WO2001002543A1 - Process for producing l-amino acid - Google Patents

Process for producing l-amino acid Download PDF

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Publication number
WO2001002543A1
WO2001002543A1 PCT/JP2000/004343 JP0004343W WO0102543A1 WO 2001002543 A1 WO2001002543 A1 WO 2001002543A1 JP 0004343 W JP0004343 W JP 0004343W WO 0102543 A1 WO0102543 A1 WO 0102543A1
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Prior art keywords
lysine
producing
enolase
amino acid
acid
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PCT/JP2000/004343
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French (fr)
Japanese (ja)
Inventor
Masakazu Sugimoto
Hisao Ito
Osamu Kurahashi
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Ajinomoto Co., Inc.
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Application filed by Ajinomoto Co., Inc. filed Critical Ajinomoto Co., Inc.
Priority to AU57071/00A priority Critical patent/AU5707100A/en
Publication of WO2001002543A1 publication Critical patent/WO2001002543A1/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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

  • the present invention relates to a method for producing an L-amino acid by a fermentation method, and particularly to a method for producing L-lysine and L-glutamic acid.
  • L-lysine is widely used as a feed additive and the like
  • L-glutamic acid is widely used as a seasoning material and the like.
  • L-amino acids such as L-lysine and L-glutamic acid have been fermented using a coryneform bacterium belonging to the genus Brevipacterium or Corynebacterium, which has the ability to produce these L-amino acids. It is industrially produced by the law. For these coryneform bacteria, strains isolated from the natural world or artificial mutants of the strains are used in order to improve productivity.
  • diaminobi L-lysine producing ability can be obtained by successively amplifying or introducing the methacrylate dehydrogenase gene (ddh) (or the succinylase tetrahydrodipicolinate gene (dapD) and the succinyldiaminopimelate deacylase gene (dapE)). Is known to be improved (W0 95/16042).
  • ddh methacrylate dehydrogenase gene
  • dapD succinylase tetrahydrodipicolinate gene
  • dapE succinyldiaminopimelate deacylase gene
  • JP-A-63-214189 discloses that L-glutamic acid dehydrogenase gene, isocitrate dehydrogenase gene, aconitate hydratase gene, and citrate synthase gene are amplified or introduced. Techniques for increasing the ability to produce glutamate have been disclosed.
  • An object of the present invention is to provide a method for producing an L-amino acid such as L-lysine or L-glumic acid by a fermentation method which has been further improved, and a strain used therefor.
  • the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, by introducing an enolase-encoding gene into coryneform bacteria to enhance enolase activity, L-lysine or L-glutamine
  • the present inventors have found that the production of minic acid can be increased, and have completed the present invention. That is, the present invention is as follows.
  • a coryneform bacterium having enhanced enolase activity in a cell and capable of producing L-amino acid (1) A coryneform bacterium having enhanced enolase activity in a cell and capable of producing L-amino acid.
  • L-amino acid is selected from L-lysine, L-glutamic acid, L-threonine, L-isoleucine, and L-serine.
  • the coryneform bacterium according to any of (1) to (4) is cultured in a medium, L-amino acids are produced and accumulated in the culture, and L-amino acids are collected from the culture.
  • a method for producing L-amino acid is produced.
  • the coryneform bacterium of the present invention is a coryneform bacterium having L-amino acid-producing ability and enhanced enolase activity in cells.
  • L-amino acids include L-lysine, L-glutamic acid, L-threonine, L-isoleucine, L-serine and the like. Of these, L-lysine and L-glutamic acid are preferred.
  • an embodiment of the present invention will be described mainly with respect to a coryneform bacterium having L-lysine-producing ability or L-glucamic acid-producing ability, but the present invention relates to a biosynthesis system specific to an L-amino acid of interest.
  • the coryneform bacterium referred to in the present invention is a group of microorganisms defined in Purges' Manual of Determinative Bacteriology, 8th edition, p. 599 (1974). Yes, aerobic, gram positive, It is a non-acid-fast, non-spore-forming bacillus, including bacteria that were previously classified into the genus Brevipacterium, but are now integrated as corynebacteria (Int. J. Syst. Bacterid., 41, 255 (1981)), and also includes bacteria of the genus Brevipacterium and Microbatterium, which are very closely related to the genus Corynepacterium. Examples of the strains of coryneform bacteria suitably used for producing L-lysine or L-glutamic acid include, for example, those shown below.
  • Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539) To obtain these, for example, American Type Culture Collection, Address 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America). That is, a registration number corresponding to each microorganism is assigned, and the microorganism can be ordered by referring to this registration number. The registration number corresponding to each microorganism is listed in the catalog of the American Type Culture Collection.
  • AJ12340 strain was deposited under the Budapest Treaty with the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, Institute of Biotechnology and Industrial Technology (zip code 305-8566, 1-3 1-3 Tsukuba-Higashi, Ibaraki, Japan). Have been.
  • mutants having L-amino acid-producing ability such as L-lysine or L-glucamic acid, derived from these strains can also be used in the present invention.
  • Such artificial mutants include the following. S- (2-aminoethyl) -cysteine (hereinafter abbreviated as "AEC") resistant mutant (for example, Brevibacterium 'Lactofamentum AJ11082 (NRRL B-11470), Japanese Patent Publication No. 56-1914, Japanese Patent Publication No. No. 56-1915, No. 57-14157, No. 57-14158, No. 57-30474, No. 58-10075, No. 59-4993, No. 61-35840, No.
  • AEC S- (2-aminoethyl) -cysteine
  • JP-A-53-86089 JP-A-55-9783, JP-A-55-9759, JP-A-56-32995, JP-A-56-39778, JP-B-53-43591, and JP-A-53-43591. No. 53-1833
  • L-lysine-producing mutants requiring inositol or acetic acid Japanese Patent Application Laid-Open Nos.
  • coryneform bacteria having L-threonine-producing ability include corynebacterium 'acetacidophila AJ12318 (FERM BP-1172) (see US Pat. No. 5,188,949) and L-isoleucine.
  • coryneform bacteria having the ability to produce are Brevibacterium flavum AJ12149 (FERM BP-759) (see US Pat. No. 4,656,135) and the like.
  • the ability to produce L-amino acids such as L-lysine means that when a coryneform bacterium is cultured in a medium, a significant amount of L-amino acid such as L-lysine is accumulated in the medium.
  • a recombinant DNA is prepared by ligating a gene fragment encoding the enolase to a vector that functions in the bacterium, preferably a multicopy vector. This may be introduced into a coryneform bacterium capable of producing L-lysine or L-glucamic acid for transformation.
  • the enolase activity is enhanced as a result of an increase in the copy number of the gene encoding the enolase in the cells of the transformed strain.
  • Enola is encoded by the eno gene in Escherichia coli.
  • enolase gene a gene of a coryneform bacterium or a gene derived from another organism such as a bacterium belonging to the genus Escherichia can be used.
  • the nucleotide sequence of the Escherichia coli eno gene has already been determined (Klein, M. et al., DNA Seq. 6, 315-355 (1996), Genbank / EMBL / DDBJ accetion No. X82400).
  • a primer prepared based on the base sequence for example, the primers shown in SEQ ID NOs: 1 and 2 in the Sequence Listing, a PCR method using polymerase chain reaction (PCR: polymerase chain reaction; White, TJ et al.) Eno gene can be obtained according to Trends Genet. 5, 185 (1989)).
  • Genes encoding enolase of other microorganisms such as coryneform bacteria can be obtained in a similar manner.
  • Chromosomal DNA is obtained from the DNA donor bacterium, for example, by the method of Saito and Miura (H. Saito and K. Miura Biochem. Biophys. Acta, 72, 619 (1963)) Pp. 97-98, Baifukan, 1992).
  • the gene encoding the enolase amplified by the PCR method is connected to a vector DNA capable of autonomously replicating in the cells of Escherichia coli and / or coryneform bacteria to prepare a recombinant DNA, which is then ligated to Escherichia coli. Introduced into E. coli cells Then, the operation later becomes difficult.
  • a vector capable of autonomous replication in Escherichia coli cells plasmid vectors are preferable, and those capable of autonomous replication in host cells are preferable.For example, pUC19, pUC18, pBR322, pHSG299, pHSG399 N pHS G398, RSF1010, etc. Is mentioned.
  • Examples of a vector capable of autonomous replication in a coryneform bacterium include PAM330 (see Japanese Patent Application Laid-Open No. 58-67699), pHM1519 (see Japanese Patent Application Laid-Open No. 58-77895), and the like.
  • a DNA fragment having the ability to enable autonomous replication of plasmid in coryneform bacteria is extracted from these vectors and inserted into the Escherichia coli vector, whereby autonomously expressed in both Escherichia coli and coryneform bacteria. It can be used as any shuttle vector that is not replicable.
  • Such shuttle vectors include: Microorganisms carrying the respective vectors and the accession numbers of the international depository organizations are shown in parentheses.
  • the vector is cut with a restriction enzyme that matches the end of the gene encoding the enolase. Ligation is usually performed using a ligase such as T4DNA ligase.
  • recombinant DNA can be obtained by transforming cells of a DNA-receiving bacterium into a protoplast or spheroblast that readily incorporates the recombinant DNA, as is known for Bacillus subtilis, actinomycetes and yeast.
  • Method for introduction into DNA recipient bacteria (Chang, S. and Choen, SN, Molec. Gen. Genet., 168, 111 (1979); Bibb, MJ, Ward, JM and Hopwood, 0.A., ature, 274, 398 (1978); Hinnen, A., Hicks, JBand Fink, GR, Proc. Natl. Acad. Sci. USA, 75 1929 (1978)).
  • the transformation method used in the examples of the present invention is the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791).
  • Enolase-encoding activity can also be enhanced by having multiple copies of the enolase-encoding gene on the chromosomal DNA of the host.
  • homologous recombination is performed by using a sequence that exists in multiple copies on the chromosome DNA as a target.
  • a sequence present in multiple copies on the chromosome DNA repetitive DNA and inverted repeats present at the end of a transposable element can be used.
  • Enolase activity is enhanced not only by the above-mentioned gene amplification but also by replacing the expression regulatory sequence such as the promoter of the gene encoding the enolase on chromosome DNA or plasmid with a strong one. Is also achieved (see Japanese Patent Application Laid-Open No. Hei 1-215280). For example, la. Promoters, trp promoters, trc promoters, ac promoters, PR promoters for lambda phage, PL promoters, etc. are known as strong promoters. The substitution with these promoters enhances the enolase activity by enhancing the expression of the gene encoding the enolase.
  • the coryneform bacterium of the present invention may have an enhanced enzymatic activity by enhancing an enzyme gene such as another amino acid synthesizing pathway or a glycolytic pathway in addition to the enolase activity.
  • an enzyme gene such as another amino acid synthesizing pathway or a glycolytic pathway in addition to the enolase activity.
  • genes that can be used for the production of L-lysine include aspartokinase subunit protein or / ?, in which synergistic feedback inhibition by L-lysine and L-threonine has been substantially eliminated.
  • genes that can be used for the production of L-glutamic acid include glutamate dehydrogenase (GDH, Japanese Patent Application Laid-Open No. Sho 61-268185), glutamate synthase, and glutamate. Synthase, isoquenate dehydrogenase (Japanese Patent Application Laid-open No. Sho 62-166890, Japanese Patent Application Laid-Open No. 63-214189), aconitate hydratase (Japanese Patent Application Laid-Open No. 62-294,086), citrate synthase, pyruvate carboxylase (Japanese Patent Laid-Open No. 60-87788, Japanese Patent Laid-open No.
  • an enzyme that catalyzes a reaction that diverges from a target L-amino acid biosynthetic pathway to produce a compound other than the same L-amino acid may be reduced or lacking.
  • homoserine dehydrogenase is an enzyme that catalyzes a reaction that produces a compound other than L-lysine by branching off from the L-lysine biosynthetic pathway (see W095 / 23864).
  • enzymes that catalyze the reaction that diverges from the biosynthetic pathway of L-glutamic acid to produce compounds other than L-glutamic acid include ketoglutarate dehydrogenase, isoquenate lyase, acetyl phosphate transferase, and acetate.
  • the biotin action inhibitor can be added to a medium containing an excess amount of biotin.
  • L-glutamic acid can be produced in the absence of E. coli (see W096 / 06180).
  • Examples of such coryneform bacteria include Brevipacterium. Lactofarmentum AJ13029 described in W096 / 06180. The AJ13029 strain was registered with the Institute of Biotechnology, Industrial Science and Technology (Postal Code 305-8566, Tsukuba 1-3-1, Ibaraki, Japan) on September 2, 1994 under the accession number FERM P-14501.
  • biotin-inhibiting substance by imparting a temperature-sensitive mutation to a biotin-inhibiting substance to a coryneform bacterium capable of producing L-lysine and L-glutamic acid, the biotin-inhibiting substance can be expressed in a medium containing an excessive amount of biotin. L-Lysine and L-glutamic acid can be produced simultaneously in the absence (see W096 / 06180). Examples of such a strain include Brevipacterium. Lactofermentum AJ12993 strain described in W096 / 06180.
  • the stock was issued to the Institute of Biotechnology, Institute of Industrial Technology (Postal Code 305-8566, Tsukuba 1-3-1-3, Ibaraki, Japan) on June 3, 1994, with the accession number FERM P- Deposited at 14348, transferred to the International Deposit under the Budapest Treaty on August 1, 1995, and given accession number FERM BP-5188.
  • the phrase “enhanced activity” of an enzyme generally means that the enzyme activity in a cell is higher than that of a wild-type strain, and is modified by a gene recombination technique or the like. When a strain with enhanced enzyme activity is obtained, it means that the enzyme activity in the cell is higher than that of the strain before modification.
  • reduced activity of an enzyme usually means that the enzyme activity in a cell is lower than that of a wild-type strain, and the enzyme activity has been reduced by modification by genetic recombination technology or the like. When a strain is obtained, it means that the enzyme activity in the cell is lower than that of the strain before modification.
  • a coryneform bacterium having enhanced enolase activity and capable of producing L-amino acid When a coryneform bacterium having enhanced enolase activity and capable of producing L-amino acid is cultured in a suitable medium, the L-amino acid accumulates in the medium. If it ’s clear, Enola If a coryneform bacterium having enhanced L-lysine activity and capable of producing L-lysine acid is cultured in a suitable medium, L-lysine accumulates in the medium. In addition, when a coryneform bacterium having enhanced enolase activity and capable of producing L-glutamic acid is cultured in a suitable medium, L-glutamic acid accumulates in the medium.
  • L-lysine and L-glutamic acid accumulate in the medium.
  • the L-lysine-producing bacterium may be cultured under L-glucaminic acid-producing conditions, or a L-lysine-producing coryneform A bacterium and a coryneform bacterium capable of producing L-glutamic acid may be mixedly cultured (Japanese Patent Application Laid-Open No. 5-3793).
  • the medium used for producing L-amino acids such as L-lysine or L-glutamic acid using the microorganism of the present invention contains a carbon source, a nitrogen source, inorganic ions, and other organic micronutrients as required. This is a normal medium.
  • Carbon sources include glucose, lactose, galactose, fructos, sucrose, molasses, carbohydrates such as starch hydrolysates, alcohols such as ethanol and inositol, acetic acid, fumaric acid, citric acid, An organic acid such as succinic acid can be used.
  • Nitrogen sources include inorganic ammonium salts such as ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, ammonium acetate, ammonia, peptone, meat extract, yeast extract, yeast extract, corn 'steep' liquor, soy hydrolyzate, etc.
  • Organic nitrogen, ammonia gas, aqueous ammonia, etc. can be used.
  • inorganic ions small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions and the like are added.
  • organic trace nutrients it is desirable to include a required substance such as vitamin B1 or a yeast extract in an appropriate amount as necessary.
  • the culture is preferably carried out for 16 to 72 hours under aerobic conditions, such as shaking culture and aeration / agitation culture, at a culture temperature of 30 ° (up to 45 ° C, pH during culture). Is controlled to 5 to 9.
  • aerobic conditions such as shaking culture and aeration / agitation culture
  • a culture temperature of 30 ° (up to 45 ° C, pH during culture). Is controlled to 5 to 9.
  • an inorganic or organic acidic or alkaline substance, ammonia gas or the like can be used.
  • L-amino acid can be collected from the fermentation liquor in the same manner as in the normal L-amino acid production method. Can be done.
  • L-lysine can be collected usually by a combination of an ion exchange resin method, a precipitation method and other known methods.
  • the method of collecting L-glucamic acid may be a conventional method, for example, an ion exchange resin method, a crystallization method, or the like.
  • L-glutamic acid may be adsorbed and separated by an anion exchange resin, or may be neutralized and crystallized.
  • the nucleotide sequence of the Escherichia coli eno gene has already been determined (Klein, M. et al., DNA Seq. 6, 315-355 (1996), Genbank / EMBL / DDBJ accetion No. X82400). Based on the reported nucleotide sequence, the primers shown in SEQ ID NOs: 1 and 2 in the Sequence Listing were synthesized, and the pyruvate dehydrogenase gene was amplified by the PCR method using the chromosome DNA of Escherichia coli JM109 strain as type III.
  • SEQ ID NO: 1 corresponds to the sequence from the first to the 24th base of the base sequence of the eno gene described in Genbank / EMBL / DDBJ accetion No. X 82400
  • SEQ ID NO: 1 2 corresponds to the sequence from the 289th base to the 266th base.
  • the chromosome DNA of Escherichia coli JM109 strain was prepared by a conventional method (Biotechnological Experiments, edited by The Society of Biotechnology, Japan, pages 97-98, Baifukan, 1992).
  • PCR reaction standard reaction conditions described on page 185 of the PCR method forefront (edited by Takeo Sekiya et al., Kyoritsu Shuppan, 1989) are used.
  • the resulting PCR product was purified by a conventional method, ligated with Smal-cleaved plasmid pHC4 using a ligation kit (Takara Shuzo), and then combined with Escherichia coli JM109 (Takara Shuzo). Transformation was performed using L-culture medium containing 30 g / ml of chloram fenicol (10 g / L of Pak Tributone, The extract was applied to 5 g / L of extract, 5 g / L of NaCl, 15 g / L of agar, pH 7.2), cultured overnight, and the white colonies that appeared were picked and separated into single colonies to obtain a transformant. Plasmid was extracted from the obtained transformant to obtain a plasmid pHC4eno in which the eno gene was linked to a vector.
  • Escherichia coli which holds pHC 4, was named private number AJ12617, and on April 24, 1999, the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry (zip code 305-8566, Ibaraki, Japan) Deposit No. FE RM P—122 1 15 at Tsukuba 1-3-1, Higashi-chome) and transferred to an international deposit based on the Budapest Convention on August 26, 1999, accession no. BP— 3 5 3 2 is awarded.
  • the enolase activity of the JM109 strain and the JM109 strain retaining pHC4eno was determined by the method of Bucher, T. et al. , Meth. Enzymol. 1, 427-435 (1955).
  • the JM109 strain carrying pHC4eno exhibited about 15 times the enolase activity of the JM109 strain not carrying pHC4en0, indicating that the eno gene was expressed. confirmed.
  • Brevibacterium lactofermentum AJ13029 was transformed with the plasmid pHC4eno by the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791) to obtain the resulting transformant.
  • cultivation for producing L-glucamic acid was performed as follows.
  • the cells of the AJ13029 / pHC4eno strain obtained by culturing on a CM2B plate medium containing 5 ⁇ g / ml chloramphenicol were added to L-cells having the following composition containing 5 ⁇ g / ml chloramphenicol.
  • the medium was inoculated into a glucanic acid-producing medium, shake-cultured at 31.5, and shaken until the sugar in the medium was consumed.
  • the obtained culture was inoculated into a medium having the same composition in an amount of 5%, and cultured at 37 ° C with shaking until the sugar in the medium was consumed.
  • a strain obtained by transforming a plasmid pHC4, which can be autonomously replicated with a previously obtained corynebacterium bacterium, into the corynebacterium bacterium AJ13029 as a control by the electric pulse method and culturing in the same manner as described above did.
  • the cells of the AJ11082 / pHC4eno strain obtained by culturing in a 0 ⁇ 12B plate medium containing 5 ⁇ / 1111 chloramphenicol were added to L-cells of the following composition containing 5 g / ml of chloramphenicol.
  • a lysine production medium was inoculated and cultured with shaking at 31.5 ° C until the sugar in the medium was consumed.
  • Corynepterium as control A strain obtained by transforming the strain AJ11082 with a plasmid pHC4 capable of autonomously replicating by a corynebacterium already obtained by the electric pulse method was cultured in the same manner as described above.
  • Brevi Pacterium 'Lactafamentum AJ11082 was established on January 31, 1981 at the Agricultural Research Culture Collection, America, Illinois, 6 1604 Peoria Northuniversity Street 18 1 5 (1815 N. University Street, Peoria, Illinois 61604 USA) and deposited under accession number NRRL B-11470.
  • Protein hydrolyzate (bean concentrate) 30 ml
  • Brevipacterium lactofermentum AJ12993 was transformed with plasmid pHC4eno by the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791) to obtain the obtained transformant.
  • culture for producing L-lysine and L-glucamic acid was performed as follows.
  • the cells of the AJ12993 / pHC4eno strain obtained by culturing in a CM2B plate medium containing 5 ⁇ g / ml chloramphenicol were added to the L-lysine production medium containing 5 / g / ml chloramphenicol. The cells were inoculated and cultured at 31.5 ° C.
  • the culture temperature was shifted to 34 ° C, and the culture was performed with shaking until the sugar in the medium was consumed.
  • a strain obtained by transforming a plasmid pHC4 capable of autonomously replicating with a previously obtained corynebacterium bacterium into an AJ12993 bacterium of the genus Corynebacterium by an electric pulse method was cultured in the same manner as described above.
  • L-anoic acid such as L-lysine or L-monoglutamic acid

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Abstract

A process for producing an L-amino acid such as L-lysine or L-glutamic acid by an improved fermentation method compared with the conventional methods which comprises transferring a gene encoding enolase into a coryneform bacterium capable of producing an L-amino acid such as L-lysine or L-glutamic acid and thus enhancing the enolase activity to thereby elevate the L-amino acid productivity; and strains to be used in this method.

Description

明細書  Specification
L一アミノ酸の製造法 技術分野 本発明は、 発酵法による L—アミノ酸の製造法、 特に L一リジン及び Lーグル 夕ミン酸の製造法に関する。 L一リジンは飼料添加物等として、 L一グルタミン 酸は調味料原料等として広く用いられている。 景技術 従来、 L一リジン及び L一グルタミン酸等の L—アミノ酸は、 これらの Lーァ ミノ酸生産能を有するブレビパクテリゥム属ゃコリネバクテリゥム属に属するコ リネ型細菌を用いて発酵法により工業生産されている。 これらのコリネ型細菌は、 生産性を向上させるために、 自然界から分離した菌株または該菌株の人工変異株 が用いられている。 TECHNICAL FIELD The present invention relates to a method for producing an L-amino acid by a fermentation method, and particularly to a method for producing L-lysine and L-glutamic acid. L-lysine is widely used as a feed additive and the like, and L-glutamic acid is widely used as a seasoning material and the like. BACKGROUND ART Conventionally, L-amino acids such as L-lysine and L-glutamic acid have been fermented using a coryneform bacterium belonging to the genus Brevipacterium or Corynebacterium, which has the ability to produce these L-amino acids. It is industrially produced by the law. For these coryneform bacteria, strains isolated from the natural world or artificial mutants of the strains are used in order to improve productivity.
また、 組換え D N A技術により Lーァミノ酸の生合成酵素活性を増強すること によって、 L—アミノ酸の生産能を増加させる種々の技術が開示されている。 例 えば、 L一リジン生産能を有するコリネ型細菌において、 L一リジン及び Lース レオニンによるフィ一ドバック阻害が解除されたァスパル卜キナーゼをコ一ドす る遺伝子 （変異型 lysC) 、 ジヒ ドロジピコリン酸レダク夕ーゼ遺伝子 （dapB) 、 ジヒ ドロジピコ リン酸シン夕ーゼ遺伝子 （dapA) 、 ジアミノピメ リン酸デカルボ キシラーゼ遺伝子 （lysA) 、 及びジアミノピメ リン酸デヒ ドロゲナーゼ遺伝子 ( ddh) (W096/40934) 、 lysA及び ddh (特開平 9— 322774号） 、 lysC、 iysA及びホ スホェノールピルビン酸カルボキシラーゼ遺伝子 （ppC) (特開平 10- 165180号） 、 変異型 lysC、 dapB、 dapA、 lysA及びァスパラギン酸アミノ トランスフェラ一ゼ遺 伝子 （aspC) (特開平 10-215883号） を導入することにより、 同細菌の L一リジン 生産能が向上することが知られている。 In addition, various techniques for increasing L-amino acid producing ability by enhancing L-amino acid biosynthetic enzyme activity by recombinant DNA technology have been disclosed. For example, in a coryneform bacterium capable of producing L-lysine, a gene (mutant lysC) encoding aspartokinase that has been released from feedback inhibition by L-lysine and L-threonine, and dihydro- Dipicolinate reductase gene (dapB), dihydrodipicophosphate synthase gene (dapA), diaminopimephosphate decarboxylase gene (lysA), and diaminopimephosphate dehydrogenase gene (ddh) (W096 / 40934) , lysA and ddh (JP-A-9- 322774), lysC, iysA and host scan Hoe Nord carboxylase gene (pp C) (JP-A-10- 165180), mutant lysC, dapB, dapA, lysA and Asuparagin acid amino It is known that the introduction of transferase gene (aspC) (Japanese Patent Laid-Open No. 10-215883) improves the L-lysine production ability of the bacterium. ing.
また、 ェシエリヒア属細菌においては、 dapA、 変異型 lysC、 dapB、 ジアミノ ビ メリン酸デヒ ドロゲナ一ゼ遺伝子 （ddh) (又はテトラヒ ドロジピコリン酸スクシ 二ラ一ゼ遺伝子 （dapD ) 及びスクシニルジアミノピメ リン酸デアシラーゼ遺伝子 ( dapE ) ) を順次増幅又は導入すると L—リジン生産能が向上することが知られ ている （W0 95/16042 ) 。 尚、 W0 95八 6042ではテ トラヒ ドロジピコリン酸スクシ ニラ一ゼがスクシニルジアミノビメ リン酸トランスアミナーゼと誤記されている。 一方、 コリネバクテリゥム属またはブレビバタテリゥム属細菌において、 ェシ エリヒア · コリ又はコリネバクテリゥム · グル夕ミクム由来のクェン酸シン夕一 ゼをコ一ドする遺伝子の導入が、 L—グル夕 ミン酸生産能の増強に効果的であつ たことが報告されている （特公平 7- 121228号） 。 また、 特開昭 61- 268185号公報に は、 コリネバクテリゥム属細菌由来のグルタ ミン酸デヒ ドロゲナーゼ遺伝子を含 む組換え体 D N Aを保有した細胞が開示されている。 さらに、 特開昭 63- 214189号 公報には、 グルタミン酸デヒ ドロゲナーゼ遺伝子、 イソクェン酸デヒ ドロゲナ一 ゼ遺伝子、 アコニッ ト酸ヒ ドラターゼ遺伝子、 及びクェン酸シンターゼ遺伝子を 増幅又は導入することによって、 L一グルタ ミン酸の生産能を増加させる技術が 開示されている。 For bacteria belonging to the genus Escherichia, dapA, mutant lysC, dapB, diaminobi L-lysine producing ability can be obtained by successively amplifying or introducing the methacrylate dehydrogenase gene (ddh) (or the succinylase tetrahydrodipicolinate gene (dapD) and the succinyldiaminopimelate deacylase gene (dapE)). Is known to be improved (W0 95/16042). In addition, in WO 95/86042, tetrahydrodipicolinate succinylase is erroneously described as succinyldiaminobimephosphate transaminase. On the other hand, in a bacterium belonging to the genus Corynebacterium or Brevibataterium, the introduction of a gene encoding citrate synthase derived from Escherichia coli or Corynebacterium gulurium is introduced into L- It has been reported that it was effective in enhancing glutamate-mi- nate-producing ability (Japanese Patent Publication No. 7-121228). Also, Japanese Patent Application Laid-Open No. 61-268185 discloses a cell having a recombinant DNA containing a glutamate dehydrogenase gene derived from a bacterium belonging to the genus Corynebacterium. Further, JP-A-63-214189 discloses that L-glutamic acid dehydrogenase gene, isocitrate dehydrogenase gene, aconitate hydratase gene, and citrate synthase gene are amplified or introduced. Techniques for increasing the ability to produce glutamate have been disclosed.
しかし、 エノラーゼをコ一ドする遺伝子の構造はコリネ型細菌では報告されて おらず、 エノラーゼをコ一ドする遺伝子をコリネ型細菌の育種に利用することも 知られていない。 発明の開示 本発明は、 従来よりもさらに改良された発酵法による L—リジン又は Lーグル 夕 ミン酸等の L—アミノ酸の製造法、 及びそれに用いる菌株を提供することを課 題とする。  However, the structure of the enolase-encoding gene has not been reported for coryneform bacteria, and the use of the enolase-encoding gene for breeding of coryneform bacteria is not known. DISCLOSURE OF THE INVENTION An object of the present invention is to provide a method for producing an L-amino acid such as L-lysine or L-glumic acid by a fermentation method which has been further improved, and a strain used therefor.
本発明者等は、 上記課題を解決するために鋭意検討を行った結果、 エノラーゼ をコードする遺伝子をコ リネ型細菌に導入し、 エノラーゼ活性を増強することに より、 Lーリジン又は L—グル夕 ミン酸の生産量を増大させることができること を見出し、 本発明を完成するに至った。 すなわち本発明は、 以下のとおりである。 The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, by introducing an enolase-encoding gene into coryneform bacteria to enhance enolase activity, L-lysine or L-glutamine The present inventors have found that the production of minic acid can be increased, and have completed the present invention. That is, the present invention is as follows.
( 1 ) 細胞中のエノラーゼ活性が増強され、 かつ L—アミノ酸生産能を有するコ リネ型細菌。  (1) A coryneform bacterium having enhanced enolase activity in a cell and capable of producing L-amino acid.
( 2 ) 前記 L一アミノ酸が、 L—リジン、 L一グルタミン酸、 L—スレオニン、 L—イソロイシン、 L—セリンから選ばれる （ 1 ) のコリネ型細菌。  (2) The coryneform bacterium according to (1), wherein the L-amino acid is selected from L-lysine, L-glutamic acid, L-threonine, L-isoleucine, and L-serine.
( 3 ) 前記エノラーゼ活性の増強が、 前記細菌細胞内のエノラーゼをコードする 遺伝子のコピー数を高めることによるものである前記 （ 1 ) のコリネ型細菌。 (3) The coryneform bacterium according to (1), wherein the enolase activity is enhanced by increasing the copy number of a gene encoding enolase in the bacterial cell.
( 4 ) 前記エノラ一ゼをコ一ドする遺伝子がェシエリヒア属細菌由来である ( 3 ) のコリネ型細菌。 (4) The coryneform bacterium according to (3), wherein the gene encoding the enolase is derived from a bacterium belonging to the genus Escherichia.
( 5 ) 前記 （ 1 ) 〜 （4 ) のいずれかのコリネ型細菌を培地に培養し、 該培養物 中に L一アミノ酸を生成蓄積せしめ、 該培養物から L一アミノ酸を採取すること を特徴とする Lーァミノ酸の製造法。  (5) The coryneform bacterium according to any of (1) to (4) is cultured in a medium, L-amino acids are produced and accumulated in the culture, and L-amino acids are collected from the culture. A method for producing L-amino acid.
( 6 ) 前記 L—アミノ酸が、 L—リジン、 L—グルタミン酸、 Lースレオニン、 L—イソロイシン、 Lーセリンから選ばれる （ 5 ) の方法。 発明を実施するための最良の形態 以下、 本発明を詳細に説明する。  (6) The method according to (5), wherein the L-amino acid is selected from L-lysine, L-glutamic acid, L-threonine, L-isoleucine, and L-serine. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
< 1 〉本発明のコリネ型細菌 <1> Coryneform bacterium of the present invention
本発明のコリネ型細菌は、 L—アミノ酸生産能を有し、 細胞中のエノラーゼ活 性が増強されたコリネ型細菌である。 L一アミノ酸としては、 L—リジン、 L— グルタミン酸、 Lースレオニン、 L一イソロイシン、 Lーセリン等が挙げられる。 これらの中では、 L一リジン及び L一グルタミン酸が好ましい。 以下、 本発明の 実施の形態を、 主として Lーリジン生産能又は L—グル夕ミン酸生産能を有する コリネ型細菌について説明するが、 本発明は、 目的とする L一アミノ酸固有の生 合成系がエノラーゼよりも下流に位置するものについては同様に適用され得る。 本発明でいうコリネ型細菌は、 パージ一ズ · マニュアル . ォブ . デターミネィ ティブ ' ノ クテリオロジ一 ( Bergey' s Manual of Determinative Bacteriology) 第 8版 599頁 （1974) に定義されている一群の微生物であり、 好気性、 グラム陽性、 非抗酸性で、 胞子形成能を有しない桿菌であり、 従来ブレビパクテリゥム属に分 類されていたが現在コリネバクテリゥム属細菌として統合された細菌を含み （In t . J. Syst. Bacterid . , 41 , 255 ( 1981 ) ) 、 またコリネパクテリゥム属と非常 に近縁なブレビパクテリゥム属細菌及びミクロバテリゥム属細菌を含む。 L—リ ジン又は L一グルタミン酸の製造に好適に用いられるコリネ型細菌の菌株として は、 例えば以下に示すものが挙げられる。 The coryneform bacterium of the present invention is a coryneform bacterium having L-amino acid-producing ability and enhanced enolase activity in cells. Examples of L-amino acids include L-lysine, L-glutamic acid, L-threonine, L-isoleucine, L-serine and the like. Of these, L-lysine and L-glutamic acid are preferred. Hereinafter, an embodiment of the present invention will be described mainly with respect to a coryneform bacterium having L-lysine-producing ability or L-glucamic acid-producing ability, but the present invention relates to a biosynthesis system specific to an L-amino acid of interest. The same applies to those located downstream of the enolase. The coryneform bacterium referred to in the present invention is a group of microorganisms defined in Purges' Manual of Determinative Bacteriology, 8th edition, p. 599 (1974). Yes, aerobic, gram positive, It is a non-acid-fast, non-spore-forming bacillus, including bacteria that were previously classified into the genus Brevipacterium, but are now integrated as corynebacteria (Int. J. Syst. Bacterid., 41, 255 (1981)), and also includes bacteria of the genus Brevipacterium and Microbatterium, which are very closely related to the genus Corynepacterium. Examples of the strains of coryneform bacteria suitably used for producing L-lysine or L-glutamic acid include, for example, those shown below.
コリネバクテリゥム · ァセ トァシドフィルム ATCC13870  Corynebacterium acetate film ATCC13870
コリネバクテリウム · ァセ トグルタミクム ATCC15806  Corynebacterium acetoglutamicum ATCC15806
コリネバクテリゥム · カルナェ ATCC15991  Corynebacterium carnaet ATCC15991
コリネバクテリゥム · グル夕ミクム ATCC13032  Corynebacterium · Guru Yu Mikum ATCC13032
(ブレビバクテリウム · ディバリカタム） ATCC14020  (Brevibacterium divaricatum) ATCC14020
(ブレビバクテリゥム · ラク トフアーメンタム） ATCC13869  (Brevibacterium lactofamentum) ATCC13869
(コリネバクテリゥム . リ リゥム） ATCC15990  (Corynebacterium ream) ATCC15990
(ブレビバクテリゥム · フラバム） ATCC14067  (Brevibacterium flavum) ATCC14067
コリネバクテリゥム メラセコーフ ATCC17965  Corynebacterium Melasekov ATCC17965
ブレビバクテリウム サッカロリティ クム ATCC14066  Brevibacterium saccharinity comb ATCC14066
ブレビパクテリゥム インマリオフィルム ATCC14068  Brevi Pacterium Inmario Film ATCC14068
ブレビパクテリゥム ロゼゥム ATCC13825  Brevi Pacterium Rosemze ATCC13825
ブレビパクテリゥム チォゲ二夕リス ATCC19240  Brevi Pacterium Choge Niyu Squirrel ATCC19240
ミクロバクテリゥム アンモニアフィラム ATCC15354  Microbacterium Ammonia Filum ATCC15354
コリネバクテリゥム サーモアミノゲネス AJ12340(FERM BP-1539 ) これらを入手するには、 例えばァメ リカン · タイプ · カルチャー · コレクショ ン (American Type Culture Col lection, 住所 12301 Parklawn Drive, Rockvi l l e， Maryland 20852, United States of America) より分譲を受けることができ る。 すなわち、 各微生物ごとに対応する登録番号が付与されており、 この登録番 号を引用して分譲を受けることができる。 各微生物に対応する登録番号はァメ リ カン · タイプ . カルチャー · コレクションのカタログに記載されている。 また、 AJ12340株は、 通商産業省工業技術院生命工学工業技術研究所 （郵便番号 305- 856 6 日本国茨城県つくば巿東一丁目 1番 3号） にブダペス ト条約に基づいて寄託さ れている。 Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539) To obtain these, for example, American Type Culture Collection, Address 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America). That is, a registration number corresponding to each microorganism is assigned, and the microorganism can be ordered by referring to this registration number. The registration number corresponding to each microorganism is listed in the catalog of the American Type Culture Collection. In addition, AJ12340 strain was deposited under the Budapest Treaty with the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, Institute of Biotechnology and Industrial Technology (zip code 305-8566, 1-3 1-3 Tsukuba-Higashi, Ibaraki, Japan). Have been.
また、 上記菌株以外にも、 これらの菌株から誘導された L—リジン又は Lーグ ル夕ミン酸等の L一アミノ酸生産能を有する変異株等も、 本発明に利用できる。 この様な人工変異株としては次の様なものがある。 S— （ 2—アミノエチル） 一 システィン （以下、 「AEC」 と略記する） 耐性変異株 （例えば、 ブレビバクテリウ ム ' ラク トフアーメンタム AJ11082 (NRRL B-11470) 、 特公昭 56- 1914号、 特公昭 56- 1915号、 特公昭 57-14157号、 特公昭 57-14158号、 特公昭 57- 30474号、 特公昭 5 8-10075号、 特公昭 59- 4993号、 特公昭 61-35840号、 特公昭 62- 24074号、 特公昭 62 - 36673号、 特公平 5- 11958号、 特公平 7- 112437号、 特公平 7-112438号参照） 、 その 成長に L—ホモセリン等のアミノ酸を必要とする変異株 （特公昭 48-28078号、 特 公昭 56- 6499号） 、 AECに耐性を示し、 更に L一口イシン、 L一ホモセリン、 L— プロリン、 L—セリン、 L一アルギニン、 Lーァラニン、 L—パリン等のアミノ 酸を要求する変異株 （米国特許第 3708395号及び第 3825472号） 、 D L—ひ一アミ ノー ε—力プロラクタム、 ひ一ァミノ一ラウリルラクタム、 ァスパラギン酸ーァ ナログ、 スルファ剤、 キノイ ド、 Ν—ラウロイルロイシンに耐性を示す L—リジ ン生産変異株、 ォキザ口酢酸脱炭酸酵素 （デカルポキシラーゼ） または呼吸系酵 素阻害剤の耐性を示す L一リジン生産変異株 （特開昭 50- 53588号、 特開昭 50-310 93号、 特開昭 52-102498号、 特開昭 53-9394号、 特開昭 53-86089号、 特開昭 55- 978 3号、 特開昭 55- 9759号、 特開昭 56- 32995号、 特開昭 56- 39778号、 特公昭 53-43591 号、 特公昭 53- 1833号） 、 イノシトールまたは酢酸を要求する L一リジン生産変異 株 （特開昭 55- 9784号、 特開昭 56- 8692号） 、 フルォロピルビン酸または 34°C以上 の温度に対して感受性を示す L—リジン生産変異株 （特開昭 55-9783号、 特開昭 5 3 - 86090号） 、 エチレングリコールに耐性を示し、 L—リジンを生産するブレビバ クテリウム属またはコリネパクテリゥム属の生産変異株 （米国特許第 4411997号） 。 また、 L—スレオニン生産能を有するコリネ型細菌としては、 コリネパクテリ ゥム ' ァセ トァシ ドフィ ラム AJ12318 (FERM BP- 1172 ) (米国特許第 5, 188， 949号参 照） 等が、 L—イソロイシン生産能を有するコリネ型細菌としてはブレビバクテ リウム . フラバム AJ12149 ( FERM BP-759) (米国特許第 4， 656， 135号参照) 等が挙 げられる。 なお、 本明細書において 「L—リジン等の L—アミノ酸生産能」 とは、 コリネ 型細菌を培地に培養したときに、 培地中に有意な量の L—リジン等の Lーァミノ 酸を蓄積する能力、 又は菌体中の L—リジン等のアミノ酸含量を増加させる能力 をいう。 In addition to the above strains, mutants having L-amino acid-producing ability, such as L-lysine or L-glucamic acid, derived from these strains can also be used in the present invention. Such artificial mutants include the following. S- (2-aminoethyl) -cysteine (hereinafter abbreviated as "AEC") resistant mutant (for example, Brevibacterium 'Lactofamentum AJ11082 (NRRL B-11470), Japanese Patent Publication No. 56-1914, Japanese Patent Publication No. No. 56-1915, No. 57-14157, No. 57-14158, No. 57-30474, No. 58-10075, No. 59-4993, No. 61-35840, No. 61-35840, No. Nos. 62-24074, JP-B 62-36673, JP-B 5-11958, JP-B 7-112437, and JP-B 7-112438), mutants that require amino acids such as L-homoserine for their growth. (JP-B-48-28078, JP-B-56-6499), showing resistance to AEC, and furthermore, L-mouth isin, L-homoserine, L-proline, L-serine, L-arginine, L-alanine, L-parin, etc. Mutants that require amino acids (US Pat. Nos. 3,708,395 and 3,825,472), DL-amino ε- L-lysine-producing mutants resistant to caprolactam, hyaminoaminolauryl lactam, aspartic acid-analog, sulfa drugs, quinoids, Ν-lauroylleucine, oxaxate acetate decarboxylase (decarboxylase) Alternatively, L-lysine-producing mutants showing resistance to respiratory enzyme inhibitors (Japanese Patent Application Laid-Open Nos. 50-53588, 50-31093, 52-102498, 52-102498, 53-9394) JP-A-53-86089, JP-A-55-9783, JP-A-55-9759, JP-A-56-32995, JP-A-56-39778, JP-B-53-43591, and JP-A-53-43591. No. 53-1833), L-lysine-producing mutants requiring inositol or acetic acid (Japanese Patent Application Laid-Open Nos. 55-9784 and 56-8692), sensitive to fluoropyruvic acid or a temperature of 34 ° C or higher L-lysine-producing mutants (JP-A-55-9783, JP-A-53-86090), ethylene glycol Call resistant, producing L- lysine Burebiba Kuteriumu genus or Corynebacterium Park Teri © beam genus producing mutant strain (U.S. Patent No. 4,411,997). Examples of coryneform bacteria having L-threonine-producing ability include corynebacterium 'acetacidophila AJ12318 (FERM BP-1172) (see US Pat. No. 5,188,949) and L-isoleucine. Examples of coryneform bacteria having the ability to produce are Brevibacterium flavum AJ12149 (FERM BP-759) (see US Pat. No. 4,656,135) and the like. As used herein, “the ability to produce L-amino acids such as L-lysine” means that when a coryneform bacterium is cultured in a medium, a significant amount of L-amino acid such as L-lysine is accumulated in the medium. Ability or ability to increase the content of amino acids such as L-lysine in cells.
< 2 >エノラーゼ活性の増強  <2> Enolase activity enhancement
コリネ型細菌細胞中のエノラーゼ活性を増強するには、 エノラーゼをコ一ドす る遺伝子断片を、 該細菌で機能するベクター、 好ましくはマルチコピー型のべク ターと連結して組み換え D N Aを作製し、 これを L—リジン又は L—グル夕ミン 酸生産能を有するコリネ型細菌に導入して形質転換すればよい。 形質転換株の細 胞内のエノラーゼをコ一ドする遺伝子のコピー数が上昇する結果、 エノラーゼ活 性が増強される。 エノラ一ゼは、 ェシエリヒア ' コリでは eno遺伝子にコードされ ている。  To enhance the enolase activity in coryneform bacterial cells, a recombinant DNA is prepared by ligating a gene fragment encoding the enolase to a vector that functions in the bacterium, preferably a multicopy vector. This may be introduced into a coryneform bacterium capable of producing L-lysine or L-glucamic acid for transformation. The enolase activity is enhanced as a result of an increase in the copy number of the gene encoding the enolase in the cells of the transformed strain. Enola is encoded by the eno gene in Escherichia coli.
エノラ一ゼ遺伝子は、 コリネ型細菌の遺伝子を用いることも、 ェシヱリヒア属 細菌等の他の生物由来の遺伝子のいずれも使用することができる。  As the enolase gene, a gene of a coryneform bacterium or a gene derived from another organism such as a bacterium belonging to the genus Escherichia can be used.
ェシエリヒア · コリの eno遺伝子の塩基配列は既に明らかにされている （Klein, M. et al .， DNA Seq. 6， 315-355 ( 1996年）， Genbank/EMBL/DDBJ accetion No. X82400) ので、 その塩基配列に基づいて作製したブライマー、 例えば配列表配列 番号 1及び 2に示すプライマ一を用いて、 ェシエリヒア ' コリ染色体 D N Aを銪 型とする P C R法 （P C R ： polymerase chain reaction； White, T. J. et al ； Trends Genet. 5， 185( 1989)参照） によって、 eno遺伝子を取得することができる。 コリネ型細菌等の他の微生物のエノラーゼをコ一ドする遺伝子も、 同様にして取 得され得る。  The nucleotide sequence of the Escherichia coli eno gene has already been determined (Klein, M. et al., DNA Seq. 6, 315-355 (1996), Genbank / EMBL / DDBJ accetion No. X82400). Using a primer prepared based on the base sequence, for example, the primers shown in SEQ ID NOs: 1 and 2 in the Sequence Listing, a PCR method using polymerase chain reaction (PCR: polymerase chain reaction; White, TJ et al.) Eno gene can be obtained according to Trends Genet. 5, 185 (1989)). Genes encoding enolase of other microorganisms such as coryneform bacteria can be obtained in a similar manner.
染色体 D N Aは、 D N A供与体である細菌から、 例えば、 斎藤、 三浦の方法 (H. Saito and K. Miura Biochem. Biophys. Acta, 72, 619 ( 1963 )、 生物工学 実験書、 日本生物工学会編、 9 7〜9 8頁、 培風館、 1 9 9 2年参照） 等により 調製することができる。  Chromosomal DNA is obtained from the DNA donor bacterium, for example, by the method of Saito and Miura (H. Saito and K. Miura Biochem. Biophys. Acta, 72, 619 (1963)) Pp. 97-98, Baifukan, 1992).
P C R法により増幅されたエノラーゼをコ一ドする遺伝子は、 ェシェリヒア · コリ及び/又はコリネ型細菌の細胞内において自律複製可能なベクタ一 D N Aに 接続して組換え D N Aを調製し、 これをェシヱリヒア · コリ細胞に導入しておく と、 後の操作がしゃすくなる。 ェシヱリヒア ' コリ細胞内において自律複製可能 なべクタ一としては、 プラスミ ドベクタ一が好ましく、 宿主の細胞内で自立複製 可能なものが好ましく、 例えば pUC19、 pUC18、 pBR322、 pHSG299、 pHSG399N pHS G398、 RSF1010等が挙げられる。 The gene encoding the enolase amplified by the PCR method is connected to a vector DNA capable of autonomously replicating in the cells of Escherichia coli and / or coryneform bacteria to prepare a recombinant DNA, which is then ligated to Escherichia coli. Introduced into E. coli cells Then, the operation later becomes difficult. As a vector capable of autonomous replication in Escherichia coli cells, plasmid vectors are preferable, and those capable of autonomous replication in host cells are preferable.For example, pUC19, pUC18, pBR322, pHSG299, pHSG399 N pHS G398, RSF1010, etc. Is mentioned.
コリネ型細菌の細胞内において自律複製可能なベクターとしては、 PAM330 (特 開昭 58-67699号公報参照） 、 pHM1519 (特開昭 58-77895号公報参照） 等が挙げられ る。 また、 これらのベクターからコリネ型細菌中でプラスミ ドを自律複製可能に する能力を持つ DN A断片を取り出し、 前記ェシエリヒア · コリ用のベクターに 挿入すると、 ェシエリヒア · コリ及びコリネ型細菌の両方で自律複製可能ないわ ゆるシャトルベクターとして使用することができる。 このようなシャトルべクタ —としては、 以下のものが挙げられる。 尚、 それそれのベクターを保持する微生 物及び国際寄託機関の受託番号をかっこ内に示した。  Examples of a vector capable of autonomous replication in a coryneform bacterium include PAM330 (see Japanese Patent Application Laid-Open No. 58-67699), pHM1519 (see Japanese Patent Application Laid-Open No. 58-77895), and the like. In addition, a DNA fragment having the ability to enable autonomous replication of plasmid in coryneform bacteria is extracted from these vectors and inserted into the Escherichia coli vector, whereby autonomously expressed in both Escherichia coli and coryneform bacteria. It can be used as any shuttle vector that is not replicable. Such shuttle vectors include: Microorganisms carrying the respective vectors and the accession numbers of the international depository organizations are shown in parentheses.
PAJ655 ェシエリヒア 'コリ AJ11882(FERM BP-136)  PAJ655 Escherichia Cori AJ11882 (FERM BP-136)
コリネハ"クテリゥム 'ク、 'ルタミクム SR8201(ATCC39135)  Korineha "Certium", "Rutamicum SR8201 (ATCC39135)
PAJ1844 工シエリヒア'コリ AJ11883(FERM BP- 137)  PAJ1844 Engineering Koerihikari 'Kori' AJ11883 (FERM BP-137)
コリネハ、、クテリゥム，ク、、ルタミクム SR8202(ATCC39136)  Coryneha, Cterium, C, Rutamicum SR8202 (ATCC39136)
PAJ611 ェシエリヒア ':iDAJ11884(FERM BP-138)  PAJ611 Escherichia ': iDAJ11884 (FERM BP-138)
PAJ3148 コリネハ、、クテリゥム'ク'、ルタミクム SR8203(ATCC39137)  PAJ3148 Coryneha, Cterium 'K', Rutamikum SR8203 (ATCC39137)
PAJ440 、チルス ·ΓΓチリス AJ11901(FERM BP- 140)  PAJ440, chillsPetiris AJ11901 (FERM BP-140)
pHC4 Iシエリヒア'コリ AJ12617(FERM BP- 3532)  pHC4 I Shierihia 'coli AJ12617 (FERM BP-3532)
エノラーゼをコードする遺伝子とコリネ型細菌で機能するべクタ一を連結して 組み換え D N Aを調製するには、 エノラーゼをコードする遺伝子の末端に合うよ うな制限酵素でベクタ一を切断する。 連結は、 T 4 D N Aリガ一ゼ等のリガーゼ を用いて行うのが普通である。  In order to prepare recombinant DNA by linking the gene encoding the enolase to the vector that functions in coryneform bacteria, the vector is cut with a restriction enzyme that matches the end of the gene encoding the enolase. Ligation is usually performed using a ligase such as T4DNA ligase.
上記のように調製した組み換え D N Aをコリネ型細菌に導入するには、 これま でに報告されている形質転換法に従って行えばよい。 例えば、 ェシエリヒア *コ リ K一 1 2について報告されているような、 受容菌細胞を塩化カルシウムで処 理して D N Aの透過性を増す方法 (Mandel,M.and Higa,A.,J. Mol. Biol., 53, 159 (1970)) があり、 バチルス 'ズブチリスについて報告されているような、 増 殖段階の細胞からコンビテン トセルを調製して DNAを導入する方法 （ Duncan, C.H. , Wilson, G. A. and Young, F.E., Gene, 1, 153 (1977)) がある。 あるいは、 バ チルス · ズブチリス、 放線菌類及び酵母について知られているような、 DNA受 容菌の細胞を、 組換え DN Aを容易に取り込むプロ トプラス トまたはスフェロブ ラス トの状態にして組換え DNAを D NA受容菌に導入する方法 （ Chang, S. and Choen,S.N.,Molec. Gen. Genet., 168, 111 ( 1979) ; Bibb, M. J. , Ward, J. M. and H opwood,0.A., ature, 274, 398 ( 1978 );Hinnen, A., Hicks, J.B.and Fink,G.R.,Pr oc. Natl. Acad. Sci. USA, 75 1929 (1978)) も応用できる。 本発明の実施例で 用いた形質転換の方法は、 電気パルス法 （特開平 2— 20779 1号公報参照） である。 To introduce the recombinant DNA prepared as described above into a coryneform bacterium, it may be carried out according to the transformation method reported so far. For example, a method of increasing the permeability of DNA by treating recipient cells with calcium chloride, as reported for Escherichia * coli K-112 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)), with increased numbers as reported for Bacillus' subtilis. There is a method of preparing a competent cell from cells at the stage of breeding and introducing DNA (Duncan, CH, Wilson, GA and Young, FE, Gene, 1, 153 (1977)). Alternatively, recombinant DNA can be obtained by transforming cells of a DNA-receiving bacterium into a protoplast or spheroblast that readily incorporates the recombinant DNA, as is known for Bacillus subtilis, actinomycetes and yeast. Method for introduction into DNA recipient bacteria (Chang, S. and Choen, SN, Molec. Gen. Genet., 168, 111 (1979); Bibb, MJ, Ward, JM and Hopwood, 0.A., ature, 274, 398 (1978); Hinnen, A., Hicks, JBand Fink, GR, Proc. Natl. Acad. Sci. USA, 75 1929 (1978)). The transformation method used in the examples of the present invention is the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791).
エノラーゼをコードする活性の増強は、 エノラーゼをコ一ドする遺伝子を上記 宿主の染色体 DN A上に多コピー存在させることによつても達成できる。 コリネ 型細菌に属する微生物の染色体 D N A上にエノラーゼをコ一ドする遺伝子を多コ ピーで導入するには、 染色体 D N A上に多コピー存在する配列を標的に利用して 相同組換えにより行う。 染色体 D N A上に多コピー存在する配列としては、 レぺ ッティ ブ DNA、 転移因子の端部に存在するインバーティ ッ ド · リピートが利用 できる。 あるいは、 特開平 2— 1 09985号公報に開示されているように、 ェ ノラ一ゼをコ一ドする遺伝子を トランスポゾンに搭載してこれを転移させて染色 体 D N A上に多コピー導入することも可能である。 いずれの方法によっても形質 転換株内のエノラーゼをコ一ドする遺伝子のコピー数が上昇する結果、 エノラー ゼ活性が増強される。  Enolase-encoding activity can also be enhanced by having multiple copies of the enolase-encoding gene on the chromosomal DNA of the host. In order to introduce a gene that encodes an enolase into a chromosome DNA of a microorganism belonging to a coryneform bacterium in multiple copies, homologous recombination is performed by using a sequence that exists in multiple copies on the chromosome DNA as a target. As a sequence present in multiple copies on the chromosome DNA, repetitive DNA and inverted repeats present at the end of a transposable element can be used. Alternatively, as disclosed in Japanese Patent Application Laid-Open No. 2-110985, it is also possible to mount a gene encoding enolase on a transposon, transfer this gene, and introduce multiple copies into chromosomal DNA. It is possible. Either method increases the copy number of the gene encoding the enolase in the transformant, resulting in enhanced enolase activity.
エノラーゼ活性の増強は、 上記の遺伝子増幅による以外に、 染色体 DN A上又 はプラスミ ド上のエノラーゼをコ一ドする遺伝子のプロモーター等の発現調節配 列を強力なものに置換することによつても達成される （特開平 1一 2 15280 号公報参照） 。 たとえば、 l a。プロモーター、 t r pプロモータ一、 t r cプ 口モーター、 a cプロモーター、 ラムダファージの P Rプロモータ一、 PLプ ロモ一夕一等が強力なプロモーターとして知られている。 これらのプロモーター への置換により、 エノラーゼをコ一ドする遺伝子の発現が強化されることによつ てエノラーゼ活性が増強される。 また、 本発明のコリネ型細菌は、 エノラーゼ活性に加えて、 他のアミノ酸生合 成経路又は解糖系等の酵素遺伝子を強化することによって、 それらの酵素活性が 増強されてもよい。 例えば、 L一リジンの製造に利用可能な遺伝子の例としては、 Lーリジン及び L—スレオニンによる相乗的なフィ一ドバック阻害が実質的に解 除されたァスパルトキナーゼひサブュニッ ト蛋白質又は/?サブユニッ ト蛋白質を コードする遺伝子 （W094/25605国際公開パンフレッ ト） 、 コリネホルム細菌由来 の野生型ホスホエノ一ルビルビン酸カルボキシラーゼ遺伝子 （特開昭 60- 87788号 公報） 、 コリネホルム細菌由来の野生型ジヒ ドロジピコリン酸合成酵素をコード する遺伝子 （特公平 6- 55149号公報） 等が知られている。 Enolase activity is enhanced not only by the above-mentioned gene amplification but also by replacing the expression regulatory sequence such as the promoter of the gene encoding the enolase on chromosome DNA or plasmid with a strong one. Is also achieved (see Japanese Patent Application Laid-Open No. Hei 1-215280). For example, la. Promoters, trp promoters, trc promoters, ac promoters, PR promoters for lambda phage, PL promoters, etc. are known as strong promoters. The substitution with these promoters enhances the enolase activity by enhancing the expression of the gene encoding the enolase. In addition, the coryneform bacterium of the present invention may have an enhanced enzymatic activity by enhancing an enzyme gene such as another amino acid synthesizing pathway or a glycolytic pathway in addition to the enolase activity. For example, examples of genes that can be used for the production of L-lysine include aspartokinase subunit protein or / ?, in which synergistic feedback inhibition by L-lysine and L-threonine has been substantially eliminated. Gene encoding subunit protein (W094 / 25605 International Publication Pamphlet); wild-type phosphoenolpyruvate carboxylase gene derived from coryneform bacteria (JP-A-60-87788); wild-type dihydropicolinic acid derived from coryneform bacteria A gene encoding a synthase (Japanese Patent Publication No. 6-55149) and the like are known.
また、 L—グルタ ミン酸の製造に利用可能な遺伝子の例としては、 グルタミン 酸デヒ ドロゲナーゼ （G D H、 特開昭 6 1— 2 6 8 1 8 5号） 、 グル夕 ミンシン テターゼ、 グルタ ミン酸シン夕ーゼ、 イソクェン酸デヒ ドロゲナーゼ （特開昭 6 2— 1 6 6 8 9 0号、 特開昭 6 3— 2 1 4 1 8 9号） 、 アコニッ ト酸ヒ ドラター ゼ （特開昭 6 2— 2 9 4 0 8 6号） 、 クェン酸シン夕ーゼ、 ピルビン酸カルボキ シラーゼ （特開昭 6 0— 8 7 7 8 8号、 特開昭 6 2— 5 5 0 8 9号） 、 ホスホェ ノールピルビン酸カルボキシラーゼ、 ホスホエノ一ルビルビン酸シン夕一ゼ、 ェ ノラ一ゼ、 ホスホグリセロムターゼ、 ホスホグリセリン酸キナ一ゼ、 グリセルァ ルデヒ ドー 3—リン酸デヒ ドロゲナ一ゼ、 トリオ一スリン酸イソメラ一ゼ、 フル トースビスリン酸アルドラ一ゼ、 ホスホフルク トキナーゼ （特開昭 6 3 - 1 0 2 6 9 2号） 、 グルコースリン酸イソメラ一ゼ等がある。  Examples of genes that can be used for the production of L-glutamic acid include glutamate dehydrogenase (GDH, Japanese Patent Application Laid-Open No. Sho 61-268185), glutamate synthase, and glutamate. Synthase, isoquenate dehydrogenase (Japanese Patent Application Laid-open No. Sho 62-166890, Japanese Patent Application Laid-Open No. 63-214189), aconitate hydratase (Japanese Patent Application Laid-Open No. 62-294,086), citrate synthase, pyruvate carboxylase (Japanese Patent Laid-Open No. 60-87788, Japanese Patent Laid-open No. 62-55089) , Phosphoenolpyruvate carboxylase, phosphoenolpyruvate synthase, enolase, phosphoglyceromutase, phosphoglycerate quinase, glyceraldehyde 3-phosphate dehydrogenase, triosulfonate isomera Aldose, fructose bisphosphate phosphate , Hosuhofuruku Tokinaze (JP 6 3 - 1 0 2 6 9 2 No.), there are glucose phosphate Isomera Ichize like.
さらに、 目的とする Lーァミノ酸の生合成経路から分岐して同 L一アミノ酸以 外の化合物を生成する反応を触媒する酵素の活性が低下または欠損していてもよ い。 例えば、 L—リジンの生合成経路から分岐して L一リジン以外の化合物を生 成する反応を触媒する酵素としては、 ホモセリンデヒ ドロゲナーゼがある （W0 9 5/23864参照） 。 また、 L一グルタミン酸の生合成経路から分岐して Lーグルタミ ン酸以外の化合物を生成する反応を触媒する酵素としては、 ひケ トグルタール酸 デヒ ドロゲナーゼ、 イソクェン酸リアーゼ、 リン酸ァセチルトランスフェラーゼ、 酢酸キナーゼ、 ァセ トヒ ドロキシ酸シンターゼ、 ァセ ト乳酸シン夕一ゼ、 ギ酸ァ セチルトランスフヱラーゼ、 乳酸デヒ ドロゲナーゼ、 グルタミン酸デカルボキシ ラーゼ、 1—ピロリン酸デヒドロゲナ一ゼ、 等がある。 Furthermore, the activity of an enzyme that catalyzes a reaction that diverges from a target L-amino acid biosynthetic pathway to produce a compound other than the same L-amino acid may be reduced or lacking. For example, homoserine dehydrogenase is an enzyme that catalyzes a reaction that produces a compound other than L-lysine by branching off from the L-lysine biosynthetic pathway (see W095 / 23864). In addition, enzymes that catalyze the reaction that diverges from the biosynthetic pathway of L-glutamic acid to produce compounds other than L-glutamic acid include ketoglutarate dehydrogenase, isoquenate lyase, acetyl phosphate transferase, and acetate. Kinase, acetohydroxylate synthase, acetolactate synthase, acetyl formate transferase, lactate dehydrogenase, glutamate decarboxylate And 1-pyrophosphate dehydrogenase.
さらに、 L—グルタミン酸生産能を有するコリネ型細菌に、 界面活性剤等のビ ォチン作用抑制物質に対する温度感受性変異を付与することにより、 過剰量のビ ォチンを含有する培地中にてピオチン作用抑制物質の非存在下で L—グル夕ミン 酸を生産させることができる （W096/06180号参照） 。 このようなコリネ型細菌と しては、 W096/06180号に記載されているブレビパクテリゥム . ラクトフアーメン タム AJ 13029が挙げられる。 AJ13029株は、 1994年 9月 2日付けで工業技術院生命 工学工業技術研究所 （郵便番号 305- 8566 日本国茨城県つくば巿東一丁目 1番 3 号） に、 受託番号 FERM P-14501として寄託され、 1 9 9 5年 8月 1日にブダぺス ト条約に基づく国際寄託に移管され、 受託番号 FERM BP-5189が付与されている。 また、 L—リジン及び L一グルタミン酸生産能を有するコリネ型細菌に、 ピオ チン作用抑制物質に対する温度感受性変異を付与することにより、 過剰量のピオ チンを含有する培地中にてピオチン作用抑制物質の非存在下で Lーリジン及び L —グルタミン酸を同時生産させることができる （W096/06180号参照） 。 このよう な菌株としては、 W096/06180号に記載されているブレビパクテリゥム . ラクトフ ァ一メンタム AJ12993株が挙げられる。 同株は 1 9 9 4年 6月 3日付けで工業技術 院生命工学工業技術研究所 （郵便番号 305- 8566 日本国茨城県つくば巿東一丁目 1 番 3号） に、 受託番号 FERM P- 14348で寄託され、 1 9 9 5年 8月 1日にブダぺス ト条約に基づく国際寄託に移管され、 受託番号 FERM BP-5188が付与されている。 なお、 本明細書において、 酵素の 「活性が増強されている」 とは、 通常には、 野生株よりも細胞内のその酵素活性が高いことを意味し、 遺伝子組換え技術等に よる改変によりその酵素活性が増強された菌株を得た場合には、 改変前の菌株よ りも細胞内のその酵素活性が高いことを意味する。 また、 酵素の 「活性が低下し ている」 とは、 通常には、 野生株よりも細胞内のその酵素活性が低いことを意味 し、 遺伝子組換え技術等による改変によりその酵素活性が低下した菌株を得た場 合には、 改変前の菌株よりも細胞内のその酵素活性が低いことを意味する。  Furthermore, by imparting a temperature-sensitive mutation to a biotin action inhibitor such as a surfactant to a coryneform bacterium capable of producing L-glutamic acid, the biotin action inhibitor can be added to a medium containing an excess amount of biotin. L-glutamic acid can be produced in the absence of E. coli (see W096 / 06180). Examples of such coryneform bacteria include Brevipacterium. Lactofarmentum AJ13029 described in W096 / 06180. The AJ13029 strain was registered with the Institute of Biotechnology, Industrial Science and Technology (Postal Code 305-8566, Tsukuba 1-3-1, Ibaraki, Japan) on September 2, 1994 under the accession number FERM P-14501. Deposited, transferred to an international deposit under the Budapest Treaty on August 1, 1995, and given accession number FERM BP-5189. In addition, by imparting a temperature-sensitive mutation to a biotin-inhibiting substance to a coryneform bacterium capable of producing L-lysine and L-glutamic acid, the biotin-inhibiting substance can be expressed in a medium containing an excessive amount of biotin. L-Lysine and L-glutamic acid can be produced simultaneously in the absence (see W096 / 06180). Examples of such a strain include Brevipacterium. Lactofermentum AJ12993 strain described in W096 / 06180. The stock was issued to the Institute of Biotechnology, Institute of Industrial Technology (Postal Code 305-8566, Tsukuba 1-3-1-3, Ibaraki, Japan) on June 3, 1994, with the accession number FERM P- Deposited at 14348, transferred to the International Deposit under the Budapest Treaty on August 1, 1995, and given accession number FERM BP-5188. As used herein, the phrase “enhanced activity” of an enzyme generally means that the enzyme activity in a cell is higher than that of a wild-type strain, and is modified by a gene recombination technique or the like. When a strain with enhanced enzyme activity is obtained, it means that the enzyme activity in the cell is higher than that of the strain before modification. The term “reduced activity” of an enzyme usually means that the enzyme activity in a cell is lower than that of a wild-type strain, and the enzyme activity has been reduced by modification by genetic recombination technology or the like. When a strain is obtained, it means that the enzyme activity in the cell is lower than that of the strain before modification.
< 3 > L一アミノ酸の生産 <3> Production of L-amino acid
エノラーゼ活性が増強され、 かつ、 L—アミノ酸生産能を有するコリネ型細菌 を好適な培地で培養すれば、 同 L一アミノ酸が培地に蓄積する。 冽えば、 ェノラ ーゼ活性が増強され、 かつ Lーリジン酸生産能を有するコリネ型細菌を好適な培 地で培養すれば、 L—リジンが培地に蓄積する。 また、 エノラーゼ活性が増強さ れ、 かつ L一グルタ ミン酸生産能を有するコリネ型細菌を好適な培地で培養すれ ば、 L—グルタ ミン酸が培地に蓄積する。 When a coryneform bacterium having enhanced enolase activity and capable of producing L-amino acid is cultured in a suitable medium, the L-amino acid accumulates in the medium. If it ’s clear, Enola If a coryneform bacterium having enhanced L-lysine activity and capable of producing L-lysine acid is cultured in a suitable medium, L-lysine accumulates in the medium. In addition, when a coryneform bacterium having enhanced enolase activity and capable of producing L-glutamic acid is cultured in a suitable medium, L-glutamic acid accumulates in the medium.
さらに、 エノラーゼ活性が増強され、 かつ L一リジン及び L—グルタ ミン酸生 産能を有するコリネ型細菌を培地で培養すれば、 L—リジン及び L—グル夕ミン 酸が培地に蓄積する。 Lーリジンと L—グル夕ミン酸を同時に K酵生産する場合 には、 Lーリジン生産菌を L一グル夕 ミン酸の生産条件下で培養してもよいし、 あるいは Lーリジン生産能を有するコリネ型細菌と L—グルタ ミン酸生産能を有 するコリネ型細菌を混合培養してもよい （特開平 5 — 3 7 9 3号公報） 。  Furthermore, when a coryneform bacterium having enhanced enolase activity and capable of producing L-lysine and L-glutamic acid is cultured in the medium, L-lysine and L-glutamic acid accumulate in the medium. When L-lysine and L-glucaminic acid are simultaneously produced in the K enzyme, the L-lysine-producing bacterium may be cultured under L-glucaminic acid-producing conditions, or a L-lysine-producing coryneform A bacterium and a coryneform bacterium capable of producing L-glutamic acid may be mixedly cultured (Japanese Patent Application Laid-Open No. 5-3793).
本発明の微生物を用いて L一リジン又は L—グルタ ミン酸等の L—アミノ酸を 製造するのに用いる培地は、 炭素源、 窒素源、 無機イオン及び必要に応じその他 の有機微量栄養素を含有する通常の培地である。 炭素源としては、 グルコース、 ラク トース、 ガラク トース、 フラク ト一ス、 シュクロース、 廃糖蜜、 澱粉加水分 解物などの炭水化物、 エタノールやイノシトールなどのアルコール類、 酢酸、 フ マール酸、 クェン酸、 コハク酸等の有橾酸類を用いることができる。  The medium used for producing L-amino acids such as L-lysine or L-glutamic acid using the microorganism of the present invention contains a carbon source, a nitrogen source, inorganic ions, and other organic micronutrients as required. This is a normal medium. Carbon sources include glucose, lactose, galactose, fructos, sucrose, molasses, carbohydrates such as starch hydrolysates, alcohols such as ethanol and inositol, acetic acid, fumaric acid, citric acid, An organic acid such as succinic acid can be used.
窒素源としては、 硫酸アンモニゥム、 硝酸アンモニゥム、 塩化アンモニゥム、 リン酸アンモニゥム、 酢酸アンモニゥム等の無機アンモニゥム塩、 アンモニア、 ペプトン、 肉エキス、 酵母エキス、 酵母エキス、 コーン ' スティープ ' リカー、 大豆加水分解物などの有機窒素、 アンモニアガス、 アンモニア水等を用いること ができる。  Nitrogen sources include inorganic ammonium salts such as ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, ammonium acetate, ammonia, peptone, meat extract, yeast extract, yeast extract, corn 'steep' liquor, soy hydrolyzate, etc. Organic nitrogen, ammonia gas, aqueous ammonia, etc. can be used.
無機イオンとしては、 リン酸カリウム、 硫酸マグネシウム、 鉄イオン、 マンガ ンイオン等が少量添加される。 有機微量栄養素としては、 ビタミン B 1などの要 求物質または酵母エキス等を必要に応じ適量含有させることが望ましい。  As inorganic ions, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions and the like are added. As organic trace nutrients, it is desirable to include a required substance such as vitamin B1 or a yeast extract in an appropriate amount as necessary.
培養は、 振とう培養、 通気撹拌培養等による好気的条件下で 1 6〜 7 2時間実 施するのがよく、 培養温度は 3 0 ° (：〜 4 5 °Cに、 培養中 p Hは 5〜 9に制御する。 尚、 p H調整には無機あるいは有機の酸性あるいはアルカリ性物質、 更にアンモ ニァガス等を使用することができる。  The culture is preferably carried out for 16 to 72 hours under aerobic conditions, such as shaking culture and aeration / agitation culture, at a culture temperature of 30 ° (up to 45 ° C, pH during culture). Is controlled to 5 to 9. For pH adjustment, an inorganic or organic acidic or alkaline substance, ammonia gas or the like can be used.
発酵液からの L—ァミノ酸の採取は、 通常の L—ァミノ酸の製造法と同様にし て行うことができる。 例えば、 L—リジンの採取は、 通常イオン交換樹脂法、 沈 澱法その他の公知の方法を組み合わせることにより実施できる。 また、 L—グル 夕ミン酸を採取する方法も常法によって行えばよく、 例えばイオン交換樹脂法、 晶析法等によることができる。 具体的には、 L—グルタミン酸を陰イオン交換樹 脂により吸着、 分離させるか、 または中和晶析させればよい。 L一リジン及び L —グル夕ミン酸の両方を製造する場合、 これらを混合物として用いる場合には、 これらのアミノ酸を相互に分離することは不要である。 実施例 以下、 本発明を実施例によりさらに具体的に説明する。 L-amino acid can be collected from the fermentation liquor in the same manner as in the normal L-amino acid production method. Can be done. For example, L-lysine can be collected usually by a combination of an ion exchange resin method, a precipitation method and other known methods. The method of collecting L-glucamic acid may be a conventional method, for example, an ion exchange resin method, a crystallization method, or the like. Specifically, L-glutamic acid may be adsorbed and separated by an anion exchange resin, or may be neutralized and crystallized. When producing both L-lysine and L-glucamic acid, when these are used as a mixture, it is not necessary to separate these amino acids from each other. Examples Hereinafter, the present invention will be described more specifically with reference to Examples.
く 1〉ェシエリヒア ' コリ JM109株の eno遺伝子のクローニング <1> Cloning of the eno gene of Escherichia coli JM109
ェシエリヒア · コリの eno遺伝子の塩基配列は既に明らかにされている （Kl ein, M. et al .， DNA Seq. 6 , 315-355 ( 1996年）， Genbank/EMBL/DDBJ accetion No . X82400) 。 報告されている塩基配列に基づいて配列表配列番号 1及び 2に示すブ ライマ一を合成し、 ェシエリヒア · コリ JM109株の染色体 D N Aを铸型にして P C R法によりピルビン酸デヒドロゲナーゼ遺伝子を増幅した。  The nucleotide sequence of the Escherichia coli eno gene has already been determined (Klein, M. et al., DNA Seq. 6, 315-355 (1996), Genbank / EMBL / DDBJ accetion No. X82400). Based on the reported nucleotide sequence, the primers shown in SEQ ID NOs: 1 and 2 in the Sequence Listing were synthesized, and the pyruvate dehydrogenase gene was amplified by the PCR method using the chromosome DNA of Escherichia coli JM109 strain as type III.
合成したプライマーの内、 配列番号 1は、 Genbank/EMBL/DDBJ acceti on No . X 82400に記載されている eno遺伝子の塩基配列の 1番目から 2 4番目の塩基に至る 配列に相当し、 配列番号 2は、 2 0 8 9番目から 2 0 6 6番目の塩基に至る配列 に相当する。  Among the synthesized primers, SEQ ID NO: 1 corresponds to the sequence from the first to the 24th base of the base sequence of the eno gene described in Genbank / EMBL / DDBJ accetion No. X 82400, and SEQ ID NO: 1 2 corresponds to the sequence from the 289th base to the 266th base.
ェシヱリヒア · コリ JM109株の染色体 D N Aの調製は常法によった （生物工学実 験書、 日本生物工学会編、 9 7〜9 8頁、 培風館、 1 9 9 2年） 。 また、 P C R 反応は、 P C R法最前線 （関谷剛男ほか編、 共立出版社、 1 9 8 9年） 1 8 5頁 に記載されている標準反応条件を用いた。  The chromosome DNA of Escherichia coli JM109 strain was prepared by a conventional method (Biotechnological Experiments, edited by The Society of Biotechnology, Japan, pages 97-98, Baifukan, 1992). For the PCR reaction, standard reaction conditions described on page 185 of the PCR method forefront (edited by Takeo Sekiya et al., Kyoritsu Shuppan, 1989) are used.
生成した P C R産物を常法により精製後、 Smalで切断したプラスミ ド p H C 4 と、 ライゲーシヨンキット （宝酒造社製） を用いて連結した後、 ェシエリヒア ' コリ JM109のコンビテン トセル （宝酒造社製） を用いて形質転換を行い、 クロラム フエ二コール 3 0〃g/mlを含む L培地 （パク ト ト リ ブトン 10g/L、 パクトイ一ス ト エキス トラク ト 5g/L、 NaCl 5g/L、 寒天 15g/L、 pH7.2) に塗布し、 一晩培養後、 出現した白色のコロニーを釣り上げ、 単コロニー分離し、 形質転換株を得た。 取 得した形質転換体よりプラスミ ドを抽出し、 ベクタ一に eno遺伝子が結合したブ ラスミ ド p H C 4 e n oを得た。 The resulting PCR product was purified by a conventional method, ligated with Smal-cleaved plasmid pHC4 using a ligation kit (Takara Shuzo), and then combined with Escherichia coli JM109 (Takara Shuzo). Transformation was performed using L-culture medium containing 30 g / ml of chloram fenicol (10 g / L of Pak Tributone, The extract was applied to 5 g / L of extract, 5 g / L of NaCl, 15 g / L of agar, pH 7.2), cultured overnight, and the white colonies that appeared were picked and separated into single colonies to obtain a transformant. Plasmid was extracted from the obtained transformant to obtain a plasmid pHC4eno in which the eno gene was linked to a vector.
pHC 4を保持するェシエリヒア · コリは、 プライベートナンバー AJ12617と命 名され、 1 99 1年 4月 24日に、 通商産業省工業技術院生命工学工業技術研究 所 （郵便番号 305- 8566 日本国茨城県つくば巿東一丁目 1番 3号） に受託番号 FE RM P— 1 22 1 5として寄託され、 1 99 1年 8月 26曰に、 ブタペス ト条 約に基く国際寄託に移管され、 受託番号 FERM BP— 3 5 3 2が付与されて いる。  Escherichia coli, which holds pHC 4, was named private number AJ12617, and on April 24, 1999, the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry (zip code 305-8566, Ibaraki, Japan) Deposit No. FE RM P—122 1 15 at Tsukuba 1-3-1, Higashi-chome) and transferred to an international deposit based on the Budapest Convention on August 26, 1999, accession no. BP— 3 5 3 2 is awarded.
次に、 クローニングされた D N A断片がエノラーゼ活性を有するタンパク質を コードしていることを確認するため、 JM 1 09株及び、 pHC 4 e noを保持 する JM 1 09株のエノラーゼ活性を Bucher, T., Meth. Enzymol . 1， 427-435 ( 1955)に記載の方法により測定した。 その結果、 pHC 4 e n oを保持する JM 1 09株は、 p H C 4 e n 0を保持しない J M 1 0 9株の約 1 5倍のエノラーゼ 活性を示すことから、 eno遺伝子が発現していることを確認した。  Next, in order to confirm that the cloned DNA fragment encodes a protein having enolase activity, the enolase activity of the JM109 strain and the JM109 strain retaining pHC4eno was determined by the method of Bucher, T. et al. , Meth. Enzymol. 1, 427-435 (1955). As a result, the JM109 strain carrying pHC4eno exhibited about 15 times the enolase activity of the JM109 strain not carrying pHC4en0, indicating that the eno gene was expressed. confirmed.
< 2 >コリネ型細菌の L一グル夕ミン酸生産株への p H C 4 e n oの導入と L一 グルタ ミ ン酸生産 <2> Introduction of pH4eno into coryneform bacteria producing L-glucaminic acid and production of L-glutamate
ブレビパクテリゥム · ラク トフアーメンタム AJ13029を電気パルス法 （特開平 2 -207791号公報参照） によりプラスミ ド pHC4enoで形質転換し、 得られた形質転換 株を得た。 得られた形質転換株 AJ13029/pHC4enoを用いて L一グル夕ミン酸生産の ための培養を以下のように行った。 5〃g/mlのクロラムフエ二コールを含む CM 2 Bプレート培地にて培養して得た AJ13029/pHC4eno株の菌体を、 5〃 g/mlのクロラ ムフエ二コールを含む下記組成を有する L—グル夕 ミン酸生産培地に接種し、 31. 5てにて振とう培養し、 培地中の糖が消費されるまで振とう培養した。 得られた培 養物を、 同じ組成の培地に 5 %量接種し、 37°Cにて培地中の糖が消費されるまで 振とう培養した。 コン トロールとしてコリネパクテリゥム属細菌 AJ13029株に、 既 に取得されているコリネパクテリゥム属細菌で自律複製可能なプラスミ ド pHC 4を電気パルス法により形質転換した菌株を上記と同様にして培養した。 〔L一グル夕ミン酸生産培地〕 Brevibacterium lactofermentum AJ13029 was transformed with the plasmid pHC4eno by the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791) to obtain the resulting transformant. Using the obtained transformant AJ13029 / pHC4eno, cultivation for producing L-glucamic acid was performed as follows. The cells of the AJ13029 / pHC4eno strain obtained by culturing on a CM2B plate medium containing 5 μg / ml chloramphenicol were added to L-cells having the following composition containing 5 μg / ml chloramphenicol. The medium was inoculated into a glucanic acid-producing medium, shake-cultured at 31.5, and shaken until the sugar in the medium was consumed. The obtained culture was inoculated into a medium having the same composition in an amount of 5%, and cultured at 37 ° C with shaking until the sugar in the medium was consumed. A strain obtained by transforming a plasmid pHC4, which can be autonomously replicated with a previously obtained corynebacterium bacterium, into the corynebacterium bacterium AJ13029 as a control by the electric pulse method and culturing in the same manner as described above did. [L-glucamic acid production medium]
下記成分 （ 1 L中） を溶解し、 K0Hで pH8.0に調製し、 115°Cで 15分殺菌する。 グルコース 150g  Dissolve the following components (in 1 L), adjust to pH 8.0 with K0H, and sterilize at 115 ° C for 15 minutes. Glucose 150g
Figure imgf000015_0001
Figure imgf000015_0001
大豆蛋白加水分解液 50ml  Soy protein hydrolyzate 50ml
ピオチン 2mg  Piotin 2mg
サイアミン塩酸塩 3mg  Siamin hydrochloride 3mg
培養終了後、 培養液中の L—グル夕ミン酸蓄積量を旭化成工業社製バイオテツ クアナライザ一 A S— 2 1 0により測定した。 このときの結果を表 1に示した。 表 1 菌 株 L一グルタミン酸生成量（g/L )  After the completion of the culture, the amount of accumulated L-glucamic acid in the culture solution was measured using a Biotech Analyzer AS210 manufactured by Asahi Kasei Corporation. Table 1 shows the results. Table 1. Strain L-glutamic acid production (g / L)
AJ13029/pHC4 2 0 . 9 AJ13029 / pHC4 20.9
AJ13029/pHC4eno 2 3 . 4  AJ13029 / pHC4eno 23.4
< 3 >コリネ型細菌の L一リジン生産株への pHC4enoの導入と L一リジン生産 ブレビパクテリゥム . ラクトフアーメンタム AJ11082を電気パルス法 （特開平 2 -207791号公報参照） によりプラスミ ド pHC4enoで形質転換し、 得られた形質転換 株を得た。 得られた形質転換株 AJ11082/pHC4enoを用いて Lーリジン生産のための 培養を以下のように行った。 5〃 /1111のクロラムフェニコ一ルを含む0 ^1 2 Bブレ ート培地にて培養して得た AJ11082/pHC4eno株の菌体を、 5 g/mlのクロラムフエ 二コールを含む下記組成の L—リジン生産培地に接種し、 31. 5°Cにて培地中の糖 が消費されるまで振とう培養した。 コントロールとしてコリネパクテリゥム属細 菌 AJ11082株に、 既に取得されているコリネバクテリゥム属細菌で自律複製可能な プラスミ ド pHC4を電気パルス法により形質転換した菌株を上記と同様にして培養 した。 <3> Introduction of pHC4eno into L-lysine-producing strain of coryneform bacterium and production of L-lysine Brevipacterium. Lactofamentum AJ11082 was converted to plasmid pHC4eno by the electric pulse method (see JP-A-2-207791). After transformation, the resulting transformant was obtained. Using the obtained transformant AJ11082 / pHC4eno, culture for L-lysine production was performed as follows. The cells of the AJ11082 / pHC4eno strain obtained by culturing in a 0 ^ 12B plate medium containing 5〃 / 1111 chloramphenicol were added to L-cells of the following composition containing 5 g / ml of chloramphenicol. A lysine production medium was inoculated and cultured with shaking at 31.5 ° C until the sugar in the medium was consumed. Corynepterium as control A strain obtained by transforming the strain AJ11082 with a plasmid pHC4 capable of autonomously replicating by a corynebacterium already obtained by the electric pulse method was cultured in the same manner as described above.
ブレビパクテリゥム ' ラク トファ一メンタム AJ11082は、 1981年 1月 31日に農学 研究菌培養収集所 (Agricultural Research Culture Collectionヽ ァメリカ合衆 国 イ リノイ州 6 1604ピオリア ノースュニバーシティ通り 18 1 5 (1815 N. University Street, Peoria, Illinois 61604 U.S.A.)) に国際寄託され、 受 託番号 NRRL B- 11470が付与されている。  Brevi Pacterium 'Lactafamentum AJ11082 was established on January 31, 1981 at the Agricultural Research Culture Collection, America, Illinois, 6 1604 Peoria Northuniversity Street 18 1 5 (1815 N. University Street, Peoria, Illinois 61604 USA) and deposited under accession number NRRL B-11470.
〔Lーリジン生産培地〕  (L-lysine production medium)
炭酸カルシウム以外の下記成分 （ 1 L中） を溶解し、 K0Hで pH8.0に調製し、 11 5°Cで 15分殺菌した後、 別に乾熱殺菌した炭酸カルシウムを 50 g加える。  Dissolve the following ingredients (in 1 L) other than calcium carbonate, adjust to pH 8.0 with K0H, sterilize at 115 ° C for 15 minutes, and add 50 g of dry-heat-killed calcium carbonate.
グルコース 100 g 100 g glucose
Figure imgf000016_0001
Figure imgf000016_0001
KH2P 04 1 SKH 2 P 0 4 1 S
Figure imgf000016_0002
Figure imgf000016_0002
ピオチン 500 も  Piotin 500 too
チアミン 2000 g
Figure imgf000016_0003
Thiamine 2000 g
Figure imgf000016_0003
ニコチンアミ ド 5 mg  Nicotinamide 5 mg
蛋白質加水分解物 （豆濃） 30 ml  Protein hydrolyzate (bean concentrate) 30 ml
炭酸カルシウム 50 g  50 g calcium carbonate
培養終了後、 培養液中の L一リジン蓄積量を旭化成工業社製バイォテックアナ ライザ一 AS— 2 10により測定した。 このときの結果を表 2に示した。 表 2 菌 株 Lーリジン生成量（g/L) After the completion of the culture, the amount of L-lysine accumulated in the culture solution was measured with a Biotech Analyzer AS-210 manufactured by Asahi Kasei Corporation. Table 2 shows the results. Table 2 Strain L-Lysine production (g / L)
AJ11082/pHC4 2 9 . 5 AJ11082 / pHC4 29.5
AJ11082/pHC4eno 3 2 . 4  AJ11082 / pHC4eno 32.4
く 4 >コリネ型細菌の L一リジン及び L—グル夕ミン酸生産株への pHC4enoの導入 と Lーリジン及び L—グルタ ミン酸同時生産 Introduction of pHC4eno into L-lysine and L-glutamic acid producing strains of coryneform bacteria and simultaneous production of L-lysine and L-glutamic acid
ブレビパクテリゥム · ラク トフアーメンタム AJ12993を電気パルス法 （特開平 2 -207791号公報参照） によりプラスミ ド pHC4enoで形質転換し、 得られた形質転換 株を得た。 得られた形質転換株 AJ12993/pHC4enoを用いて Lーリジン及び L—グル 夕ミン酸生産のための培養を以下のように行った。 5〃g/mlのクロラムフエニコ一 ルを含む C M 2 Bブレート培地にて培養して得た AJ12993/pHC4eno株の菌体を、 5 / g/mlのクロラムフエ二コールを含む前記 L—リジン生産培地に接種して 31 . 5°C にて培養した。 培養を開始してから 1 2時間後に培養温度を 3 4 °Cにシフ ト し、 培地中の糖が消費されるまで振とう培養した。 コン トロールとしてコリネバクテ リゥム属細菌 AJ12993株に、 既に取得されているコリネバクテリゥム属細菌で自律 複製可能なプラスミ ド pHC4を電気パルス法により形質転換した菌株を上記と同様 にして培養した。  Brevipacterium lactofermentum AJ12993 was transformed with plasmid pHC4eno by the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791) to obtain the obtained transformant. Using the obtained transformant AJ12993 / pHC4eno, culture for producing L-lysine and L-glucamic acid was performed as follows. The cells of the AJ12993 / pHC4eno strain obtained by culturing in a CM2B plate medium containing 5 μg / ml chloramphenicol were added to the L-lysine production medium containing 5 / g / ml chloramphenicol. The cells were inoculated and cultured at 31.5 ° C. After 12 hours from the start of the culture, the culture temperature was shifted to 34 ° C, and the culture was performed with shaking until the sugar in the medium was consumed. As a control, a strain obtained by transforming a plasmid pHC4 capable of autonomously replicating with a previously obtained corynebacterium bacterium into an AJ12993 bacterium of the genus Corynebacterium by an electric pulse method was cultured in the same manner as described above.
培養終了後、 培養液中の L—リジン及び L一グルタミン酸蓄積量を旭化成工業 社製バイオテックアナライザ一 A S— 2 1 0により測定した。 このときの結果を 表 3に した。 表 3 菌 株 L—リジン生成量（g/L) L—グルタミン酸生成量（g/L) After completion of the culture, the accumulated amounts of L-lysine and L-glutamic acid in the culture solution were measured using a Biotech Analyzer AS-210 manufactured by Asahi Kasei Corporation. Table 3 shows the results. Table 3 Strain L-Lysine production (g / L) L-Glutamic acid production ( g / L)
AJ12993/pHC4 10. 2 1 9. 1 AJ12993 / pHC4 10.2 19.1
AJ12993/pHC4eno 12. 4 2 1. 5  AJ12993 / pHC4eno 12.4 21.5
産業上の利用可能性 本発明により、 コリネ型細菌の L—リジン又は L一グル夕ミン酸等の Lーァ ノ酸の生産能を向上させることができる。 INDUSTRIAL APPLICABILITY According to the present invention, the ability of coryneform bacteria to produce L-anoic acid such as L-lysine or L-monoglutamic acid can be improved.

Claims

請求の範囲 The scope of the claims
1 . 細胞中のエノラーゼ活性が増強され、 かつ L—アミノ酸生産能を有するコ リネ型細菌。 1. A coryneform bacterium with enhanced enolase activity in cells and L-amino acid producing ability.
2 . 前記 L—アミノ酸が、 L—リジン、 L—グルタミン酸、 Lースレオニン、 L—イソロイシン、 Lーセリンから選ばれる請求項 1記載のコリネ型細菌。 2. The coryneform bacterium according to claim 1, wherein the L-amino acid is selected from L-lysine, L-glutamic acid, L-threonine, L-isoleucine, and L-serine.
3 . 前記エノラーゼ活性の増強が、 前記細菌細胞内のエノラーゼをコードする 遺伝子のコピー数を高めることによるものである請求項 1記載のコリネ型細菌。 3. The coryneform bacterium according to claim 1, wherein the enolase activity is enhanced by increasing the copy number of a gene encoding enolase in the bacterial cell.
4 . 前記エノラーゼをコ一ドする遺伝子がェシエリヒア属細菌由来である請求 項 3記載のコリネ型細菌。 4. The coryneform bacterium according to claim 3, wherein the gene encoding the enolase is derived from a bacterium belonging to the genus Escherichia.
5 . 請求項 1〜4のいずれか一項に記載のコリネ型細菌を培地に培養し、 該培 養物中に L一アミノ酸を生成蓄積せしめ、 該培養物から L—アミノ酸を採取する ことを特徴とする L一アミノ酸の製造法。 5. culturing the coryneform bacterium according to any one of claims 1 to 4 in a medium, producing and accumulating L-amino acid in the culture, and collecting L-amino acid from the culture. Characteristic method for producing L-amino acids.
6 . 前記 L—アミノ酸が、 L一リジン、 L—グルタミン酸、 Lースレオニン、 L一イソロイシン、 Lーセリンから選ばれる請求項 5記載の方法。 6. The method according to claim 5, wherein the L-amino acid is selected from L-lysine, L-glutamic acid, L-threonine, L-isoleucine, and L-serine.
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