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US20100112647A1 - Method for producing an acidic substance having a carboxyl group - Google Patents

Method for producing an acidic substance having a carboxyl group Download PDF

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US20100112647A1
US20100112647A1 US12/579,577 US57957709A US2010112647A1 US 20100112647 A1 US20100112647 A1 US 20100112647A1 US 57957709 A US57957709 A US 57957709A US 2010112647 A1 US2010112647 A1 US 2010112647A1
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acid
gene
strain
seq
microorganism
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Yoshihiko Hara
Keita Fukui
Yoshinori Tajima
Kazue Kawamura
Yoshihiro Usuda
Kazuhiko Matsui
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Ajinomoto Co Inc
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Assigned to AJINOMOTO CO., INC. reassignment AJINOMOTO CO., INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FUKUI, KEITA, HARA, YOSHIHIKO, TAJIMA, YOSHINORI, KAWAMURA, KAZUE, USUDA, YOSHIHIRO, MATSUI, KAZUHIKO
Publication of US20100112647A1 publication Critical patent/US20100112647A1/en
Priority to US14/723,776 priority Critical patent/US9822385B2/en
Priority to US15/787,861 priority patent/US20180044703A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/14—Glutamic acid; Glutamine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/265—Enterobacter (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/20—Aspartic acid; Asparagine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/48—Tricarboxylic acids, e.g. citric acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/50—Polycarboxylic acids having keto groups, e.g. 2-ketoglutaric acid

Definitions

  • the present invention relates to a method for producing an acidic substance having a carboxyl group.
  • L-Glutamic acid and L-aspartic acid are widely used as raw materials in making seasonings and so forth.
  • Succinic acid is widely used as a raw material in making seasonings and biodegradable plastics.
  • L-Glutamic acid is mainly produced by fermentation utilizing L-glutamic acid-producing bacteria of the so-called coryneform bacteria belonging to the genus Brevibacterium, Corynebacterium or Microbacterium , or their mutant strains (see, for example, Kunihiko Akashi et al., Amino Acid Fermentation, Japan Scientific Societies Press (Gakkai Shuppan Center), pp. 195-215, 1986). Methods are known for producing L-glutamic acid by fermentation using other bacterial strains, including microorganisms belonging to the genera Bacillus, Streptomyces, Penicillium or the like (see, for example, U.S. Pat. No.
  • microorganisms belonging to the genera Pseudomonas, Arthrobacter, Serratia, Candida or the like see, for example, U.S. Pat. No. 3,563,857
  • microorganisms belonging to the genera Bacillus, Pseudomonas, Serratia, Aerobacter aerogenes (currently referred to as Enterobacter aerogenes ) or the like see, for example, Japanese Patent Publication (KOKOKU) No. 32-9393), a mutant strain of Escherichia coli (see, for example, Japanese Patent Laid-open (KOKAI) No. 5-244970), and the like.
  • Methods for improving the production of target substances are also known, including by modifying the uptake or secretion systems of target substances.
  • Such methods include, for example, by deleting or attenuating the system for uptake of a target substance into cells.
  • known methods to improve production of L-glutamic acid include deleting the gluABCD operon, or a part thereof, to eliminate or attenuate uptake of L-glutamic acid into cells (see, for example, European Patent Application Laid-open No. 1038970), and enhancing production of purine nucleotides by attenuating uptake of purine nucleotides into cells (see, for example, European Patent Application Laid-open No. 1004663), and the like.
  • methods of enhancing the secretion system for a target substance and methods of deleting or attenuating the secretion system for an intermediate or substrate in the biosynthetic system of a target substance are known.
  • Known methods of enhancing the secretion system of a target substance include, for example, production of L-lysine by utilizing a Corynebacterium strain in which the L-lysine secretion gene (lysE) is enhanced (see, for example, WO2001/5959), and production of L-glutamic acid by using an enterobacterium in which the L-glutamic acid secretion system gene (yhfK) is enhanced (see, for example, Japanese Patent Laid-open No. 2005-278643 (U.S.
  • L-glutamic acid production efficiency can be improved in Escherichia bacteria by enhancing the expression of genes thought to participate in secretion of L-amino acids such as yfiK (see, for example, Japanese Patent Laid-open No. 2000-189180 (U.S. Pat. No. 6,979,560)).
  • L-glutamic acid-producing ability can be improved by enhancing expression of the yhfK gene (see, for example, Japanese Patent Laid-open No. 2005-278643 (U.S. Patent Published Application No. 2005196846)).
  • L-glutamic acid by culturing a microorganism under acidic conditions to precipitate the L-glutamic acid (see, for example, Japanese Patent Laid-open No. 2001-333769 (U.S. Patent Published Application No. 2007134773)).
  • the pH is kept low, L-glutamic acid is precipitated, and the ratio of L-glutamic acid in free form with no electrical charge increases. As a result, the L-glutamic acid easily penetrates cell membranes.
  • L-glutamic acid When L-glutamic acid is taken up into cells, it is converted into an intermediate of the TCA cycle, 2-oxoglutaric acid, in one step by glutamate dehydrogenase, and therefore it is generally thought that L-glutamic acid taken up into cells is easily metabolized.
  • 2-oxoglutarate dehydrogenase activity can be deleted or attenuated, or the like, in the fermentative production of L-glutamic acid (for example, Japanese Patent Laid-open No. 2001-333769 (U.S. Patent Published Application No. 2007134773), Japanese Patent Laid-open No. 7-203980 (U.S. Pat. No.
  • anaerobic bacteria including those belonging to the genus Anaerobiospirillum or Actinobacillus are usually used (U.S. Pat. Nos. 5,142,834 and 5,504,004, Guettler, M. V. et al., 1999, International Journal of Systematic Bacteriology, 49:207-216).
  • organic nitrogen sources such as corn steep liquor (CSL) into the culture medium.
  • CSL corn steep liquor
  • Escherichia coli which is a facultative anaerobic gram negative bacterium
  • methods for producing a non-amino organic acid by culturing it once under aerobic conditions, and then allowing the cells to rest in the absence of supplied oxygen, resulting in an anaerobically produced non-amino organic acid
  • E. coli can be aerobically cultured to aerobically produce the non-amino organic acid (U.S. Patent Published Application No. 20050170482).
  • Escherichia coli is a gram negative bacterium, it is vulnerable to osmotic pressure, and there remains room for improvement in productivity per cell etc.
  • the ybjL gene is located on the genome of Escherichia coli (see, for example, Blattner, F. R. et al., 1997, Science, 277(5331):1453-74), and it is also thought to code for a transporter on the basis of the motifs, topology etc. of the deduced amino acid sequence.
  • cloning of the gene, as well as expression of the gene and analysis of the expression product have not been reported, and the actual functions of the gene remained unknown.
  • An aspect of the present invention is to provide a bacterial strain that can efficiently produce an acidic substance having a carboxyl group, especially L-glutamic acid, L-aspartic acid, and succinic acid, and to provide a method for efficiently producing an acidic substance having a carboxyl group by using such a strain.
  • the ybjL gene was isolated and shown to be involved in L-glutamic acid resistance. Also, it has been found that when the expression of the ybjL gene is enhanced, L-glutamic acid fermentation yield is improved and the production rate or yield of succinic acid is improved.
  • the ybjL gene encodes a protein: (A) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4 and 87; (B) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4 and 87, but wherein one or several amino acid residues are substituted, deleted, inserted or added, and the protein improves the ability of the microorganism to produce an acidic substance having a carboxyl group when expression of the gene encoding the protein is enhanced in the microorganism.
  • microorganism is a bacterium belonging to the family Enterobacteriaceae.
  • microorganism is a rumen bacterium.
  • microorganism is Mannheimia succiniciproducens.
  • the acidic substance is an organic acid selected from the group consisting of succinic acid, fumaric acid, malic acid, oxalacetic acid, citric acid, isocitric acid, ⁇ -ketoglutaric acid, and combinations thereof.
  • It is a further aspect of the present invention to provide a method for producing an acidic substance having a carboxyl group comprising culturing the aforementioned microorganism in a medium to produce and accumulate the acidic substance having a carboxyl group in the medium, and collecting the acidic substance having a carboxyl group from the medium.
  • the acidic substance is an organic acid selected from the group consisting of succinic acid, fumaric acid, malic acid, oxalacetic acid, citric acid, isocitric acid, ⁇ -ketoglutaric acid, and combinations thereof.
  • the acidic substance is L-glutamic acid and/or L-aspartic acid.
  • It is a further aspect of the present invention to provide a method for producing an acidic substance having a carboxyl group comprising: A) allowing a substance to act on an organic raw material in a reaction mixture containing carbonate ions, bicarbonate ions, or carbon dioxide gas, wherein the substance is selected from the group consisting of i) the microorganism as described above, ii) a product obtained by processing the microorganism of i), and iii) combinations thereof, and collecting the acidic substance having a carboxyl group.
  • the acidic substance is an organic acid selected from the group consisting of succinic acid, fumaric acid, malic acid, oxalacetic acid, citric acid, isocitric acid, ⁇ -ketoglutaric acid, and combinations thereof.
  • FIG. 1 shows the structure of helper plasmid RSF-Red-TER.
  • FIG. 2 shows construction of helper plasmid RSF-Red-TER.
  • FIG. 3 shows the growth of the ybjL-amplified strain in the presence of a high concentration L-glutamic acid.
  • FIG. 4 shows accumulation of succinic acid obtained with the ybjL-amplified strain of Escherichia bacterium.
  • FIG. 5 shows accumulation of succinic acid obtained with the ybjL-amplified strain of Enterobacter bacterium.
  • the microorganism in accordance with the presently disclosed subject matter has an ability to produce an acidic substance having a carboxyl group and has been modified so that expression of the ybjL gene is enhanced.
  • the term “ability to produce an acidic substance having a carboxyl group” can mean the ability of a microorganism to produce and cause accumulation of an acidic substance having a carboxyl group in a medium or cells to such a degree that the acidic substance having a carboxyl group can be collected from the cells or medium when the microorganism of the present invention is cultured in the medium.
  • the microorganism can originally have the ability to produce an acidic substance having a carboxyl group, or the ability to produce an acidic substance can be obtained by modifying a microorganism such as those described below using mutation or recombinant DNA techniques. Also, a microorganism can be imparted with the ability to produce an acidic substance having a carboxyl group, or the ability to produce an acidic substance having a carboxyl group can be enhanced by introducing the gene described herein.
  • the “acidic substance having a carboxyl group” can mean an organic compound having one or more carboxyl groups and which is acidic when the substance is in the free form, and not in the salt form.
  • examples include organic acids and L-amino acids having two carboxyl groups, for example, acidic amino acids.
  • the L-amino acids include L-glutamic acid and L-aspartic acid
  • examples of the organic acids include succinic acid, fumaric acid, malic acid, oxalacetic acid, citric acid, isocitric acid, ⁇ -ketoglutaric acid, and the like.
  • the parent strain which can be used to derive the microorganism in accordance with the presently disclosed subject matter is not particularly limited, and can include bacteria, for example, Enterobacteriaceae, rumen bacteria, and coryneform bacteria.
  • the Enterobacteriaceae family encompasses bacteria belonging to the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Raoultella, Salmonella, Serratia, Shigella, Morganella, Yersinia , and the like.
  • bacteria belonging to the genus Escherichia, Enterobacter, Raoultella, Pantoea, Klebsiella , or Serratia are particular examples.
  • a “bacterium belonging to the genus Escherichia ” means that the bacterium is classified into the genus Escherichia according to the classification known to a person skilled in the art of microbiology, although the bacterium is not particularly limited.
  • Examples of the bacterium belonging to the genus Escherichia include, but are not limited to, Escherichia coli ( E. coli ).
  • Other examples include, for example, the bacteria of the phyletic groups described in the work of Neidhardt et al. (Neidhardt F. C. Ed., 1996, Escherichia coli and Salmonella : Cellular and Molecular Biology/Second Edition, pp.
  • strains are available from, for example, the American Type Culture Collection (Address: 12301 10801 University Boulevard, Manassas, Va. 20108, United States of America). That is, registration numbers are given to each of the strains, and the strains can be ordered by using these numbers. The registration numbers of the strains are listed in the catalogue of the American Type Culture Collection.
  • Pantoea, Erwinia , and Enterobacter bacteria are classified as ⁇ -proteobacteria, and they are taxonomically very close to one another (J. Gen. Appl. Microbiol., 1997, 43, 355-361; Int. J. Syst. Bacteriol., 1997, 43, 1061-1067).
  • some bacteria belonging to the genus Enterobacter were reclassified as Pantoea agglomerans, Pantoea dispersa , or the like, on the basis of DNA-DNA hybridization experiments etc. (Int. J. Syst. Bacteriol., 1989, 39:337-345).
  • some bacteria belonging to the genus Erwinia were reclassified as Pantoea ananas or Pantoea stewartii (refer to Int. J. Syst. Bacteriol., 1993, 43:162-173).
  • Enterobacter bacteria examples include Enterobacter agglomerans, Enterobacter aerogenes , and the like.
  • the strains exemplified in European Patent Application Laid-open No. 952221 can be used.
  • Typical strains of the genus Enterobacter include Enterobacter agglomerans ATCC 12287, Enterobacter aerogenes ATCC 13048, Enterobacter aerogenes NBRC 12010 (Biotechnol Bioeng., 2007, Mar. 27; 98(2):340-348), and Enterobacter aerogenes AJ110637 (FERM ABP-10955), and the like.
  • the AJ110637 strain was obtained from the soil at the seashore of Susuki Kaigan, Makinohara-shi, Shizuoka-ken on March, 2006 by cumulative liquid culture using glycerol as the carbon source.
  • the full-length 16S rDNA sequence was then determined, and it was found to be 99.9% homologous to that from Enterobacter aerogenes NCTC 10006.
  • the strain gave results similar to the prototype species of Enterobacter aerogenes , and therefore it was identified as Enterobacter aerogenes .
  • This strain was deposited at International Patent Organism Depository, Agency of Industrial Science and Technology (Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Aug. 22, 2007, and assigned an accession number of FERM P-21348. Then, the deposit was converted to an international deposit based on the Budapest Treaty on Mar. 13, 2008, and assigned an accession number of FERM ABP-10955.
  • Pantoea bacteria include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans , and Pantoea citrea . Specific examples include the following strains:
  • Pantoea ananatis AJ13355 (FERM BP-6614, European Patent Laid-open No. 0952221)
  • Pantoea ananatis AJ13356 (FERM BP-6615, European Patent Laid-open No. 0952221)
  • Erwinia bacteria examples include Erwinia amylovora and Erwinia carotovora
  • examples of the Klebsiella bacteria examples include Klebsiella planticola
  • examples of the Raoultella bacteria include Raoultella planticola . Specific examples include the following strains:
  • Klebsiella planticola AJ13399 strain (FERM BP-6600, European Patent Laid-open No. 955368)
  • Klebsiella planticola AJ13410 strain (FERM BP-6617, European Patent Laid-open No. 955368).
  • rumen bacteria examples include Mannheimia, Actinobacillus, Anaerobiospirillum, Pyrobacterium , and Selenomonas .
  • Bacteria including Mannheimia succiniciproducens, Actinobacillus succinogenes, Selenomonas ruminantium, Veillonella parvula, Wolnella succinogenes, Anaerobiospirillum succiniciproducens , and the like, can be used.
  • Specific strains include Mannheimia sp. 55E (KCTC0769BP strain, U.S. Patent Published Application No. 20030113885, International Patent Publication WO2005/052135).
  • the expression of one or two or more enzymes involved in the biosynthesis of an acidic substance having a carboxyl group can be enhanced. Furthermore, the impartation of properties such as auxotrophic mutation, analogue resistance, or metabolic regulation mutation can be combined with the enhancement of the biosynthetic enzymes.
  • a mutant strain auxotrophic for an acidic substance having a carboxyl group, a strain resistant to an analogue of an acidic substance having a carboxyl group, or a metabolic regulation mutant strain can be obtained by subjecting a parent or wild-type strain to a conventional mutagenesis, such as exposure to X-rays or UV irradiation, or a treatment with a mutagen such as N-methyl-N′-nitro-N-nitrosoguanidine, and then selecting bacteria which exhibit an autotrophy, analogue resistance, or metabolic regulation mutation and which also have the ability to produce an acidic substance having a carboxyl group.
  • a conventional mutagenesis such as exposure to X-rays or UV irradiation
  • a mutagen such as N-methyl-N′-nitro-N-nitrosoguanidine
  • Examples of the method of modifying a microorganism to impart L-glutamic acid-producing ability to the microorganism include, for example, enhancing expression of a gene coding for an enzyme involved in L-glutamic acid biosynthesis.
  • Examples of an enzyme involved in L-glutamic acid biosynthesis include glutamate dehydrogenase (“GDH”)(gdh), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (“CS”) (gltA), methylcitrate synthase (hereinafter also referred to as “PRPC” (prpC), phosphoenolpyruvate carboxylase (“PEPC”) (ppc), pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, lpd
  • the first method is to increase the copy number of the target gene by cloning the target gene on an appropriate plasmid and transforming the chosen host bacterium with the obtained plasmid.
  • the target gene is the gene coding for CS (gltA gene), the gene coding for PEPC (ppc gene) or the gene coding for GDH (gdhA gene)
  • nucleotide sequences of these genes from Escherichia bacteria and Corynebacterium bacteria have already been reported (Ner, S. et al., 1983, Biochemistry, 22:5243-5249; Fujita, N. et al., 1984, J. Biochem., 95:909-916; Valle, F.
  • a phage DNA can also be used as the vector instead of a plasmid.
  • Examples of plasmids for simultaneously enhancing the activities of CS or PRPC, PEPC, and GDH as described above include RSFCPG which includes the gltA gene, ppc gene, and gdhA gene (refer to European Patent Laid-open No. 0952221), and RSFPPG which is the same as RSFCPG, but the gltA gene is replaced with the prpC gene.
  • transformation methods include treating recipient cells with calcium chloride so to increase permeability for DNA, which has been reported for Escherichia coli K-12 (Mandel, M. and Higa, A., 1970, J. Mol. Biol., 53:159-162), and preparing competent cells from cells which are in the growth phase, followed by transformation with DNA, which has been reported for Bacillus subtilis (Duncan, C. H., et al., 1977, Gene, 1:153-167).
  • a method of making potential host cells into protoplasts or spheroplasts which can easily take up recombinant DNA, followed by introducing the recombinant DNA into the cells.
  • microorganisms can also be transformed by the electric pulse method (Japanese Patent Laid-open No. 2-207791).
  • the copy number of a target gene can also be increased by introducing multiple copies of the gene into the chromosomal DNA of the microorganism. Multiple copies of a gene can be introduced into the chromosomal DNA by homologous recombination (Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory) using multiple copies of a sequence as targets in the chromosomal DNA. Sequences, which are present in multiple copies on the chromosomal DNA, repetitive DNAs, and inverted repeats present at the end of a transposable element, are exemplary. Also, as disclosed in Japanese Patent Laid-open No.
  • target gene it is possible to incorporate a target gene into a transposon, and allow it to be transferred to introduce multiple copies of the gene into the chromosomal DNA.
  • the target gene can also be introduced into the bacterial chromosome by using Mu phage (Japanese Patent Laid-open No. 2-109985).
  • the second method is to increase expression of the target gene by replacing an expression regulatory sequence of the target gene, such as promoter, on the chromosomal DNA or plasmid with a stronger promoter.
  • a stronger promoter such as promoter
  • the lac promoter, trp promoter, trc promoter, etc. are known as strong promoters.
  • Substitution of an expression regulatory sequence can be performed, for example, in the same manner as for gene substitution using a temperature-sensitive plasmid.
  • vectors having a temperature-sensitive replication origin and are usable for Escherichia coli and Pantoea ananatis include, for example, the pMAN997 plasmid described in International Publication WO99/03988, and the like.
  • an expression regulatory sequence can also be substituted by the method called “Red-driven integration” using Red recombinase of ⁇ phage (Datsenko, K. A. and Wanner, B. L., 2000, Proc. Natl. Acad. Sci. USA.
  • a strain resistant to a ⁇ Red gene product for example, Pantoea ananatis SC17(0)
  • the SC17(0) strain was deposited at the Russian National Collection of Industrial Microorganisms (VKPM), GNII Genetika (Russia, 117545 Moscow 1, Dorozhny proezd. 1) on Sep. 21, 2005 under an accession number of VKPM B-9246.
  • microorganisms modified by the method described above so that expression of citrate synthase gene, methylcitrate synthase gene, phosphoenolpyruvate carboxylase gene and/or glutamate dehydrogenase gene are enhanced include the microorganisms disclosed in Japanese Patent Laid-open Nos. 2001-333769, 2000-106869, 2000-189169, 2000-333769, 2006-129840, WO2006/051660, and the like.
  • L-glutamic acid-producing ability can also be imparted by enhancing the 6-phosphogluconate dehydratase activity, 2-keto-3-deoxy-6-phosphogluconate aldolase activity, or both.
  • Examples of the microorganism in which 6-phosphogluconate dehydratase activity and 2-keto-3-deoxy-6-phosphogluconate aldolase activity are increased include the microorganism disclosed in Japanese Patent Laid-open No. 2003-274988.
  • L-glutamic acid-producing ability can also be imparted by decreasing or eliminating the activity of an enzyme that catalyzes a reaction that branches off from the L-glutamic acid biosynthesis pathway, producing a compound other than L-glutamic acid.
  • examples of these include 2-oxoglutarate dehydrogenase (sucA), isocitrate lyase (aceA), phosphate acetyltransferase (pta), acetate kinase (ack), acetohydroxy acid synthase (ilvG), acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), glutamate decarboxylase (gadAB), 1-pyrroline-5-carboxylate dehydrogenase (putA), and the like.
  • Gene names are indicated in the parentheses after the enzyme names. The activity of 2-oxoglutarate dehydrogenas
  • a decrease in the intracellular activity of the target enzyme and the degree by which the activity is decreased, including if it is completely eliminated, can be confirmed by measuring the enzyme activity of a cell extract or a purified fraction thereof obtained from a candidate strain and comparing it with that of a wild-type strain.
  • 2-oxoglutarate dehydrogenase activity can be measured by the method of Reed et al. (Reed L. J. and Mukherjee B. B., 1969, Methods in Enzymology, 13, pp. 55-61).
  • Escherichia bacteria which are deficient in 2-oxoglutarate dehydrogenase activity or in which the 2-oxoglutarate dehydrogenase activity is decreased include the following strains (U.S. Pat. Nos. 5,378,616 and 5,573,945).
  • E. coli W3110sucA::Kmr is obtained by disrupting the 2-oxoglutarate dehydrogenase gene (sucA gene) of Escherichia coli W3110. This strain is completely deficient in 2-oxoglutarate dehydrogenase.
  • bacteria wherein the activity of 2-oxoglutarate dehydrogenase is deleted or decreased include the following strains:
  • Pantoea ananatis AJ13601 (FERM BP-7207, European Patent Laid-open No. 1078989)
  • Pantoea ananatis AJ13356 (FERM BP-6615, U.S. Pat. No. 6,331,419)
  • Pantoea ananatis SC17sucA (FERM BP-8646, WO2005/085419)
  • Klebsiella planticola AJ13410 strain (FERM BP-6617, U.S. Pat. No. 6,197,559)
  • the SC17sucA strain is obtained by obtaining a low phlegm production mutant strain (SC17) from the AJ13355 strain, which was isolated from nature as a strain that could proliferate in a medium containing L-glutamic acid and a carbon source at low pH, and disrupting the 2-oxoglutarate dehydrogenase gene (sucA) in the mutant strain.
  • SC17 low phlegm production mutant strain
  • the AJ13601 strain is obtained by introducing the plasmid RSFCPG containing the gltA, ppc and gdhA genes derived from Escherichia coli and the plasmid pSTVCB containing the gltA gene derived from Brevibacterium lactofermentum into the SC17sucA strain to obtain the SC17sucA/RSFCPG+pSTVCB strain, and selecting a high concentration L-glutamic acid-resistant strain at a low pH and a strain showing a high proliferation degree and a high L-glutamic acid-producing ability (European Patent Laid-open No. 0952221).
  • the AJ13356 strain was obtained by deleting the ⁇ KGDH-E1 subunit gene (sucA) from the AJ13355 strain.
  • the SC17sucA strain was assigned a private number of AJ417, deposited in the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, postal code: 305-8566) on Feb. 26, 2004, and assigned an accession number of FERM BP-08646.
  • the AJ13410 strain was deposited in the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, postal code: 305-8566) on Feb. 19, 1998, and assigned an accession number of FERM P-16647. The deposit was then converted to an international deposition under the provisions of Budapest Treaty on Jan. 11, 1999 and assigned an accession number of FERM BP-6617.
  • Pantoea ananatis AJ13355, AJ13356, and AJ13601 strains and the Klebsiella planticola AJ13399 strain have an ability to accumulate L-glutamic acid in a concentration which exceeds the saturation concentration of L-glutamic acid in a liquid medium when it is cultured under acid conditions.
  • the method of deleting the arcA gene (U.S. Pat. No. 7,090,998), and the method of amplifying the yhfK gene, which is a glutamic acid secretion gene (WO2005/085419) can also be used.
  • examples of coryneform bacteria having L-glutamic acid-producing ability include the following strains:
  • Corynebacterium melassecola DH344 (FERM P-11117, refer to Japanese Patent Laid-open No. 3-232497)
  • Microorganisms having L-glutamic acid-producing ability include the Brevibacterium lactofermentum AJ13029 strain (FERM BP-5189, refer to WO96/06180), which was made to be able to produce L-glutamic acid in a medium containing an excessive amount of biotin in the absence of biotin action inhibitor, such as surfactant, by introducing a mutation which imparts temperature sensitivity to the biotin action inhibitor.
  • Examples further include microorganisms that belong to the genus Alicyclobacillus and are resistant to a L-glutamic acid antimetabolite (Japanese Patent Laid-open No. 11-262398).
  • the microorganism having a L-glutamic acid producing ability can also be unable to degrade L-glutamic acid, or express the maleate synthase (aceB)•isocitrate lyase (aceA)•isocitrate dehydrogenase kinase/phosphatase (aceK) operon (henceforth abbreviated as “ace operon”) constitutively.
  • aceB maleate synthase
  • aceA isocitrate lyase
  • aceK isocitrate dehydrogenase kinase/phosphatase
  • ace operon constitutively.
  • Examples of such microorganisms include, for example, the following:
  • the former is a mutant strain in which 2-oxoglutarate dehydrogenase activity is decreased and expression of the ace operon has become constitutive.
  • the latter is a mutant strain in which 2-oxoglutarate dehydrogenase activity is decreased and the ability to degrade L-glutamic acid is decreased (refer to French Patent No. 2680178).
  • a mutation can be imparted that results in production of less extracellular phlegm as compared to a wild-type strain when it is cultured in a medium containing a saccharide.
  • bacterial strains can be screened for their ability to produce viscous materials on the solid medium (Japanese Patent Laid-open No. 2001-333769), and the ams operon that is involved in polysaccharide synthesis can be disrupted.
  • the nucleotide sequence of the ams operon is shown in SEQ ID NO: 66, and the amino acid sequences of AmsH, I, A, C and B encoded by this operon are shown in SEQ ID NOS: 67, 68, 69, 70 and 71, respectively.
  • strains which are unable to form acetic acid, lactic acid, ethanol, and formic acid can be used, and specific examples include the Escherichia coli SS373 strain (International Patent Publication WO99/06532).
  • Strains deficient in their abilities to form acetic acid, lactic acid, ethanol and formic acid can be obtained by using a strain that cannot assimilate acetic acid and lactic acid in a minimal medium, or by decreasing the activities of the lactic acid or acetic acid biosynthetic enzymes described below (International Patent Publication WO2005/052135).
  • strains as described above can also be obtained by imparting monofluoroacetic acid resistance (U.S. Pat. No. 5,521,075).
  • kits for obtaining a strain with improved succinic acid-producing ability include culturing a strain which is deficient in both formate lyase and lactate dehydrogenase and cannot assimilate pyruvic acid in a glucose-enriched medium under anaerobic conditions, and isolating a mutant strain having the ability to assimilate pyruvic acid (International Patent Publication WO97/16528).
  • the ability to produce succinic acid can also be imparted by amplifying a gene encoding an enzyme which is involved in the succinic acid biosynthesis system described below, or deleting a gene encoding an enzyme which catalyzes a reaction which branches away from the succinic acid biosynthesis system to produce another compound.
  • the ability to produce succinic acid can also be imparted by modifying a microorganism to decrease the enzymatic activity of lactate dehydrogenase (LDH), which is a lactic acid biosynthesis system enzyme (International Patent Publications WO2005/052135, WO2005/116227, U.S. Pat. No. 5,770,435, U.S. Patent Published Application No. 20070054387, International Patent Publication WO99/53035, Alam, K. Y. and Clark, D. P., 1989, J. Bacteriol., 171:6213-6217).
  • Some microorganisms can have both L-lactate dehydrogenase and D-lactate dehydrogenase, and can be modified to decrease the activity of either one of the enzymes, or the activities of both the enzymes, in another example.
  • the ability to produce succinic acid can also be imparted by modifying a microorganism to decrease the enzymatic activity of the formic acid biosynthesis system enzyme, pyruvate-formate lyase (PFL) (U.S. Patent Published Application No. 20070054387, International Patent Publications WO2005/116227, WO2005/52135, Donnelly, M. I. et al., 1998, Appl. Biochem. Biotechnol., 70-72:187-198).
  • PFL pyruvate-formate lyase
  • the ability to produce succinic acid can also be imparted by modifying a microorganism to decrease the enzymatic activities of phosphate acetyltransferase (PTA), acetate kinase (ACK), pyruvate oxidase (POXB), acetyl-CoA synthetase (ACS) and acetyl-CoA hydrolase (ACH), which are acetic acid biosynthesis system enzymes (U.S. Patent Published Application No. 20070054387, International Patent Publications WO2005/052135, WO99/53035, WO2006/031424, WO2005/113745, and WO2005/113744).
  • PTA phosphate acetyltransferase
  • ACK acetate kinase
  • POXB pyruvate oxidase
  • ACS acetyl-CoA synthetase
  • ACH acetyl-CoA hydrolase
  • the ability to produce succinic acid can also be enhanced by modifying a microorganism to decrease the enzymatic activity of alcohol dehydrogenase (ADH), which is an ethanol biosynthesis system enzyme (refer to International Patent Publication WO2006/031424).
  • ADH alcohol dehydrogenase
  • the ability to produce succinic acid can also be enhanced by decreasing the activities of pyruvate kinase, glucose PTS (ptsG), ArcA protein, IclR protein (iclR), glutamate dehydrogenase (gdh) and/or glutamine synthetase (glnA), and glutamate synthase (gltBD) (International Patent Publication WO2006/107127, WO2007007933, Japanese Patent Laid-open No. 2005-168401).
  • the gene names are indicated in the parentheses following the enzyme names.
  • the ability to produce succinic acid can also be imparted by enhancing a biosynthesis system enzyme involved in the succinic acid production.
  • the ability to produce succinic acid can also be enhanced by increasing the enzymatic activities of pyruvate carboxylase, malic enzyme, phosphoenolpyruvate carboxylase, fumarase, fumarate reductase, malate dehydrogenase and phosphoenolpyruvate carboxykinase (Japanese Patent Laid-open No. 11-196888, International Patent Publication WO99/53035, Hong, S. H., and S. Y. Lee, 2001, Biotechnol. Bioeng., 74:89-95, Millard, C. S. et al., 1996, Appl. Environ.
  • Microbiol., 62:1808-1810 International Patent Publication WO2005/021770, Japanese Patent Laid-open No. 2006-320208, Kim, P. et al., 2004, Appl. Environ. Microbiol., 70:1238-1241).
  • Increasing the enzymatic activities of these target enzymes can be performed by referring to the methods for enhancing expression of a target gene described herein, and the methods for enhancing expression of ybjL gene described later.
  • succinic acid-producing bacteria belonging to the family Enterobacteriaceae include the following strains:
  • Escherichia coli AFP184 strain (International Patent Publication WO2005/116227)
  • Escherichia coli SBS100MG strain, SBS110MG strain, SBS440MG strain, SBS550MG strain, and SBS660MG strain International Patent Publication WO2006/031424.
  • succinic acid-producing bacteria belonging to coryneform bacteria include the following strains.
  • succinic acid-producing rumen bacteria examples include the following strains.
  • Anaerobiospirillum succiniciproducens FZ10 U.S. Pat. No. 5,521,075
  • Anaerobiospirillum succiniciproducens FZ53 (U.S. Pat. No. 5,573,931)
  • a microorganism that has an ability to produce an acidic substance having a carboxyl group can be modified such as those described above so that expression of the ybjL gene is enhanced.
  • the modification to enhance expression of the ybjL gene is performed first, and then the ability to produce an acidic substance having a carboxyl group can be imparted.
  • the microorganism can already have the ability to produce an acidic substance having a carboxyl group, or the ability to produce an acidic substance having a carboxyl group can be enhanced by amplification of the ybjL gene.
  • the “ybjL gene” refers to the ybjL gene of Escherichia coli or a homologue thereof, the ybjL gene of Pantoea ananatis or a homologue thereof, or the ybjL gene of Enterobacter aerogenes or a homologue thereof.
  • Examples of the ybjL gene of Escherichia coli include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 4, for example, a gene having the nucleotide sequence of the nucleotides 101 to 1783 in SEQ ID NO: 3.
  • examples of the ybjL gene derived from Pantoea ananatis include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 2, for example, a gene having the nucleotide sequence of the nucleotides 298 to 1986 in SEQ ID NO: 1.
  • examples of the ybjL gene derived from the Enterobacter aerogenes AJ110673 strain include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 87, for example, a gene having the nucleotide sequence of the nucleotides 19 to 1704 in SEQ ID NO: 86.
  • examples of the ybjL gene derived from Salmonella typhimurium include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 25 (SEQ ID NO: 24).
  • examples of the ybjL gene derived from Yersinia pestis include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 27 (SEQ ID NO: 26).
  • Examples of the ybjL gene derived from Erwinia carotovora include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 29 (SEQ ID NO: 28).
  • Examples of the ybjL gene derived from Vibrio cholerae include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 31 (SEQ ID NO: 30).
  • Examples of the ybjL gene derived from Aeromonas hydrophila include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 33 (SEQ ID NO: 32).
  • Examples of the ybjL gene derived from Photobacterium profundum include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 35 (SEQ ID NO: 34).
  • the ybjL gene can be cloned from a coryneform bacterium such as Corynebacterium glutamicum and Brevibacterium lactofermentum, Pseudomonas bacterium such as Pseudomonas aeruginosa, Mycobacterium bacterium such as Mycobacterium tuberculosis or the like, on the basis of homology to the genes exemplified above.
  • coryneform bacterium such as Corynebacterium glutamicum and Brevibacterium lactofermentum
  • Pseudomonas bacterium such as Pseudomonas aeruginosa
  • Mycobacterium bacterium such as Mycobacterium tuberculosis or the like
  • amino acid sequences of the proteins encoded by the ybjL genes of Salmonella typhimurium, Yersinia pestis, Erwinia carotovora, Vibrio cholerae, Aeromonas hydrophila and Photobacterium profundum described above are 96%, 90%, 88%, 64%, 60% and 68% homologous to the amino acid sequence of SEQ ID NO: 4, respectively, and 86%, 90%, 84%, 63%, 60% and 67% homologous to the amino acid sequence of SEQ ID NO: 2, respectively.
  • the amino acid sequences of SEQ ID NOS: 2 and 4 are 86% homologous.
  • the consensus sequence of SEQ ID NOS: 2 and 4 is shown in SEQ ID NO: 5.
  • amino acid sequences of SEQ ID NOS: 4 and 87 re 92% homologous, and the amino acid sequences of SEQ ID NOS: 2 and 87 are 83% homologous.
  • sequence of the ybjL gene from the Enterobacter aerogenes AJ110637 strain SEQ ID NO: 86
  • sequence of the encoded amino acid sequence SEQ ID NO: 87
  • the consensus sequence for the amino acid sequences of SEQ ID NOS: 2, 4 and 87 is shown in SEQ ID NO: 88.
  • ybjL gene homologue refers to a gene derived from a microorganism other than Escherichia coli, Pantoea ananatis , and Enterobacter aerogenes , which exhibits high structural similarity to the ybjL gene of Escherichia coli, Pantoea ananatis , or Enterobacter aerogenes and codes for a protein that improves an ability of a microorganism to produce an acidic substance having a carboxyl group when expression of the gene is enhanced in the microorganism.
  • Examples of ybjL gene homologues include genes coding for a protein having a homology of 70% or more, 80% or more in another example, 90% or more in another example, 95% or more in another example, 97% or more in another example, to the entire amino acid sequence of SEQ ID NO: 2, 4 or 87 or the amino acid sequence of SEQ ID NO: 2, 4 or 87, and which improves an ability to produce an acidic substance having a carboxyl group of a microorganism when expression of the gene is enhanced in the microorganism.
  • the ybjL gene homologue can code for a protein having a homology of 70% or more, 80% or more in another example, 90% or more in another example, 95% or more in another example, or 97% or more in another example, to any of the aforementioned amino acid sequences, and the consensus sequences of the SEQ ID NOS: 5 or 88.
  • Homology of the amino acid sequences and nucleotide sequences can be determined by using, for example, the algorithm BLAST of Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) or FASTA (Methods Enzymol., 183, 63 (1990)). Programs called BLASTN and BLASTX were developed on the basis of the algorithm BLAST (refer to www.ncbi.nlm.nih.gov). The term “homology” can also be used to mean “identity”.
  • the ybjL gene can have one or more conservative mutations, and can be artificially modified, so long as enhancement of the tbjL gene's expression results in an improved ability to produce a target substance by a microorganism. That is, the ybjL gene can encode for an amino acid sequence of a known protein, for example, a protein having an amino acid sequence of SEQ ID NO: 2, 4, 25, 27, 29, 31, 33, 35 or 87, but which includes a conservative mutation, specifically, substitution, deletion, insertion or addition, of one or several amino acid residues at one or several positions. Although the number meant by the term “several” can differ depending on the position in the three-dimensional structure of the protein, or the type of amino acid residue.
  • the ybjL gene can encode for a protein having a homology of 70% or more, 80% or more in another example, 90% or more in another example, 95% or more in another example, or 97% or more in another example, to the entire amino acid sequence of SEQ ID NO: 2, 4, 25, 27, 29, 31, 33, 35 or 87, and which improves an ability to produce a target substance by a microorganism when expression of the gene is enhanced in the microorganism.
  • the aforementioned conservative substitution can be neutral, in that the function of the protein is not changed.
  • the conservative mutation can take place mutually among Phe, Tip and Tyr, if the substitution site is an aromatic amino acid; among Leu, Ile and Val, if the substitution site is a hydrophobic amino acid; between Gln and Asn, if it is a polar amino acid; among Lys, Arg and His, if it is a basic amino acid; between Asp and Glu, if it is an acidic amino acid; and between Ser and Thr, if it is an amino acid having hydroxyl group.
  • substitution considered conservative substitution include: substitution of Ser or Thr for Ala; substitution of Gln, His or Lys for Arg; substitution of Glu, Gln, Lys, His or Asp for Asn; substitution of Asn, Glu or Gln for Asp; substitution of Ser or Ala for Cys; substitution of Asn, Glu, Lys, His, Asp or Arg for Gln; substitution of Gly, Asn, Gln, Lys or Asp for Glu; substitution of Pro for Gly; substitution of Asn, Lys, Gln, Arg or Tyr for H is; substitution of Leu, Met, Val or Phe for Ile; substitution of Ile, Met, Val or Phe for Leu; substitution of Asn, Glu, Gln, His or Arg for Lys; substitution of Ile, Leu, Val or Phe for Met; substitution of Trp, Tyr, Met, Ile or Leu for Phe; substitution of Thr or Ala for Ser; substitution of Ser or Ala for Thr; substitution of P
  • Such a gene can be obtained by modifying the nucleotide sequence of the nucleotides 298 to 1986 in SEQ ID NO: 1, the nucleotide sequence of the nucleotides 101 to 1783 in SEQ ID NO: 3, the nucleotide sequence of the nucleotides 19 to 1704 in SEQ ID NO: 86, or the nucleotide sequence of SEQ ID NO: 24, 26, 28, 30, 32 or 34 by, for example, site-specific mutagenesis, so that substitution, deletion, insertion or addition of an amino acid residue or residues occurs at a specific site in the encoded protein.
  • such a gene can also be obtained by a known mutation treatment, examples of which include treating a gene having any of the nucleotide sequences described above with hydroxylamine or the like in vitro, and treating a microorganism, for example, an Escherichia bacterium, containing the gene with ultraviolet ray irradiation or a mutagen used in a usual mutation treatment, such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or EMS (ethyl methanesulfonate).
  • NTG N-methyl-N′-nitro-N-nitrosoguanidine
  • EMS ethyl methanesulfonate
  • the mutation for the substitutions, deletions, insertions, additions, inversions or the like of amino acid residues described above also includes a naturally occurring mutation based on individual difference, difference in species of microorganisms that contain the ybjL gene (mutant or variant), and the like.
  • the ybjL gene can also be a DNA hybridizable with a complementary strand of DNA having the nucleotide sequence of the nucleotides 298 to 1986 in SEQ ID NO: 1, a DNA having the nucleotide sequence of the nucleotides 101 to 1783 in SEQ ID NO: 3, a DNA having the nucleotide sequence of the nucleotides 19 to 1704 in SEQ ID NO: 86, or a DNA having the nucleotide sequence of SEQ ID NO: 1, 3, 24, 26, 28, 30, 32, 34 or 86, or a probe that can be prepared from the DNAs having these sequences under stringent conditions and which codes for a protein which improves an ability to produce a substance of a microorganism when expression of the gene is enhanced in the microorganism.
  • the “stringent conditions” can be conditions under which a so-called specific hybrid is formed, and non-specific hybrid is not formed. It is difficult to clearly express this condition by using any numerical value.
  • the stringent conditions include, for example, when DNAs having high homology to each other, for example, DNAs having a homology of, for example, not less than 80%, not less than 90% in another example, not less than 95% in another example, or not less than 97% in another example, hybridize with each other, and DNAs having homology lower than the above level do not hybridize with each other, and when washing in ordinary Southern hybridization, i.e., washing once, twice or three times in another example, at salt concentrations and temperature of 1 ⁇ SSC, 0.1% SDS at 60° C., 0.1 ⁇ SSC, 0.1% SDS at 60° C. in another example, 0.1 ⁇ SSC, 0.1% SDS at 65° C., 0.1 ⁇ SSC in another example, or 0.1% SDS at 68° C. in another example.
  • the probe can have a partial sequence of the ybjL gene.
  • a probe can be produced by PCR using oligonucleotides prepared on the basis of the nucleotide sequence of the gene and a DNA fragment including the gene as the template and performed in a manner well known to those skilled in the art.
  • the washing conditions after hybridization can be exemplified by 2 ⁇ SSC, 0.1% SDS at 50° C.
  • the expression of the ybjL gene can be enhanced by increasing the copy number of the ybjL gene, modifying an expression control sequence of the ybjL gene, amplifying a regulator that increases expression of the ybjL gene, or deleting or attenuating a regulator that decreases expression of the ybjL gene.
  • Expression can be enhanced or increased using transformation or homologous recombination performed in the same manner as the methods for enhancing expression of a target gene described above for L-glutamic acid-producing bacteria.
  • an activity for secreting an acidic substance having a carboxyl group can be improved by enhancing the expression of the ybjL gene. Whether “the activity for secreting an acidic substance having a carboxyl group is improved” can be confirmed by comparing the amount of the acidic substance having a carboxyl group which has been secreted into the medium in which the microorganism is cultured with the amount obtained by culturing a control microorganism into which the ybjL gene is not introduced.
  • the “improvement in the activity for secreting the acidic substance having a carboxyl group” is observed as an increase in the concentration of the acidic substance having a carboxyl group in the medium in which the microorganism is cultured as compared to the concentration obtained with a control microorganism. Furthermore, the “improvement in the activity for secreting an acidic substance having a carboxyl group” is also observed as a decrease in intracellular concentration of the acidic substance having a carboxyl group in the microorganism.
  • the intracellular concentration of the acidic substance having a carboxyl group can be decreased by 10% or more, 20% or more in another example, 30% or more in another example, as compared to the concentration in a strain in which expression of the ybjL gene is not enhanced.
  • the absolute “activity for secreting an acidic substance having a carboxyl group” of a microorganism can be detected by measuring the difference between the intracellular and extracellular concentrations of the acidic substance having a carboxyl group.
  • the “activity for secreting an acidic substance having a carboxyl group” can also be detected by measuring the activity for taking up amino acids into the cells using reverted membrane vesicles with a radioisotope (J. Biol. Chem., 2002, vol. 277, No. 51, pp. 49841-49849).
  • the activity can be measured by preparing reverted membrane vesicles from cells in which the ybjL gene is expressed, adding a substrate which can act as a driving force such as ATP, and measuring the uptake activity for RI-labeled glutamic acid.
  • the activity can also be measured by detecting an exchange reaction rate of a labeled acidic substance having a carboxyl group and unlabeled acidic substance having a carboxyl group in live bacteria.
  • the microorganism can have an ability to accumulate L-glutamic acid in the liquid medium in an amount which is more than the amount at the saturation concentration of L-glutamic acid when it is cultured under acidic conditions (this ability is also referred to as the “L-glutamic acid accumulation ability under acidic conditions”).
  • Such a microorganism can have the L-glutamic acid accumulation ability under acidic conditions by enhancing the expression of the ybjL gene.
  • the microorganism can have a native ability to accumulate L-glutamic acid under acidic conditions.
  • microorganisms having L-glutamic acid accumulation ability under acidic conditions include the aforementioned Pantoea ananatis AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207) (for these, refer to Japanese Patent Laid-open No. 2001-333769), and the like.
  • Pantoea ananatis AJ13355 and AJ13356 strains were deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology, Address: Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Feb. 19, 1998 and given accession numbers of FERM P-16644 and FERM P-16645. The deposits were then converted to international deposits under the provisions of Budapest Treaty on Jan. 11, 1999 and given accession numbers of FERM BP-6614 and FERM BP-6615.
  • the AJ13601 strain was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently, International Patent Organism Depository, National Institute of Advanced Industrial Science and Technology) on Aug. 18, 1999 and given an accession number of FERM P-17516. The deposit was converted to an international deposit under the provisions of the Budapest Treaty on Jul. 6, 2000 and given an accession number of FERM BP-7207. These strains were identified as Enterobacter agglomerans when they were isolated and deposited as Enterobacter agglomerans AJ13355, AJ13356, and AJ13601 strains. However, they were recently re-classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and the like.
  • an organic acid especially succinic acid
  • a microorganism which is modified to decrease the activities of one or more enzymes among lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), and pyruvate formate lyase (PFL), in addition to increasing the expression of the ybjL gene, is more effective.
  • LDH lactate dehydrogenase
  • ADH alcohol dehydrogenase
  • PFL pyruvate formate lyase
  • the expression “modified so that lactate dehydrogenase activity is decreased” can mean that the lactate dehydrogenase activity is decreased as compared to that of a strain in which lactate dehydrogenase is unmodified.
  • the lactate dehydrogenase activity can be decreased to 10% per cell or lower as compared to that of a lactate dehydrogenase-unmodified strain.
  • the lactate dehydrogenase activity can also be completely deleted. Decrease of the lactate dehydrogenase activity can be confirmed by measuring the lactate dehydrogenase activity by a known method (Kanarek, L. and Hill, R. L., 1964, J. Biol. Chem., 239:4202).
  • the method for producing a mutant strain of Escherichia coli in which the lactate dehydrogenase activity is decreased include the method described in Alam, K. Y., and Clark, D. P., 1989, J. Bacteriol., 171:6213-6217, and the like.
  • the microorganism with decreased lactate dehydrogenase activity and enhanced expression of the ybjL gene can be obtained by, for example, preparing a microorganism in which the LDH gene is disrupted, and transforming this microorganism with a recombinant vector containing the ybjL gene, as described in Example 1.
  • LDH is encoded by the ldhA and lldD genes.
  • the DNA sequence of the ldhA gene is shown in SEQ ID NO: 36
  • the amino acid sequence encoded thereby is shown in SEQ ID NO: 37
  • the DNA sequence of the lldD gene is shown in SEQ ID NO: 38
  • the amino acid sequence encoded thereby is shown in SEQ ID NO: 39.
  • a mutation that decreases or deletes the intracellular activity of LDH can be introduced into the LDH gene on the chromosome by a known mutagenesis method.
  • the gene coding for LDH on the chromosome can be deleted, or an expression control sequence such as a promoter and the Shine-Dalgarno (SD) sequence can be modified by gene recombination.
  • SD Shine-Dalgarno
  • a mutation can also be introduced to cause an amino acid substitution (missense mutation), a stop codon (nonsense mutation), or a frame shift mutation that adds or deletes one or two nucleotides into the coding region of the LDH on the chromosome, or a part of, or the entire gene can be deleted (Qiu Z.
  • the LDH activity can also be decreased or deleted by gene disruption, for example, by deleting the coding region of the LDH gene in a DNA construct, and replacing the normal LDH gene with the DNA construct on the chromosome by homologous recombination or the like, or by introducing a transposon or IS factor into the gene.
  • the chromosomal LDH gene can be replaced with a mutant gene by preparing a mutant LDH gene in which a partial sequence of the LDH gene is modified so that it does not produce an enzyme that can function normally, and transforming a bacterium with a DNA containing the mutant gene to cause homologous recombination between the mutant gene and the gene on a chromosome.
  • Such site-specific mutagenesis based on gene substitution utilizing homologous recombination has been already reported, and include a method called Red driven integration developed by Datsenko and Wanner (Datsenko, K. A, and Wanner, B.
  • the expression “modified so that alcohol dehydrogenase activity is decreased” can mean that the alcohol dehydrogenase activity is decreased as compared to that of a strain in which alcohol dehydrogenase is unmodified.
  • the alcohol dehydrogenase activity can be decreased to 10% per cell or lower as compared to an alcohol dehydrogenase-unmodified strain.
  • the alcohol dehydrogenase activity can also be completely deleted. Decrease in the alcohol dehydrogenase activity can be confirmed by measuring the alcohol dehydrogenase activity by a known method (Lutstorf, U. M. et al., 1970, Eur. J. Biochem., 17:497-508).
  • the method for producing a mutant strain of Escherichia coli in which the alcohol dehydrogenase activity is decreased include the method described in Sanchez A. M. et al., 2005, Biotechnol. Prog., 21:358-365, and the like.
  • the microorganism in which alcohol dehydrogenase activity is decreased and expression of the ybjL gene is enhanced can be obtained by, for example, preparing a microorganism in which the gene coding for alcohol dehydrogenase (ADH) is disrupted, and transforming this microorganism with a recombinant vector containing the ybjL gene.
  • the alcohol dehydrogenase activity can be decreased by a method similar to that for decreasing the lactate dehydrogenase activity described above.
  • the nucleotide sequence (partial sequence) of the ADH gene from the Enterobacter aerogenes AJ110637 strain (FERM ABP-10955) is shown in SEQ ID NO: 74.
  • the entire nucleotide sequence of this gene can be determined by, for example, isolating the ADH gene (adhE) from the chromosomal DNA of Enterobacter aerogenes on the basis of this partial sequence.
  • the expression “modified so that pyruvate formate lyase activity is decreased” can mean that the pyruvate formate lyase activity is decreased as compared to that of a strain in which the pyruvate formate lyase is unmodified.
  • the pyruvate formate lyase activity can be decreased to 10% per cell or lower as compared to a pyruvate formate lyase-unmodified strain.
  • the pyruvate formate lyase activity can also be completely deleted.
  • a decrease in the pyruvate formate lyase activity can be confirmed by measuring the pyruvate formate lyase activity by a known method (Knappe, J.
  • the microorganism in which pyruvate formate lyase activity is decreased and expression of the ybjL gene is enhanced can be obtained by, for example, preparing a microorganism in which the PFL gene is disrupted, and transforming this microorganism with a recombinant vector containing the ybjL gene. However, either the modification for decreasing the PFL activity or the modification for enhancing expression of ybjL can be performed first.
  • the pyruvate formate lyase activity can be decreased by a method similar to the method for decreasing the lactate dehydrogenase activity described above.
  • a bacterium modified so that the pyruvate carboxylase (PC) activity is enhanced, in addition to the enhanced the expression of the ybjL gene, can also be used to produce an organic acid, especially succinic acid. Enhancing the pyruvate carboxylase activity can be combined with decreasing the lactate dehydrogenase activity, alcohol dehydrogenase activity, and/or pyruvate formate lyase activity.
  • the expression “modified so that pyruvate carboxylase activity is enhanced” can mean that the pyruvate carboxylase activity is increased as compared to that of an unmodified strain such as a wild-type strain or parent strain.
  • the pyruvate carboxylase activity can be measured by, for example, a method of measuring a decrease of NADH as described later.
  • the nucleotide sequence of the PC gene can be a reported sequence or can be obtained by isolating a DNA fragment encoding a protein having the PC activity from the chromosome of a microorganism, animal, plant, or the like, and then the nucleotide sequence can be determined. After the nucleotide sequence is determined, a gene synthesized on the basis of that sequence can also be used.
  • the PC gene can be derived from a coryneform bacterium, such as Corynebacterium glutamicum (Peters-Wendisch, P. G. et al., 1998, Microbiology, vol. 144:915-927). Furthermore, so long as the functions of the encoded PC protein, such as its involvement in carbon dioxide fixation, are not substantially degraded, nucleotides in the PC gene can be replaced with other nucleotides or deleted, or other nucleotides can be inserted into the sequence. Alternatively, a part of the nucleotide sequence can be transferred.
  • PC genes obtained from bacteria other than Corynebacterium glutamicum can also be used.
  • sequences of PC genes derived from other microorganisms, animals and plants described below are known (references are indicated below), and they can be obtained by hybridization or amplification by PCR of the ORF portions in the same manner as described above.
  • Yeast Saccharomyces cerevisiae [Mol. Gen. Genet., 229, 307-315, (1991)], Schizosaccharomyces pombe [DDBJ Accession No.; D78170]
  • Rhizobium etli J. Bacteriol., 178, 5960-5970, (1996)
  • the PC gene can be enhanced in the same manner as when enhancing expression of a target gene described above for L-glutamic acid-producing bacteria and enhancing expression of the ybjL gene.
  • An acidic substance having a carboxyl group can be produced by culturing the microorganism in a medium to produce and cause accumulation of an acidic substance having a carboxyl group in the medium and collecting the substance from the medium. Furthermore, the acidic substance having a carboxyl group can also be produced by allowing the microorganism, or a product obtained by processing the microorganism, to act on an organic raw material in a reaction mixture containing carbonate ions, bicarbonate ions, or carbon dioxide gas to produce the acidic substance having a carboxyl group, and collecting the substance.
  • the former method is exemplary for producing an acidic amino acid.
  • the latter method is exemplary for producing an organic acid.
  • further examples methods for producing an acidic amino acid and an organic acid will be exemplified.
  • An acidic amino acid can be produced by culturing the microorganism in a medium to produce and cause accumulation of an acidic amino acid in the medium and collecting the acidic amino acid from the medium.
  • the medium used for the culture a known medium containing a carbon source, nitrogen source, and inorganic salts, as well as trace amounts of organic nutrients such as amino acids and vitamins as required can be used. Either a synthetic medium or natural medium can be used.
  • the carbon source and nitrogen source used in the medium can be of any type so long as substances that can be utilized by the chosen strain to be cultured.
  • the carbon source saccharides such as glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolysate and molasses can be used.
  • alcohols such as ethanol can be used independently or in combination with another carbon source.
  • organic acids such as acetic acid and citric acid other than the target substance can also be used as the carbon source.
  • ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate and ammonium acetate, nitrates and the like can be used.
  • amino acids amino acids, vitamins, fatty acids, nucleic acids, those containing these substances such as peptone, casamino acid, yeast extract and soybean protein decomposition products
  • amino acids vitamins, fatty acids, nucleic acids, those containing these substances such as peptone, casamino acid, yeast extract and soybean protein decomposition products
  • the required nutrient can be supplemented.
  • mineral salts phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like can be used.
  • the culture can be performed as an aeration culture, while the fermentation temperature can be controlled to be 20 to 45° C., and pH to be 3 to 9.
  • the medium can be neutralized by addition of, for example, calcium carbonate, or with an alkali such as ammonia gas.
  • An acidic amino acid can accumulate in the culture broth, for example, after 10 to 120 hours of culture under such conditions as described above.
  • L-glutamic acid can precipitate into the medium by using a liquid medium, the pH of which is adjusted so that L-glutamic acid precipitates.
  • L-glutamic acid can be collected from the culture medium by any known collection method. For example, after cells are removed from the culture medium, L-glutamic acid can be collected by concentrating the culture medium so it crystallizes, or by ion exchange chromatography, or the like. When the culture is performed so that L-glutamic acid precipitates, the L-glutamic acid that has precipitated in the medium can be collected by centrifugation, filtration, or the like. In this case, it is also possible to crystallize L-glutamic acid that has dissolved in the medium, and then collect the precipitated L-glutamic acid in the culture broth together with the crystallized L-glutamic acid.
  • An organic acid can be produced by allowing the microorganism, or a product obtained by processing the microorganism, to act on an organic raw material in a reaction mixture containing carbonate ions, bicarbonate ions, or carbon dioxide gas, and collecting the organic acid.
  • the medium can be the reaction mixture.
  • Proliferation of the microorganism and production of the organic acid can simultaneously occur, or there can be a period during the culture in which proliferation of the microorganism mainly occurs, and a period in which production of the organic acid mainly occurs.
  • an organic acid can be produced.
  • a product obtained by processing the cells of the microorganism can also be used.
  • Examples of the product obtained by processing cells include, for example, immobilized cells obtained with acrylamide, carragheenan, or the like, a disrupted cellular product, a centrifugation supernatant of the disrupted product, a fraction obtained by partial purification of the supernatant by ammonium sulfate treatment or the like, and the like.
  • bacteria can be obtained on a solid medium such as an agar medium by slant culture, bacteria previously cultured in a liquid medium (seed culture) are examples.
  • a medium usually used for culture of microorganisms can be used.
  • a typical medium obtained by adding natural nutrients such as meat extract, yeast extract and peptone, to a composition comprising inorganic salts such as ammonium sulfate, potassium phosphate and magnesium sulfate can be used.
  • the carbon source added to the medium also serves as the organic raw material for the production of the organic acid.
  • the cells after the culture are collected by centrifugation, membrane separation, or the like, and used for the organic acid production reaction.
  • the organic raw material is not particularly limited so long as the chosen microorganism can assimilate it to produce succinic acid.
  • fermentable carbohydrates including carbohydrates such as galactose, lactose, glucose, fructose, glycerol, sucrose, saccharose, starch and cellulose, polyalcohols such as glycerin, mannitol, xylitol and ribitol, and the like are typically used.
  • Glucose, fructose and glycerol are examples, and glucose is a particular example.
  • succinic acid fumaric acid or the like can be added in order to efficiently produce succinic acid as described in Japanese Patent Laid-open No. 5-68576, and malic acid can be added instead of fumaric acid.
  • concentration of the aforementioned organic raw material is not particularly limited, it is more advantageous that the concentration is as high as possible and within such a range that the culture of the microorganism and production of the organic acid are not inhibited.
  • concentration of the organic raw material in the medium is generally in the range of 5 to 30% (w/v), or 10 to 20% (w/v) in another example.
  • concentration of the organic raw material in the reaction mixture is generally in the range of 5 to 30% (w/v), or 10 to 20% (w/v) in another example.
  • the aforementioned reaction mixture containing carbonate ions, bicarbonate ions, or carbon dioxide gas and the organic raw material is not particularly limited, and it can be, for example, a typical medium for culturing microorganisms, or it can be a buffer such as phosphate buffer.
  • the reaction mixture can be an aqueous solution containing a nitrogen source, inorganic salts, and the like.
  • the nitrogen source is not particularly limited so long the chosen microorganism can assimilate it to produce an organic acid, and specific examples include various organic or inorganic nitrogen compounds such as ammonium salts, nitrates, urea, soybean hydrolysate, casein degradation products, peptone, yeast extract, meat extract, and corn steep liquor.
  • inorganic salts examples include various phosphates, sulfates, and metallic salts such as those of magnesium, potassium, manganese, iron, and zinc. If necessary, growth-promoting factors including vitamins such as biotin, pantothenic acid, inositol, and nicotinic acid, nucleotides, amino acids and the like can be added. In order to suppress foaming during the reaction, an appropriate amount of a commercially available antifoam can be added to the medium.
  • the pH of the reaction mixture can be adjusted by adding sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide, or the like. Since the pH for the reaction is usually 5 to 10, or 6 to 9.5 in another example, the pH of the reaction mixture can be adjusted to be within the aforementioned range with an alkaline substance, carbonate, urea, or the like, even during the reaction, if needed.
  • Water, buffer, medium or the like can be used as the reaction mixture, but a medium is a particular example.
  • the medium can contain, for example, the aforementioned organic raw material, and carbonate ions, bicarbonate ions, or carbon dioxide gas, and the reaction can be performed under anaerobic conditions.
  • Magnesium carbonate, sodium carbonate, sodium bicarbonate, potassium carbonate, or potassium bicarbonate can be the source of the carbonate or bicarbonate ions, and these can also be used as a neutralizing agent.
  • carbonate or bicarbonate ions can also be supplied from carbonic acid or bicarbonic acid or salts thereof or carbon dioxide gas.
  • salts of carbonic acid or bicarbonic acid include, for example, magnesium carbonate, ammonium carbonate, sodium carbonate, potassium carbonate, ammonium bicarbonate, sodium bicarbonate, potassium bicarbonate, and the like.
  • Carbonate ions or bicarbonate ions can be added at a concentration of 0.001 to 5 M, 0.1 to 3 M in another example, or 1 to 2 M in another example.
  • carbon dioxide gas When carbon dioxide gas is present, it can be in an amount of 50 mg to 25 g, 100 mg to 15g in another example, or 150 mg to 10g in another example, per liter of the solution.
  • the optimal growth temperature of the chosen microorganism is generally in the range of 25 to 40° C. Therefore, the reaction temperature is generally in the range of 25 to 40° C., or in the range of 30 to 37° C. in another example.
  • the amount of bacterial cells in the reaction mixture is, although it is not particularly limited, 1 to 700 g/L, 10 to 500 g/L in another example, or 20 to 400 g/L in another example.
  • the reaction time can be 1 to 168 hours, or 3 to 72 hours in another example.
  • the reaction can be performed batchwise or on a column.
  • the culture of the bacteria can be performed under aerobic conditions.
  • the organic acid production reaction can be performed under aerobic conditions, microaerobic conditions, or anaerobic conditions.
  • the reaction can be performed in a sealed reaction vessel without aeration, with a supplied inert gas such as nitrogen gas, or with a supplied inert gas containing carbon dioxide gas, and the like.
  • the organic acid can accumulate in the reaction mixture (culture medium) and can be separated and purified from the reaction mixture in a conventional manner. Specifically, solids such as bacterial cells can be removed by centrifugation, filtration, or the like, then the resulting solution can be desalted with an ion exchange resin or the like, and the organic acid can be separated and purified from the solution by crystallization or column chromatography.
  • the target organic acid is succinic acid
  • a polymerization reaction can be carried out by using the produced succinic acid as a raw material to produce a polymer containing succinic acid.
  • the produced succinic acid can be converted into polymers such as polyesters and polyamides and used (Japanese Patent Laid-open No. 4-189822).
  • succinic acid-containing polymers include succinic acid polyesters obtained by polymerizing a diol such as butanediol and ethylene glycol and succinic acid, succinic acid polyamides obtained by polymerizing a diamine such as hexamethylenediamine and succinic acid, and the like.
  • succinic acid and succinic acid-containing polymers obtained by the production method described herein, and compositions containing these can be used for food additives, pharmaceutical agents, cosmetics, and the like.
  • This plasmid can be used in a wide range of hosts having different genetic backgrounds. This is because 1) this plasmid has the replicon of the RSF1010 wide host spectrum plasmid (Scholz, et al., 1989, Gene, 75:271-288; Buchanan-Wollaston et al., 1987, Nature, 328:172-175), which is stably maintained by many types of gram negative and gram positive bacteria, and even plant cells, 2) the ⁇ Red genes, gam, bet and exo, are under the control of the PlacUV5 promoter, which is recognized by the RNA polymerases of many types of bacteria (for example, Brunschwig, E.
  • the autoregulation factor PlacUV5-lacI and the p-non-dependent transcription terminator (TrrnB) of the rrnB operon of Escherichia coli lower the basal expression level of the ⁇ Red genes (Skorokhodova, A. Y. et al, 2004, Biotekhnologiya (Rus), 5, 3-21).
  • the RSF-Red-TER plasmid contains the levansucrase gene (sacB), and by the expression of this gene, the plasmid can be collected from cells in a medium containing sucrose.
  • the frequency of integration of a PCR-generated DNA fragment along with the short flanking region provided by the RSF-Red-TER plasmid is as high as the frequency obtained when using the pKD46 helper plasmid (Datsenko, K. A., Wanner, B. L., 2000, Proc. Natl. Acad. Sci. USA, 97, 6640-6645).
  • expression of the ⁇ Red genes is toxic to Pantoea ananatis .
  • a variant strain of Pantoea ananatis which is resistant to expression of all three of the ⁇ Red genes was selected.
  • the RSF-Red-TER plasmid was introduced into the Pantoea ananatis SC17 strain (U.S. Pat. No. 6,596,517) by electroporation. After an 18-hour culture, about 10 6 transformants were obtained, and among these, 10 clones formed colonies of a large size, and the remainder formed extremely small colonies. After an 18 hour culture, the large colonies were about 2 mm, and the small colonies were about 0.2 mm. While the small colonies did not grow any more even when the culture was extended up to 24 hours, the large colonies continued to grow.
  • One of the large colony Pantoea ananatis mutant strains which was resistant to expression of all three of the ⁇ Red genes was used for further analysis.
  • the RSF-Red-TER plasmid DNA was isolated from one clone of the large colony clones, and from several clones of the small colony clones, and transformed again into Escherichia coli MG1655 to examine the ability of the plasmid to synthesize an active Red gene product.
  • a control experiment for Red-dependent integration in the obtained transformants was used to demonstrate that only the plasmid isolated from the large colony clone induced expression of the ⁇ Red genes required for the Red-dependent integration.
  • electroporation was performed using a linear DNA fragment produced by PCR.
  • This fragment was designed so that it contains a Km R marker and a flanking region of 40 by homologous to the hisD gene, and is integrated into the hisD gene of Pantoea ananatis at the SmaI recognition site.
  • Two small colony clones were used as control.
  • the nucleotide sequence of the hisD gene of Pantoea ananatis is shown in SEQ ID NO: 40.
  • the oligonucleotides of SEQ ID NOS: 41 and 42 were used as primers, and the pMW118-( ⁇ att-Km r - ⁇ att) plasmid was used as the template.
  • the RSF-Red-TER plasmid can induce expression of the Red genes by the lad gene carried on the plasmid.
  • Two kinds of induction conditions were investigated. In the first group, IPTG (1 mM) was added 1 hour before the electroporation, and in the second group, IPTG was added at the start of the culture to prepare cells in which electroporation is possible.
  • the growth rate of the progeny of the SC17 strain harboring RSF-Red-TER derived from the large colony clone was not significantly lower than that of a strain not having the RSF-Red-TER plasmid. The addition of IPTG only slightly decreased the growth rate of these cultures.
  • the RSF-Red-TER-introduced SC17 strain derived from the progeny of the small colony clones grew extremely slowly even without the addition of IPTG, and after induction, growth was substantially arrested.
  • electroporation of RSF-Red-TER isolated from the cells of the progeny of the large colony clone many Km R clones grew (18 clones after a short induction time, and about 100 clones after an extended induction time). All of the 100 clones that were investigated had a His ⁇ phenotype, and about 20 clones were confirmed by PCR to have the expected structure of the chromosome in the cells.
  • an integrated strain was not obtained.
  • the large colony clone was grown on a plate containing 7% sucrose to eliminate the plasmid, and transformed again with RSF-Red-TER.
  • the strain without the plasmid was designated SC17(0).
  • This strain was deposited at the Russian National Collection of Industrial Microorganisms (VKPM), GNII Genetica (Address: 1 Dorozhny proezd., 1 Moscow 117545, Russia) on Sep. 21, 2005, and assigned an accession number of VKPM B-9246.
  • the scheme for constructing the helper plasmid RSF-Red-TER is shown in FIG. 2 .
  • an RSFsacBPlacMCS vector was designed.
  • DNA fragments containing the cat gene of the pACYC184 plasmid and the structural region of the sacB gene of Bacillus subtilis were amplified by PCR using the oligonucleotides of SEQ ID NOS: 43 and 44, and 45 and 46, respectively.
  • These oligonucleotides contained the BglII, Sad, XbaI and BamHI restriction enzyme sites in the 5′ end regions. These sites are required and convenient for further cloning.
  • the obtained sacB fragment of 1.5 kb was cloned into the previously obtained pMW119-P lac lacI vector at the XbaI-BamHI site.
  • This vector was constructed in the same manner as that described for the pMW118-P lac lacI vector (Skorokhodova, A. Y. et al, 2004, Biotekhnologiya (Rus), 5:3-21). However, this vector contained a polylinker moiety derived from pMW219 instead of the pMW218 plasmid.
  • the aforementioned cat fragment of 1.0 kb was treated with BglII and Sad, and cloned into the RSF-P lac lacIsacB plasmid obtained in the previous step at the BamHI-SacI site.
  • the obtained plasmid pMW-P lac lacIsacBcat contained the PlacUV5-lacI-sacB-cat fragment.
  • pMW-P lac lacIsacBcat was digested with BglII, blunt-ended with DNA polymerase I Klenow fragment, and successively digested with Sad.
  • a DNA fragment containing the PlacUV5 promoter was amplified by PCR using the oligonucleotides of SEQ ID NOS: 47 and 48 as primers and the pMW119-P lac lacI plasmid as a template.
  • the obtained fragment of 146 by was digested with SacI and NotI, and ligated with the SacI-NotI large fragment of the RSFsacB plasmid.
  • a ⁇ -dependent transcription terminator of the rrnB operon of Escherichia coli was inserted at a position between the cat gene and the PlacUV5 promoter.
  • a DNA fragment containing the PlacUV5 promoter and the TrrnB terminator was amplified by PCR using the oligonucleotides of SEQ ID NOS: 51 and 48 as primers and the chromosome of Escherichia coli BW3350 as the template. These obtained fragments were treated with KpnI and ligated.
  • the 0.5 kb fragment containing both PlacUV5 and TrrnB was amplified by PCR using the oligonucleotides of SEQ ID NOS: 48 and 52 as primers.
  • the obtained DNA fragment was digested with EcoRI, blunt-ended by a treatment with DNA polymerase I Klenow fragment, digested with BamHI, and ligated with the Ecl136II-BamHI large fragment of the RSFsacBPlacMCS vector.
  • the obtained plasmid was designated RSF-Red-TER.
  • the pMW118-( ⁇ attL-Km r - ⁇ attR) plasmid was constructed from the pMW118-attL-Tc-attR (WO2005/010175) plasmid by replacing the tetracycline resistance marker gene with the kanamycin resistance gene of the pUC4K plasmid.
  • the EcoRI-HindIII large fragment from pMW118-attL-Tc-attR was ligated to two fragments from the pUC4K plasmid: the HindIII-PstI fragment (676 bp) and EcoRI-HindIII fragment (585 bp).
  • Basic pMW118-attL-Tc-attR was obtained by ligation of the following four fragments.
  • the BglII-EcoRI fragment (114 bp) including attL (SEQ ID NO: 55) which was obtained by PCR amplification of the region corresponding to attL of the Escherichia coli W3350 (containing ⁇ prophage) chromosome using the primers P1 and P2 (SEQ ID NOS: 53 and 54) (these primers contained the subsidiary recognition sites for BglII and EcoRI).
  • L-glutamic acid secretion gene A search for an L-glutamic acid secretion gene was performed as follows. Since L-glutamic acid is converted into an intermediate of the tricarboxylic acid cycle, 2-oxoglutarate, in one step by glutamate dehydrogenase, L-glutamic acid is thought to be easily metabolized in many microorganisms having glutamate dehydrogenase or the tricarboxylic acid cycle. However, since a strain in which 2-oxoglutarate dehydrogenase is deleted cannot degrade L-glutamic acid, growth of the cells is inhibited in the presence of a high concentration glutamic acid.
  • the SC17sucAams strain derived from the Pantoea ananatis SC17sucA strain (refer to Japanese Patent Laid-open No. 2001-333769) is deficient in the extracellular polysaccharide biosynthesis system, and is also deficient in 2-oxoglutarate dehydrogenase. Therefore, this strain was used to try to obtain an L-glutamic acid excretion gene utilizing resistance to a high concentration of L-glutamic acid as a marker.
  • PCR was performed by using pMW118- ⁇ attL-Km r - ⁇ attR as the template and the primers of SEQ ID NOS: 6 and 7 to amplify a gene fragment containing a kanamycin resistance gene, attL and attR sequences of ⁇ phage at the both ends of the resistance gene, and 50 by upstream sequence of amsI and 50 by downstream sequence of the amsC gene added to the outer ends of the ⁇ phage sequences.
  • This fragment was purified by using Wizard PCR Prep DNA Purification System (Promega).
  • SC17(0) strain was transformed with RSF-Red-TER to obtain an SC17(0)/RSF-Red-TER strain.
  • This strain was cultured overnight in L medium (10 g of Bacto tryptone, 5 g of yeast extract and 5 g of NaCl in 1 L of pure water, pH 7.0) containing 25 mg/L of chloramphenicol, and then the culture medium after the overnight culture was inoculated in 1/100 volume into 100 mL of the L medium containing 25 mg/L of chloramphenicol and 1 mM isopropyl- ⁇ -D-thiogalactopyranoside, and culture was performed at 34° C. for 3 hours.
  • L medium 10 g of Bacto tryptone, 5 g of yeast extract and 5 g of NaCl in 1 L of pure water, pH 7.0
  • the culture medium after the overnight culture was inoculated in 1/100 volume into 100 mL of the L medium containing 25 mg/L of chloramphenicol and 1 mM isopropyl
  • the cells prepared as described above were collected, washed three times with ice-cooled 10% glycerol, and finally suspended in 0.5 mL of 10% glycerol.
  • the suspended cells were used as competent cells, and 100 ng of the PCR fragment prepared in the above section was introduced into the cells by using GENE PULSER II (BioRad) with a field strength of 18 kV/cm, capacitor capacity of 25 ⁇ F and resistance of 200 ⁇ .
  • Ice-cooled SOC medium (20 g/L of Bacto tryptone, 5 g/L of yeast extract, 0.5 g/L of NaCl, and 10 g/L of glucose) was added to the cell suspension, and culture was performed at 34° C. for 2 hours with shaking.
  • the culture was applied to a medium prepared by adding ingredients of minimal medium (5 g of glucose, 2 mM magnesium sulfate, 3 g of monopotassium phosphate, 0.5 g of sodium chloride, 1 g of ammonium chloride and 6 g of disodium phosphate in 1 L) and 40 mg/L of kanamycin to the L medium (10 g of Bacto tryptone, 5 g of yeast extract, 5 g of NaCl and 15 g of agar in 1 L of pure water, pH 7.0). The colonies that appeared were purified with the same medium, and then it was confirmed by PCR that the ams gene had been replaced with the kanamycin resistance gene.
  • the chromosome was extracted from the ams gene-deficient strain using a Bacterial Genomic DNA Purification Kit (Edge Biosystems). Separately, the SC17sucA strain was cultured overnight on an agar medium obtained by adding the ingredients of the minimal medium described above to the L medium. The cells were scraped with a loop, washed three times with ice-cooled 10% glycerol, and finally suspended in 10% glycerol to a final volume of 500 ⁇ l.
  • the suspended cells were used as competent cells, and 600 ng of the aforementioned chromosome DNA was introduced into the competent cells using GENE PULSER II (BioRad) with a field strength of 17.5 kV/cm, capacitor capacity of 25 ⁇ F and resistance of 200 ⁇ . Ice-cooled SOC medium was added to the cell suspension, and culture was performed at 34° C. for 2 hours with shaking. Then, the culture was applied on an agar medium prepared by adding ingredients of the minimal medium described above and 40 mg/L of kanamycin to the L medium. The colonies that appeared were purified with the same medium, and then it was confirmed by PCR that the ams gene had been replaced with the kanamycin resistance gene. This strain was designated as SC17sucAams.
  • Chromosomal DNA extracted from the Pantoea ananatis AJ13355 strain was partially digested with the restriction enzyme Sau3AI. Then, fragments of about 10 kb were collected and introduced into the BamHI site of pSTV28 (Takara Bio) to prepare a plasmid library. This plasmid library was introduced into competent cells of the SC17sucAams strain prepared in a conventional manner by electroporation.
  • the transformants were cultured at 34° C. for 3 days, and 64 clones among the colonies that appeared were allowed to again form single colonies on the same plate. As a result, 11 clones were found to form colonies after 48 hours, and the remaining colonies formed colonies after 72 hours.
  • the genes inserted into the vectors harbored by the transformants were analyzed. Plasmids were extracted from the transformants, and nucleotide sequences were determined. It was found that among the 11 clones that formed colonies within 48 hours, 10 clones had the same loci, and all contained genes showing homology to ybjL, which was an Escherichia coli gene of unknown function. In addition, this ybjL gene could not be obtained under the same conditions that the glutamic acid secretion system gene described in WO2005/085419, yhfK, was obtained, and conversely, the yhfK gene could not be obtained under the selection conditions used in this example.
  • the ybjL gene was cloned. PCR was performed using the chromosomal DNA of AJ13355 strain as the template and oligonucleotides ybjL-F1 and ybjL-R2 shown in SEQ ID NOS: 8 and 9 to amplify the fragment of about 1.8 kb containing the ybjL gene of P. ananatis .
  • This fragment was purified using Wizard PCR Prep DNA Purification System (Promega), and then treated with the restriction enzymes KpnI and SphI, and the product was ligated with pSTV28 (Takara Bio) which had been treated with the same enzymes to obtain pSTV-PanybjL.
  • the SC17sucA strain was transformed with this pSTV-PanybjL plasmid, and using pSTV28 as a control for comparison (Takara Bio) to construct the SC17sucA/pSTV-PanybjL and SC17sucA/pSTV28 strains.
  • the SC17sucA/pSTV-ybjL strain was plated on minimal medium (5 g of glucose or sucrose, 2 mM magnesium sulfate, 3 g of monopotassium phosphate, 0.5 g of sodium chloride, 1 g of ammonium chloride, 6 g of disodium phosphate, 0.2 M sodium L-glutamate, 100 mg/L each of lysine, methionine and diaminopimelic acid and 15 g of agar in 1 L of pure water) containing glutamic acid. Culture was performed at 34° C. for 2 days. As a result, it was confirmed that whereas the control vector-introduced strain, SC17sucA/pSTV28, could not form colonies, SC17sucA/pSTV-ybjL could form colonies on the minimal medium.
  • minimal medium 5 g of glucose or sucrose, 2 mM magnesium sulfate, 3 g of monopotassium phosphate, 0.5 g of
  • the SC17sucA/pSTV-PanybjL strain was cultured in liquid minimal medium (5 g of glucose, 2 mM magnesium sulfate, 3 g of monopotassium phosphate, 0.5 g of sodium chloride, 1 g of ammonium chloride, 6 g of disodium phosphate, 0.2 M sodium L-glutamate, 100 mg/L each of lysine, methionine and diaminopimelic acid in 1 L of pure water) containing glutamic acid to examine growth of the strain in the presence of a high concentration of L-glutamic acid.
  • liquid minimal medium 5 g of glucose, 2 mM magnesium sulfate, 3 g of monopotassium phosphate, 0.5 g of sodium chloride, 1 g of ammonium chloride, 6 g of disodium phosphate, 0.2 M sodium L-glutamate, 100 mg/L each of lysine, methionine and diaminopimelic acid in 1 L of pure
  • the plasmid for ybjL amplification, pSTV-PanybjL was introduced into the L-glutamic acid producing bacterium SC17sucA/RSFCPG having the plasmid for L-glutamic acid production, RSFCPG, shown in SEQ ID NO: 10 (refer to Japanese Patent Laid-open No. 2001-333769), and L-glutamic acid productivity thereof was examined.
  • pSTV-PanybjL and the control plasmid, pSTV28 were each introduced into SC17sucA/RSFCPG by electroporation, and transformants were obtained using chloramphenicol resistance as a marker.
  • the strain with the plasmid for ybjL amplification was designated SC17sucA/RSFCPG+pSTV-PanybjL
  • the strain with pSTV29 was designated SC17sucA/RSFCPG+pSTV28.
  • SC17sucA/RSFCPG+pSTV-PanybjL and the control strain SC17sucA/RSFCPG+pSTV28 were cultured to examine the L-glutamic acid producing ability of the strains.
  • the medium had the following composition:
  • section A Sucrose 30 g/L MgSO 4 •7H 2 O 0.5 g/L [[Group B: KH 2 PO 4 2.0 g/L Yeast Extract 2.0 g/L FeSO 4 •7H 2 O 0.02 g/L MnSO 4 •5H 2 O 0.02 g/L L-Lysine hydrochloride 0.2 g/L DL-Methionine 0.2 g/L Diaminopimelic acid 0.2 g/L (adjusted to pH 7.0 with KOH) Group C: CaCO 3 20 g/L
  • the ingredients of groups A and B were sterilized at 115° C. for 10 minutes by autoclaving, and the ingredient of group C was sterilized at 180° C. for 3 hours with dry heat. Then, the ingredients of the three groups were mixed, and 12.5 mg/L of tetracycline hydrochloride and 25 mg/L of chloramphenicol were added to the mixture.
  • SC17sucA/RSFCPG+pSTV29 and SC17sucA/RSFCPG+pSTV-PanybjL were each precultured on a medium obtained by adding ingredients of the minimal medium (medium containing 5 g of glucose, 2 mM magnesium sulfate, 3 g of monopotassium phosphate, 0.5 g of sodium chloride, 1 g of ammonium chloride and 6 g of disodium phosphate in 1 L of pure water), 25 mg/L of chloramphenicol and 12.5 mg/L tetracycline to the L medium (medium containing 10 g of Bacto tryptone, 5 g of yeast extract, 5 g of NaCl and 15 g of agar in 1 L of pure water, pH 7.0), and inoculated into an appropriate amount to 5 ml of the aforementioned medium in a test tube.
  • the minimal medium medium containing 5 g of glucose, 2 mM magnesium sulfate, 3 g of monopot
  • the cells were cultured for 17.5 hours, and then cell density, L-glutamic acid concentration, and the amount of residual saccharide in the culture medium were measured.
  • the cell density was examined by measuring turbidity at 620 nm of the medium diluted 51 times using a spectrophotometer (U-2000A, Hitachi).
  • the L-glutamic acid concentration was measured in culture supernatant appropriately diluted with water by using Biotech Analyzer (AS-210, Sakera SI). The results are shown in Table 1.
  • L-Glutamic acid accumulation was increased by about 3 g/L, and the yield based on saccharide increased by about 9% in the ybjL-amplified strain, SC17sucA/RSFPCPG+pSTV-PanybjL, as compared to the control strain, SC17sucA/RSFCPG+pSTV28.
  • PCR was performed using the oligonucleotides shown in SEQ ID NOS: 11 and 12 prepared on the basis of the sequence of the ybjL of Escherichia con registered at GeneBank as AP009048 (SEQ ID NO: 3) and the chromosome from the Escherichia coli W3110 strain (ATCC 27325) as the template to obtain a fragment of about 1.7 kb containing the ybjL gene. This fragment was treated with SphI and KpnI, and ligated with pSTV28 (Takara Bio) at the corresponding site.
  • the plasmid for amplification of ybjL of Escherichia coli was designated as pSTV-EcoybjL.
  • the plasmid pSTV-EcoybjL for ybjL amplification was introduced into the aforementioned SC17sucA/RSFCPG strain by electroporation, and a transformant was obtained using chloramphenicol resistance as a marker.
  • the obtained Escherichia coli ybjL gene-amplified strain was designated as SC17sucA/RSFCPG+pSTV-EcoybjL.
  • the medium had the following composition.
  • Group A Sucrose 30 g/L MgSO 4 •7H 2 O 0.5 g/L
  • Group B KH 2 PO 4 2.0 g/L Yeast Extract 2.0 g/L FeSO 4 •7H 2 O 0.02 g/L MnSO 4 •5H 2 O 0.02 g/L L-Lysine hydrochloride 0.2 g/L DL-Methionine 0.2 g/L Diaminopimelic acid 0.2 g/L (adjusted to pH 7.0 with KOH)
  • Group C CaCO 3 20 g/L
  • the ingredients of groups A and B were sterilized at 115° C. for 10 minutes by autoclaving, and the ingredient of group C was sterilized at 180° C. for 3 hours with dry heat. Then, the ingredients of the three groups were mixed, and 12.5 mg/L of tetracycline hydrochloride and 25 mg/L of chloramphenicol were added to the mixture.
  • SC17sucA/RSFCPG+pSTV28 and SC17sucA/RSFCPG+pSTV-EcoybjL were each precultured on a medium obtained by adding ingredients of the minimal medium (5 g of glucose, 2 mM magnesium sulfate, 3 g of monopotassium phosphate, 0.5 g of sodium chloride, 1 g of ammonium chloride and 6 g of disodium phosphate in 1 L of pure water), 25 mg/L of chloramphenicol, and 12.5 mg/L tetracycline to the L medium (10 g of Bacto tryptone, 5 g of yeast extract, 5 g of NaCl and 15 g of agar in 1 L of pure water, pH 7.0), and inoculated in an appropriate amount to 5 ml of the aforementioned medium in a test tube.
  • the minimal medium 5 g of glucose, 2 mM magnesium sulfate, 3 g of monopotassium phosphate, 0.5 g of
  • the cells were cultured for 17.5 hours, and then cell density and L-glutamic acid concentration in the culture medium were measured in the same manner as shown in Example 2. The results are shown in Table 2.
  • L-Glutamic acid accumulation was increased by about 2 g/L, and the yield based on saccharide increased by about 7% in the ybjL-amplified strain, SC17sucA/RSFCPG+pSTV-EcoybjL, as compared to the control strain, SC17sucA/RSFCPG+pSTV28.
  • the ybjL gene derived from Pantoea ananatis and the ybjL gene derived from Escherichia coli were each introduced into Escherichia coli , and the effect of the amplification was examined.
  • the aforementioned vector for amplification of the ybjL gene derived from Pantoea ananatis , pSTV-PanybjL, vector for amplification of ybjL gene derived from Escherichia coli , pSTV-EcoybjL, and the control plasmid pSTV28 were each introduced into an Escherichia coli wild-type strain, W3110, by electroporation to obtain transformants resistant to chloramphenicol.
  • the strain in which ybjL derived from Pantoea ananatis had been amplified was designated W3110/pSTV-PanybjL
  • the strain in which ybjL derived from Escherichia coli had been amplified was designated W3110/pSTV-EcoybjL
  • the control strain with pSTV28 was designated W3110/pSTV28.
  • W3110/pSTV-PanybjL, W3110/pSTV-EcoybjL, and W3110/pSTV28 were each precultured on L medium (10 g of Bacto tryptone, 5 g of yeast extract, 5 g of NaCl and 15 g of agar in 1 L of pure water, pH 7.0) containing chloramphenicol, and one loop of cells were inoculated by using a 1-mL volume loop (Nunc) to 5 mL of a medium having the following composition in a test tube, and cultured at 37° C. for 11.5 hours with shaking. Cell density and L-glutamic acid concentration in the culture medium were measured in the same manners as those in Example 2. The results are shown in Table 3.
  • Group A Glucose 30 g/L MgSO 4 •7H 2 O 0.5 g/L
  • Group B (NH 4 ) 2 SO 4 20 g/L KH 2 PO 4 2.0 g/L Yeast Extract 2.0 g/L FeSO 4 •7H 2 O 20 mg/L MnSO 4 •5H 2 O 20 mg/L (adjusted to pH 7.0 with KOH)
  • Group C Calcium carbonate 20 g/L
  • the L-glutamic acid producing ability was markedly improved in the Escherichia coli W3110/pSTV-PanybjL strain, which is a Pantoea ananatis in which the ybjL gene has been amplified, and the Escherichia coli W3110/pSTV-EcoybjL strain, which is an Escherichia coli in which the ybjL gene has been amplified, as compared to the control strain W3110/pSTV28 strain.
  • W3110/pSTV-PanybjL, and the control strain W3110/pSTV28 were precultured in L medium (10 g of Bacto tryptone, 5 g of yeast extract, 5 g of NaCl and 15 g of agar in 1 L of pure water, pH 7.0) containing chloramphenicol, and one loop of cells were inoculated by using a 5-mL volume loop (Nunc) to 5 mL of a medium having the following composition in a test tube, and cultured at 37° C. for 7 hours with shaking. Cell densities and L-amino acid concentrations in the culture medium were measured in the same manner as in Example 2. The results are shown in Table 4.
  • Group A Glucose 40 g/L MgSO 4 •7H 2 O 0.5 g/L
  • Group B (NH 4 ) 2 SO 4 20 g/L KH 2 PO 4 2.0 g/L Yeast Extract 2.0 g/L FeSO 4 •7H 2 O 20 mg/L MnSO 4 •5H 2 O 20 mg/L (adjusted to pH 7.0 with KOH)
  • Group C Calcium carbonate 30 g/L
  • the ybjL gene derived from Pantoea ananatis was introduced into Klebsiella planticola , and the effect of the amplification of the gene was examined.
  • the aforementioned vector for amplification of ybjL gene derived from Pantoea ananatis , pSTV-PanybjL, and the control plasmid pSTV28 were each introduced into a Klebsiella planticola L-glutamic acid-producing strain, AJ13410 (Japanese Patent Application No. 11-68324), by electroporation to obtain transformants which are resistant to chloramphenicol.
  • the strain in which ybjL derived from Pantoea ananatis had been amplified was designated as AJ13410/pSTV-PanybjL
  • the control strain with pSTV28 was designated as AJ13410/pSTV28.
  • Group A Sucrose 30 g/L MgSO 4 •7H 2 O 0.5 g/L
  • Group B KH 2 PO 4 2.0 g/L Yeast Extract 2.0 g/L FeSO 4 •7H 2 O 0.02 g/L MnSO 4 •5H 2 O 0.02 g/L L-Lysine hydrochloride 0.2 g/L DL-Methionine 0.2 g/L Diaminopimelic acid 0.2 g/L (adjusted to pH 7.0 with KOH)
  • Group C CaCO 3 20 g/L
  • L-glutamic acid-producing ability was markedly improved in the Klebsiella planticola AJ13410/pSTV-PanybjL, which is a Pantoea ananatis ybjL gene-amplified strain, as compared to the control strain, AJ13410/pSTV28.
  • a gene-disrupted strain in a single step by using a PCR product obtained with synthetic oligonucleotide primers in which a part of the target gene is designed in the 5′ end and a part of an antibiotic resistance gene is designed in the 3′ end. Furthermore, by using the ⁇ phage excision system in combination, the antibiotic resistance gene which is integrated into the gene-disrupted strain can be removed.
  • PCR was performed by using synthetic oligonucleotides having sequences corresponding to parts of the ldhA gene in the 5′ end and sequences corresponding to both ends of attL and attR of ⁇ phage at the 3′ end as primers and plasmid pMW118-attL-Cm-attR as the template.
  • pMW118-attL-Cm-attR is a plasmid obtained by inserting attL and attR genes, which are the attachment sites for ⁇ phage, and the cat gene, which is an antibiotic resistance gene, into pMW118 (Takara Bio), and the genes are inserted in the order of attL-cat-attR.
  • the sequences of the synthetic oligonucleotides used as the primers are shown in SEQ ID Nos. 13 and 14.
  • the amplified PCR product was purified on an agarose gel and introduced into Escherichia coli MG1655 strain containing the plasmid pKD46, which is capable of temperature-sensitive replication by electroporation.
  • PCR was performed by using the synthetic oligonucleotides shown in SEQ ID NOS: 15 and 16 as primers. Whereas the PCR product obtained for the parent strain was about 1.2 kb, the deficient strain was about 1.9 kb.
  • the strain was transformed with helper plasmid pMW-intxis-ts, and an ampicillin resistant strain was selected.
  • the pMW-intxis-ts contains the ⁇ phage integrase (Int) gene and excisionase (Xis) gene and shows temperature-sensitive replication. Then, the strain in which att-cat and pMW-intxis-ts had been eliminated was confirmed by PCR on the basis of ampicillin sensitivity and chloramphenicol sensitivity. PCR was performed by using the synthetic oligonucleotides shown in SEQ ID NOS: 15 and 16 as primers.
  • a strain was constructed in the same manner as that of the construction of the ldhA gene-deficient strain. PCR was performed using synthetic oligonucleotides having sequences corresponding to parts of the lldD gene in the 5′ end and sequences corresponding to both ends of attL and attR of ⁇ phage in the 3′ end as primers and plasmid pMW118-attL-Cm-attR as the template. The sequences of the synthetic oligonucleotides used as the primers are shown in SEQ ID Nos. 17 and 18.
  • the amplified PCR product was purified on an agarose gel and introduced into the Escherichia coli MG1655 ⁇ ldhA strain containing plasmid pKD46 capable of temperature-sensitive replication by electroporation. Then, an ampicillin-sensitive strain not harboring pKD46 was obtained, and the deletion of the lldD gene was confirmed by PCR. PCR was performed by using the synthetic oligonucleotides shown in SEQ ID NOS: 19 and 20 as primers. Whereas the PCR product obtained for the parent strain was about 1.4 kb, a band of about 1.9 kb was observed for the deficient strain.
  • the strain was transformed with helper plasmid pMW-intxis-ts, and an ampicillin resistant strain was selected. Then, the strain from which att-cat and pMW-intxis-ts had been eliminated was obtained and confirmed by PCR on the basis of ampicillin sensitivity and chloramphenicol sensitivity. PCR was performed by using the synthetic oligonucleotides shown in SEQ ID NOS: 19 and 20 as primers. Whereas the PCR product obtained for the strain in which att-cat remained was about 1.9 kb, a band of about 0.3 kb was observed for the strain in which att-cat was eliminated.
  • the lldD deficient strain obtained as described above was designated as MG1655 ⁇ ldhA ⁇ lldD strain.
  • plasmid pMW219::Pthr was used.
  • This plasmid corresponds to the vector pMW219 (Nippon Gene) having the promoter region of the threonine operon (thrLABC) in the genome of the Escherichia coli shown in SEQ ID NO: 21 at the HindIII site and BamHI site, and enables amplification of a gene by cloning the gene at a position in the plasmid downstream from that promoter.
  • PCR was performed by using the synthetic oligonucleotide having a BamHI site shown in SEQ ID NO: 22 as a 5′ primer, and the synthetic oligonucleotide having a BamHI site shown in SEQ ID NO: 23 as a 3′ primer, which were prepared on the basis of the nucleotide sequence of the ybjL gene in the genome sequence of Escherichia coli (GenBank Accession No. U00096), and the genomic DNA of Escherichia coli MG1655 strain as the template.
  • the product was treated with the restriction enzyme BamHI to obtain a gene fragment containing the ybjL gene.
  • the PCR product was purified and ligated with the vector pMW219::Pthr which had been treated with BamHI to construct plasmid pMW219::Pthr::ybjL for ybjL amplification.
  • pMW219::Pthr::ybjL obtained in ⁇ 6-2> described above and pMW219 were used to transform the Escherichia coli MG1655 ⁇ ldhA ⁇ lldD strain by the electric pulse method, and the transformants were applied to the LB agar medium containing 25 ⁇ g/ml of kanamycin, and cultured at 37° C. for about 18 hours. The colonies which appeared were purified, and plasmids were extracted from them in a conventional manner to confirm introduction of the target plasmid.
  • the obtained strains were designated as MG1655 ⁇ ldhA ⁇ lldD/pMW219::Pthr:ybjL and MG1655 ⁇ ldhA ⁇ lldD/pMW219, respectively.
  • the Enterobacter aerogenes AJ110637 strain was also transformed with pMW219::Pthr::ybjL and pMW219 by the electric pulse method, and the transformants were applied to the LB agar medium containing 50 ⁇ g/ml of kanamycin, and cultured at 37° C. for about 18 hours. The colonies which appeared were purified, and plasmids were extracted from them in a conventional manner to confirm introduction of the target plasmid.
  • the obtained strains were designated as AJ110637/pMW219::Pthr::ybjL and AJ110637/pMW219, respectively.
  • This cell suspension in a volume of 100 gland a production medium (10 g/l of glucose, 10 g/l 2Na malate, 45.88 g/l of TES, 6 g/l of Na 2 HPO 4 , 3 g/l of KH 2 PO 4 , 1 g/l of NH 4 Cl, adjusted to pH 7.3 with KOH and filtered) in a volume of 1.3 ml in which the dissolved gases in the medium were replaced with nitrogen gas by bubbling nitrogen gas beforehand were put into 1.5-ml volume micro tubes, and the cells were cultured at 31.5° C. for 10 hours by using a stirrer for micro tubes. After the culture, the amount of succinic acid which had accumulated in the medium was analyzed by liquid chromatography.
  • This cell suspension in a volume of 1000 and a production medium (10 g/l of glucose, 10 g/l 2Na malate, 45.88 g/l of TES, 6 g/l of Na 2 HPO 4 , 3 g/l of KH 2 PO 4 , 1 g/l of NH 4 Cl, adjusted to pH 7.3 with KOH and filtered) in a volume of 1.3 ml in which the dissolved gases in the medium were replaced with nitrogen gas by bubbling nitrogen gas beforehand were put into 1.5-ml volume micro tubes, and the cells were cultured at 31.5° C. for 6 hours by using a stirrer for micro tubes. After the culture, the amount of succinic acid which had accumulated in the medium was analyzed by liquid chromatography.
  • a gene fragment for deleting adhE was prepared by PCR using the plasmid pMW-attL-Tc-attR described in WO2005/010175 as the template and oligonucleotides of SEQ ID NOS: 72 and 73 as primers.
  • pMW118-attL-Tc-attR is a plasmid obtained by inserting attL and attR genes, which are the attachment sites of ⁇ phage, and the Tc gene, which is a tetracycline resistance gene, into pMW118 (Takara Bio), and the genes are inserted in the order of attL-Tc-attR.
  • a gene fragment containing a tetracycline resistance gene, attL and attR sites of ⁇ phage at the both ends of the resistance gene, and 60 by upstream sequence and 59 by downstream sequence of the adhE gene added to the outer ends of the ⁇ phage sequences was amplified. This fragment was purified by using Wizard PCR Prep DNA Purification System (Promega).
  • the Enterobacter aerogenes AJ110637 strain was transformed with RSF-Red-TER to obtain the Enterobacter aerogenes AJ110637/RSF-Red-TER strain.
  • This strain was cultured overnight in LB medium containing 40 ⁇ g/mL of chloramphenicol, the culture medium was inoculated in a 1/100 volume to 50 mL of the LB medium containing 40 ⁇ g/mL of chloramphenicol and 0.4 mM isopropyl- ⁇ -D-thiogalactopyranoside, and culture was performed at 31° C. for 4 hours.
  • the cells were collected, washed three times with ice-cooled 10% glycerol, and finally suspended in 0.5 mL of 10% glycerol.
  • the suspended cells were used as competent cells, and the PCR fragment prepared in the above section was introduced into the cells by using GENE PULSER II (BioRad) under the conditions of a field strength of 20 kV/cm, capacitor capacity of 25 ⁇ F and resistance of 200 ⁇ .
  • Ice-cooled LB medium was added to the cell suspension, and culture was performed at 31° C. for 2 hours with shaking. Then, the culture was applied to a LB plate containing 25 ⁇ g/mL of tetracycline. The colonies that appeared were purified with the same plate, and then it was confirmed by PCR that the adhE gene had been replaced with the tetracycline resistance gene.
  • PCR was performed by using the synthetic oligonucleotide having an SacI site shown in SEQ ID NO: 75 as a 5′ primer, the synthetic oligonucleotide shown in SEQ ID NO: 76 as a 3′ primer, and the genomic DNA of Escherichia coli MG1655 strain (ATCC 47076, ATCC 700926) as the template to obtain a threonine operon promoter fragment (A) (SEQ ID NO: 77).
  • a pyc gene fragment derived from the Corynebacterium glutamicum 2256 strain was obtained.
  • PCR was performed by using the synthetic oligonucleotide shown in SEQ ID NO: 78 as a 5′ primer, the synthetic oligonucleotide having an SacI site shown in SEQ ID NO: 79 as a 3′ primer, and the genomic DNA of the Corynebacterium glutamicum 2256 strain (ATCC 13869) as a template to obtain a pyc gene fragment (B) (SEQ ID NO: 80).
  • PCR was performed by using the fragments (A) and (B) as templates, the primers of SEQ ID NOS: 75 and 79 to obtain a gene fragment (C) including the fragments (A) and (B) ligated to each other.
  • This gene fragment (C) was treated with the restriction enzyme SacI, and purified, and the product was ligated with the plasmid vector pSTV28 (Takara Bio) which had been digested with the restriction enzyme Sad to construct a plasmid pSTV28::Pthr::pyc for pyc amplification.
  • the plasmid pSTV28::Pthr::pyc for pyc amplification was introduced into the aforementioned Enterobacter aerogenes AJ110637 ⁇ adhE strain by electroporation to obtain a transformant which are resistant to tetracycline and chloramphenicol.
  • This pyc-amplified strain of Enterobacter aerogenes AJ110637 ⁇ adhE was designated as AJ110637 ⁇ adhE/pSTV28::Pthr::pyc.
  • PCR amplification of the promoter region of the threonine operon (thrLABC) of Escherichia coli Escherichia coli K-12 strain was performed.
  • PCR was performed by using the synthetic oligonucleotide shown in SEQ ID NO: 81 as a 5′ primer, the synthetic oligonucleotide shown in SEQ ID NO: 82 as a 3′ primer, and the genomic DNA of Escherichia coli MG1655 strain (ATCC 47076, ATCC 700926) as a template to obtain a threonine operon promoter fragment (A) (SEQ ID NO: 83).
  • PCR was performed by using the synthetic oligonucleotide shown in SEQ ID NO: 84 as a 5′ primer, the synthetic oligonucleotide shown in SEQ ID NO: 85 as a 3′ primer, and the genomic DNA of the Enterobacter aerogenes AJ110637 strain as the template to obtain a ybjL gene fragment (B) (SEQ ID NO: 86).
  • PCR was performed by using the fragments (A) and (B) as templates, and the primers of SEQ ID NOS: 81 and 85 to obtain a gene fragment (C) with the fragments (A) and (B) ligated to each other.
  • This gene fragment (C) was blunt-ended by using TaKaRa BKL Kit (Takara Bio), and the 5′ end was phosphorylated.
  • the fragment was digested with the restriction enzyme SmaI, and the product was ligated with the plasmid vector pMW218 dephosphorylated with alkaline phosphatase to construct a plasmid pMW218::Pthr::Ent-ybjL for ybjL amplification.
  • the aforementioned vector pMW218::Pthr::Ent-ybjL for amplification of the ybjL gene derived from the Enterobacter aerogenes AJ110637 strain, and the control plasmid pMW218 were each introduced into the Enterobacter aerogenes AJ110637 ⁇ adhE/pSTV28::Pthr::pyc strain by electroporation to obtain transformants which are resistant to tetracycline, chloramphenicol and kanamycin.
  • the ybjL-amplified strain derived from the Enterobacter aerogenes AJ110637 ⁇ adhE/pSTV28::Pthr::pyc strain was designated as AJ110637 ⁇ adhE/pSTV28::Pthr::pyc/pMW218::Pthr::Ent-ybjL, and the control strain as with pMW218 was designated as AJ110637 ⁇ adhE/pSTV28::Pthr:pyc/pMW218.
  • AJ110637 ⁇ adhE/pSTV28::Pthr::pyc/pMW218::Pthr::Ent-ybjL, and AJ110637 ⁇ adhE/pSTV28::Pthr:pyc/pMW218 were each uniformly applied to an LB plate containing 50 ⁇ g/ml of kanamycin, 25 ⁇ g/ml of tetracycline, and 40 ⁇ g/ml of chloramphenicol, and cultured at 31.5° C. for 16 hours.
  • the cells were inoculated into 3 ml of a seed medium (20 g/l of Bacto tryptone, 10 g/l of yeast extract, 20 g/L of NaCl) in a test tube, and cultured at 31.5° C. for 16 hours with shaking.
  • a succinic acid production medium (100 g/l of glucose, 50 g/L of calcium carbonate subjected to hot air sterilization for 3 hours or more) in a volume of 3 ml was added to the medium obtained above, then the tube was sealed with a silicone stopper, and culture was performed at 31.5° C. for 24 hours with shaking. After the culture, the amount of succinic acid which had accumulated in the medium was analyzed by liquid chromatography.
  • SEQ ID NO: 1 ybjL gene of Pantoea ananatis
  • SEQ ID NO: 2 YbjL of Pantoea ananatis
  • SEQ ID NO: 3 ybjL gene of Escherichia coli
  • SEQ ID NO: 4 YbjL of Escherichia coli
  • SEQ ID NO: 5 Consensus sequence of YbjL of Pantoea ananatis and Escherichia coli
  • SEQ ID NO: 6 Primer for ams gene disruption
  • SEQ ID NO: 7 Primer for ams gene disruption
  • SEQ ID NO: 8 Primer for amplification of ybjL of P. ananatis
  • SEQ ID NO: 9 Primer for amplification of ybjL of P. ananatis
  • SEQ ID NO: 10 Sequence of RSFCPG plasmid
  • SEQ ID NO: 11 Primer for amplification of ybjL of E. coli W3110
  • SEQ ID NO: 12 Primer for amplification of ybjL of E. coli W3110
  • SEQ ID NO: 13 Primer for deletion of ldhA
  • SEQ ID NO: 14 Primer for deletion of ldhA
  • SEQ ID NO: 15 Primer for confirming deletion of ldhA
  • SEQ ID NO: 16 Primer for confirming deletion of ldhA
  • SEQ ID NO: 17 Primer for deletion of lldD
  • SEQ ID NO: 18 Primer for deletion of lldD
  • SEQ ID NO: 19 Primer for confirming deletion of lldD
  • SEQ ID NO: 20 Primer for confirming deletion of lldD
  • SEQ ID NO: 21 Threonine promoter sequence
  • SEQ ID NO: 22 Primer for amplification of ybjL of E. coli MG1655
  • SEQ ID NO: 23 Primer for amplification of ybjL of E. coli MG1655
  • SEQ ID NO: 24 ybjL gene of Salmonella typhimurium
  • SEQ ID NO: 25 YbjL of Salmonella typhimurium
  • SEQ ID NO: 26 ybjL gene of Yersinia pestis
  • SEQ ID NO: 27 YbjL of Yersinia pestis
  • SEQ ID NO: 28 ybjL gene of Erwinia carotovora
  • SEQ ID NO: 29 YbjL of Erwinia carotovora
  • SEQ ID NO: 30 ybjL gene of Vibrio cholerae
  • SEQ ID NO: 31 YbjL of Vibrio cholerae
  • SEQ ID NO: 32 ybjL gene of Aeromonas hydrophia
  • SEQ ID NO: 33 YbjL of Aeromonas hydrophia
  • SEQ ID NO: 34 ybjL gene of Photobacterium profundum
  • SEQ ID NO: 35 YbjL of Photobacterium profundum
  • SEQ ID NO: 36 ldhA gene of Escherichia coli
  • SEQ ID NO: 37 LdhA of Escherichia coli
  • SEQ ID NO: 38 lldD gene of Escherichia coli
  • SEQ ID NO: 39 LldD of Escherichia coli
  • SEQ ID NO: 40 Nucleotide sequence of hisD gene of Pantoea ananatis
  • SEQ ID NO: 41 Primer for amplification of fragment for incorporation of Km r gene into hisD gene
  • SEQ ID NO: 42 Primer for amplification of fragment for incorporation of Km r gene into hisD gene
  • SEQ ID NO: 43 Primer for amplification of cat gene
  • SEQ ID NO: 44 Primer for amplification of cat gene
  • SEQ ID NO: 45 Primer for amplification of sacB gene
  • SEQ ID NO: 46 Primer for amplification of sacB gene
  • SEQ ID NO: 47 Primer for amplification of DNA fragment containing PlacUV5 promoter
  • SEQ ID NO: 48 Primer for amplification of DNA fragment containing PlacUV5 promoter
  • SEQ ID NO: 49 Primer for amplification of DNA fragment containing ⁇ Red ⁇ gene and tL3
  • SEQ ID NO: 50 Primer for amplification of DNA fragment containing ⁇ Red ⁇ gene and tL3
  • SEQ ID NO: 51 Primer for amplification of DNA fragment containing PlacUV5 promoter and TrrnB
  • SEQ ID NO: 52 Primer for amplification of DNA fragment containing PlacUV5 promoter and TrrnB
  • SEQ ID NO: 53 Primer for amplification of attL
  • SEQ ID NO: 54 Primer for amplification of attL
  • SEQ ID NO: 55 Nucleotide sequence of attL
  • SEQ ID NO: 56 Primer for amplification of attR
  • SEQ ID NO: 57 Primer for amplification of attR
  • SEQ ID NO: 58 Nucleotide sequence of attR
  • SEQ ID NO: 59 Primer for amplification of DNA fragment containing bla gene
  • SEQ ID NO: 60 Primer for amplification of DNA fragment containing bla gene
  • SEQ ID NO: 61 Primer for amplification of DNA fragment containing ter_rrnB
  • SEQ ID NO: 62 Primer for amplification of DNA fragment containing ter_rrnB
  • SEQ ID NO: 63 Nucleotide sequence of the DNA fragment containing ter_thrL terminator
  • SEQ ID NO: 64 Primer for amplification of DNA fragment containing ter_thrL terminator
  • SEQ ID NO: 65 Primer for amplification of DNA fragment containing ter_thrL terminator
  • SEQ ID NO: 66 ams operon of Pantoea ananatis
  • SEQ ID NO: 67 AmsH of Pantoea ananatis
  • SEQ ID NO: 68 AmsI of Pantoea ananatis
  • SEQ ID NO: 69 AmsA of Pantoea ananatis
  • SEQ ID NO: 70 AmsC of Pantoea ananatis
  • SEQ ID NO: 71 AmsB of Pantoea ananatis
  • SEQ ID NO: 72 Nucleotide sequence of primer for deletion of adhE
  • SEQ ID NO: 73 Nucleotide sequence of primer for deletion of adhE
  • SEQ ID NO: 74 Nucleotide sequence of adhE of Enterobacter aerogenes AJ110637 partial sequence
  • SEQ ID NO: 75 Primer for amplification of threonine promoter
  • SEQ ID NO: 76 Primer for amplification of threonine promoter
  • SEQ ID NO: 77 Threonine promoter gene fragment
  • SEQ ID NO: 78 Primer for amplification of pyruvate carboxylase gene
  • SEQ ID NO: 79 Primer for amplification of pyruvate carboxylase gene
  • SEQ ID NO: 80 Pyruvate carboxylase gene fragment
  • SEQ ID NO: 81 Primer for amplification of threonine promoter
  • SEQ ID NO: 82 Primer for amplification of threonine promoter
  • SEQ ID NO: 83 Threonine promoter gene fragment
  • SEQ ID NO: 84 Nucleotide sequence of primer for amplification of ybjL of Enterobacter aerogenes AJ110637
  • SEQ ID NO: 85 Nucleotide sequence of primer for amplification of ybjL of Enterobacter aerogenes AJ110637
  • SEQ ID NO: 86 Nucleotide sequence of ybjL of Enterobacter aerogenes AJ110637
  • SEQ ID NO: 87 Amino acid sequence of YbjL of Enterobacter aerogenes AJ110637
  • SEQ ID NO: 88 Consensus sequence of YbjL of Escherichia, Pantoea and Enterobacter
  • Production efficiency of an acidic substance having a carboxyl group can be improved by using the microorganism of the present invention.

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Cited By (15)

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US20100297716A1 (en) * 2007-12-06 2010-11-25 Yoshinori Tajima Method for producing an organic acid
US20110014663A1 (en) * 2008-01-23 2011-01-20 Shigeo Suzuki Method for producing an l-amino acid
US20110117613A1 (en) * 2008-05-22 2011-05-19 Yasushi Hoshino Method for production of l-amino acid
US20110143403A1 (en) * 2004-10-22 2011-06-16 Konstantin Vyacheslavovich Rybak Method for producing l-amino acids using bacteria of the enterobacteriaceae family
US20110212496A1 (en) * 2008-09-08 2011-09-01 Rie Takikawa L-amino acid-producing microorganism and a method for producing an l-amino acid
US8192963B2 (en) 2008-09-05 2012-06-05 Ajinomoto Co., Inc. Bacterium capable of producing L-amino acid and method for producing L-amino acid
US8497104B2 (en) 2009-05-27 2013-07-30 Ajinomoto Co., Inc. Method for producing an organic acid
RU2537003C2 (ru) * 2010-08-30 2014-12-27 Корея Эдванст Инститьют Оф Сайенс Энд Текнолоджи Мутантный микроорганизм, продуцирующий янтарную кислоту, способ его получения и способ получения янтарной кислоты (варианты).
US8932834B2 (en) 2009-08-28 2015-01-13 Ajinomoto Co., Inc. Method for producing an L-amino acid in a cultured bacterium having reduced activity of the arginine succinyltransferase pathway
US8951760B2 (en) 2010-12-10 2015-02-10 Ajinomoto Co., Inc. Method for producing an L-amino acid
US8975045B2 (en) 2010-02-08 2015-03-10 Ajinomoto Co., Inc. Mutant RpsA gene and method for producing L-amino acid
US9447443B2 (en) 2012-05-29 2016-09-20 Ajinomoto Co., Inc. Method for producing 3-acetylamino-4-hydroxybenzoic acid
US9587259B2 (en) 2012-02-29 2017-03-07 Ajinomoto Co., Inc. Escherichia coli capable of producing 3-amino-4-hydroxybenzoic acid
US9822385B2 (en) 2007-04-17 2017-11-21 Ajinomoto Co., Inc. Method for producing an L-glutamic acid and L-aspartic acid using a recombinant microorganism having enhanced expression of a ybjL protein
US12152234B2 (en) 2021-03-22 2024-11-26 Hitachi, Ltd. Method of screening CO2-assimilating microorganism

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0673920B2 (ja) 1991-12-04 1994-09-21 鐘淵化学工業株式会社 電気用硬質積層体の連続製造方法
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EP3861109A1 (fr) 2018-10-05 2021-08-11 Ajinomoto Co., Inc. Procédé de production d'une substance cible par fermentation bactérienne
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US20240117393A1 (en) 2022-09-30 2024-04-11 Ajinomoto Co., Inc. Method for producing l-amino acid

Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020115159A1 (en) * 2000-09-15 2002-08-22 Degussa Ag Nucleotide sequences coding for the ATR61protein
US20020142404A1 (en) * 2000-09-15 2002-10-03 Mike Farwick Nucleotide sequences which code for the atr43 gene
US20020155556A1 (en) * 2000-12-22 2002-10-24 Ajinomoto Co., Inc Method of producing target substance by fermentation
US20040265956A1 (en) * 2002-11-11 2004-12-30 Rie Takikawa Method for producing target substance by fermentation
US6911332B2 (en) * 2002-06-12 2005-06-28 Ajinomoto Co., Inc. Isolated polynucleotides encoding d-arabino-3-hexulose-6-phosphate synthases from Methylophilus methylotrophus
US20050196846A1 (en) * 2004-03-04 2005-09-08 Yoshihiko Hara L-glutamic acid-producing microorganism and a method for producing L-glutamic acid
US20050233308A1 (en) * 2002-03-04 2005-10-20 Yosuke Nishio Method for modifying a property of a protein
US20060003424A1 (en) * 1998-09-25 2006-01-05 Yoko Asakura Method of constructing amino acid producing bacterial strains, and method of preparing amino acids by fermentation with the constructed amino acid producing bacterial strains
US20060040365A1 (en) * 2004-08-10 2006-02-23 Kozlov Yury I Use of phosphoketolase for producing useful metabolites
US7026149B2 (en) * 2003-02-28 2006-04-11 Ajinomoto Co., Inc. Polynucleotides encoding polypeptides involved in the stress response to environmental changes in Methylophilus methylotrophus
US7029893B2 (en) * 2003-02-28 2006-04-18 Ajinomoto Co., Inc. Polynucleotides encoding polypeptides involved in amino acid biosynthesis in methylophilus methylotrophus
US20060088919A1 (en) * 2004-10-22 2006-04-27 Rybak Konstantin V Method for producing L-amino acids using bacteria of the Enterobacteriaceae family
US7060475B2 (en) * 2003-02-28 2006-06-13 Ajinomoto Co., Inc. Polynucleotides encoding polypeptides involved in intermediates metabolism of central metabolic pathway in methylophilus methylotrophus
US7090998B2 (en) * 2002-07-12 2006-08-15 Ajinomoto Co., Inc. Method for producing target substance by fermentation
US20060234357A1 (en) * 2003-05-16 2006-10-19 Yoshihiro Usuda Novel polynucleotides encoding useful polypeptides in corynebacterium glutamicum ssp. lactofermentum
US20070254345A1 (en) * 2004-11-25 2007-11-01 Keita Fukui L-Amino Acid-Producing Bacterium and a Method for Producing L-Amino Acid
US7306933B2 (en) * 2003-07-29 2007-12-11 Ajinomoto Co., Inc. Method for producing L-lysine or L-threonine
US7501282B2 (en) * 2005-02-25 2009-03-10 Ajinomoto Co., Inc. Plasmid autonomously replicable in Enterobacteriaceae family
US20090068712A1 (en) * 2007-09-04 2009-03-12 Masaru Terashita Amino acid producing microorganism and a method for producing an amino acid
US20090093029A1 (en) * 2006-03-03 2009-04-09 Yoshihiro Usuda Method for producing l-amino acid
US20090104659A1 (en) * 2006-03-24 2009-04-23 Sergey Vasilievich Smirnov Novel aldolase and production process of 4-hydroxy-l-isoleucine
US20090203090A1 (en) * 2006-07-19 2009-08-13 Leonid Romanovich Ptitsyn Method for producing an l-amino acid using a bacterium of the enterobacteriaceae family
US20090215131A1 (en) * 2006-08-18 2009-08-27 Yoshihiko Hara L-glutamic acid producing bacterium and a method for producing l-glutamic acid
US20090239269A1 (en) * 2006-10-10 2009-09-24 Yoshinori Tajima Method for production of l-amino acid
WO2009116566A1 (fr) * 2008-03-18 2009-09-24 協和発酵キリン株式会社 Micro-organisme industriellement utile
US20090246835A1 (en) * 2005-09-27 2009-10-01 Shintaro Iwatani l-amino acid-producing bacterium and a method for producing an l-amino acid

Family Cites Families (75)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS329393B1 (fr) 1954-12-25 1957-11-07
US3220929A (en) 1964-02-10 1965-11-30 Kyowa Hakko Kogyo Kk Microbiological production of amino acid by reductive amination
US3563857A (en) 1967-03-20 1971-02-16 Sanraku Ocean Co Process for producing l-glutamic acid by fermentation
JPS56151495A (en) 1980-04-25 1981-11-24 Ajinomoto Co Inc Production of l-glutamine through fermentation
JPS61202694A (ja) 1985-03-07 1986-09-08 Ajinomoto Co Inc 発酵法によるｌ−グルタミンの製造法
JPH07121228B2 (ja) 1986-11-07 1995-12-25 協和醗酵工業株式会社 Ｌ−グルタミン酸およびｌ−プロリンの製造法
FR2627508B1 (fr) 1988-02-22 1990-10-05 Eurolysine Procede pour l'integration d'un gene choisi sur le chromosome d'une bacterie et bacterie obtenue par ledit procede
JP2817157B2 (ja) 1989-01-13 1998-10-27 味の素株式会社 発酵法によるｌ‐アミノ酸の製造法
JPH02207791A (ja) 1989-02-07 1990-08-17 Ajinomoto Co Inc 微生物の形質転換法
JPH03232497A (ja) 1990-02-08 1991-10-16 Asahi Chem Ind Co Ltd 発酵法によるｌ―グルタミンの製造方法
US5142834A (en) 1990-07-12 1992-09-01 Donnelly Corporation Vehicle trim assembly and fastener therefor
JPH07108228B2 (ja) 1990-10-15 1995-11-22 味の素株式会社 温度感受性プラスミド
JP2825969B2 (ja) 1990-11-26 1998-11-18 昭和高分子株式会社 脂肪族ポリエステルの製造方法
PH30131A (en) 1991-08-07 1997-01-21 Ajinomoto Kk Process for producing l-glutamic acid by fermentation
JPH0568576A (ja) 1991-09-11 1993-03-23 Mitsubishi Petrochem Co Ltd コハク酸の製造法
JPH07121228A (ja) 1993-10-22 1995-05-12 Mazda Motor Corp 生産設備の生産情報確認方法
JP3880636B2 (ja) 1994-01-10 2007-02-14 味の素株式会社 発酵法によるｌ−グルタミン酸の製造法
CN1103819C (zh) 1994-06-14 2003-03-26 味之素株式会社 α－酮戊二酸脱氢酶基因
EP1293560B1 (fr) 1994-08-19 2007-11-14 Ajinomoto Co., Inc. Procédé de préparation de L-Lysine et L-acide glutamique par fermentation
US5521075A (en) 1994-12-19 1996-05-28 Michigan Biotechnology Institute Method for making succinic acid, anaerobiospirillum succiniciproducens variants for use in process and methods for obtaining variants
US5504004A (en) 1994-12-20 1996-04-02 Michigan Biotechnology Institute Process for making succinic acid, microorganisms for use in the process and methods of obtaining the microorganisms
US5573931A (en) 1995-08-28 1996-11-12 Michigan Biotechnology Institute Method for making succinic acid, bacterial variants for use in the process, and methods for obtaining variants
US5770435A (en) 1995-11-02 1998-06-23 University Of Chicago Mutant E. coli strain with increased succinic acid production
DE19548222A1 (de) 1995-12-22 1997-06-26 Forschungszentrum Juelich Gmbh Verfahren zur mikrobiellen Herstellung von Aminosäuren durch gesteigerte Aktivität von Exportcarriern
WO1999003988A1 (fr) 1997-07-18 1999-01-28 Ajinomoto Co., Inc. Procede de production de nucleosides de purine par fermentation
KR19990013007A (ko) 1997-07-31 1999-02-25 박원훈 형질전환된 대장균 ｓｓ373(ｋｃｔｃ 8818ｐ)과 이를 이용한숙신산의 생산방법
JP3082717B2 (ja) 1997-08-14 2000-08-28 日本電気株式会社 基板配線方式
JPH11135018A (ja) 1997-08-29 1999-05-21 Canon Inc 画像形成装置の製造方法、製造装置および画像形成装置
JP4197754B2 (ja) 1997-10-09 2008-12-17 三菱化学株式会社 乳酸又はコハク酸の製造方法
JP3967812B2 (ja) 1998-01-16 2007-08-29 三菱化学株式会社 ピルビン酸カルボキシラーゼ遺伝子組み換え菌体による有機酸の製造法
JPH11196887A (ja) 1998-01-16 1999-07-27 Mitsubishi Chemical Corp ホスホエノールピルビン酸カルボキシラーゼ遺伝子組み換え菌体による有機酸の製造法
JPH11262398A (ja) 1998-03-18 1999-09-28 Ajinomoto Co Inc 発酵法によるｌ−グルタミン酸の製造法
AU746542B2 (en) 1998-03-18 2002-05-02 Ajinomoto Co., Inc. L-glutamic acid-producing bacterium and method for producing L-glutamic acid
JP3921866B2 (ja) 1998-03-18 2007-05-30 味の素株式会社 Ｌ−グルタミン酸生産菌及びｌ−グルタミン酸の製造法
JP4144098B2 (ja) 1998-03-18 2008-09-03 味の素株式会社 Ｌ−グルタミン酸生産菌及びｌ−グルタミン酸の製造法
AU756507B2 (en) 1998-03-18 2003-01-16 Ajinomoto Co., Inc. L-glutamic acid-producing bacterium and method for producing L-glutamic acid
BR9909615A (pt) 1998-04-13 2000-12-12 Univ Georgia Res Found Superexpressão de carboxilase de piruvato para a produção intensificada de produtos bioquìmicos derivados de oxaloacetato em células microbianas
US6159738A (en) 1998-04-28 2000-12-12 University Of Chicago Method for construction of bacterial strains with increased succinic acid production
JP4144131B2 (ja) 1998-10-19 2008-09-03 味の素株式会社 Ｌ−グルタミン酸生産菌及びｌ−グルタミン酸の製造法
AU761990B2 (en) 1998-10-19 2003-06-12 Ajinomoto Co., Inc. L-glutamic acid producing bacterium and process for producing L-glutamic acid
JP2002293797A (ja) 1998-12-18 2002-10-09 Ajinomoto Co Inc Ａｂｃトランスポーター及びそれをコードする遺伝子
RU2148642C1 (ru) 1998-12-23 2000-05-10 ЗАО "Научно-исследовательский институт АДЖИНОМОТО-Генетика" (ЗАО "АГРИ") Фрагмент днк rhtc, кодирующий синтез белка rhtc, придающего повышенную устойчивость к l-треонину бактериям escherichia coli, и способ получения l-аминокислоты
RU2175351C2 (ru) 1998-12-30 2001-10-27 Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО "АГРИ") Фрагмент днк из escherichia coli, определяющий повышенную продукцию l-аминокислот (варианты), и способ получения l-аминокислот
JP2000262288A (ja) 1999-03-16 2000-09-26 Ajinomoto Co Inc コリネ型細菌の温度感受性プラスミド
JP4061769B2 (ja) 1999-03-25 2008-03-19 味の素株式会社 Ｌ−グルタミン酸の製造法
JP2000333769A (ja) 1999-05-31 2000-12-05 Ikeda Bussan Co Ltd シートバックフレーム
JP2003159065A (ja) 1999-07-19 2003-06-03 Ajinomoto Co Inc 発酵法による目的物質の製造法
JP4427878B2 (ja) 1999-08-20 2010-03-10 味の素株式会社 析出を伴う発酵法によるｌ−グルタミン酸の製造法
KR100372218B1 (ko) 2000-06-29 2003-02-14 바이오인포메틱스 주식회사 유기산을 생산하는 균주 및 이를 이용한 유기산의 생산방법
JP2002238592A (ja) 2001-02-20 2002-08-27 Ajinomoto Co Inc Ｌ−グルタミン酸の製造法
JP3932945B2 (ja) 2002-03-27 2007-06-20 味の素株式会社 Ｌ−アミノ酸の製造法
JP4451393B2 (ja) 2003-07-29 2010-04-14 財団法人地球環境産業技術研究機構 コリネ型細菌形質転換体及びそれを用いるジカルボン酸の製造方法
BRPI0413403A (pt) 2003-08-28 2006-10-17 Mitsubishi Chem Corp método para produzir ácido succìnico
US7244610B2 (en) 2003-11-14 2007-07-17 Rice University Aerobic succinate production in bacteria
EP1692271B2 (fr) 2003-11-27 2022-08-03 Korea Advanced Institute of Science and Technology Nouveaux variants de bacterie de la panse et procede de preparation d'acide succinique utilisant de tels variants
JP4428999B2 (ja) 2003-12-11 2010-03-10 三菱化学株式会社 非アミノ有機酸の製造方法
JP4665558B2 (ja) 2004-03-04 2011-04-06 味の素株式会社 Ｌ−グルタミン酸生産微生物及びｌ−グルタミン酸の製造法
DK1751292T3 (da) 2004-05-03 2010-11-01 Ut Battelle Llc Fremgangsmåde til fremstilling af ravsyre ud fra rå hydrolysater
BRPI0510921A (pt) 2004-05-20 2008-05-20 Ajinomoto Kk bactéria produtora de ácido succìnico e processo para produzir ácido succìnico
CN1989238B (zh) 2004-05-20 2010-05-26 味之素株式会社 生产琥珀酸的细菌和生产琥珀酸的方法
EP1765857A2 (fr) * 2004-07-02 2007-03-28 Metanomics GmbH Procede de production de produits chimiques fins
BRPI0514734B1 (pt) 2004-08-27 2018-02-06 Rice University VARIEDADE BACTERIANA DE E. Coli MODIFICADA
JP2006129840A (ja) 2004-11-09 2006-05-25 Ajinomoto Co Inc Ｌ−グルタミン酸の製造法
JP2008029202A (ja) 2004-11-09 2008-02-14 Ajinomoto Co Inc Ｌ−グルタミン酸の製造法
KR100630836B1 (ko) 2005-04-08 2006-10-02 한국과학기술원 인-실리코 분석을 통한 균주 개량방법
JP4760121B2 (ja) 2005-05-17 2011-08-31 三菱化学株式会社 コハク酸の製造方法
KR100655495B1 (ko) 2005-07-11 2006-12-08 한국과학기술원 대사산물들의 플럭스 섬을 이용한 인-실리코 생물 개량방법
EP2094858B1 (fr) 2006-12-11 2015-02-18 Ajinomoto Co., Inc. Procede de production d'acide l-amine
JP2010041920A (ja) 2006-12-19 2010-02-25 Ajinomoto Co Inc Ｌ−アミノ酸の製造法
KR101730837B1 (ko) 2007-01-22 2017-04-27 아지노모토 가부시키가이샤 Ｌ-아미노산을 생산하는 미생물 및 ｌ-아미노산의 제조법
JP2010110217A (ja) 2007-02-22 2010-05-20 Ajinomoto Co Inc Ｌ−アミノ酸生産菌及びｌ−アミノ酸の製造法
JP2010130899A (ja) 2007-03-14 2010-06-17 Ajinomoto Co Inc Ｌ−グルタミン酸系アミノ酸生産微生物及びアミノ酸の製造法
JP5493854B2 (ja) 2007-04-10 2014-05-14 味の素株式会社 有機酸の製造方法
WO2008133131A1 (fr) 2007-04-16 2008-11-06 Ajinomoto Co., Inc. Procédé de fabrication d'un acide organique
WO2008133161A1 (fr) 2007-04-17 2008-11-06 Ajinomoto Co., Inc. Procédé de fabrication d'une substance acide ayant un groupe carboxyle

Patent Citations (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060003424A1 (en) * 1998-09-25 2006-01-05 Yoko Asakura Method of constructing amino acid producing bacterial strains, and method of preparing amino acids by fermentation with the constructed amino acid producing bacterial strains
US20020115159A1 (en) * 2000-09-15 2002-08-22 Degussa Ag Nucleotide sequences coding for the ATR61protein
US20020142404A1 (en) * 2000-09-15 2002-10-03 Mike Farwick Nucleotide sequences which code for the atr43 gene
US20020155556A1 (en) * 2000-12-22 2002-10-24 Ajinomoto Co., Inc Method of producing target substance by fermentation
US20050233308A1 (en) * 2002-03-04 2005-10-20 Yosuke Nishio Method for modifying a property of a protein
US7192748B2 (en) * 2002-06-12 2007-03-20 Ajinomoto Co., Inc. Isolated polynucleotides encoding phosphohexuloisomerase from Methylophilus methylotrophus
US6911332B2 (en) * 2002-06-12 2005-06-28 Ajinomoto Co., Inc. Isolated polynucleotides encoding d-arabino-3-hexulose-6-phosphate synthases from Methylophilus methylotrophus
US7090998B2 (en) * 2002-07-12 2006-08-15 Ajinomoto Co., Inc. Method for producing target substance by fermentation
US20040265956A1 (en) * 2002-11-11 2004-12-30 Rie Takikawa Method for producing target substance by fermentation
US7026149B2 (en) * 2003-02-28 2006-04-11 Ajinomoto Co., Inc. Polynucleotides encoding polypeptides involved in the stress response to environmental changes in Methylophilus methylotrophus
US7029893B2 (en) * 2003-02-28 2006-04-18 Ajinomoto Co., Inc. Polynucleotides encoding polypeptides involved in amino acid biosynthesis in methylophilus methylotrophus
US7220570B2 (en) * 2003-02-28 2007-05-22 Ajinomoto Co., Inc. Polynucleotides encoding polypeptides involved in amino acid biosynthesis in Methylophilus methylotrophus
US7060475B2 (en) * 2003-02-28 2006-06-13 Ajinomoto Co., Inc. Polynucleotides encoding polypeptides involved in intermediates metabolism of central metabolic pathway in methylophilus methylotrophus
US20070249017A1 (en) * 2003-02-28 2007-10-25 Yoshihiro Usuda Polynucleotides Encoding Polypeptides Involved in Amino Acid Biosynthesis in Methylophilus Methylotrophus
US20060234356A1 (en) * 2003-05-16 2006-10-19 Yoshihiro Usuda Novel polynucleotides encoding useful polypeptides in corynebacterium glutamicum ssp. lactofermentum
US7468262B2 (en) * 2003-05-16 2008-12-23 Ajinomoto Co., Inc. Polynucleotides encoding useful polypeptides in corynebacterium glutamicum ssp. lactofermentum
US20060234357A1 (en) * 2003-05-16 2006-10-19 Yoshihiro Usuda Novel polynucleotides encoding useful polypeptides in corynebacterium glutamicum ssp. lactofermentum
US20090148915A1 (en) * 2003-07-29 2009-06-11 Stephen Van Dien Method for Producing L-Lysine or L-Threonine
US7306933B2 (en) * 2003-07-29 2007-12-11 Ajinomoto Co., Inc. Method for producing L-lysine or L-threonine
US20090226981A1 (en) * 2004-03-04 2009-09-10 Yoshihiko Hara L-Glutamic Acid-Producing Microorganism and a Method for Producing L-Glutamic Acid
US20050196846A1 (en) * 2004-03-04 2005-09-08 Yoshihiko Hara L-glutamic acid-producing microorganism and a method for producing L-glutamic acid
US7344874B2 (en) * 2004-03-04 2008-03-18 Ajinomoto Co., Inc. L-glutamic acid-producing microorganism and a method for producing L-glutamic acid
US20060040365A1 (en) * 2004-08-10 2006-02-23 Kozlov Yury I Use of phosphoketolase for producing useful metabolites
US20060088919A1 (en) * 2004-10-22 2006-04-27 Rybak Konstantin V Method for producing L-amino acids using bacteria of the Enterobacteriaceae family
US20070254345A1 (en) * 2004-11-25 2007-11-01 Keita Fukui L-Amino Acid-Producing Bacterium and a Method for Producing L-Amino Acid
US7501282B2 (en) * 2005-02-25 2009-03-10 Ajinomoto Co., Inc. Plasmid autonomously replicable in Enterobacteriaceae family
US20090246835A1 (en) * 2005-09-27 2009-10-01 Shintaro Iwatani l-amino acid-producing bacterium and a method for producing an l-amino acid
US20090093029A1 (en) * 2006-03-03 2009-04-09 Yoshihiro Usuda Method for producing l-amino acid
US20090104659A1 (en) * 2006-03-24 2009-04-23 Sergey Vasilievich Smirnov Novel aldolase and production process of 4-hydroxy-l-isoleucine
US20090203090A1 (en) * 2006-07-19 2009-08-13 Leonid Romanovich Ptitsyn Method for producing an l-amino acid using a bacterium of the enterobacteriaceae family
US20090215131A1 (en) * 2006-08-18 2009-08-27 Yoshihiko Hara L-glutamic acid producing bacterium and a method for producing l-glutamic acid
US20090239269A1 (en) * 2006-10-10 2009-09-24 Yoshinori Tajima Method for production of l-amino acid
US20090068712A1 (en) * 2007-09-04 2009-03-12 Masaru Terashita Amino acid producing microorganism and a method for producing an amino acid
WO2009116566A1 (fr) * 2008-03-18 2009-09-24 協和発酵キリン株式会社 Micro-organisme industriellement utile
US20110111458A1 (en) * 2008-03-18 2011-05-12 Kyowa Hakko Kirin Co., Ltd. Industrially useful microorganism

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
Abe et al, Plasmid-Encoded asp Operon Confers a Proton Motive Metabolic Cycle Catalyzed by an Aspartate-Alanine Exchange Reaction. JOURNAL OF BACTERIOLOGY, June 2002, p. 2906-2913 *
Andersson et al, Gene Amplification and Adaptive Evolution in Bacteria. Annu. Rev. Genet. 2009. 43:167-95. *
BRENDA 2.6.1.1 aspartate transaminase. Retreived 1/23/14. *
Galye et al, Identification of regions in interleukin-1 alpha important for activity. J Biol Chem. 1993 Oct 15;268(29):22105-11. *
KEGG Alanine, Aspartate, and Glutamate Metabolism Retrieved 1/23/14. *
Nanatani et al, Topology of AspT, the Aspartate:Alanine Antiporter of Tetragenococcus halophilus, Determined by Site-Directed Fluorescence Labeling. J. Bacteriol. 2007, 189(19):7089-97. *
Whisstock et al, Prediction of protein function from protein sequence and structure. Q Rev Biophys. 2003 Aug;36(3):307-40. Review. *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8785161B2 (en) 2004-10-22 2014-07-22 Ajinomoto Co., Inc. Method for producing L-amino acids using bacteria of the enterobacteriaceae family
US20110143403A1 (en) * 2004-10-22 2011-06-16 Konstantin Vyacheslavovich Rybak Method for producing l-amino acids using bacteria of the enterobacteriaceae family
US8728774B2 (en) 2004-10-22 2014-05-20 Ajinomoto Co., Inc. Method for producing L-amino acids using bacteria of the enterobacteriaceae family
US9822385B2 (en) 2007-04-17 2017-11-21 Ajinomoto Co., Inc. Method for producing an L-glutamic acid and L-aspartic acid using a recombinant microorganism having enhanced expression of a ybjL protein
US20100297716A1 (en) * 2007-12-06 2010-11-25 Yoshinori Tajima Method for producing an organic acid
US8247201B2 (en) 2007-12-06 2012-08-21 Ajinomoto Co., Inc. Method for producing an organic acid
US20110014663A1 (en) * 2008-01-23 2011-01-20 Shigeo Suzuki Method for producing an l-amino acid
US8728772B2 (en) 2008-01-23 2014-05-20 Ajinomoto Co., Inc. Method for producing an L-amino acid
US8354254B2 (en) 2008-01-23 2013-01-15 Ajinomoto Co., Inc. Method for producing an L-amino acid
US8389249B2 (en) 2008-05-22 2013-03-05 Ajinomoto Co., Inc. Method for production of L-amino acid
US20110117613A1 (en) * 2008-05-22 2011-05-19 Yasushi Hoshino Method for production of l-amino acid
US8192963B2 (en) 2008-09-05 2012-06-05 Ajinomoto Co., Inc. Bacterium capable of producing L-amino acid and method for producing L-amino acid
US8206954B2 (en) 2008-09-08 2012-06-26 Ajinomoto Co., Inc. L-amino acid-producing microorganism and a method for producing an L-amino acid
US20110212496A1 (en) * 2008-09-08 2011-09-01 Rie Takikawa L-amino acid-producing microorganism and a method for producing an l-amino acid
US8497104B2 (en) 2009-05-27 2013-07-30 Ajinomoto Co., Inc. Method for producing an organic acid
US8932834B2 (en) 2009-08-28 2015-01-13 Ajinomoto Co., Inc. Method for producing an L-amino acid in a cultured bacterium having reduced activity of the arginine succinyltransferase pathway
US8975045B2 (en) 2010-02-08 2015-03-10 Ajinomoto Co., Inc. Mutant RpsA gene and method for producing L-amino acid
RU2537003C2 (ru) * 2010-08-30 2014-12-27 Корея Эдванст Инститьют Оф Сайенс Энд Текнолоджи Мутантный микроорганизм, продуцирующий янтарную кислоту, способ его получения и способ получения янтарной кислоты (варианты).
US8951760B2 (en) 2010-12-10 2015-02-10 Ajinomoto Co., Inc. Method for producing an L-amino acid
US9587259B2 (en) 2012-02-29 2017-03-07 Ajinomoto Co., Inc. Escherichia coli capable of producing 3-amino-4-hydroxybenzoic acid
US9447443B2 (en) 2012-05-29 2016-09-20 Ajinomoto Co., Inc. Method for producing 3-acetylamino-4-hydroxybenzoic acid
US12152234B2 (en) 2021-03-22 2024-11-26 Hitachi, Ltd. Method of screening CO2-assimilating microorganism

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US9822385B2 (en) 2017-11-21

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