WO2001002545A1

WO2001002545A1 - Process for producing l-amino acid

Info

Publication number: WO2001002545A1
Application number: PCT/JP2000/004345
Authority: WO
Inventors: Masakazu Sugimoto; Hisao Ito; Osamu Kurahashi
Original assignee: Ajinomoto Co., Inc.
Priority date: 1999-07-02
Filing date: 2000-06-30
Publication date: 2001-01-11
Also published as: JP2003144160A; AU5707300A

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 fumarase into a coryneform bacterium capable of producing an L-amino acid such as L-lysine or L-glutamic acid and thus enhancing the fumarase activity to thereby elevate the L-amino acid productivity; and strains to be used in this method.

Description

Statement

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 obtained by using a coryneform bacterium belonging to the genus Brevipacterium or Corynebacterium having 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.

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 encoding aspartkinase (mutant lysC) in which feedback inhibition by L-lysine and L-threonine has been released, dihydropicolinic acid phosphate Evening gene (dapB), dihydroxypicolinate synthase gene (dapA), diaminopimeline decarboxylase gene (lysA), and diaminopimeline dehydrogenase gene (ddh) (W096 / 40934), lysA and ddh Kaihei 9-322774), lysCslysA and phosphoenolpyruvate carboxylase gene (pp _C ) (JP-A-10-165180), mutant lysC, dapB, dapA, lysA and aspartate aminotransferase gene ( aspC) (Japanese Unexamined Patent Publication (Kokai) No. 10-215883) has been reported to improve the L-lysine production ability of the bacterium. To have.

In Escherichia bacteria, dapA, mutant lysC, dapB, diaminobi L-lysine production ability is improved by successively amplifying or introducing the methacrylate dehydrogenase gene (ddh) (or the tetrahydrodpicolinic acid succinylase gene (dapD) and the succinyldiaminopimelate deacylase gene (dapE)). (W0 95/16042). In WO95 / 16042, tetrahydrodipicophosphate succinylase is erroneously described as succinyldiaminopimelate transaminase. On the other hand, in a bacterium of the genus Corynepacterium or Brevipacterium, the introduction of a gene encoding a citrate synthase derived from Escherichia coli or Corynebacterium glutamicum is introduced into L-glutamic acid. It was reported that it was effective in enhancing 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 Corynebacterium bacterium. Further, JP-A-63-2189 discloses that a glutamate dehydrogenase gene, an isocitrate dehydrogenase gene, an aconitate hydrase gene and a citrate synthase gene are amplified or introduced. Accordingly, a technique for increasing the ability to produce L-glucamic acid has been disclosed.

However, the structure of the gene encoding fumarase has not been reported for coryneform bacteria, and the use of the gene encoding fumarase 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-amino acid by a fermentation method which is further improved, and a strain used for the same.

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, by introducing a gene encoding fumarase into coryneform bacteria and enhancing fumarase activity, L-lysine or L-glutamine The inventors have found that the production of evening acid can be increased, and have completed the present invention. That is, the present invention is as follows.

(1) A coryneform bacterium having enhanced fumalase activity in cells and capable of producing L-amino acid.

(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) The coryneform bacterium according to (1), wherein the enhancement of the fumarase activity is due to an increase in the copy number of a gene encoding humalase in the bacterial cell.

(4) The gene encoding the humalase is derived from a bacterium belonging to the genus Escherichia.

(3) Coryneform bacterium.

(5) The coryneform bacterium of any of (1) to (4) above is cultured in a medium, L-amino acids are produced and accumulated in the culture, and L-amino acids are collected from the culture. L—A method for producing amino acids.

(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> Coryneform bacterium of the present invention

The coryneform bacterium of the present invention is a coryneform bacterium having an ability to produce L-amino acid and having enhanced fumarase activity in cells. Examples of the L-amino acid 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-glucaminic acid-producing ability, but the present invention provides a biosynthesis system specific to an L-amino acid of interest. The same applies to those located downstream. The coryneform bacteria referred to in the present invention are a group of microorganisms defined in Bergey's Manual of Determinative Bacteriology, 8th edition, p. 599 (1974). , 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. Bacteriol., 41, 255 (1981)), and also includes bacteria of the genus Brevipacterium and Micropaterims 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 acetate film ATCC13870

Corynebacterium acetoglutamicum ATCC15806

Corynebacterium carnaet ATCC15991

CorynepaterumGlutamicum ATCC13032

(Brevipactium Divarikatam) ATCC14020

(Brevipactium · Lacto Armament) ATCC13869

(Corynebacterium lilium) ATCC15990

(Brevibacterium flavum) ATCC 14067

Corynebacterium mefse: 3-f ATCC17965

Brevi Pacterium Saccharolyticum ATCC14066

Brevi Pacterium Inmario Film ATCC14068

Brevi Pacterium Rosemze ATCC13825

Brevibacterium choge niyu squirrel ATCC19240

Microbacterium Ammonia Filum ATCC15354

Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539) To obtain them, for example, American Type Culture Collective 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 numbers for each microorganism are listed on the catalog of the American Culture's 'Culture' collection. In addition, AJ12340 strain was deposited with the Institute of Biotechnology, Industrial Technology Institute of the Ministry of International Trade and Industry (Postal Code 305-856-6 1-3 1-3 Tsukuba-Higashi, Ibaraki, Japan) based on the Budapest Treaty. Have been.

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) -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. 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 their growth (JP-B-48-28078, JP-B-56-6499), shows resistance to AEC, and furthermore L-mouth isin, L-homoserine, L-proline, L-serine, L-arginine, L-alanine, L-valine, etc. Mutant strains requiring amino acids (US Pat. Nos. 3,708,395 and 3,825,472), DL—amino acid L-lysine-producing enzyme, H-amino-lauryl lactam, aspartate mono-analog, sulfa drugs, quinoids, L-lysine-producing mutants resistant to N-lauroylleucine, oxaxate acetate decarboxylase (decarboxylase) or L-lysine-producing mutants showing resistance to respiratory enzyme inhibitors (JP-A-50-53588, JP-A-50-31093, JP-A-52-102498, JP-A-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, JP-B-53-43591 53-1833), L-lysine-producing mutants that require inositol or acetic acid (JP-A-55-9784, JP-A-56-8692), and are sensitive to fluoropyruvic acid or temperatures above 34 ° C. L-lysine-producing mutant strains (JP-A-55-9783, JP-A-53-86090) 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 acetoacidophilum AJ12318 (FERM BP-1172) (see US Pat. No. 5,188,949) and L-isoleucine-producing ability. Examples of the coryneform bacterium having the above include 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 acid such as L-lysine” refers to the ability of a coryneform bacterium to accumulate a significant amount of L-amino acid such as L-lysine in the medium when cultured in the medium. Or the ability to increase the content of amino acids such as L-lysine in cells.

<2> Enhancement of humalase activity

To enhance fumarase activity in coryneform bacterium cells, a gene fragment encoding fumarase is ligated to a vector that functions in the bacterium, preferably a multicopy vector, to produce recombinant DNA. Then, this may be introduced into a coryneform bacterium capable of producing L-lysine or L-glucamic acid for transformation. An increase in the copy number of the gene encoding fumalase in the cells of the transformed strain results in enhanced fumalase activity. Humalase is encoded by the fUDl gene in Escherichia coli.

As the fumarase gene, either 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 fum gene of Escherichia coli has already been clarified (Bell, PJ et al., J. Bacteriol. 171, 3494-3503 (1989), Genbank / EMBL / DDBJ accession No. M27058). 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 Escherichia coli chromosomal DNA as type I (PCR: polymerase chain reaction; J. et al; Trends Genet. 5, 185 (1989)) to obtain the fum gene. Genes encoding fumarase of other microorganisms such as coryneform bacteria can be obtained in a similar manner.

Chromosomal DNA is obtained from bacteria that are DNA donors, for example, by the method of Saito and Miura (H. Saito and .iura Biochem. Biophys. Acta, 72, 619 (1963)), Biological Engineering Experiment, Pp. 97-98, Baifukan, 1992).

The gene encoding fumarase amplified by the PCR method is connected to a vector DNA capable of autonomous replication in the cells of Escherichia coli and / or coryneform bacteria to prepare a recombinant DNA, which is then transformed into Escherichia coli. Introduced into cells Then, the operation later becomes difficult. As a vector capable of autonomous replication in Escherichia coli cells, a plasmid vector is preferable, and a vector capable of autonomous replication in a host cell is preferable.For example, pUC19, pUC18, pBR322, pHSG299, pHSG399, pHSG398, RSF1010, etc. Is mentioned.

The vector autonomously replicable in cells of coryneform bacteria (see Japanese Patent Laid HirakiAkira 58- 67699) pAM330, _P HM1519 (see JP-A-58- 77895) and the Ru mentioned. In addition, a DNA fragment capable of autonomously replicating plasmid in coryneform bacteria is extracted from these vectors and inserted into the Escherichia coli vector, whereby autonomous replication in both Escherichia coli and coryneform bacteria occurs. It can be used as a shuttle vector that is not possible. The following are examples of such a shuttle vector. In addition, the microorganism holding each vector and the accession number of the international depository organization are shown in parentheses.

PAJ655 Escherichia, Kori AJ11882 (FERM BP-136)

Coryneha, Cterium 'K' Ruta Mikum SR8201 (ATCC39135)

PAJ1844 Escherichia Cori AJ11883 (FERM BP-137)

Coryneha, Cterium 'ku', Lutamicum SR8202 (ATCC39136)

PAJ611 Escherichia Cori AJ11884 (FERM BP-138)

PAJ3148 Coryne Λ, テ, ル, ルミ 820 820 SR8203 (ATCC39137)

PAJ440 Λ, chillsΓ Γ "Chillis AJ1190UFERM BP-140)

PHC Sherihia 'Kori AJ12617 (FERM BP-3532)

To prepare recombinant DNA by ligating the gene encoding fumarase with a vector that functions in coryneform bacteria, cut the vector with a restriction enzyme that matches the end of the gene encoding fumarase. Ligation is usually performed using a ligase such as T4 DNA ligase.

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, a method of increasing the permeability of DNA by treating recipient cells with calcium chloride, as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)), 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, the recombinant DNA is transformed into DNA by transforming the cells of a DNA-accepting 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 recipient bacteria (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 fumarase-encoding activity can also be achieved by the presence of multiple copies of the fumarase-encoding gene on the chromosome DNA of the host. In order to introduce a gene encoding fumarase into a chromosome DNA of a microorganism belonging to a coryneform bacterium in multiple copies, homologous recombination is performed by using a sequence present in multiple copies on chromosome DNA as a target. As a sequence present in multiple copies on the chromosomal DNA, it is possible to use repetitive DNA and inverted repeats present at the end of a transposable element. Alternatively, as disclosed in Japanese Patent Application Laid-Open No. 2-109985, a gene encoding fumalase is mounted on a transposon and transferred, and multiple copies are introduced into chromosomal DNA. Is also possible. Either method increases the copy number of the gene encoding fumarase in the transformant, resulting in enhanced fumarase activity.

The enhancement of fumarase activity can be achieved not only by the gene amplification described above, but also by replacing the expression regulatory sequence such as the promoter of the gene encoding fumarase on chromosome DNA or plasmid with a strong one. Is also achieved (see Japanese Patent Application Laid-Open No. H11-215280). For example, lac promoter, trp promoter, trc promoter, tac promoter, PR promoter of lambda phage, PL promoter and the like are known as strong promoters. By substituting these promoters, fumarase activity is enhanced by enhancing expression of a gene encoding humalase. In addition, the coryneform bacterium of the present invention may have enhanced enzymatic activities thereof by enhancing enzyme genes such as other amino acid synthesizing pathways or glycolytic pathways in addition to the fumarase activity. For example, examples of genes that can be used for the production of L-lysine include Assubald Kinase Hissubunit protein or / ?, in which synergistic feedback inhibition by L-lysine and L-threonine has been substantially eliminated. A gene encoding a subunit protein (W094 / 25605 International Publication Pamphlet); a wild-type phosphoenolpyruvate carboxylase gene derived from coryneform bacteria (JP-A-60-87788); a wild-type dihydropicolinic acid derived from coryneform bacteria A gene encoding a synthase (Japanese Patent Publication No. 6-55149) and the like are known.

Examples of genes that can be used for the production of L-glutamic acid include glutamate dehydrogenase (GDH, JP-A-61-268185), glutamine synthase, and glutamate synthase. Isosenate dehydrogenase (Japanese Patent Application Laid-Open No. 62-166890, Japanese Patent Application Laid-Open No. 63-214189), aconitate hydratase (Japanese Patent Application Laid-open No. — 294 886), Synthetic citrate, pyruvate carboxylase (Japanese Patent Application Laid-Open No. 60-87788, Japanese Patent Application Laid-Open No. 62-55089), Phosphoe Nol-pyruvate carboxylase, phosphoenol-pyruvate synthase, enolase, phosphoglycerome kinase, phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, triosephosphate isomerase, frutoosebisphosphate Aldraise And phosphofructokinase (Japanese Unexamined Patent Publication No. 63-102692), glucose phosphate isomerase and the like.

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 diverges from the L-lysine biosynthetic pathway to produce a compound other than L-lysine (see WO95 / 23864). Enzymes that catalyze the reaction that branches off from the L-glutamic acid biosynthetic pathway to produce compounds other than L-glutamic acid include ketoglutaric acid dehydrogenase, isoquenate lyase, and acetyl phosphate. Transferase, acetate kinase, acetate hydroxysynthase, acetate lactate synthase, acetate acetyl formate, lactate dehydrogenase, glutamate decarboxylate And 1-pyrophosphate dehydrogenase.

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, a biotin action inhibitor in a medium containing an excessive amount of biotin can be obtained. L-glutamic acid can be produced in the absence of E. coli (see W096 / 06180). Examples of such coryneform bacteria include Brevipacterium. Lactofermentum AJ13029 described in W096 / 06180. The AJ13029 strain was granted to the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (Postal Code 305-8566, 1-3 1-3 Tsukuba-Higashi, Ibaraki, Japan) on September 2, 1994 under the accession number FERM P-14501. It was deposited and transferred to an international deposit under the Budapest Treaty on August 1, 1995, and given accession number FERM BP-5189. In addition, a coryneform bacterium capable of producing L-lysine and L-glutamic acid is given a temperature-sensitive mutation against a substance that suppresses the action of biotin, thereby suppressing the action of biotin in a medium containing an excessive amount of biotin. L-lysine and L-glutamic acid can be produced simultaneously in the absence of a substance (see W096 / 06180). Examples of such strains include Brevipacterium 'lactofermentum AJ12993 strain described in W096 / 06180. The stock was issued to the Institute of Biotechnology, Institute of Biotechnology, Japan, on June 3, 1999 (Postal Code 305-8566, Tsukuba 1-chome 1-3, Ibaraki, Japan), with accession number FERM P- Deposited at 14348, 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. 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—Production of amino acids

If a coryneform bacterium having enhanced fumarase activity and capable of producing L-amino acid is cultured in a suitable medium, the L-amino acid accumulates in the medium. For example, Humara 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, if a coryneform bacterium having enhanced fumarase activity and capable of producing L-glucaminic acid is cultured in a suitable medium, L-glucaminic acid accumulates in the medium.

Furthermore, when choline-type bacteria having enhanced humalase activity and capable of producing L-lysine and L-glutamic acid are cultured in the medium, L-lysine and L-glucamic 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 it has the ability to produce L-lysine. A coryneform bacterium and a coryneform bacterium having an ability to produce L-glucaminic acid may be mixed and cultured (Japanese Patent Application Laid-Open No. 5-37993).

The culture medium used for producing L-amino acids such as L-lysine or L-glumic 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, fructose, sucrose, molasses, carbohydrates such as starch hydrolysates, alcohols such as ethanol and inositol, acetic acid, fumaric acid, citric acid, and succinic acid. And other organic acids can be used.

Nitrogen sources include ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, ammonium acetate, and other inorganic ammonium salts, ammonia, peptone, meat extract, yeast extract, yeast extract, corn 'steep' liquor, soy hydrolyzate Organic nitrogen, ammonia gas, ammonia water, and the like can be used.

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.

The cultivation is preferably carried out for 16 to 72 hours under aerobic conditions such as shaking cultivation, aeration and stirring cultivation, and the cultivation temperature is 30 ° C to 45 ° C. Control to ~ 9. For pH adjustment, 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. 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. The method for collecting L-glutamic 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-glutamic acid, when these are used as a mixture, it is unnecessary to separate these amino acids from each other. Examples Hereinafter, the present invention will be described more specifically with reference to Examples.

<1> Cloning of the fum gene of Escherichia coli JM109

The nucleotide sequence of the fum gene of Escherichia coli has already been elucidated (Bell, P. J. et al., J. Bacteriol. 171, 3494-3503 (1989), Genbank / EMBL / DDBJ accession No. M27058). Based on the reported nucleotide sequence, SEQ ID NOS: 1 and 2: The following primers were synthesized, and the pyruvate dehydrogenase gene was amplified by PCR using the chromosomal DNA of Escherichia coli JM109 strain as type III. did.

Among the synthesized primers, S sequence number 1 corresponds to the sequence from the 1st to 24th base of the base sequence of the fum gene described in Genbank / EMBL / DDBJ accetion No. M 27058; 2 corresponds to the sequence from the 3162nd base to the 3149th base.

The chromosomal DNA of Escherichia coli JM109 strain was prepared according to a conventional method (Biotechnological Experiments, edited by Biotechnology Society of Japan, pp. 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, 1998) were used.

After purifying the main PCR product by a conventional method, it was ligated with plasmid pHC4 cut with Smal using a ligation kit (Takara Shuzo), and then combined with Escherichia coli KM JM109 competent cell (Takara Shuzo). Transformation was performed using an L medium containing 10 g / ml of chloram fenicol (10 g / L of pact tribton, Bacterial yeast). Extract tractor DOO 5 g / L, was applied NaCl 5 g / L, agar 15 g / L, the _P H7.2), after overnight incubation, appeared white colonies were picked up and separated into single colonies to obtain transformants . Plasmid was extracted from the obtained transformant to obtain a plasmid pHC 4 fum in which the fum gene was linked to the vector.

Escherichia coli harboring pHC4 was named the private number AJ12617, and said on April 24, 1999, 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). Deposit No. FE RM P—122 1-5 at Tsukuba 1-chome 1-3, Ibaraki, Japan; transferred to an international deposit based on the Budapest Convention on August 26, 1999 Accession number FERM BP—3532 is assigned.

Next, in order to confirm that the cloned DNA fragment encodes a protein having a fumarase activity, the fumalase activity of the JM109 strain and the JM109 strain having pHC4 fum was determined by Kanarek, It was measured by the method described in L. et al., J. Biol. Chem., 239, 4202-4206 (1964). As a result, the JM109 strain having pHC 4 fum exhibited about 17 times the fumarase activity of the JM109 strain without pHC 4 fum, indicating that the fum gene was expressed. confirmed. <2> Introduction of pH 4 f um into L-glucamic acid-producing strain of coryneform bacteria and L-glutamic acid production

Brevipacterium lactofermentum AJ13029 was transformed with plasmid pHC4fum by the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791) to obtain the obtained transformant. Using the obtained transformant AJ13029 / pHC4fum, culture for producing L-glutamic acid was performed as follows. Cells of the AJ13029 / pHC4fum strain obtained by culturing in a CM2B plate medium containing 5 μg / ml chloramphenicol were mixed with L-cells having the following composition containing 5 μg / ml chloramphenicol. The medium was inoculated into a glucanic acid production medium, cultured at 31.5 ° C with shaking, 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-glutamic acid production medium)

Dissolve the following components (in 1 L), adjust to pH 8.0 with K0H, and sterilize at 115 ° C for 15 minutes. Glucose 150g

KH ₂ P 0 2g

Soy protein hydrolyzate 50ml

Piotin 2mg

Siamin hydrochloride 3mg

After completion of the culture, the amount of L-glucamic acid accumulated 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 19.2

<3> Introduction of pHC4fum into L-lysine-producing strain of coryneform bacterium and L-lysine production Transformation of Brevipacterium lactofermen AJ11082 with plasmid pHC4fum by the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791) The transformant was obtained. Using the obtained transformant AJ11082 / pHC4fum, culture for producing L-lysine was performed as follows. Cells of the AJ11082 / pHC4fum strain obtained by culturing in a CM2B plate medium containing 5 g / ml chloramphenicol were transformed into L-lysine having the following composition containing 5 μg / ml chloramphenicol. The production medium was inoculated and shake-cultured at 31.5 until the sugar in the medium was consumed. Corynebacterium as control A strain obtained by transforming the AJ11082 strain with a Brassmid pHC4, which can be autonomously replicated by a corynepacterium bacterium already obtained, by the electric pulse method was cultured in the same manner as described above.

Brevi Pacterium 'Lactofermen AJ11082 was established on January 31, 1981 at the Agricultural Research Culture Collection ^ America, United States of America 6 1 6 0 4 Peoria Northuniversity Street International Deposit No. 1815 (1815 N. University Street, Peoria, Illinois 61604 USA) with accession number NRRL B-11470.

(L-lysine production medium)

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.

100 g glucose

ΚΗ ₂ Ρ 0 ₄ 1

Piotin 500 g

Thiamine 2000 g

F e S〇 7 H ₂ 0 0.01 g

Mn S〇 7 H2O 0.01 g

Nicotinamide 5 mg

Protein hydrolyzate (bean concentrate) 30 ml

50 g calcium carbonate

After the completion of the culture, the amount of L-lysine accumulated in the culture solution was measured using a Biotech Analyzer AS-210 manufactured by Asahi Kasei Corporation. Table 2 shows the results. Table 2 L-Lysine production (g / L)

AJ11082 / pHC4 29.9

AJ 11082 / pHC4fura 3 3.4

Introduction of pHC4fum into L-lysine and L-glutamic acid producing strains of coryneform bacteria and simultaneous production of L-lysine and L-glutamic acid

Brevipacterium lactofermentum AJ12993 was transformed with plasmid pHC4fum by the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791) to obtain the obtained transformant. Using the obtained transformant AJ12993 / pHC4fum, culture for producing L-lysine and L-glucamic acid was carried out as follows. The cells of the AJ12993 / pHC4fum strain obtained by culturing in a CM2B plate medium containing 5 μg / ml chloramphenicol were mixed with the L-cell containing 5 // g / ml chloramphenicol. A lysine production medium was 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.

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. Table 3 shows the results. Table 3 Strains L-Lysine production (g / L) L-Glutamic acid production (g / L)

AJ12993 / pHC49, 919.5

AJ12993 / pHC4fum 1 3.1 22.8

INDUSTRIAL APPLICABILITY According to the present invention, the ability of coryneform bacteria to produce L-amino acid such as L-lysine or L-glucamic acid can be improved.

Claims

The scope of the claims

1. Coryneform bacterium with enhanced fumarase activity in cells and L-amino acid producing ability.

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. The coryneform bacterium according to claim 1, wherein the enhancement of the fumarase activity is caused by increasing the copy number of a gene encoding fumarase in the bacterial cell.

4. The coryneform bacterium according to claim 3, wherein the gene encoding the fumarase is derived from a bacterium belonging to the genus Escherichia.

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. 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.