WO2001002544A1 - Process for producing l-amino acid - Google Patents

Process for producing l-amino acid Download PDF

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Publication number
WO2001002544A1
WO2001002544A1 PCT/JP2000/004344 JP0004344W WO0102544A1 WO 2001002544 A1 WO2001002544 A1 WO 2001002544A1 JP 0004344 W JP0004344 W JP 0004344W WO 0102544 A1 WO0102544 A1 WO 0102544A1
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WIPO (PCT)
Prior art keywords
lysine
amino acid
producing
succinate dehydrogenase
acid
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PCT/JP2000/004344
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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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Publication date
Application filed by Ajinomoto Co., Inc. filed Critical Ajinomoto Co., Inc.
Priority to AU57072/00A priority Critical patent/AU5707200A/en
Publication of WO2001002544A1 publication Critical patent/WO2001002544A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/99—Oxidoreductases acting on the CH-CH group of donors (1.3) with other acceptors (1.3.99)
    • C12Y103/99001—Succinate dehydrogenase (1.3.99.1)
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • 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
    • 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

Definitions

  • the present invention relates to a method for producing an L-amino acid by a fermentation method, and more particularly to a method for producing L-lysine and L-glutamic acid.
  • L-lysine is widely used as a feed additive
  • L-glutamic acid is widely used as a seasoning material.
  • L-amino acids such as L-lysine and L-glutamic acid have been obtained by 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 fermentation. For these coryneform bacteria, strains isolated from the natural world or artificial mutants of the strains are used in order to improve productivity.
  • diaminobi Successive amplification or introduction of the mehydrogenate dehydrogenase gene (ddh) (or the tetrahydrodipicolinic acid succinylase gene (dapD) and the succinyldiaminobimelate deacylase gene (dapE)) may improve L-lysine production.
  • ddh mehydrogenate dehydrogenase gene
  • dapD tetrahydrodipicolinic acid succinylase gene
  • dapE succinyldiaminobimelate deacylase gene
  • JP-A-63-214189 discloses that a glutamate dehydrogenase gene, an isocitrate dehydrogenase gene, an aconitate hydrase gene, and a quinate synthase gene are amplified or introduced. Discloses a technique for increasing the ability to produce L-glutamic acid.
  • An object of the present invention is to provide a method for producing an L-amino acid such as L-lysine or L-glucaminic acid by a fermentation method which has been further improved than before, and a strain used therefor.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, introduced a gene encoding succinate dehydrogenase into a coryneform bacterium, thereby enhancing succinate dehydrogenase activity, thereby improving L-lysine or L-lysine.
  • the inventors have found that the production of glutamic acid can be increased, and have completed the present invention. That is, the present invention is as follows.
  • a coryneform bacterium having enhanced succinate dehydrogenase activity in cells and having L-amino acid producing ability (1) A coryneform bacterium having enhanced succinate dehydrogenase activity in cells and having L-amino acid producing ability.
  • 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. Method for producing L-amino acid.
  • the coryneform bacterium of the present invention is a coryneform bacterium having an ability to produce L-amino acid and having enhanced succinate dehydrogenase 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.
  • embodiments of the present invention will be described mainly with respect to a coryneform bacterium having L-lysine-producing ability or L-glutamate-producing ability.
  • the present invention provides a biosynthetic system specific to an L-amino acid of interest. The same applies to those that are located downstream of the drogenase.
  • the coryneform bacterium referred to in the present invention is a barges. Manual. Tibno, a group of microorganisms defined in Bergey's Manual of Determinative Bacteriology, 8th edition, p. 599 (1974), aerobic, gram-positive, nonacidic, and capable of sporulation. Bacteria that have not been classified into the genus Brevipacterium, but now include bacteria that have been integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1981)). Also, it includes bacteria of the genus Brevipacterium and Microbaterim, 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 samminoaminogenes AJ12340 (FERM BP-1539) To obtain them, for example, American Type Culture Collection, American Type Culture Collection, Address 12301 Park lawn 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 'Cultural Collection.
  • the AJ12340 strain has been deposited with the Ministry of International Trade and Industry at the National Institute of Bioscience and Human-Technology (Postal Code 305-856-6 1-3 1-3 Tsukuba-Higashi, Ibaraki, Japan) based on the Budapest Treaty.
  • mutants having L-amino acid-producing ability such as L-lysine or L-glutamic acid derived from these strains can also be used in the present invention.
  • Such artificial mutants include the following. S— (2-aminoethyl) -cystine (hereinafter abbreviated as “AEC”) resistant mutant (eg, 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 (2-aminoethyl) -cystine
  • coryneform bacteria having L-threonine-producing ability include: (See U.S. Pat. No. 5,188,949) is one example of a coryneform bacterium having the ability to produce L-isoleucine. Lium Flavam AJ12149 (FERM BP-759) (see US Pat. No. 4,656,135).
  • the ability to produce L-amino acid 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.
  • a recombinant DNA is obtained by ligating a gene fragment encoding succinate dehydrogenase to a vector that functions in the bacterium, preferably a multicopy vector. May be prepared and introduced into a coryneform bacterium having the ability to produce L-lysine or L-glucaminic acid, followed by transformation.
  • succinate dehydrogenase activity is enhanced.
  • Succinate dehydrogenase is encoded by the sdh gene in Escherichia coli.
  • succinate dehydrogenase 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 sdh gene of Escherichia coli has already been determined (Genbank / EMBL / DDBJ accetion No. J01619 K00542 MU121)
  • primers prepared based on the nucleotide sequence for example, SEQ ID NO: 1 and Using the primers shown in 2 above, the PCR method (PCR: polymerase chain reaction; White, TJ et al; Trends Genet. 5, 185 (1989)) using the Escherichia coli chromosome DNA as type I was carried out.
  • the sdh gene can be obtained.
  • the gene encoding succinate dehydrogenase amplified by the PCR method is connected to a vector DNA capable of autonomous replication in cells of Escherichia coli and / or coryneform bacteria to prepare a recombinant DNA, which is then prepared. If introduced into Escherichia coli cells, subsequent operations will be slower. Plasmid vectors are preferred as vectors capable of autonomous replication in Escherichia coli cells, and those capable of autonomous replication in host cells are preferred.For example, pUC19, pUC18, pBR322, pHSG299, pHSG399, pHSG398, RSF1010 And the like.
  • Examples of vectors capable of autonomous replication in coryneform bacterium cells include PAM330 (see JP-A-58-67699), pHM1519 (see JP-A-58-77895), and the like.
  • a DNA fragment having the ability to enable autonomous replication of a plasmid in a coryneform bacterium is extracted from these vectors and inserted into the Escherichia coli vector, which results in autonomous expression in both the Escherichia coli and the coryneform bacteria. It can be used as a replicable shuttle vector.
  • the following are examples of such shuttle vectors. Microorganisms carrying the respective vectors and the accession numbers of the international depository organizations are shown in parentheses.
  • PAJ611 Escherichia Cori AJ11884 (FERM BP-138)
  • the vector To prepare recombinant DNA by ligating a gene encoding succinate dehydrogenase and a vector that functions in coryneform bacteria, the vector must be digested with a restriction enzyme that matches the end of the gene encoding succinate dehydrogenase. Disconnect. The ligation is usually performed using a ligase such as T4 DNA ligase.
  • Escherichia A method for increasing the permeability of DNA by treating recipient cells with calcium chloride, as reported for li-112 (Mandel, M. and Higa, A., J. Mol. Biol., 53 , 159 (1970)), and a method for preparing a recombinant cell from a cell at the growth stage and introducing DNA as described in Bacillus' subtilis (Duncan, CH, Wilson, GA and Young, FE, Gene, 1, 153 (1977)).
  • recombinant DNA can be obtained by transforming cells of a DNA-accepting bacterium into protoblasts or spherovlasts that readily incorporate the recombinant DNA, as is known for Bacillus subtilis, actinomycetes and yeast. Methods for introduction into DNA recipients (Chang, S. and Choen, SN, Molec. Gen. Genet., 168, 111 (1979); Bibb, MJ, Ward, JMand Hopwood, 0.A., Nature, 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).
  • Enhancement of the activity encoding succinate dehydrogenase can also be achieved by causing multiple copies of a gene encoding succinate dehydrogenase to be present on the chromosomal DNA of the host.
  • homologous recombination is performed using a sequence present on the chromosomal DNA in a multiple copy as a target. Sequences present in multiple copies on the chromosomal DNA include repetitive DNA and invert 'repeats at the ends of transposable elements.
  • a gene encoding succinate dehydrogenase can be mounted on a transposon and transferred, and multiple copies can be introduced into chromosomal DNA, as disclosed in JP-A-2-19985. is there. Either method increases the copy number of the gene encoding succinate dehydrogenase in the transformant, resulting in enhanced succinate dehydrogenase activity.
  • Enhancement of succinate dehydrogenase activity can be achieved not only by the above-described gene amplification but also by replacing an expression regulatory sequence such as a promoter of a gene encoding succinate dehydrogenase on chromosomal DNA or plasmid with a strong one.
  • an expression regulatory sequence such as a promoter of a gene encoding succinate dehydrogenase on chromosomal DNA or plasmid with a strong one.
  • lac promotion Yuichi, trp promoter, trc promoter, tac bromo, PR promoter and PL promoter of lambda phage are known as strong promoters. Substitution with these promoters enhances the expression of the gene encoding succinate dehydrogenase, thereby enhancing succinate dehydrogenase activity.
  • the coryneform bacterium of the present invention can enhance the enzymatic genes of other amino acid biosynthetic pathways or glycolytic pathways in addition to the succinate dehydrogenase activity, thereby enhancing the enzymatic activity.
  • genes that can be used for the production of L-lysine include aspartase kinase subunit protein and / or protein, in which synergistic feedback inhibition by L-lysine and L-threonine has been substantially released.
  • genes that can be used for the production of L-glutamic acid include glutamate dehydrogenase (GDH, Japanese Patent Application Laid-Open No. 61-268185), glutamate synthetase, glutamate synthetase, and isoquene.
  • Acid dehydrogenase JP-A-62-166890, JP-A-63-214189), aconitate hydrazine (JP-A-62-294086), quenic acid Synthase, pyruvate carboxylase (JP-A-60-87788, JP-A-62-55089), phosphoenolpyruvate carboxylase, phosphoenolpyruvate synthase, enolase, phosphoglyceromase, Phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, triosephosphate isomerase, fructosebisphosphate aldolase, phosphofruc Kinase (JP 63 - 1 02 692), there is a glucose phosphate Isomera Ichize like.
  • an enzyme that catalyzes a reaction that diverges from a target L-amino acid biosynthetic pathway to produce a compound other than the 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 (W09). 5/23864).
  • Enzymes that catalyze the reaction that diverges from the L-glutamic acid biosynthetic pathway to produce a compound other than L-glutamic acid include: ketoglutarate dehydrogenase, isoquenate lyase, acetyl phosphate transferase, and the like. There are acetate kinase, acetate hydroxysynthase, acetate lactate synthase, acetyl formate transferase, lactate dehydrogenase, glutamate decarboxylase, and monopyrophosphate dehydrogenase.
  • a temperature-sensitive mutation to a biotin-inhibitory substance such as a surfactant to a coryneform bacterium capable of producing L-glutamic acid
  • a biotin-inhibitory substance such as a surfactant
  • the biotin-inducing action in a medium containing an excessive amount of biotin can be achieved.
  • L-glutamic acid can be produced in the absence of inhibitors (see W096 / 06180).
  • Examples of such coryneform bacteria include Brevibacterium lactofermentum AJ13029 described in W096 / 06180.
  • the AJ13029 strain was registered on September 2, 1994, with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (Postal Code 305-8566, 1-3-1 Tsukuba East, Ibaraki Prefecture, Japan) under the accession number FERM P-14501. Deposited and transferred to an international deposit under the Budapest Treaty on August 1, 1995, and given accession number FERM BP-5189.
  • a temperature-sensitive mutation to a substance inhibiting the action of biotin to a coryneform bacterium capable of producing L-lysine and L-glutamic acid, the action of biotin in a medium containing an excessive amount of biotin is suppressed.
  • L-lysine and L-glutamic acid can be produced simultaneously in the absence of a substance (see W096 / 06180).
  • Such strains include Brevipacterium 'Lactofamentum AJ12993 strain described in W096 / 06180.
  • the stock was granted to the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (Postal Code 305-8566, 1-3-1, Higashi 1-3-chome, Tsukuba, Ibaraki, Japan) on June 3, 1994, with accession number FERM P-14348. And transferred to an 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. Also, “reduced activity” of an enzyme usually means that the enzyme activity in a cell is lower than that of a wild-type strain. However, when a strain whose enzyme activity is reduced by modification by genetic recombination technology or the like is obtained, it means that the enzyme activity in the cell is lower than that of the strain before modification.
  • the L-amino acid When a coryneform bacterium having enhanced succinate dehydrogenase activity and capable of producing L-amino acid is cultured in a suitable medium, the L-amino acid accumulates in the medium. For example, when a coryneform bacterium having enhanced succinate dehydrogenase activity and capable of producing L-lysine acid is cultured in a suitable medium, L-lysine accumulates in the medium. Further, when a coryneform bacterium having enhanced succinate dehydrogenase activity and capable of producing L-gluminic acid is cultured in a suitable medium, L-gluminic acid accumulates in the medium.
  • L-lysine and L-glucaminic acid accumulate in the medium.
  • the L-gine-producing bacterium may be cultured under L-glucamic acid production conditions, or L-lysine-producing ability
  • a coryneform bacterium having L-glutamic acid-producing ability may be mixedly cultured (JP-A-5-37993).
  • the medium used to produce L-amino acids such as L-lysine or L-glucamic acid using the microorganism of the present invention contains a carbon source, a nitrogen source, inorganic ions, and if necessary, other organic micronutrients. This is a normal medium.
  • Carbon sources include glucose, lactose, galactose, fructose, sucrose, molasses, carbohydrates such as starch hydrolysates, alcohols such as ethanol and inositol, acetic acid, fumaric acid, citric acid, Organic acids such as succinic acid can be used.
  • nitrogen source examples include inorganic ammonium salts such as ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, and ammonium acetate, ammonia, peptone, meat extract, yeast extract, yeast extract, corn steep liquor, soybean hydrolyzate, etc.
  • organic nitrogen, ammonia gas, ammonia water, and the like can be used.
  • Potassium phosphate, magnesium sulfate, iron ion, manga A small amount of ion or the like is 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 cultivation is preferably carried out for 16 to 72 hours under aerobic conditions such as shaking cultivation and aeration / agitation cultivation.
  • aerobic conditions such as shaking cultivation and aeration / agitation cultivation.
  • pH adjustment an inorganic or organic acidic or alkaline substance, ammonia gas or the like can be used.
  • the L-amino acid can be collected from the fermentation liquor in the same manner as in the usual method for producing an L-amino acid.
  • collection of L-lysine can be usually carried out by combining an ion exchange resin method, a precipitation method and other known methods.
  • the method for collecting L-glutamic acid may be a conventional method, such as an ion exchange resin method or a crystallization method.
  • L-glutamic acid may be adsorbed and separated by an anion exchange resin, or may be neutralized and crystallized.
  • the nucleotide sequence of the sdh gene of Escherichia coli has already been elucidated (Genbank / EMBL / DDBJ accetion No. J01619 K00542 M11121). Primers shown in SEQ ID NOS: 1 and 2 in the Sequence Listing were synthesized based on the reported nucleotide sequence, 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 extends from nucleotide 199 to nucleotide 215 of the nucleotide sequence of the sdh gene described in Genbank / EMBL / DDBJ accetion No. J 01619 K00542 M11121.
  • SEQ ID NO: 2 corresponds to the sequence from the 6309th to the 286th base.
  • the resulting PCR product is purified by a conventional method, and then ligated with Smal-cleaved plasmid pHC4 using a ligation kit (Takara Shuzo Co., Ltd.). Then, Escherichia coli KM JM109 competent cells (Takara Shuzo) Transformation is performed using L medium containing 30 g / ml of chloramphenicol (10 g / L of pact tributone, 5 g / L of pak toy extract, 5 g / L of NaCl, and 15 g / L of agar). , PH 7.2), and after culture, the white colonies that appeared were picked and separated into single colonies to obtain transformed strains. Plasmid was extracted from the obtained transformant to obtain a plasmid pHC4sdh in which the sdh gene was bound to the vector.
  • Escherichia coli harboring pHC4 was named private number AJ12617, and on April 24, 1991, the Ministry of International Trade and Industry, National Institute of Industrial Science and Technology, Institute of Biotechnology and Industrial Technology (Zip code 305-8566 Japan Deposit No. FE RM P—122 15 at Tsukuba 1-3-1 Higashi, Ibaraki Pref., Japan, and transferred to an international deposit based on the Budapest Agreement on August 26, 1999.
  • FERM BP—3532 is granted.
  • the succinate dehydrogenase activity of the JM109 strain and the JM109 strain carrying pHC4sdh was determined.
  • the sdh gene was expressed because the JM109 strain carrying pHC 4 sdh exhibited about 19 times the succinate dehydrogenase activity of the JM109 strain not carrying pHC4 sdh. .
  • Brevipacterium 'lactofermentum AJ13029 was transformed with plasmid pHC4sdh by the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791) to obtain the obtained transformant.
  • Using the resulting transformant AJ13029 / pHC4sdh to produce L-glutamic acid Cultivation was performed as follows.
  • the AJ13029 / pHC4sdh strain obtained by culturing on a CM2B plate medium containing chloramphenicol in L-glutamic acid producing medium containing 5 ⁇ g / ml chloramphenicol and having the following composition Then, the cells were shake-cultured at 31.5 ° C and shake-cultured 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 capable of autonomously replicating with a previously obtained Corynebacterium bacterium into an AJ13029 strain of Corynebacterium bacterium by the electric pulse method was cultured in the same manner as described above. did.
  • the cells of the AJ11082 / pHC4sdh strain obtained by culturing in ⁇ 2B plate medium containing chloramphenicol were inoculated into an L-lysine production medium having the following composition containing 5 / ml chloramphenicol, and Shaking culture was performed at ° C 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 by the electric pulse method was cultured in the corynebacterium bacterium AJ11082 strain in the same manner as described above.
  • Protein hydrolyzate (bean concentrate) 30 ml 50 g calcium carbonate
  • Brevipacterium lactofermentum AJ12993 was transformed with plasmid pHC4sdh 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.
  • AJ12993 / pHC4sdh strain cells obtained by culturing in a CM2B plate medium containing 5 ⁇ g / ml chloramphenicol were inoculated into the L-lysine production medium containing 5 ig / ml chloramphenicol. 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 belonging to the genus Corynebacterium by the electric pulse method was cultured in the same manner as described above.
  • L-anoic acid such as L-lysine or L-glutamic acid

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 succinate dehydrogenase into a coryneform bacterium capable of producing an L-amino acid such as L-lysine or L-glutamic acid and thus enhancing the succinate dehydrogenase 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一アミノ酸の製造法、 特に L一リジン及び L—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—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。 背景技術 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 more particularly to a method for producing L-lysine and L-glutamic acid. L-lysine is widely used as a feed additive, and L-glutamic acid is widely used as a seasoning material. BACKGROUND ART Conventionally, L-amino acids such as L-lysine and L-glutamic acid have been obtained by 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 fermentation. 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一ア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ードバック阻害が解除されたァスバルト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一ゼ遺伝子 （dapB) 、 ジヒ ドロジピコリン酸シンターゼ遺伝子 （dapA) 、 ジアミノピメ リン酸デカルボ キシラ一ゼ遺伝子 （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 9一 322774号） 、 lysC、 lysA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—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 coding for aspartokinase (mutant lysC) whose feedback inhibition by L-lysine and L-threonine has been released, dihydropicolin, Acid reductase gene (dapB), dihydropicolinate synthase gene (dapA), diaminopimephosphate decarboxylase gene (lysA), and diaminopimephosphate dehydrogenase gene (ddh) (W096 / 40934), lysA and ddh (Japanese Patent Laid-Open 9, item 322774), lysC, lysA and host scan Hoe Nord carboxylase gene (pp C) (JP-A-10- 165180), mutant lysC, dapB, dapA, lysA and Asuparagin acid aminotransferase It has been known that the introduction of the Izeze gene (aspC) (Japanese Patent Application Laid-Open No. Hei 10-215883) improves the L-lysine producing ability of the bacterium. There.
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 二ラーゼ遺伝子 （dapD ) 及びスクシニルジアミノビ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 95/16042ではテ トラヒ ドロジピコリン酸スクシ 二ラーゼがスクシ二ルジァミノピメ リン酸トランスアミナーゼと誤記されている。 一方、 コリネパクテリゥム属またはブレビパク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ウム · グル夕 ミクム由来のクェン酸シン夕一 ゼをコードする遺伝子の導入が、 L一グル夕 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夕 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 ドロゲナー ゼ遺伝子、 アコニッ ト酸ヒドラ夕ーゼ遺伝子、 及びクェン酸シン夕一ゼ遺伝子を 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 Successive amplification or introduction of the mehydrogenate dehydrogenase gene (ddh) (or the tetrahydrodipicolinic acid succinylase gene (dapD) and the succinyldiaminobimelate deacylase gene (dapE)) may improve L-lysine production. Known (W0 95/16042). In WO95 / 16042, tetrahydropicolinic acid succinylase is erroneously described as succinyl diaminopimephosphate transaminase. On the other hand, in a bacterium of the genus Corynepacterium or Brevipacterium, introduction of a gene encoding citrate synthase derived from Escherichia coli or Corynebacterium gulp It was reported that it was effective in enhancing acid production capacity (Japanese Patent Publication No. 7-121228). 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 a glutamate dehydrogenase gene, an isocitrate dehydrogenase gene, an aconitate hydrase gene, and a quinate synthase gene are amplified or introduced. Discloses a technique for increasing the ability to produce L-glutamic acid.
しかし、 スクシネートデヒ ドロゲナ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、 スクシネートデヒ ドロゲナーゼをコードする遺伝子 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ーリジン又は L—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 gene encoding succinate dehydrogenase has not been reported in coryneform bacteria, and the use of the gene encoding succinate dehydrogenase 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-glucaminic acid by a fermentation method which has been further improved than before, 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、 スクシネー トデヒ ドロゲナーゼをコードする遺伝子をコリネ型細菌に導入し、 スクシネート デヒ ドロゲナーゼ活性を増強することにより、 Lーリジン又は L—グルタミン酸 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 to solve the above problems, and as a result, introduced a gene encoding succinate dehydrogenase into a coryneform bacterium, thereby enhancing succinate dehydrogenase activity, thereby improving L-lysine or L-lysine. —The inventors have found that the production of glutamic acid can be increased, and have completed the present invention. That is, the present invention is as follows.
( 1 ) 細胞中のスクシネートデヒ ドロゲナーゼ活性が増強され、 かつ L—ァミノ 酸生産能を有するコリネ型細菌。  (1) A coryneform bacterium having enhanced succinate dehydrogenase activity in cells and having L-amino acid producing ability.
( 2 ) 前記 L一アミノ酸が、 L—リジン、 L一グルタミン酸、 Lースレオニン、 L一イソロイシン、 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 ) 前記スクシネートデヒ ドロゲナ一ゼ活性の増強が、 前記細菌細胞内のスク シネートデヒドロゲナーゼをコ一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 enhancement of the succinate dehydrogenase activity is caused by increasing the copy number of a gene encoding succinate dehydrogenase in the bacterial cell.
( 4 ) 前記スクシネートデヒ ドロゲナーゼをコ一ドする遺伝子がェシヱリヒア属 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 succinate dehydrogenase 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一アミノ酸の製造法。  (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. Method for producing L-amino acid.
( 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 （ 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一アミノ酸生産能を有し、 細胞中のスクシネート デヒ ドロゲナ一ゼ活性が増強されたコリネ型細菌である。 L—アミノ酸としては、 L一リジン、 L一グルタ ミン酸、 L—スレオニン、 L—イソロイシン、 L—セリ ン等が挙げられる。 これらの中では、 L—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—リジン生産能又は Lーグルタミ 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 —アミノ酸固有の生合成系がスクシネートデヒ ドロゲナーゼょりも下流に位置す るものについては同様に適用され得る。  The coryneform bacterium of the present invention is a coryneform bacterium having an ability to produce L-amino acid and having enhanced succinate dehydrogenase 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, embodiments of the present invention will be described mainly with respect to a coryneform bacterium having L-lysine-producing ability or L-glutamate-producing ability. However, the present invention provides a biosynthetic system specific to an L-amino acid of interest. The same applies to those that are located downstream of the drogenase.
本発明でいうコリネ型細菌は、 バージーズ . マニュアル . ォブ . デターミネィ ティブ · ノ、'クテリオロジー (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 . Bacteriol . , 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 referred to in the present invention is a barges. Manual. Tibno, a group of microorganisms defined in Bergey's Manual of Determinative Bacteriology, 8th edition, p. 599 (1974), aerobic, gram-positive, nonacidic, and capable of sporulation. Bacteria that have not been classified into the genus Brevipacterium, but now include bacteria that have been integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1981)). Also, it includes bacteria of the genus Brevipacterium and Microbaterim, 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
コリネパクテリゥム · ァセ トグルタミクム ATCC15806  Corynepaterum case Toggle tamukum ATCC15806
コリネバクテリウム · カルナェ ATCC15991  Corynebacterium carnaet ATCC15991
コリネパクテリゥム · グル夕 ミクム ATCC13032  Corynepaterum Guru Yu Mikum ATCC13032
(ブレビパクテリゥム · ディバリカ夕ム） ATCC 020  (Brevipactium / Dibarika evening) ATCC 020
(ブレビパクテリゥム · ラク トフアーメンタム） ATCC13869  (Brevipactium · Lacto Armament) ATCC13869
(コリネバクテリゥム · リ リウム） ATCC15990  (Corynebacterium lilium) 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 メフセコーラ ATCC17965  Corynebacterium mehsekora ATCC17965
ブレビバクテリウム サッカロリティクム ATCC14066  Brevibacterium saccharolyticum 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 チォゲニタリス ATCC19240  Brevipacterium Chogenitalis 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 サ一モアミノゲネス AJ12340(FERM BP-1539 ) これらを入手するには、 例えばァメ リカン · 夕イブ · カルチヤ一 · コレクショ ン (American Type Culture Col lection^ 住所 12301 Park lawn Drive, Rockvi l le, 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 ' カルチヤ一 · 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 samminoaminogenes AJ12340 (FERM BP-1539) To obtain them, for example, American Type Culture Collection, American Type Culture Collection, Address 12301 Park lawn 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 'Cultural Collection. Also, The AJ12340 strain has been deposited with the Ministry of International Trade and Industry at the National Institute of Bioscience and Human-Technology (Postal Code 305-856-6 1-3 1-3 Tsukuba-Higashi, Ibaraki, Japan) based on the Budapest Treaty.
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 ルタミン酸等の 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—ロイシン、 L一ホモセリン、 L一 プロリン、 L—セリン、 L—アルギニン、 Lーァラニン、 Lーバリン等のアミノ 酸を要求する変異株 （米国特許第 3708395号及び第 3825472号） 、 D L— a—ァミ ノー £一カブロラクタム、 ひ一アミノーラウリルラクタム、 ァスパラギン酸ーァ ナログ、 スルファ剤、 キノイ ド、 N—ラウロイルロイシンに耐性を示す Lーリジ ン生産変異株、 ォキザ口酢酸脱炭酸酵素 （デカルボキシラーゼ） 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—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一リジン生産変異株 （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一リジンを生産するブレビバ クテリゥ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） c また、 Lー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。 In addition to the above strains, mutants having L-amino acid-producing ability such as L-lysine or L-glutamic acid derived from these strains can also be used in the present invention. Such artificial mutants include the following. S— (2-aminoethyl) -cystine (hereinafter abbreviated as “AEC”) resistant mutant (eg, 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. 61-840, No. No. 62-24074, Japanese Patent Publication No. 62-36673, Japanese Patent Publication No. 5-11958, Japanese Patent Publication No. 7-112437, Japanese Patent Publication No. 7-112438), mutants that require amino acids such as L-homoserine for growth. (Japanese Patent Publication No. 48-28078, Japanese Patent Publication No. 56-6499), resistant to AEC, and furthermore, L-leucine, L-homoserine, L-proline, L-serine, L-arginine, L-alanine, L-valine, etc. Mutants that require amino acids (US Pat. Nos. 3,708,395 and 3,825,472), DL-a-amino Brolactam, H-amino-lauryl lactam, aspartic acid-analog, sulfa drugs, quinoids, L-lysine-producing mutants resistant to N-lauroylleucine, oxalate acetic acid decarboxylase (decarboxylase) or respiratory enzymes L-lysine-producing mutants that exhibit resistance to elemental inhibitors (Japanese Patent Application Laid-Open Nos. 50-53588, 50-31093, 52-102498, 53-9394, and 53-9394, 53-86089, JP-A-55-9783, JP-A-55-9759, JP-A-56-32995, JP-A-56-39778, JP-B-53-43591, JP-B-53-1833 L-lysine-producing mutants that require inositol or acetic acid (JP-A-55-9784 and JP-A-56-8692), L-lysine that is sensitive to fluoropyruvic acid or a temperature of 34 ° C or higher. Lysine-producing mutants (JP-A-55-9783, JP-A-53-86090), ethyleneglycol And a mutant strain of the genus Brevibacterium or Corynepacterium which is resistant to L-lysine and produces L-lysine (US Patent No. 4411997). Also, coryneform bacteria having L-threonine-producing ability include: (See U.S. Pat. No. 5,188,949) is one example of a coryneform bacterium having the ability to produce L-isoleucine. Lium Flavam AJ12149 (FERM BP-759) (see US Pat. No. 4,656,135).
なお、 本明細書において 「L一リジン等の L一アミノ酸生産能」 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ーリジン等の L—アミノ 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。  As used herein, “the ability to produce L-amino acid 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 >スクシネートデヒ ドロゲナーゼ活性の増強  <2> Enhancement of succinate dehydrogenase activity
コリネ型細菌細胞中のスクシネートデヒ ドロゲナ一ゼ活性を増強するには、 ス クシネートデヒ ドロゲナーゼをコードする遺伝子断片を、 該細菌で機能するべク ター、 好ましくはマルチコピー型のベクタ一と連結して組み換え DNAを作製し、 これを L—リジン又は L一グル夕 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スクシネートデヒ ドロゲナーゼをコ 一ドする遺伝子のコピー数が上昇する結果、 スクシネートデヒ ドロゲナーゼ活性 が増強される。 スクシネートデヒ ドロゲナ一ゼは、 ェシヱリヒア · コリでは sdh遺 伝子にコ一ドされている。  To enhance succinate dehydrogenase activity in coryneform bacterium cells, a recombinant DNA is obtained by ligating a gene fragment encoding succinate dehydrogenase to a vector that functions in the bacterium, preferably a multicopy vector. May be prepared and introduced into a coryneform bacterium having the ability to produce L-lysine or L-glucaminic acid, followed by transformation. As a result of an increase in the copy number of a gene encoding succinate dehydrogenase in the cells of the transformed strain, succinate dehydrogenase activity is enhanced. Succinate dehydrogenase is encoded by the sdh gene in Escherichia coli.
スクシネートデヒ ドロゲナーゼ遺伝子は、 コリネ型細菌の遺伝子を用いること も、 ェシエ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 succinate dehydrogenase 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 sdh遺伝子の塩基配列は既に明らかにされている （Genba nk/EMBL/DDBJ accetion No. J01619 K00542 MU121) ので、 その塩基配列に基づ いて作製したプライマー、 例えば配列表配列番号 1及び 2に示すプライマーを用 いて、 ェシエリヒア · コリ染色体 DNAを銪型とする P CR法 （P CR ： polyme rase chain reaction； White, T.J. et al ； Trends Genet. 5， 185( 1989)参照) に よって、 sdh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スク シネートデヒドロゲナーゼをコ一ドする遺伝子も、 同様にして取得され得る。 染色体 DNAは、 DN 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、 97〜98頁、 培風館、 1 992年参照） 等により 調製することができる。 P CR法により増幅されたスクシネートデヒ ドロゲナーゼをコードする遺伝子 は、 ェシエリヒア ' 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 なべクタ一 DN Aに接続して組換え DN Aを調製し、 これをェシエリヒア ' コリ 細胞に導入しておく と、 後の操作がしゃすくなる。 ェシエリヒア · コリ細胞内に おいて自律複製可能なベクターとしては、 プラスミ ドベクターが好ましく、 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、 pHS G299、 pHSG399、 pHSG398、 RSF1010等が挙げられる。 Since the nucleotide sequence of the sdh gene of Escherichia coli has already been determined (Genbank / EMBL / DDBJ accetion No. J01619 K00542 MU121), primers prepared based on the nucleotide sequence, for example, SEQ ID NO: 1 and Using the primers shown in 2 above, the PCR method (PCR: polymerase chain reaction; White, TJ et al; Trends Genet. 5, 185 (1989)) using the Escherichia coli chromosome DNA as type I was carried out. The sdh gene can be obtained. Genes encoding succinate dehydrogenase of other microorganisms such as coryneform bacteria can be obtained in a similar manner. Chromosomal DNA was obtained from bacteria that are DNA donors, for example, by the method of Saito and Miura (H. Saito and K. Miura Biochem. Biophys. Acta, 72, 619 (1963), Bioengineering Experiments, Japan Society for Biotechnology. Ed., Pp. 97-98, Baifukan, 19992). The gene encoding succinate dehydrogenase amplified by the PCR method is connected to a vector DNA capable of autonomous replication in cells of Escherichia coli and / or coryneform bacteria to prepare a recombinant DNA, which is then prepared. If introduced into Escherichia coli cells, subsequent operations will be slower. Plasmid vectors are preferred as vectors capable of autonomous replication in Escherichia coli cells, and those capable of autonomous replication in host cells are preferred.For example, pUC19, pUC18, pBR322, pHSG299, pHSG399, pHSG398, RSF1010 And the like.
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 vectors capable of autonomous replication in coryneform bacterium cells include PAM330 (see JP-A-58-67699), pHM1519 (see JP-A-58-77895), and the like. In addition, a DNA fragment having the ability to enable autonomous replication of a plasmid in a coryneform bacterium is extracted from these vectors and inserted into the Escherichia coli vector, which results in autonomous expression in both the Escherichia coli and the coryneform bacteria. It can be used as a replicable shuttle vector. The following are examples of such shuttle vectors. 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)  Coryneha, Cterium 'K', Rutamikum SR8201 (ATCC39135)
PAJ1844 ェシエリヒア ·]リ AJ11883(FERM BP - 137)  PAJ1844 Escherichia ·] Re AJ11883 (FERM BP-137)
コリネハ、、クテリゥム'ク'、ルタミクム SR8202(ATCC39136)  Coryneha, Cterium 'K', Rutamicum SR8202 (ATCC39136)
PAJ611 ェシエリヒア'コリ AJ11884(FERM BP- 138)  PAJ611 Escherichia Cori AJ11884 (FERM BP-138)
PAJ3148 コリネハ"クテリウム 'ク"ルタミクム SR8203(ATCC39137)  PAJ3148 Korineha "Cuterium 'C" Rutamicum SR8203 (ATCC39137)
PAJ440 Γ'チルス ·Γフ、、チリス AJ1190UFERM BP-140)  (PAJ440 チ ル 's chills, chillis AJ1190UFERM BP-140)
pHC4 Iシエリヒア 'コリ AJ12617(FERM BP- 3532)  pHC4 I Shierihia 'Coli AJ12617 (FERM BP-3532)
スクシネートデヒ ドロゲナーゼをコ一ドする遺伝子とコリネ型細菌で機能する ベクタ一を連結して組み換え DN Aを調製するには、 スクシネートデヒ ドロゲナ —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 DNAリガーゼ等のリガーゼを用いて行うのが普通である。  To prepare recombinant DNA by ligating a gene encoding succinate dehydrogenase and a vector that functions in coryneform bacteria, the vector must be digested with a restriction enzyme that matches the end of the gene encoding succinate dehydrogenase. Disconnect. The ligation is usually performed using a ligase such as T4 DNA ligase.
上記のように調製した組み換え DN 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 DNA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 ' ズプチリス、 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 D N Aを容易に取り込むプロ トブラス トまたはスフヱロブ ラス トの状態にして組換え DN Aを DN A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., Nature, 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— 2077 9 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 a transformation method reported so far. For example, Escherichia A method for increasing the permeability of DNA by treating recipient cells with calcium chloride, as reported for li-112 (Mandel, M. and Higa, A., J. Mol. Biol., 53 , 159 (1970)), and a method for preparing a recombinant cell from a cell at the growth stage and introducing DNA as described in Bacillus' subtilis (Duncan, CH, Wilson, GA and Young, FE, Gene, 1, 153 (1977)). Alternatively, recombinant DNA can be obtained by transforming cells of a DNA-accepting bacterium into protoblasts or spherovlasts that readily incorporate the recombinant DNA, as is known for Bacillus subtilis, actinomycetes and yeast. Methods for introduction into DNA recipients (Chang, S. and Choen, SN, Molec. Gen. Genet., 168, 111 (1979); Bibb, MJ, Ward, JMand Hopwood, 0.A., Nature, 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).
スクシネートデヒ ドロゲナーゼをコードする活性の増強は、 スクシネートデヒ ドロゲナーゼをコ一ドする遺伝子を上記宿主の染色体 DN A上に多コピ一存在さ 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 DN A上 にスクシネートデヒ ドロゲナ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 DNA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 DN 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一ティ 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、 スクシネートデヒ ドロゲナ —ゼをコー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 株内のスクシネートデヒ ドロゲナーゼをコ一ドする遺伝子のコピー数が上昇する 結果、 スクシネートデヒ ドロゲナーゼ活性が増強される。  Enhancement of the activity encoding succinate dehydrogenase can also be achieved by causing multiple copies of a gene encoding succinate dehydrogenase to be present on the chromosomal DNA of the host. In order to introduce multiple copies of a gene encoding succinate dehydrogenase onto the chromosome DNA of a microorganism belonging to a coryneform bacterium, homologous recombination is performed using a sequence present on the chromosomal DNA in a multiple copy as a target. Sequences present in multiple copies on the chromosomal DNA include repetitive DNA and invert 'repeats at the ends of transposable elements. Alternatively, a gene encoding succinate dehydrogenase can be mounted on a transposon and transferred, and multiple copies can be introduced into chromosomal DNA, as disclosed in JP-A-2-19985. is there. Either method increases the copy number of the gene encoding succinate dehydrogenase in the transformant, resulting in enhanced succinate dehydrogenase activity.
スクシネートデヒ ドロゲナーゼ活性の増強は、 上記の遺伝子増幅による以外に、 染色体 DNA上又はプラスミ ド上のスクシネートデヒ ドロゲナーゼをコードする 遺伝子のプロモータ一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 1 5 280号公報参照） 。 たとえば、 l a cプロモー 夕一、 t r pプロモー夕一、 t r cプロモータ一、 t acブロモ一夕一、 ラムダ ファージの P Rプロモーター、 P Lプロモーター等が強力なプロモータ一として 知られている。 これらのプロモーターへの置換により、 スクシネートデヒ ドロゲ ナ一ゼをコードする遺伝子の発現が強化されることによってスクシネートデヒ ド 口ゲナ一ゼ活性が増強される。 Enhancement of succinate dehydrogenase activity can be achieved not only by the above-described gene amplification but also by replacing an expression regulatory sequence such as a promoter of a gene encoding succinate dehydrogenase on chromosomal DNA or plasmid with a strong one. (See Japanese Patent Application Laid-Open No. H11-215280). For example, lac promotion Yuichi, trp promoter, trc promoter, tac bromo, PR promoter and PL promoter of lambda phage are known as strong promoters. Substitution with these promoters enhances the expression of the gene encoding succinate dehydrogenase, thereby enhancing succinate dehydrogenase activity.
また、 本発明のコリネ型細菌は、 スクシネートデヒドロゲナーゼ活性に加えて、 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ースレオニンによる相乗的なフィードバッ ク阻害が実質的に解除されたァスパル卜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ェノールピ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。  In addition, the coryneform bacterium of the present invention can enhance the enzymatic genes of other amino acid biosynthetic pathways or glycolytic pathways in addition to the succinate dehydrogenase activity, thereby enhancing the enzymatic activity. Good. For example, examples of genes that can be used for the production of L-lysine include aspartase kinase subunit protein and / or protein, in which synergistic feedback inhibition by L-lysine and L-threonine has been substantially released. Gene coding for Bunitite protein (W094 / 25605 international publication pamphlet), wild-type phosphoenolpyruvate carboxylase gene derived from coryneform bacterium (JP-A-60-87788), wild-type dihydrodipicolin derived from coryneform bacterium Genes encoding acid synthase (JP-B-6-55149) are known.
また、 L一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 （GDH、 特開昭 6 1— 2 68 1 85号） 、 グル夕ミンシン テターゼ、 グルタミン酸シン夕一ゼ、 イソクェン酸デヒ ドロゲナーゼ （特開昭 6 2— 1 66890号、 特開昭 6 3— 2 1 4 1 8 9号） 、 アコニッ ト酸ヒ ドラ夕一 ゼ （特開昭 62— 294086号） 、 クェン酸シン夕一ゼ、 ピルビン酸カルボキ シラーゼ （特開昭 60— 87788号、 特開昭 62— 55089号） 、 ホスホェ ノールピルビン酸カルボキシラーゼ、 ホスホェノールピルビン酸シン夕一ゼ、 ェ ノラーゼ、 ホスホグリセロム夕ーゼ、 ホスホグリセリン酸キナーゼ、 グリセルァ ルデヒ ドー 3—リン酸デヒ ドロゲナーゼ、 ト リオースリン酸イソメラーゼ、 フル トースビスリン酸アルドラーゼ、 ホスホフルク トキナーゼ （特開昭 63 - 1 02 692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. 61-268185), glutamate synthetase, glutamate synthetase, and isoquene. Acid dehydrogenase (JP-A-62-166890, JP-A-63-214189), aconitate hydrazine (JP-A-62-294086), quenic acid Synthase, pyruvate carboxylase (JP-A-60-87788, JP-A-62-55089), phosphoenolpyruvate carboxylase, phosphoenolpyruvate synthase, enolase, phosphoglyceromase, Phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, triosephosphate isomerase, fructosebisphosphate aldolase, phosphofruc Kinase (JP 63 - 1 02 692), there is a glucose phosphate Isomera Ichize like.
さらに、 目的とする L一ア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—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、 イソクェン酸リア一ゼ、 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 セチルトランスフェラーゼ、 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 一ピロリン酸デヒ ドロゲナーゼ、 等がある。 Further, 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 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 (W09). 5/23864). Enzymes that catalyze the reaction that diverges from the L-glutamic acid biosynthetic pathway to produce a compound other than L-glutamic acid include: ketoglutarate dehydrogenase, isoquenate lyase, acetyl phosphate transferase, and the like. There are acetate kinase, acetate hydroxysynthase, acetate lactate synthase, acetyl formate transferase, lactate dehydrogenase, glutamate decarboxylase, and monopyrophosphate dehydrogenase.
さらに、 L一グルタ 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バクテリウム . ラク トファーメン タム AJ13029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 曰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—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一リジン及び L 一グルタ 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 アーメンタム 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 曰本国茨城県つくば市東一丁目 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-inhibitory substance such as a surfactant to a coryneform bacterium capable of producing L-glutamic acid, the biotin-inducing action in a medium containing an excessive amount of biotin can be achieved. L-glutamic acid can be produced in the absence of inhibitors (see W096 / 06180). Examples of such coryneform bacteria include Brevibacterium lactofermentum AJ13029 described in W096 / 06180. The AJ13029 strain was registered on September 2, 1994, with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (Postal Code 305-8566, 1-3-1 Tsukuba East, Ibaraki Prefecture, Japan) under the accession number FERM P-14501. Deposited and 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 substance inhibiting the action of biotin to a coryneform bacterium capable of producing L-lysine and L-glutamic acid, the action of biotin in a medium containing an excessive amount of biotin is suppressed. L-lysine and L-glutamic acid can be produced simultaneously in the absence of a substance (see W096 / 06180). Such strains include Brevipacterium 'Lactofamentum AJ12993 strain described in W096 / 06180. The stock was granted to the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (Postal Code 305-8566, 1-3-1, Higashi 1-3-chome, Tsukuba, Ibaraki, Japan) on June 3, 1994, with accession number FERM P-14348. And transferred to an 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. Also, “reduced activity” of an enzyme usually means that the enzyme activity in a cell is lower than that of a wild-type strain. However, when a strain whose enzyme activity is reduced by modification by genetic recombination technology or the like is obtained, it means that the enzyme activity in the cell is lower than that of the strain before modification.
< 3 > L—アミノ酸の生産 <3> L-amino acid production
スクシネートデヒ ドロゲナーゼ活性が増強され、 かつ、 L一アミノ酸生産能を 有するコリネ型細菌を好適な培地で培養すれば、 同 L—アミノ酸が培地に蓄積す る。 例えば、 スクシネートデヒ ドロゲナ一ゼ活性が増強され、 かつ L一リジン酸 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ーリジンが培地に蓄 積する。 また、 スクシネートデヒ ドロゲナ一ゼ活性が増強され、 かつ L一グル夕 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—グル夕ミン 酸が培地に蓄積する。  When a coryneform bacterium having enhanced succinate dehydrogenase activity and capable of producing L-amino acid is cultured in a suitable medium, the L-amino acid accumulates in the medium. For example, when a coryneform bacterium having enhanced succinate dehydrogenase activity and capable of producing L-lysine acid is cultured in a suitable medium, L-lysine accumulates in the medium. Further, when a coryneform bacterium having enhanced succinate dehydrogenase activity and capable of producing L-gluminic acid is cultured in a suitable medium, L-gluminic acid accumulates in the medium.
さらに、 スクシネートデヒ ドロゲナーゼ活性が増強され、 かつ L一リジン及び L一グル夕 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一リジン 及び L一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 に醚酵生産する場合には、 L一りジン生産菌を 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ーグル 夕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 succinate dehydrogenase activity and capable of producing L-lysine and L-glucaminic acid is cultured in the medium, L-lysine and L-glucaminic acid accumulate in the medium. When L-lysine and L-glutamic acid are simultaneously produced by fermentation, the L-gine-producing bacterium may be cultured under L-glucamic acid production conditions, or L-lysine-producing ability And a coryneform bacterium having L-glutamic acid-producing ability may be mixedly cultured (JP-A-5-37993).
本発明の微生物を用いて Lーリジン又は L—グル夕ミン酸等の 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 ト一ス、 ガラク トース、 フラク トース、 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、 コハク酸等の有機酸類を用いることができる。  The medium used to produce L-amino acids such as L-lysine or L-glucamic acid using the microorganism of the present invention contains a carbon source, a nitrogen source, inorganic ions, and if necessary, other organic micronutrients. This is a normal medium. Carbon sources include glucose, lactose, galactose, fructose, sucrose, molasses, carbohydrates such as starch hydrolysates, alcohols such as ethanol and inositol, acetic acid, fumaric acid, citric acid, Organic acids 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 . リカ一、 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。  Examples of the nitrogen source include inorganic ammonium salts such as ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, and ammonium acetate, ammonia, peptone, meat extract, yeast extract, yeast extract, corn steep liquor, soybean hydrolyzate, etc. Organic nitrogen, ammonia gas, ammonia water, and the like 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。 Potassium phosphate, magnesium sulfate, iron ion, manga A small amount of ion or the like is 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て〜 4 5 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 cultivation is preferably carried out for 16 to 72 hours under aerobic conditions such as shaking cultivation and aeration / agitation cultivation. To control. 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—アミノ酸の採取は、 通常の L一ア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一リジンの採取は、 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ーグル 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一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—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。  The L-amino acid can be collected from the fermentation liquor in the same manner as in the usual method for producing an L-amino acid. For example, collection of L-lysine can be usually carried out by combining an ion exchange resin method, a precipitation method and other known methods. Further, the method for collecting L-glutamic acid may be a conventional method, such as an ion exchange resin method or a crystallization method. Specifically, L-glutamic acid may be adsorbed and separated by an anion exchange resin, or may be neutralized and crystallized. When both L-lysine and L-glucamic acid are produced, 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 sdh遺伝子のクローニング  Cloning of the sdh gene of Escherichia coli JM109
ェシエリヒア 'コリの sdh遺伝子の塩基配列は既に明らかにされている （Genba nk/EMBL/DDBJ accetion No. J01619 K00542 M11121 ) 。 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に示すプライマーを合成し、 ェシエリヒア · コ リ JM109株の染色体 D N Aを踌型にして 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 sdh gene of Escherichia coli has already been elucidated (Genbank / EMBL / DDBJ accetion No. J01619 K00542 M11121). Primers shown in SEQ ID NOS: 1 and 2 in the Sequence Listing were synthesized based on the reported nucleotide sequence, 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 . J 01619 K00542 M11121に記載されている sdh遺伝子の塩基配列の 1 9 9 2番目から 2 0 1 5番目の塩基に至る配列に相当し、 配列番号 2は、 6 3 0 9番目から 6 2 8 6番目の塩基に至る配列に相当する。  Among the synthesized primers, SEQ ID NO: 1 extends from nucleotide 199 to nucleotide 215 of the nucleotide sequence of the sdh gene described in Genbank / EMBL / DDBJ accetion No. J 01619 K00542 M11121. SEQ ID NO: 2 corresponds to the sequence from the 6309th to the 286th base.
ェシエリヒア '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、 97〜 98頁、 培風館、 1992年） 。 また、 P CR 反応は、 P CR法最前線 （関谷剛男ほか編、 共立出版社、 1 989年） 185頁 に記載されている標準反応条件を用いた。 Preparation of chromosomal DNA of Escherichia coli E. coli JM109 was carried out by a conventional method. Test book, 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 front line of the PCR method (edited by Takeo Sekiya et al., Kyoritsu Shuppan, 1989) were used.
生成した P CR産物を常法により精製後、 Smalで切断したプラスミ ド pHC4 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 フエニコ一ル 30 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、 ー晚培養後、 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、 ベクターに sdh遺伝子が結合したブ ラスミ ド pHC4 s d hを得た。  The resulting PCR product is purified by a conventional method, and then ligated with Smal-cleaved plasmid pHC4 using a ligation kit (Takara Shuzo Co., Ltd.). Then, Escherichia coli KM JM109 competent cells (Takara Shuzo) Transformation is performed using L medium containing 30 g / ml of chloramphenicol (10 g / L of pact tributone, 5 g / L of pak toy extract, 5 g / L of NaCl, and 15 g / L of agar). , PH 7.2), and after culture, the white colonies that appeared were picked and separated into single colonies to obtain transformed strains. Plasmid was extracted from the obtained transformant to obtain a plasmid pHC4sdh in which the sdh gene was bound to the vector.
p H C 4を保持するェシエリヒア ' コリは、 プライべ一トナンバー AJ12617と命 名され、 199 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— 122 1 5として寄託され、 199 1年 8月 26日に、 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— 3532が付与されて いる。  Escherichia coli harboring pHC4 was named private number AJ12617, and on April 24, 1991, the Ministry of International Trade and Industry, National Institute of Industrial Science and Technology, Institute of Biotechnology and Industrial Technology (Zip code 305-8566 Japan Deposit No. FE RM P—122 15 at Tsukuba 1-3-1 Higashi, Ibaraki Pref., Japan, and transferred to an international deposit based on the Budapest Agreement on August 26, 1999. FERM BP—3532 is granted.
次に、 クローニングされた DNA断片がスクシネートデヒ ドロゲナ一ゼ活性を 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、 JM1 09株及び、 p HC4 s dhを保持するJM l 09株のスクシネートデヒ ドロゲナ一ゼ活性を Ac kerll, B. A. C. et al., Meth. Enzymol. 53， 466-483 (1978)に記載の方法によ り測定した。 その結果、 pHC 4 s dhを保持する JM 109株は、 pHC4 s d hを保持しない JM 109株の約 1 9倍のスクシネ一トデヒ ドロゲナーゼ活性 を示すことから、 sdh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 succinate dehydrogenase activity, the succinate dehydrogenase activity of the JM109 strain and the JM109 strain carrying pHC4sdh was determined. Was measured by the method described in Accell, BAC et al., Meth. Enzymol. 53, 466-483 (1978). As a result, it was confirmed that the sdh gene was expressed because the JM109 strain carrying pHC 4 sdh exhibited about 19 times the succinate dehydrogenase activity of the JM109 strain not carrying pHC4 sdh. .
< 2 >コリネ型細菌の L—グル夕ミン酸生産株への pHC4 s (！ の導入と ー グルタ ミン酸生産  <2> Introduction of pHC4 s (!) Into L-glutamic acid-producing strain of coryneform bacterium and glutamate production
ブレビパクテリゥム ' ラク トファーメンタム 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 pHC4sdh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/pHC4sdhを用いて L一グルタミン酸生産の ための培養を以下のように行った。 のクロラムフエ二コールを含む C M 2 Bプレート培地にて培養して得た AJ13029/pHC4sdh株の菌体を、 5〃g/mlのクロラ ムフヱ二コールを含む下記組成を有する L—グルタミン酸生産培地に接種し、 31. 5°C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バクテリゥム属細菌で自律複製可能なプラスミ ド p H C 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。 Brevipacterium 'lactofermentum AJ13029 was transformed with plasmid pHC4sdh by the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791) to obtain the obtained transformant. Using the resulting transformant AJ13029 / pHC4sdh to produce L-glutamic acid Cultivation was performed as follows. Of the AJ13029 / pHC4sdh strain obtained by culturing on a CM2B plate medium containing chloramphenicol in L-glutamic acid producing medium containing 5〃g / ml chloramphenicol and having the following composition Then, the cells were shake-cultured at 31.5 ° C and shake-cultured 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. As a control, a strain obtained by transforming a plasmid pHC4 capable of autonomously replicating with a previously obtained Corynebacterium bacterium into an AJ13029 strain of Corynebacterium bacterium by the electric pulse method was cultured in the same manner as described above. did.
〔L一グルタミン酸生産培地〕  (L-glutamic 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 AS-210 manufactured by Asahi Kasei Corporation. Table 1 shows the results. Table 1 Strain L-glutamic acid production ( g / L)
AJ13029/pHC4 1 9 . 9 AJ13029 / pHC4 1 9.9
AJ13029/pHC4sdh 2 1 . 5 < 3 >コリネ型細菌の Lーリジン生産株への pHC4sdhの導入と Lーリジン生産 ブレビパクテリゥム · ラクトファ一メン夕ム 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 PHC4sdh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/pHC4sdh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。
Figure imgf000016_0001
のクロラムフェニコ一ルを含む〇 2 Bブレ 一ト培地にて培養して得た AJ11082/pHC4sdh株の菌体を、 5 /mlのクロラムフエ 二コールを含む下記組成の Lー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。 AJ13029 / pHC4sdh 2 1.5 <3> Coryneform bacterium of L Rijin pHC4sdh introduction and L Rijin production Brevibacillus Park Terri ©-time Rakutofa one member Yumu AJ11082 the electric pulse method to producing strain (JP-A-2 see Japanese Patent -207791) by plasmid P HC4sdh And the obtained transformant was obtained. Using the obtained transformant AJ11082 / pHC4sdh, culture for L-lysine production was performed as follows.
Figure imgf000016_0001
The cells of the AJ11082 / pHC4sdh strain obtained by culturing in 〇2B plate medium containing chloramphenicol were inoculated into an L-lysine production medium having the following composition containing 5 / ml chloramphenicol, and Shaking culture was performed at ° C 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 by the electric pulse method was cultured in the corynebacterium bacterium AJ11082 strain in the same manner as described above.
ブレビパクテリゥム ' ラク トフアーメンタム AJ11082は、 1981年 1月 31日に農学 研究菌培養収集所 （Agricultural Research Culture Collection, ァメリカ合衆 国 イ リノイ州 6 1 6 0 4ピオリア ノースュニバーシティ通り 1 8 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 'Lactofermentum AJ11082 was established on January 31, 1981, at the Agricultural Research Culture Collection, Amerika, United States. International Deposit No. 1815 (1815 N. University Street, Peoria, Illinois 61604 USA) with 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_0002
Figure imgf000016_0002
ピオチン 500 jug  Piotin 500 jug
チアミン 2000 jus
Figure imgf000016_0003
Thiamine 2000 jus
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 炭酸カルシウム 50 g Protein hydrolyzate (bean concentrate) 30 ml 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 A S— 2 1 0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—リジン生成量（g/L)  After completion of the culture, the accumulated amount of L-lysine in the culture solution was measured with a Biotech Analyzer-AS210 manufactured by Asahi Kasei Corporation. Table 2 shows the results. Table 2 Strain L-Lysine production (g / L)
AJ11082/pHC4 2 9 . 9 AJ11082 / pHC4 29.9
AJ11082/pHC4sdh 3 3 . 1  AJ11082 / pHC4sdh 3 3 .1
< 4〉コリネ型細菌の L—リジン及び L一グル夕ミン酸生産株への pHC4sdhの導入 と L—リジン及び L一グルタミン酸同時生産 <4> Introduction of pHC4sdh 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 pHC4sdh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/pHC4sdhを用いて Lーリジン及び Lーグル 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プレート培地にて培養して得た AJ12993/pHC4sdh株の菌体を、 5 ig/mlのクロラムフエ二コールを含む前記 L一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 リウム属細菌 AJ12993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 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 pHC4sdh by the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791) to obtain the obtained transformant. Using the obtained transformant AJ12993 / pHC4sdh, culture for producing L-lysine and L-glucamic acid was performed as follows. AJ12993 / pHC4sdh strain cells obtained by culturing in a CM2B plate medium containing 5 μg / ml chloramphenicol were inoculated into the L-lysine production medium containing 5 ig / ml chloramphenicol. 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 belonging to the genus Corynebacterium by the electric pulse method was cultured in the same manner as described above.
培養終了後、 培養液中の Lーリジン及び L一グル夕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ー A S— 2 1 0により測定した。 このときの結果を ¾ 0に； した。 表 3 株 L—リジン生成量（g/L) L—グル夕ミン酸生成量（g/L) After completion of the culture, the accumulated amounts of L-lysine and L-glucamic acid in the culture solution were measured using a Biotech Analyzer AS-210 manufactured by Asahi Kasei Corporation. The result at this time was set to ¾0. Table 3 Strain L-Lysine production (g / L) L-Gluminic acid production (g / L)
AJ12993/pHC4 10. 1 20. 0 AJ12993 / pHC4 10.1 20.0
AJ12993/pHC4sdh 1 1. 8 22. 3  AJ12993 / pHC4sdh 1 1.8 22.3
産業上の利用可能性 本発明により、 コリネ型細菌の Lーリジン又は 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-glutamic acid can be improved.

Claims

請求の範囲 The scope of the claims
1 . 細胞中のスクシネートデヒ ドロゲナーゼ活性が増強され、 かつ L—ァミノ 酸生産能を有するコリネ型細菌。 1. Coryneform bacterium having enhanced succinate dehydrogenase 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—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 . 前記スクシネートデヒ ドロゲナ一ゼ活性の増強が、 前記細菌細胞内のスク シネートデヒ ドロゲナーゼをコ一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 enhancement of the succinate dehydrogenase activity is caused by increasing the copy number of a gene encoding succinate dehydrogenase in the bacterial cell.
4 . 前記スクシネートデヒ ドロゲナ一ゼをコー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 succinate dehydrogenase 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一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—リジン、 L一グルタミン酸、 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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EP1106684A1 (en) * 1999-12-10 2001-06-13 Degussa AG Polynucleotide sequences from Corynebacterium glutamicum coding for succinate dehydrogenase subunits (sdhA, sdhB, sdhC)
EP1382686A1 (en) * 2002-07-12 2004-01-21 Ajinomoto Co., Inc. Method for producing target substance by fermentation using a bacterial strain lacking the ArcA gene

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