gene derived from Chlorobium limicola, the flavodoxin-NADP reductase gene derived from Escherichia coli, and the flavodoxin gene derived from Escherichia coli prepared in Example 7 and the control plasmid pMWPthr were introduced into the L-glutamic acid-producing bacterium Pantoea ananatis, and the bacteria were cultured. +

[0419] <11-1>Construction of the L-glutamic acid-producing bacterium Pantoea ananatis

[0420] The plasmid RSFCPG (refer to the specification of European Patent Application Publication No. 1233068) carrying the citrate synthase gene (gltA), phosphoenolpyruvate carboxylase gene (ppc), and glutamate dehydrogenase gene (gdhA) derived from Escherichia coli was treated with BglII and KpnI, made blunt-ended, and then ligated to remove the gltA gene. The resulting plasmid was used to transform Escherichia coli JM109 strain. The plasmid was extracted from the obtained transformant and named pRSF-ppc-gdhA. The plasmid pRSF-ppc-gdhA was introduced into the L-glutamic acid-producing bacterium Pantoea ananatis NP106 strain to construct NP106 / pRSF-ppc-gdhA.

[0421] ​​The NP106 strain was obtained as follows: The Pantoeaananatis AJ13601 strain from the previous example was cultured overnight in LBGM9 liquid medium at 34°C with shaking. The culture was then diluted to 100–200 colonies per plate and plated onto LBGM9 plates containing 12.5 mg / L tetracycline. Colonies that appeared were copied onto LBGM9 plates containing 12.5 mg / L tetracycline and 25 mg / L chloramphenicol. Chloramphenicol-sensitive strains were selected, thus obtaining the pSTVCB-exfoliated strain, which was named G106S. Furthermore, the G106S strain was cultured overnight in LBGM9 liquid medium at 34°C with shaking. The culture was then diluted to 100–200 colonies per plate and plated onto drug-free LBGM9 plates. The resulting colonies were cloned onto LBGM9 plates containing 12.5 mg / L tetracycline and LBGM9 plates without the drug. Tetracycline-sensitive strains were selected, thus obtaining strains with detached RSFCPG plasmids, which were named NP106. The NP106 strain obtained in this way does not carry the RSFCPG and pSTVCB plasmids carried by strain AJ13601.

[0422] <11-2> Incorporate α-ketoglutarate synthase gene from *Sulphurella multocida* and flavin-NADP from *Escherichia coli*. + The reductase gene and flavin reductase (fldA) gene were expressed by introducing a plasmid into the NP106 / pRSF-ppc-gdhA strain.

[0423] Electroporation was used to introduce, respectively, α-ketoglutarate synthase gene enhancer and E. coli-derived flavin-NADP into NP106 / pRSF-ppc-gdhA. + The reductase gene and the flavin-oxidoreductin-ferroreductin gene from *E. coli*, expressed by the plasmid pMWPthr-KGS-fpr-fldA, along with the control plasmid pMWPthr, were used to obtain transformants, with resistance to both kanamycin and tetracycline as indicators. After confirming the introduction of the plasmids, the introduced strains were named NP106 / pRSF-ppc-gdhA / pMWPthr-KGS-fpr-fldA and NP106 / pRSF-ppc-gdhA / pMWPthr, respectively.

[0424] <11-3> Using glycerol as a carbon source, α-ketoglutarate synthase gene from *S. green sulfur* and flavin-NADP from *E. coli* were cultured under aerobic conditions. + The reductase gene and flavin reductin (fldA) gene amplification plasmid were introduced into the strain.

[0425] For the two strains constructed in <11-1>, a pre-culture was performed overnight at 37°C using LBMG medium (LB medium supplemented with 0.5 g / L glucose, 2 mM MgSO4, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, and 6 g / L Na2HPO4). One-quarter plate volume of bacterial cells was inoculated into 20 ml of pantothenic acid glycerol medium (the following composition) in a 500 ml Sakaguchi flask and incubated at 34°C for 48 hours at 120 rpm. After incubation, the accumulated L-glutamate in the medium was determined using a Biotech Analyzer (Asahi Kasei Corporation).

[0426] [Composition of glycerol culture medium for pan-based bacteria]

[0427] Glycerin 40g / L

[0428] MgSO4·7H2O 1.0 g / L

[0429] (NH4)2SO4 20g / L

[0430] KH2PO4 2.0g / L

[0431] Yeast extract 2.0g / L

[0432] FeSO4·7H2O 0.02g / L

[0433] MnSO4·5H2O 0.02g / L

[0434] Thiamine hydrochloride 0.01 g / L

[0435] L-Lysine 0.2g / L

[0436] DL-Diaminopimelic acid 0.2 g / L

[0437] L-methionine 0.2 g / L

[0438] Kanamycin 40 mg / L

[0439] Tetracycline 2.5 mg / L

[0440] Calcium carbonate 20g / L

[0441] Sterilization conditions: pH 7.0 (adjusted with KOH), 120℃, 20 minutes.

[0442] The results are shown in Table 7. Compared with the control NP106 / pRSF-ppc-gdhA / pMWPthr, the α-ketoglutarate synthase gene from *Thiobacillus aeruginosa* and the flavin-NADP gene from *Escherichia coli* showed significant differences. +In the reductase gene and the flavodoxin gene expression-enhanced strain NP106 / pRSF-ppc-gdhA / pMWPthr-KGS-fpr-fldA derived from Escherichia coli, it was confirmed that the L-glutamic acid yield increased by about 3%.

[0443] Table 7

[0444]

[0445] 〔Example 12〕<Effect of the α-ketoglutarate synthase gene expression-enhanced strain derived from Chlorobium on the L-glutamic acid production ability under aerobic conditions when using glucose as a carbon source>

[0446] Using the strain MG1655ΔsucAΔldhA / pMWPthr-KGS in which only the α-ketoglutarate synthase gene derived from Chlorobium was enhanced and the control strain MG1655ΔsucAΔldhA / pMWPthr prepared in Example 8, the L-glutamic acid production ability under aerobic conditions when using glucose as a carbon source was investigated.

[0447] The cells of each strain equivalent to 1 / 4 plate amount of the plate obtained by pre-culturing overnight at 37°C in LB medium were inoculated into 20 mL of glucose + methionine medium (in the glucose medium described in Example 5, the glucose concentration was 35.0 g / L, 40 mg / L of kanamycin was added instead of chloramphenicol, and 0.2 g / L of DL-methionine was added) in a 500 mL Sakaguchi flask, and cultured at 37°C and a stirring speed of 120 rpm for 23 hours. After the culture was completed, the L-glutamic acid accumulated in the medium was measured using a Biotech Analyzer (manufactured by Asahi Kasei Corporation).

[0448] The results are shown in Table 8. Compared with the control MG1655ΔsucAΔldhA / pMWPthr, in the strain MG1655ΔsucAΔldhA / pMWPthr-KGS in which only the expression of the α-ketoglutarate synthase gene derived from Chlorobium was enhanced, it was confirmed that the yield increased by about 6%.

[0449] Table 8

[0450]

[0451] 〔Example 13〕<Effect of the α-ketoglutarate synthase gene-enhanced strain derived from Chlorobium in Pantoea ananatis on the L-glutamic acid production ability under aerobic culture conditions when using glycerol as a carbon source>

[0452] To investigate the effect of enhanced α-ketoglutarate synthase activity in Pantoeaananatis on L-glutamate production, the α-ketoglutarate synthase gene expression plasmid pMWPthr-KGS from *Sulphurella vulgaris* prepared in Example 7 and the control plasmid pMWPthr were introduced into *Pantoeaananatis* L-glutamate producing bacteria and cultured.

[0453] The α-ketoglutarate synthase gene expression enhancement plasmid pMWPthr-KGS and the control plasmid pMWPthr, prepared in Example 7, were introduced into NP106 / pRSF-ppc-gdhA prepared in Example 11 via electroporation. Transformants were obtained using resistance to both kanamycin and tetracycline as indicators. After confirming successful plasmid introduction, the introduced strains were named NP106 / pRSF-ppc-gdhA / pMWPthr-KGS and NP106 / pRSF-ppc-gdhA / pMWPthr, respectively.

[0454] NP106 / pRSF-ppc-gdhA / pMWPthr-KGS and NP106 / pRSF-ppc-gdhA / pMWPthr were cultured overnight at 37°C using LBMG. One-quarter plate volume of the bacterial cells was inoculated into 20 mL of pantothenic acid glycerol medium (the following composition) in a 500 mL Sakaguchi flask and incubated at 34°C for 66 hours at 100 rpm. After incubation, the accumulated L-glutamate in the medium was determined using a Biotech Analyzer (Asahi Kasei Corporation).

[0455] [Composition of glycerol culture medium for pan-based bacteria]

[0456] 30g / L of glycerin

[0457] MgSO4·7H2O 0.5 g / L

[0458] (NH4)2SO4 20g / L

[0459] KH2PO4 2.0g / L

[0460] Yeast extract 2.0g / L

[0461] FeSO4·7H2O 0.02g / L

[0462] MnSO4·5H2O 0.02g / L

[0463] Calcium pantothenate 18 mg / L

[0464] Thiamine hydrochloride 0.01 g / L

[0465] L-Lysine 0.2g / L

[0466] DL-Diaminopimelic acid 0.2 g / L

[0467] L-Methionine 0.2 g / L

[0468] Kanamycin 40 mg / L

[0469] Tetracycline 12.5 mg / L

[0470] Calcium carbonate 20 g / L

[0471] pH 7.0 (adjusted with KOH) Sterilization conditions: 115 °C, 10 minutes

[0472] The results are shown in Table 9. Compared with the control NP106 / pRSF-ppc-gdhA / pMWPthr, in the enhanced strain of α-ketoglutarate synthase gene expression derived from Chlorobium limicola, NP106 / pRSF-ppc-gdhA / pMWPthr-KGS, it was confirmed that the L-glutamic acid yield increased by about 8%.

[0473] Table 9

[0474]

[0475] 〔Example 14〕<Effect of an enhanced strain of α-ketoglutarate synthase gene derived from Chlorobium limicola in Pantoea ananatis on L-glutamic acid production ability under conditions of limited oxygen concentration when using glycerol as a carbon source>

[0476] To study the effect of only enhanced α-ketoglutarate synthase activity in Pantoea ananatis on L-glutamic acid production in microaerobic culture using glycerol as a carbon source, microaerobic culture of NP106 / pRSF-ppc-gdhA / pMWPthr-KGS and NP106 / pRSF-ppc-gdhA / pMWPthr prepared in Example 13 was carried out using glycerol as a carbon source.

[0477] NP106 / pRSF-ppc-gdhA / pMWPthr-KGS and NP106 / pRSF-ppc-gdhA / pMWPthr were pre-cultured overnight at 37 °C in LBMG medium. Bacterial cells equivalent to 1 / 4 of a plate amount were inoculated into 20 mL of the following Pantoea glycerol medium in a 500 mL Sakaguchi flask and cultured at 100 rpm at 34 °C for 90 hours. After the culture was completed, the L-glutamic acid accumulated in the medium was measured using a Biotech Analyzer (Asahi Kasei Corporation).

[0478] 〔Composition of Pantoea glycerol medium〕

[0479] 30g / L of glycerin

[0480] MgSO4·7H2O 0.5 g / L

[0481] (NH4)2SO4 20g / L

[0482] KH2PO4 2.0g / L

[0483] Yeast extract 2.0g / L

[0484] FeSO4·7H2O 0.02g / L

[0485] MnSO4·5H2O 0.02g / L

[0486] Thiamine hydrochloride 0.01 g / L

[0487] Kanamycin 40 mg / L

[0488] Tetracycline 12.5 mg / L

[0489] Calcium carbonate 20g / L

[0490] Sterilization conditions: pH 7.0 (adjusted with KOH), 115℃, 10 minutes.

[0491] The results are shown in Table 10. Compared with the control NP106 / pRSF-ppc-gdhA / pMWPthr, the L-glutamate yield was confirmed to be increased by about 30% in the α-ketoglutarate synthase gene-enhanced strain NP106 / pRSF-ppc-gdhA / pMWPthr-KGS derived from *Sulphurella multocida*.

[0492] Table 10

[0493]

[0494] [Example 15] <Effect of α-ketoglutarate synthase gene expression strain from *Blastopirellulamarina* on L-glutamate production capacity using glycerol as a carbon source>

[0495] To investigate the effect of α-ketoglutarate synthase activity expression from Blastopirellulamarina on L-glutamate production using glycerol as the carbon source, a plasmid for expressing the α-ketoglutarate synthase gene from Blastopirellulamarina was constructed and introduced into Escherichia coli MG1655ΔsucAΔldhA for culture.

[0496] <15-1> Construction of plasmid for expression of α-ketoglutarate synthase gene derived from Blastopirellulamarina

[0497] Using genomic DNA from Blastopirellulamarina DSM3645 (ATCC49069) as a template, PCR was performed using the oligonucleotides shown in SEQ ID NO: 61 and 62 to amplify gene fragments containing the α and β subunits of α-ketoglutarate synthase. The resulting gene fragments were cut with KpnI and EcoRI and inserted into fragments obtained by cutting pMWPthr with KpnI and EcoRI, thus constructing a plasmid for expressing the α-ketoglutarate synthase gene from Blastopirellulamarina, named pMWPthr-BlaKGS.

[0498] <15-2> Culture of α-ketoglutarate synthase gene expression strains derived from *Blastopirellulamarina* under aerobic conditions with glycerol as a carbon source.

[0499] Transformants were obtained by electroporation of the α-ketoglutarate synthase gene expression strain pMWPthr-BlaKGS and the control strain pMWPthr, respectively, with kanamycin resistance as an indicator. After confirming plasmid introduction, the α-ketoglutarate synthase gene expression strain derived from Blastopirellulamarina was named MG1655ΔsucAΔldhA / pMWPthr-BlaKGS, and the control strain was named MG1655ΔsucAΔldhA / pMWPthr.

[0500] The MG1655ΔsucAΔldhA / pMWPthr-BlaKGS strain and the control strain MG1655ΔsucAΔldhA / pMWPthr were inoculated into LB medium and pre-cultured overnight at 37°C. Half the volume of the bacterial culture was then inoculated into 20 mL of the following glycerol medium in a 500 mL volumetric flask and aerobically cultured at 37°C and 120 rpm for 22 hours. After culture, the accumulated L-glutamate in the medium was determined using a Biotech Analyzer (Asahi Kasei Corporation).

[0501] [Composition of glycerol culture medium]

[0502] 30g / L of glycerin

[0503] MgSO4·7H2O 1.0 g / L

[0504] (NH4)2SO4 20g / L

[0505] KH2PO4 1.0 g / L

[0506] Yeast extract 2.0 g / L

[0507] FeSO4·7H2O 0.01 g / L

[0508] MnSO4·5H2O 0.01 g / L

[0509] Thiamine hydrochloride 0.01 g / L

[0510] Kanamycin 40 mg / L

[0511] Calcium carbonate 30 g / L

[0512] pH 7.0 (adjusted with KOH) Sterilization conditions: 120 °C, 20 minutes

[0513] The results are shown in Table 11. Compared with the control MG1655ΔsucAΔldhA / pMWPthr, in the expression vector MG1655ΔsucAΔldhA / pMWPthr-BlaKGS into which only the α-ketoglutarate synthase gene derived from Blastopirellula marina was introduced, the L-glutamate yield relative to glycerol consumption increased by about 8%.

[0514] Table 11

[0515]

[0516] 〔Example 16〕<Effect of the α-ketoglutarate synthase gene expression-enhanced strain derived from Blastopirellula marina on the L-glutamate production ability under aerobic conditions in a glucose carbon source>

[0517] Using the α-ketoglutarate synthase gene expression-enhanced strain MG1655ΔsucAΔldhA / pMWPthr-BlaKGS derived from Blastopirellula marina prepared in Example 15 and the control strain MG1655ΔsucAΔldhA / pMWPthr, the L-glutamate production ability when using glucose as the carbon source was investigated.

[0518] Half the volume of each strain of bacteria, equivalent to one plate obtained from an overnight LB culture at 37°C, was inoculated into 20 mL of glucose + methionine medium (the glucose medium described in Example 5, with a glucose concentration of 30.0 g / L, 40 mg / L kanamycin instead of chloramphenicol, and 0.2 g / L DL-methionine) in a 500 mL Sakaguchi flask and cultured at 37°C and 120 rpm for 24 hours. After culture, the L-glutamate accumulated in the medium was determined using a Biotech Analyzer (manufactured by Asahi Kasei Corporation).

[0519] The results are shown in Table 12. Compared with the control MG1655ΔsucAΔldhA / pMWPthr, the yield was confirmed to be about 6% higher in the strain MG1655ΔsucAΔldhA / pMWPthr-KGS, which showed enhanced expression of the α-ketoglutarate synthase gene from Blastopirellulamarina.

[0520] Table 12

[0521]

[0522] [Example 17] <Construction of strain 2256ΔldhΔsucA from wild-type lactobacillus>

[0523] A strain with a gene deletion of sucA (odhA) encoding the E1o subunit of α-ketoglutarate dehydrogenase was constructed from *Corynebacterium glutamicum* strain 2256Δldh (refer to WO2005 / 113744). The 2256Δldh strain was an ldhA-deficient strain obtained from *Corynebacterium glutamicum* strain 2256 (ATCC13869).

[0524] Based on the publicly available base sequence (SEQ ID NO: 51) of the E1o subunit gene (hereinafter referred to as the sucA gene) of *Corynebacterium glutamicum* ATCC13032 (GenBank accession number NC_003450), DNA was designed and synthesized as primers. Crossover PCR was used to obtain the α-ketoglutarate dehydrogenase gene fragment from *Lactobacillus fermentum* strain 2256, which lacks the ORF of this gene. Specifically, using the genomic DNA of *Lactobacillus fermentum* strain 2256 as a template and the synthesized DNA of SEQ ID NO: 63 and 64 as primers, PCR was performed using conventional methods to obtain the N-terminal amplification product of the sucA gene. On the other hand, to obtain the C-terminal amplification product of the sucA gene, PCR was performed using the genomic DNA of *Lactobacillus fermentum* strain 2256 as a template and the synthesized DNA of SEQ ID NO: 65 and 66 as primers, using conventional methods. SEQ ID NO: 64 and 65 are complementary.

[0525] Then, the N- and C-terminal gene products of sucA were mixed in essentially equimolar amounts as a template. Using the synthetic DNA of SEQ ID NO: 63 and 66 as primers, PCR was performed using conventional methods to obtain an amplified sucA gene product with most of its internal sequence missing. The generated PCR product was purified using conventional methods, digested with BamHI, and then inserted into the SalI site of pBS3 as described in International Publication WO2005 / 113744. This DNA was used to transform competent E. coli JM109 cells (manufactured by Takara Bio Inc.), and plated on LB medium containing 100 μM IPTG, 40 μg / mL X-Gal, and 25 μg / mL Km, and cultured overnight. Then, white colonies were picked and isolated as single colonies to obtain transformants. Plasmids were extracted from the obtained transformants, and the plasmid containing the target PCR product was named pΔsucA56-1.

[0526] Because pΔldh56-1 lacks a region enabling autonomous intracellular replication in Corynebacterium-type bacteria, strains that integrate the plasmid into their genomic DNA via homologous recombination will appear as transformants when Corynebacterium-type bacteria are transformed using this plasmid, although at a very low frequency. High concentrations of plasmid pΔldh56-1 were used to transform *Brugia lactis* strain 2256 using the electroporation method. The transformed strains were plated on CM-Dex medium containing 25 μg / mL kanamycin and incubated at 31.5°C for approximately 30 hours. In the strains grown on this medium, homologous recombination occurred between the ldh gene fragment of the plasmid and the same gene in the genome of *Brugia lactis* strain 2256, resulting in the insertion of the kanamycin resistance gene and the sacB gene derived from the plasmid into the genome.

[0527] [CM-Dex medium]

[0528] glucose 5g / L

[0529] Multipeptone 10g / L

[0530] Yeast extract 10g / L

[0531] KH2PO4 1g / L

[0532] MgSO4·7H2O 0.4 g / L

[0533] FeSO4·7H2O 0.01g / L

[0534] MnSO4·7H2O 0.01g / L

[0535] urea 3g / L

[0536] Soybean hydrolysate 1.2 g / L

[0537] Biotin 10 μg / L

[0538] Sterilization conditions: pH 7.5 (adjusted with NaOH), 120℃, 20 minutes.

[0539] Then, these first-round recombinants were cultured overnight at 31.5°C in kanamycin-free CM-Dex liquid medium. After appropriate dilution, they were spread onto kanamycin-free Dex-S10 medium containing 10% sucrose (a medium prepared by replacing the glucose in CM-Dex medium with 10 g / L sucrose) and cultured at 31.5°C for approximately 30 hours. Approximately 60 strains were obtained, and it was considered that these 60 strains became sucrose-insensitive by shedding the sacB gene through a second round of homologous recombination.

[0540] Among the strains obtained in this way were strains with the sucA gene replaced by a defective strain derived from pΔsucA56-1, and strains with the sucA gene restored to wild-type. Whether the sucA gene is mutant or wild-type can be easily confirmed by directly performing PCR on bacterial cells cultured in Dex-S10 agar medium to detect the sucA gene. Such strains were used as sucA-deficient strains in the following experiments: when analyzed using primers (SEQ ID NO: 63 and SEQ ID NO: 66) for PCR amplification of the sucA gene, the size of the PCR product was smaller than that obtained using the genomic DNA of strain 2256 as a template. As a result of the analysis of sucrose-insensitive strains using the above method, a strain possessing only the defective sucA gene was selected and named strain 2256ΔldhΔsucA.

[0541] [Example 18] <Effect of an α-ketoglutarate synthase gene expression strain derived from *Lactobacillus aeruginosa* on L-glutamate production capacity under oxygen-limited conditions with glucose as the carbon source>

[0542] To investigate the effect of α-ketoglutarate synthase expression from *S. green sulfur* on L-glutamate production in *Brevibacterium lactis* using glucose as the carbon source, a plasmid for expressing the α-ketoglutarate synthase gene from *S. green sulfur* was constructed and introduced into *Brevibacterium lactis* strain 2256ΔldhΔsucA for culture.

[0543] <18-1> Construction of a plasmid for expressing the α-ketoglutarate synthase gene from *Sulphurella multocida*

[0544] DNA was designed and synthesized as primers based on the base sequences near the tuf and KGS genes of Corynebacterium glutamicum ATCC13032 (GenBank Database accession number NC_003450), and crossover PCR was performed to obtain the KGS gene fragment with the upstream of the gene replaced by the tuf promoter.

[0545] Specifically, using pMWPthr-KGS-fpr-fldA as a template (pMWPthr-KGS-fpr-fldA is the gene constructed in Example 7 for expressing the α-ketoglutarate synthase gene from *Sulphurella multocida* and enhancing the ferricopentaprotein-NADP from *Escherichia coli*), + Using the primers shown in SEQ ID NO: 67 and 68, the KGS fragment was amplified by PCR. This KGS fragment was then treated with PstI and XbaI and inserted into the PstI and XbaI sites of pVK9 to construct the plasmid pVKKGS carrying KGS. A portion of the KGS ORF can be excised from this plasmid by treating it with PstI and AatII.

[0546] Furthermore, using the plasmid pMWPthr-KGS-fpr-fldA constructed in Example 7 as a template, the fragment (A) containing the N-terminus of KGS was amplified by PCR using primers shown in SEQ ID NO: 69 and 70. On the other hand, using the DNA of plasmid pVKPtuf as a template, the tuf promoter fragment (B) was amplified by PCR using primers shown in SEQ ID NO: 71 and 72. SEQ ID NO: 69 and 72 are complementary to each other.

[0547] Then, using fragments (A) and (B) as templates, and primers shown in SEQ ID NO: 73 and 74, a fragment containing the KGSN terminal side of the natural promoter replaced by the tuf promoter was constructed via cross-PCR. The generated PCR product was purified using conventional methods and treated with PstI and AatII, and then inserted into the PstI and AatII sites of pVKKGS, thus constructing the plasmid pVKPtuf-KGS for amplifying KGS with the natural promoter replaced by the tuf gene promoter.

[0548] Furthermore, pVK9 is a shuttle vector obtained as follows: the AvaII site of pHSG299 (TAKARABio) is blunt-terminated, and a fragment containing a region in pHK4 (TEKP 05-007491) capable of autonomous replication within Corynebacterium-type bacteria is cut out using BamHI and KpnI and blunt-terminated. In addition, the comparative control pVKPtuf is a plasmid constructed by inserting a tuf promoter fragment into the PstI site of pVK: the tuf promoter fragment was obtained by PCR amplification using the genome of *Bacillus flavus* strain MJ-233 (FERMBP-1497) as a template, using primers shown in SEQ ID NO: 75 and 76. The tuf promoter sequence of *Bacillus flavus* strain MJ-233 is shown in SEQ ID NO: 77. The tuf promoter fragment can also be obtained similarly using the genome of *Corynebacterium glutamicum* strain ATCC13032 as a template.

[0549] <18-2> The α-ketoglutarate synthase gene from *Sulphurella multocida* was introduced into strain 2256ΔldhAΔsucA.

[0550] Transformants were obtained by electroporation of pVKPtuf-KGS and the control vector pVKPtuf into 2256ΔldhAΔsucA, with kanamycin resistance as an indicator. After confirming plasmid introduction, the α-ketoglutarate synthase gene expression strain from *Thiopyridamole* was named 2256ΔldhAΔsucA / pVKPtuf-KGS, and the control strain was named 2256ΔldhAΔsucA / pVKPtuf.

[0551] <18-3> Culture of a strain of α-ketoglutarate synthase gene expression derived from *Sulphurella multocida* under anaerobic conditions with glucose as the carbon source.

[0552] The L-glutamate production capacity under anaerobic culture conditions using the α-ketoglutarate synthase gene expression strain 2256ΔldhAΔsucA / pVKPtuf-KGS (constructed in <18-2>) and the control strain 2256ΔldhAΔsucA / pVKPtuf, with glucose as the carbon source, was investigated. Cells obtained from overnight pre-culture in CM-Dex agar at 31.5°C were inoculated into 3 mL of seed culture medium and cultured aerobically in test tubes at 31.5°C with shaking for approximately 16 hours.

[0553] [Seed Culture Medium]

[0554] glucose 10g / L

[0555] (NH4)2SO4 2.5g / L

[0556] KH2PO4 0.5g / L

[0557] MgSO4·7H2O 0.25g / L

[0558] urea 2g / L

[0559] FeSO4·7H2O 0.01g / L

[0560] MnSO4·7H2O 0.01g / L

[0561] Biotin 50 μg / L

[0562] VB1·HCl 100μg / L

[0563] Protocatechuic acid 15mg / L

[0564] CuSO4 0.02 mg / L

[0565] CaCl2 10 mg / L

[0566] Sterilization conditions: pH 7.0 (adjusted with KOH), 115℃, 10 minutes.

[0567] Add 3 mL of main culture medium to 3 mL of seed culture broth. To prevent aeration, seal the test tube with a silicone stopper and then incubate with shaking at 31.5 °C. After 66 hours, stop the culture and determine the accumulated L-glutamate in the culture medium using a Biotech Analyzer (manufactured by Asahi Kasei Corporation).

[0568] [Main Culture Medium]

[0569] Glucose 60g / L

[0570] (NH4)2SO4 30g / L

[0571] KH2PO4 4g / L

[0572] urea 6g / L

[0573] FeSO4·7H2O 0.02g / L

[0574] MnSO4·7H2O 0.02g / L

[0575] Biotin 400 μg / L

[0576] VB1·HCl 400μg / L

[0577] MgCO4 50g / L

[0578] Sterilization conditions: pH 6.8 (adjusted with NaOH), 115℃, 10 minutes.

[0579] The results are shown in Table 13. Compared with the control 2256ΔldhAΔsucA / pVKPtuf, the L-glutamate yield relative to glucose consumption was increased by 0.6% in 2256ΔldhAΔsucA / pVKPtuf-KGS, which was infused with an expression vector containing only the α-ketoglutarate synthase gene from *Thiopyrrolizum aureum*.

[0580] Table 13

[0581] strain OD620 Residual glucose (g / L) L-glutamic acid yield (%) 2256ΔldhΔsucA / pVKPtuf 7.6 0.48 3.1 2256ΔldhΔsucA / pVKPtuf-KGS 6.6 6.02 3.7

[0582] [Sequence List Description]

[0583] SEQ ID NO: 1: Base sequence of the α-ketoglutarate synthase α-subunit gene of *Sulphurella multocida*

[0584] SEQ ID NO: 2: Amino acid sequence of the α-subunit of α-ketoglutarate synthase from *Sulphurella multocida*

[0585] SEQ ID NO: 3: Base sequence of the α-ketoglutarate synthase β-subunit gene of *Sulphurella multocida*

[0586] SEQ ID NO: 4: Amino acid sequence of the β-subunit of α-ketoglutarate synthase from *Sulphurella multocida*

[0587] SEQ ID NO: 5: Base sequence of the fpr gene in Escherichia coli

[0588] SEQ ID NO: 6: Amino acid sequence encoded by the fpr gene of Escherichia coli

[0589] SEQ ID NO: 7: Base sequence of the pyruvate synthase gene of *Sulphurella vulgaris*

[0590] SEQ ID NO: 8: Amino acid sequence of pyruvate synthase from *Sulphurella multocida*

[0591] SEQ ID NO: 9: Base sequence of the fdx gene in Escherichia coli

[0592] SEQ ID NO: 10: Amino acid sequence encoded by the fdx gene of Escherichia coli

[0593] SEQ ID NO: 11: Base sequence of the yfhL gene in Escherichia coli

[0594] SEQ ID NO: 12: Amino acid sequence encoded by the yfhL gene in Escherichia coli

[0595] SEQ ID NO: 13: Base sequence of the fldA gene in Escherichia coli

[0596] SEQ ID NO: 14: Amino acid sequence encoded by the fldA gene of Escherichia coli

[0597] SEQ ID NO: 15: Base sequence of the fldB gene in Escherichia coli

[0598] SEQ ID NO: 16: Amino acid sequence encoded by the fldB gene of Escherichia coli

[0599] SEQ ID NO: 17: Base sequence of the ferricoxin I gene of *Sulphurella vulgaris*

[0600] SEQ ID NO: 18: Amino acid sequence encoded by the ferricoxin I gene of *Sulphurella vulgaris*

[0601] SEQ ID NO: 19: Base sequence of the ferroredoxin II gene of *Sulphurella multocida*

[0602] SEQ ID NO: 20: Amino acid sequence encoded by the ferricoxin II gene of *Sulphurella vulgaris*

[0603] SEQ ID NO: 21: Primer 1 for amplifying the α-ketoglutarate synthase gene of *Sulphurella vulgaris*

[0604] SEQ ID NO: 22: Primer 2 for amplifying the α-ketoglutarate synthase gene of *Sulphurella vulgaris*

[0605] SEQ ID NO: 23: Primer 1 for amplifying the pyruvate synthase gene of *Sulphurella vulgaris*

[0606] SEQ ID NO: 24: Primer 2 for amplifying the pyruvate synthase gene of *Sulphurella vulgaris*

[0607] SEQ ID NO: 25: Primer 1 for amplifying the ferricoxin I gene of *Sulphurella vulgaris*

[0608] SEQ ID NO: 26: Primer 2 for amplifying the ferricoxin I gene of *Sulphurella vulgaris*

[0609] SEQ ID NO: 27: Primer 1 for ldhA in defective Escherichia coli

[0610] SEQ ID NO: 28: Primer 2 for ldhA in defective Escherichia coli

[0611] SEQ ID NO: 29: Primer 1 used to confirm ldhA deficiency in Escherichia coli

[0612] SEQ ID NO: 30: Primer 2 used to confirm ldhA deficiency in Escherichia coli

[0613] SEQ ID NO: 31: Threonine operon promoter sequence of Escherichia coli

[0614] SEQ ID NO: 32: Flavin-NADP of Escherichia coli + Primer 1 for reductase gene amplification

[0615] SEQ ID NO: 33: Flavin-NADP of Escherichia coli + Primer 2 for reductase gene amplification

[0616] SEQ ID NO: 34: Primer 1 for amplification of the fldA gene in Escherichia coli

[0617] SEQ ID NO: 35: Primer 2 for amplification of the fldA gene in Escherichia coli

[0618] SEQ ID NO: 36: Primer 1 for amplification of the fldB gene in Escherichia coli

[0619] SEQ ID NO: 37: Primer 2 for amplification of the fldB gene in Escherichia coli

[0620] SEQ ID NO: 38: Primer 1 for amplification of the fdx gene in Escherichia coli

[0621] SEQ ID NO: 39: Primer 2 for amplification of the fdx gene in Escherichia coli

[0622] SEQ ID NO: 40: Primer 1 for amplification of the yhfL gene in Escherichia coli

[0623] SEQ ID NO: 41: Primer 2 for amplification of the yfhL gene in Escherichia coli

[0624] SEQ ID NO: 42: PCR primers for amplification of the N-terminal fragment of the sucA gene in Escherichia coli

[0625] SEQ ID NO: 43: PCR primers for amplification of the N-terminal fragment of the sucA gene in Escherichia coli

[0626] SEQ ID NO: 44: PCR primers for amplification of the C-terminal fragment of the sucA gene in Escherichia coli

[0627] SEQ ID NO: 45: PCR primers for amplification of the C-terminal fragment of the sucA gene in Escherichia coli

[0628] SEQ ID NO: 46: Base sequence of the α-KGDH subunit gene and neighboring genes of Pantoea ananatis

[0629] sdhB: 2~121

[0630] sucA: 322~3129

[0631] sucB: 3145~4368

[0632] sucC: 4437~4556

[0633] SEQ ID NO: 47: Partial amino acid sequence of succinate dehydrogenase iron-sulfur protein of Pantoea ananatis.

[0634] SEQ ID NO: 48: Amino acid sequence of the α-KGDHE1o subunit of Pantoea ananatis

[0635] SEQ ID NO: 49: Amino acid sequence of the α-KGDHE2o subunit of Pantoea ananatis

[0636] SEQ ID NO: 50: Part of the β subunit of succinyl-CoA synthase in Pantoea ananatis

[0637] SEQ ID NO: 51: Base sequence of the sucA (odhA) gene in *Brucea lactis*.

[0638] SEQ ID NO: 52: Amino acid sequence of the E1o subunit encoded by sucA (odhA) of *Lactobacillus fermentum*.

[0639] SEQ ID NO: 53: Base sequence of the gene encoding the E2o subunit of *Brevibacterium lactis* (Genbank accession number NC_003450, NCgl2126).

[0640] SEQ ID NO: 54: Amino acid sequence of the E2o subunit encoded by NCgl2126 of *Lactobacillus fermentum*.

[0641] SEQ ID NO: 55: Base sequence of the sucA gene in Escherichia coli

[0642] SEQ ID NO: 56: Amino acid sequence of the α-KGDHE1 subunit encoded by the sucA gene in Escherichia coli.

[0643] SEQ ID NO: 57: Base sequence of the α-ketoglutarate synthase α-subunit gene of *Blastopirellulamarina*

[0644] SEQ ID NO: 58: Amino acid sequence of the α-ketoglutarate synthase α-subunit of Blastopirellulamarina

[0645] SEQ ID NO: 59: Base sequence of the α-ketoglutarate synthase β-subunit gene of *Blastopirellulamarina*

[0646] SEQ ID NO: 60: Amino acid sequence of the α-ketoglutarate synthase β-subunit of Blastopirellulamarina

[0647] SEQ ID NO: 61: Primer 1 for amplifying the α-ketoglutarate synthase gene of Blastopirellulamarina

[0648] SEQ ID NO: 62: Primer 2 for amplifying the α-ketoglutarate synthase gene of Blastopirellulamarina

[0649] SEQ ID NO: 63: Primer 1 for amplifying the N-terminal fragment of the sucA gene in *Lactobacillus fermentum*.

[0650] SEQ ID NO: 64: Primer 2 for amplifying the N-terminal fragment of the sucA gene in *Lactobacillus fermentum*.

[0651] SEQ ID NO: 65: Primer 1 for amplifying the C-terminal fragment of the sucA gene in *Brucea lactis*.

[0652] SEQ ID NO: 66: Primer 2 for amplifying the C-terminal fragment of the sucA gene in *Brucea lactis*

[0653] SEQ ID NO: 67: Primers 1 for full-length amplification of the α-ketoglutarate synthase gene of *Sulphurella vulgaris*

[0654] SEQ ID NO: 68: Primers 2 for full-length amplification of the α-ketoglutarate synthase gene of *Sulphurella vulgaris*

[0655] SEQ ID NO: 69: Primer 1 for amplifying the N-terminal fragment of the α-ketoglutarate synthase gene from *S. aeruginosa*

[0656] SEQ ID NO: 70: Primer 2 for amplifying the N-terminal fragment of the α-ketoglutarate synthase gene from *S. aeruginosa*.

[0657] SEQ ID NO: 71: Primer 1 for amplifying the tuf promoter of *Bacillus flavus*

[0658] SEQ ID NO: 72: Primer 2 for amplifying the tuf promoter of *Bacillus flavus*

[0659] SEQ ID NO: 73: Ptuf-KGS fragment amplification primer 1

[0660] SEQ ID NO: 74: Ptuf-KGS fragment amplification primer 2

[0661] SEQ ID NO: 75: Primer 3 for amplifying the tuf promoter of *Bacillus flavus*.

[0662] SEQ ID NO: 76: Primer 4 for amplifying the tuf promoter of *Bacillus flavus*.

[0663] SEQ ID NO: 77: Base sequence of the tuf gene promoter of *Bacillus flavus*

[0664] Industrial applicability

[0665] By using the microorganisms of this invention, the fermentation production of L-glutamic acid amino acids can be carried out efficiently. Furthermore, the method of this invention reduces carbon dioxide emissions by inhibiting decarbonation and utilizing carbon dioxide fixation reactions, making it an environmentally friendly approach.

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Examples

Embodiment 1

[0297] [Example 1]

[0298] Chlorothiobacter is a medium-high-temperature autotrophic bacterium with an optimum growth temperature of 48°C. The genome sequence of the TLS strain has been elucidated by Eisen et al. ). The α-ketoglutarate synthase gene, pyruvate synthase gene and ferredoxin gene were isolated from the strain, and a plasmid expressing these three genes simultaneously was constructed.

[0299] Construction of α-ketoglutarate synthase gene expression plasmid derived from green sulfur bacteria

[0300] Using the genomic DNA of Sulfur chlorosis TLS strain (ATCC49652) as a template, the oligonucleotides shown in SEQ ID NO: 21 and 22 were used for PCR to amplify the α- and β-subunits of α-ketoglutarate synthase base gene segment. The resulting gene fragment was cut with BamHI and inserted into the BamHI site of pSTV28 (manufactured by TAKARA Bio) to construct a plasmid for expressing the α-ketoglutarate synthase gene, which was named pSTV-KGS. In this plasmid, th...

Embodiment 2

[0305] [Example 2]

[0306] Construction of Escherichia coli sucA gene disruption plasmid

[0307] A strain in which the sucA gene encoding the E1 subunit of α-ketoglutarate dehydrogenase was disrupted was prepared from Escherichia coli MG1655 strain. Primers were synthesized based on the base sequence of the sucA gene located at base number 757929-760730 in the genome sequence (Genbank accession number. U00096), and the N of the sucA gene was amplified by PCR using the genomic DNA of Escherichia coli MG1655 strain as a template. terminal and C-terminal fragments. The base sequence of the sucA gene of Escherichia coli is shown in SEQ ID NO: 55, and the amino acid sequence of the E1 subunit encoded by the gene is shown in SEQ ID NO: 56. The oligonucleotides of SEQ ID NO: 42 and 43 were used as PCR primers for N-terminal fragment amplification, and the oligonucleotides of SEQ ID NO: 44 and 45 were used as PCR primers for C-terminal fragment amplification. The oligonucleotid...

Embodiment 3

[0311] [Example 3]

[0312]Lactate dehydrogenase is an enzyme that produces lactate from pyruvate using NADH as a coenzyme. In order to inhibit the production of lactic acid in Escherichia coli cultured under oxygen-limited conditions, a deficient strain of the ldhA gene encoding lactate dehydrogenase was constructed. Deletion of this gene utilizes a method called "Red-driven integration" developed by Datsenko and Wanner (Datsenko, K.A. and Wanner, B.L. 2000.Proc.Natl.Acad.Sci.USA.97:6640-6645) Performed with a lambda phage-derived excision system (Cho, E.H. et al. 2002. J. Bacteriol. 184:5200-5203). By this method, a synthetic oligonucleotide in which a part of the gene of interest is designed on the 5' side and a part of the antibiotic resistance gene is designed on the 3' side is used as a primer to obtain a PCR product, and the use of such a PCR product can One-step construction of gene disruption strains. The antibiotic resistance gene introduced into the gene-disrupt...

Claims

1. A method for producing L-amino acids, comprising culturing microorganisms in a culture medium at 25 to 40°C to generate and accumulate L-amino acids in the culture medium or within the cells of the microorganisms, and collecting the L-amino acids from the culture medium or cells, wherein the microorganisms are capable of producing one or more L-amino acids selected from L-glutamic acid, L-glutamine, L-proline, L-ornithine, L-citrulline, and L-arginine, wherein the microorganisms are *Escherichia coli*, *Pantoea ananatis*, or *Corynebacterium glutamicum*. The microorganisms described herein are modified to increase the activity of α-ketoglutarate synthase by increasing the copy number of the gene encoding α-ketoglutarate synthase. The gene encoding α-ketoglutarate synthase encodes a polypeptide selected from (A) and (B) and a polypeptide selected from (C) and (D): (A) A polypeptide consisting of the amino acid sequence of SEQ ID NO:

2. (B) A polypeptide consisting of the amino acid sequence of SEQ ID NO:

58. (C) A polypeptide consisting of the amino acid sequence of SEQ ID NO:

4. (D) A polypeptide consisting of the amino acid sequence of SEQ ID NO:

60. The microorganisms described therein were modified to reduce α-ketoglutarate dehydrogenase activity by deleting the gene encoding α-ketoglutarate dehydrogenase. Among them, *E. coli* and *Corynebacterium glutamicum* were modified to reduce lactate dehydrogenase activity by deleting the gene encoding lactate dehydrogenase, and Pantoeaananatis was modified to increase the expression of the phosphoenolpyruvate carboxylase gene and the glutamate dehydrogenase gene.

2. The method of claim 1, wherein, The gene encoding α-ketoglutarate synthase comprises DNA selected from (a) and (b), and DNA selected from (c) and (d): (a) DNA consisting of the nucleotide sequence of SEQ ID NO: 1; (b) DNA consisting of the nucleotide sequence of SEQ ID NO: 57; (c) DNA consisting of the nucleotide sequence of SEQ ID NO: 3; (d) DNA consisting of the nucleotide sequence of SEQ ID NO:

59.

3. The method of claim 1 or 2, wherein, This microorganism was modified to increase ferroredoxin-NADP. + The activity of reductase.

4. The method of claim 1 or 2, wherein, The microorganism was modified to increase the activity of pyruvate synthase.

5. The method of claim 1 or 2, wherein, The microorganism was modified to enhance its ability to produce ferrugin or flavin ferrugin.

6. The method for producing L-amino acids according to claim 1 or 2, characterized in that, The microorganisms were cultured under aerobic conditions.

7. The method for producing L-amino acids according to claim 1 or 2, characterized in that, The culture medium is a medium containing carbonate ions, bicarbonate ions or carbon dioxide gas, and the microorganisms are cultured under anaerobic or microaerobic conditions.

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