WO2000056859A1

WO2000056859A1 - Process for producing l-amino acid

Info

Publication number: WO2000056859A1
Application number: PCT/JP2000/001655
Authority: WO
Inventors: Masakazu Sugimoto; Hisao Ito; Osamu Kurahashi
Original assignee: Ajinomoto Co., Inc.
Priority date: 1999-03-19
Filing date: 2000-03-17
Publication date: 2000-09-28
Also published as: AU3194300A

Abstract

A coryneform bacterium having a potentiated phosphoenolpyruvate synthase activity in the cell and being capable of producing an L-amino acid; and a process for producing the L-amino acid characterized by comprising culturing this coryneform bacterium in a medium, thus forming and accumulating the L-amino acid in the medium, and then collecting the L-amino acid from the culture medium.

Description

Specification

Manufacturing method of L-amino acid

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 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 produced by fermentation using a coryneform bacterium belonging to the genus Brevibacterium or Corynebacterium having the ability to produce L-amino acid. It is industrially produced. As these coryneform bacteria, strains isolated from the natural world or human mutants of the strains are used in order to improve productivity.

Also, various techniques have been disclosed for increasing the activity of producing L-amino acids by enhancing the activity of L-amino acid biosynthetic enzymes by recombinant DNA technology. For example, in a coryneform bacterium capable of producing L-lysine, a gene encoding aspartokinase (mutant lysC) in which feedback inhibition by L-lysine and L-threonine has been released, a dihydrodipicolinate reductase gene ( dapB), the dihydrodibicolinate synthase gene (dapA), the diaminopimelate decarboxylase gene (lysA), and the diaminopimelate dehydrogenase gene (ddh) (W096 / 40934), LysA and DDH (JP-A-9-1322774). No.), LysC-LysA and phosphoenolpyruvate carboxylase gene (ppc) (Japanese Patent Application Laid-Open No. 10-165180), mutant lysC, dapB, dapA, lysA and aspartic acid aminotransferase gene (aspC) (Kaihei 10-215883) is known to improve the L-lysine-producing ability of the bacterium. .

In the bacterium belonging to the genus Escherichia, dapA, mutant lysC, dapB, diaminobimeric dehydrogenase gene (ddh) (or tetrahydroxypicolinic acid succinylase gene (dapD), and succinyldiaminopimelate deacylase gene are also available. It is known that L-lysine-producing ability is improved by sequentially increasing the number of offspring (dapE)) (W095 / 16042). In WO95 / 16042, tetrahydrodipicolinate succinylase is erroneously described as succinyldiaminopimelate transaminase.

On the other hand, in a bacterium belonging to the genus Corynepacterium or Brevipacterium, introduction of a gene encoding citrate synthase derived from Escherichia coli or Corynebacterium gulurica is introduced by L-glutamic acid. It was reported that it was effective in enhancing production capacity (Japanese Patent Publication No. 7-121228). Also, Japanese Patent Application Laid-Open No. 61-268185 discloses a cell having a recombinant DNA containing a glutamate dehydrogenase gene derived from a bacterium belonging to the genus Corynebacterium. Further, Japanese Patent Application Laid-Open No. 63-214189 discloses that the glutamate dehydrogenase gene, the disoquatate dehydrogenase gene, the aconitate hydrase enzyme, and the citrate synthase gene are enhanced. A technique for increasing the ability to produce L-glucamic acid has been disclosed.

However, the structure of the gene encoding phosphoenolpyruvate synthase has not been reported in coryneform bacteria, and the gene encoding phosphoenolpyruvate synthase is used for breeding of coryneform bacteria. Not even 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-glutamic 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 phosphoeno-rubyruvic acid synthase into coryneform bacteria, It has been found that the production of L-lysine or L-glucaminic acid can be increased by enhancing the zease activity, thereby completing the present invention.

That is, the present invention is as follows.

(1) A coryneform bacterium which has enhanced phosphoenolpyruvate synthase activity in cells and has the ability to produce amino acids. (2) The coryneform bacterium according to (1), wherein the L-amino acid is selected from L-lysine and L-glutamic acid.

(3) The enhancement of phosphoenol pyruvate synthase activity is due to an increase in the number of genes encoding phosphoenol pyruvate synthase in the bacterial cells. ) Coryneform bacteria.

(4) The coryneform bacterium according to (3), wherein the gene encoding the phosphoenol pyruvate synthase is derived from a bacterium belonging to the genus Escherichia.

(5) The coryneform bacterium according to any one of (1) to (4) is cultured in a medium, L-amino acid is produced and accumulated in the culture, and L-amino acid is collected from the culture. Method for producing L-amino acid.

(6) The method according to (5), wherein said 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 L-amino acid-producing ability and enhanced phosphoenorubyruvate synthase activity in cells. Examples of the L-amino acid include L-lysine, L-glutamic acid, L-threonine, L-isoleucine, L-serine and the like. Among these, L-lysine and L-glutamic acid are preferred. Hereinafter, the power of describing an embodiment of the present invention mainly for a coryneform bacterium having an L-lysine-producing ability or an L-glucamic acid-producing ability The present invention provides a biosynthesis system specific to an L-amino acid. Phosphoenolpyruvate synthase can be similarly applied to those located downstream.

As the coryneform bacteria referred to in the present invention, a group defined in Bergey's Manual of Determinative Bacteriology, 8th edition, p. 599 (1974) It is an aerobic, gram-positive, non-acid-fast, non-sporulating bacillus that was previously classified as Brevibacterium, but is now integrated as Corynepacterium. Bactenol., 41, 255 (1981), and also includes bacteria of the genus Brevibacterium and Micropaterium, which are closely related to the genus Corynebacterium. The strains of coryneform bacteria suitably used for the production of L-lysine or L-glutamic acid include, for example, those shown below.

Corynebacterium acetate film ATCC13870

Corynebacterium case Toggle evening Mikum ATCC15806

Corynebacterium '' Carnaet ATCC15991

Corynebacterium · Guru Yu Mikum ATCC13032

(Brevibacterium divaricatum) ATCC14020

(Brevipactium / LactoFammentum) ATCC13869

(Corynebacterium ream) ATCC15990

(Brevibacterium flavum) ATCC14067

Corynebacterium mefsekoff ATCC17965

Brevi Pacterium Saccharolyticum ATCC14066

Brevibacterium in Mario film ATCC14068

Brevi Pacterium Rosemze ATCC13825

Brevi Pacterium Choge Niyu Squirrel ATCC19240

Micropacterium ammonia ammonia ATCC15354

Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539)

These can be obtained, for example, from the American 'Type' Culture Collection. 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 microbe is listed in the American Type 'Culture Collection Log. The AJ12340 strain has been deposited under the Budapest Treaty with the Institute of Biotechnology and Industrial Technology, the Ministry of International Trade and Industry.

In addition to the above strains, mutants having L-lysine-producing ability or L-glutamic acid-producing ability derived from these strains can also be used in the present invention. Such human mutants include the following. S— (2-aminoethyl) monocysteine (hereinafter abbreviated as “AEC”) resistant mutant (for example, Brevibacterium Tof amentum AJ11082 (NRRL B-11470), JP-B 56-1914, JP-B 56-1915, JP-B 57-14157, JP-B 57-14158, JP-B 57-30474, JP-B 58- 10075, JP-B-59-4993, JP-B-61-35840, JP-B-62-24074, JP-B-62-36673, JP-B-5-11958, JP-B7-11-112437, JP-B7-112438 Mutants that require amino acids such as L-homoserine for their growth (Japanese Patent Publication No. 48-28078, Japanese Patent Publication No. 56-6499), are resistant to AEC, and furthermore are L-mouth isine, L-homoserine, Mutants requiring amino acids such as L-proline, L-serine, L-arginine, L-alanine and L-valine (US Pat. Nos. 3,708,395 and 3,825,472), DL-amino- £ -caprolactam, hyamino Monolauryl lactam, monoaspartic acid, sulfa drugs, quinoids, N-lauroy L-lysine-producing mutants that are resistant to leucine, L-lysine-producing mutants that are resistant to oxazate acetic acid decarboxylase (decarboxylase) or respiratory enzyme inhibitors (Japanese Patent Application Laid-Open No. 5058853/1983) 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-55-9759 JP-A-56-32995, JP-A-56-39778, JP-B-53-43591, JP-B-53-1833), L-lysine-producing mutants requiring inositol or acetic acid (JP-A-55-9784, JP-A-56-8692), Fluoropyruvate or an L-lysine-producing mutant which is sensitive to a temperature of 34 ° C or higher (JP-A-55-9783, JP-A-53-86090), ethylene glycol Mutants of the genus Brevibacterium or Corynepacterium which are resistant to L-lysine and produce L-lysine (US Patent No. 4411997) . As used herein, the term “L-amino acid-producing ability” refers to the ability of a coryneform bacterium to accumulate a significant amount of L-amino acid in a medium when cultured in the medium, or the L-amino acid in the cells. Refers to the ability to increase amino acid content.

<2> Enhancement of phosphoenol pyruvate synthase activity

To enhance phosphoenolpyruvate synthase activity in coryneform bacterium cells, a gene fragment encoding phosphoenolpyruvate synthase must be isolated from a vector, preferably a multicopy vector, which functions in the bacterium. Ligation may be performed to produce a recombinant DNA, which 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 phosphoenol pyruvate synthase in the cells of the transformed strain resulted in phosphoenol Rubilate synthase activity is enhanced. Phosphoenorubyruvate synthase is encoded by the pps gene in Escherichia coli.

As the phosphoenol pyruvate synthase 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 pps gene of Escherichia coli has already been determined (Mol. Gen. Genet., 231 (2), 332-336 (1992), Genbank / EMBL / DDBJ accession No. M69116). Using a primer prepared based on the sequence, for example, the primers shown in SEQ ID NOs: 1 and 2 in the Sequence Listing, a PCR method using Escherichia coli chromosome DNA as type III (PCR: polymerase chain reaction; White, TJ et al. ., Trends Genet. 5, 185 (1989)) to obtain the pps gene. Genes encoding phosphoenolpyruvate synthase of other microorganisms such as coryneform bacteria can be obtained in a similar manner.

Chromosomal DNA can be 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, Edited by the Society, pp. 97-98, Baifukan, 1992).

The gene encoding phosphoenolpyruvate synthase amplified by the PCR method is connected to a vector DNA that can be replicated autonomously in cells of Escherichia coli and / or coryneform bacteria to transform the recombinant DNA. If prepared and introduced into Escherichia coli cells, subsequent operations will be 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, HSF 1010 and the like.

Examples of vectors that can replicate autonomously in coryneform bacteria cells include PAM330 (see Japanese Patent Application Laid-Open No. 58-67699) and PHM1519 (see Japanese Patent Application Laid-Open No. 58-77895). 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, and then autonomously expressed in both Escherichia coli and coryneform bacteria. Cannot be duplicated It can be used as a so-called shuttle vector. The following are examples of such shuttle shuttles. The accession number of the international depository organization of the microorganisms holding each vector is shown in parentheses.

PAJ655 Escherichia ·] Re AJ11882 (FERM BP-136)

Coryneha "Cterium" Rutamikum SR8201 (ATCC39135)

PAJ1844 Escherichia ·] Re AJ11883 (FERM BP-137)

Coryneha ', Cterium' K 'Ruta Mikum SR8202 (ATCC39136)

PAJ611 Engineering Serihir 'Kori AJ11884 (FERM BP-138)

pA J3148 Coryneha, Cterium-g, Rutamicum SR8203 (ATCC 39137)

PAJ440 C, Chills, Chillis AJ11901 (FEM BP-140)

pHC4 I.] AJ12617 (FERM BP-3532)

In order to prepare recombinant DNA by ligating a gene encoding phosphoenolpyruvate synthase and a vector that functions in coryneform bacteria, it is necessary to match the end of the gene encoding phosphoenol pyruvate synthase. Cut the enzyme with such a restriction enzyme. 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. Upon cooling, treatment of recipient cells with calcium chloride to increase DNA permeability, as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)) and a method for preparing DNA from transgenic cells and introducing DNA as described in Bacillus subtilis (Duncan, CH, Wilson). , GA and Young, FE, Gene, 1, 153 (1977)). Alternatively, cells of DNA recipients, such as those known for Bacillus subtilis, actinomycetes and yeast, can be transformed into protoplasts or spheroplasts that readily incorporate the recombinant DNA and the recombinant DNA can be converted to DNA. Method for introduction into recipient bacteria (Chang, S. and Choen, SN, Molec. Gen. Genet., 168, 111 (1979); Bibb'MJ, Ward, JM and Hopwood, OA, Nature, 274, 398 (1978) Hinnen, A., Hicks. JB and Fink, GR, Proc. Natl. Acad. Sci. USA, 751929 (1978)). Implementation of the present invention The transformation method used in the examples is the electric pulse method (see Japanese Patent Application Laid-Open No. Hei 2-20771).

Enhancement of phosphoenol pyruvate synthase-encoding activity can also be achieved by allowing the gene encoding phosphoenolpyruvate synthase to be present in multiple copies on the chromosomal DNA of the host. In order to introduce multiple copies of the gene encoding phosphoenolpyruvate synthase into the chromosomal DNA of microorganisms belonging to coryneform bacteria, multiple copies of the gene on chromosomal DNA can be used as a target. Performed by homologous recombination. As a sequence present in multiple copies on the chromosomal DNA, a relative DNA and an inverted repeat at the end of a transposable element can be used. Alternatively, as disclosed in Japanese Patent Application Laid-Open No. 2-109985, a gene encoding phosphoenol pyruvate synthase is mounted on a transposon, transferred, and placed on chromosomal DNA. It is also possible to introduce multiple copies. Either method results in an increase in the number of coenzymes encoding phosphoenolpyruvate synthase in the transformed strain, resulting in an increase in phosphoenolpyruvate synthase activity.

Enhancement of phosphoenolpyruvate synthase activity can be achieved not only by the gene amplification described above, but also by the expression control sequences such as the promoter of the gene encoding phosphoenolpyruvate synthase on chromosomal DNA or plasmid. It can also be achieved by replacing it with a powerful one. For example, lac promoter, trp promoter evening -, trc promoter one, tac promoter, P _R promoter one evening one lambda phage, P _L promoter Isseki Chief is known as a powerful promoter evening one. Substitution with these promoters enhances phosphoenol pyruvate synthase activity by enhancing the expression of genes encoding phosphoenol pyruvate synthase.

In addition, the coryneform bacterium of the present invention is characterized in that, by introducing or amplifying an enzyme gene of another amino acid biosynthetic pathway or glycolytic pathway in addition to the phosphoenol pyruvate synthase activity, the enzyme activity of the coryneform bacterium is increased. May be enhanced. For example, examples of genes that can be used for the production of L-lysine include aspartokinase subunits in which synergistic feedback inhibition by L-lysine and L-threonine has been substantially eliminated. Gene encoding a cut protein or a / 5-subunit protein (W094 / 25605, International Publication Pamphlet); a wild-type phosphoenolpyruvate carboxylase gene derived from a coryneform bacterium (JP-A-60-87788); a gene derived from a coryneform bacterium And a gene encoding a wild type dihydrodipicolinate synthase (Japanese Patent Publication No. 6-55149).

Examples of genes that can be used for the production of L-glutamic acid include glycosylated phosphofructokinase (PFK, Japanese Patent Application Laid-Open No. 63-169292), and phosphoenolpyruvine in the anaplerotidic pathway. Acid carboxylase (PEPC, JP-A-60-87788, JP-A-62-55089), citrate synthase of the TCA cycle (CS, JP-A-62-2010) No. 1985, JP-A-63-119968, a. Conidic acid hydra-yuzide (ACO, JP-A No. 62-294,086), isocitrate dehydrogenate (CDH, JP-A-62-166890, JP-A-63-214189), glutamate dehydrogenase (GDH, 6 1—2 6 8 1 8 5).

Furthermore, by imparting a temperature-sensitive mutation to a biotin action inhibitor such as a surfactant to a coryneform bacterium capable of producing L-glutamic acid, the biotin action inhibitor in a medium containing an excessive amount of biotin can be added. L-glutamic acid can be produced in the absence of E. coli (see W096 / 06180). An example of such a coryneform bacterium is Brevipacterium 'lactofarmentum AJ13029 described in W096 / 06180. The AJ13029 strain was registered on September 2, 1994 with the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry (zip code 305-8566, 1-3 1-3 Higashi, Tsukuba, Ibaraki, Japan) and the accession number FERM. Deposited as P-14501, transferred to an international deposit under the Budapest Treaty on August 1, 1995, and given accession number FERM BP-5189. In addition, by imparting a temperature-sensitive mutation to a biotin-inhibiting substance to a coryneform bacterium capable of producing L-lysine and L-glutamic acid, the biotin-inhibiting substance can be expressed in a medium containing an excessive amount of biotin. L-Lysine and L-glutamic acid can be produced simultaneously in the absence (see W096 / 06180). Examples of such a strain include Brevibacterium 'Lactofamentum AJ12993 strain described in W096 / 06180. The shares were issued by the Ministry of International Trade and Industry on June 3, 1994. Deposited at the National Institute of Advanced Industrial Science and Technology (Postal Code: 305-8566, 1-3 1-3 Higashi, Tsukuba, Ibaraki, Japan) under the accession number FERM P-14348. It has been transferred to an international deposit under the United Nations Convention and has been assigned the 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 genetic recombination technology 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.

<3> Production of L-amino acids

When a coryneform bacterium having enhanced phosphoenorubyruvate synthase activity and capable of producing L-amino acid is cultured in a suitable medium, L-amino acid accumulates in the medium. For example, when a coryneform bacterium having enhanced phosphoenolpyruvate synthase activity and capable of producing L-lysine acid is cultured in a suitable medium, L-lysine is accumulated in the medium. In addition, when a coryneform bacterium having enhanced phosphoenol pyruvate synthase activity and having L-gluminic acid producing ability is cultured in a suitable medium, L-gluminic acid accumulates in the medium.

Furthermore, when coryneform bacteria having enhanced phosphoenol pyruvate synthase activity and L-lysine and L-glutamic acid producing ability are cultured in the medium, L-lysine and L-glucamic acid accumulate in the medium. I do. When L-lysine and L-glucaminic acid are simultaneously produced by fermentation, the L-lysine-producing bacterium may be cultured under L-glucaminic acid production conditions or L-lysine-producing ability. And a coryneform bacterium having an ability to produce L-glucamic acid may be mixed and cultured (Japanese Patent Application Laid-Open No. 5-37993).

The medium used to produce L-amino acids using the microorganism of the present invention is a normal medium containing a carbon source, a nitrogen source, inorganic ions and, if necessary, other organic micronutrients. Carbon sources include glucose, lactose, galactose, fructose, sucrose, molasses, carbohydrates such as starch hydrolysates, alcohols such as ethanolinositol, acetic acid, fumaric acid, and citric acid. And organic acids such as succinic acid.

Nitrogen sources include ammonium sulfate, ammonium nitrate, ammonium chloride, Inorganic ammonium salts such as ammonium phosphate and ammonium acetate, ammonia, peptone, meat extract, yeast extract, yeast extract, organic nitrogen such as soybean hydrolysate, soybean hydrolyzate, ammonia gas, ammonia water, etc. 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 a vitamin 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. To control. For 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. For example, L-lysine can be usually carried out by a combination of 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 both L-lysine and L-glutamic acid are produced, when these are used as a mixture, it is not necessary to separate these amino acids from each other. Example

Hereinafter, the present invention will be described more specifically with reference to examples.

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

The nucleotide sequence of the pps gene of Escherichia coli has already been elucidated (Mol. Gen. Genet., 231 (2), 332-336 (1992), Genbank / EMBL / DDBJ accession No. M69116). Based on the reported nucleotide sequence, the primers shown in SEQ ID NOs: 1 and 2 in the Sequence Listing were synthesized, and the chromosomal DNA of Escherichia coli 'JM109 strain was type III, and the phosphoenolpyruvate synthase gene was subjected to PCR by PCR. Was amplified.

Among the synthesized primers, SEQ ID NO: 1 is Genbank / EMBL / DDBJ accession No. It corresponds to the sequence from the first to the 23rd base of the base sequence of the PPS gene described in M69116, and SEQ ID NO: 2 corresponds to the sequence from the 366 to the 3640th base.

The chromosome DNA of Escherichia coli JM109 strain was prepared by a conventional method (Bioengineering 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 front line (Takeo Sekiya et al., Edited by Kyoritsu Shuppan, 1989) were used.

The purified PCR product is purified by a conventional method, and then ligated with Smal-cleaved plasmid pHC4 using the Reigession Kit (Takara Shuzo). Then, the Escherichia coli KM JM109 Combinent Cell (Takara Shuzo) ) And transformed into L medium containing 30 zg / ml of chloramphenicol (Bacto Tryptone 10 g / L, Pactoist Extract 5 g / L NaCl 5 g / L, Agar 15 g / L, pH 7.2) ), And after overnight culture, appeared white colonies were picked up and separated into single colonies to obtain transformed strains. Plasmid was extracted from the obtained transformant to obtain a plasmid pHC4 pps in which the pps gene was bound to the vector.

Escherichia coli harboring pHC4 was named private number AJ12617, and on April 24, 1991, the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry (zip code 305-8566, Ibaraki, Japan) Deposit No. FERM P-12215 at Tsukuba Higashi 1-chome 1-3), transferred to an international deposit based on the Budapest Treaty on August 26, 1991, and given a deposit number FERM BP-35332. ing.

Next, to confirm that the cloned DNA fragment encodes a protein having phosphoenolpyruvate synthase activity, the JM109 strain and the JM109 strain retaining pH pps 4 pps were used. Was measured by the method described in Methods in Enzymol., 13, 309 (1969). As a result, the JM109 strain having pHC of 4 pps showed about 15 times the phosphoenolylrubic acid synthase activity of the JM109 strain not having pHC of 4 pps. Was confirmed to be expressed.

<2> Introduction of pH C 4 pps into L-glucamic acid-producing strain of coryneform bacterium and L-glucamic acid production Brevipacterium lactofermentum AJ13029 was transformed with plasmid pHC 4 pps by the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791) to obtain the resulting transformant. Using the obtained transformant AJ13029 / pHC4pps, cultivation for producing L-glucaminic acid was performed as follows. Cells of the AJ13029 / pHC4pps strain obtained by culturing in a CM2B plate medium containing 5 g / ml chloramphenicol were mixed with L-gluminamine having the following composition containing 5 g / ml chloramphenicol. An acid production medium was inoculated, 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 corynebacterium bacterium AJ13029 strain was cultured in the same manner as described above, and a strain transformed with an already obtained corynebacterium bacterium autonomously replicating plasmid pHC4 by the electric pulse method was cultured as described above. .

(L-glutamic acid production medium)

Dissolve the following ingredients (in 1 L), adjust to ρΗ80 with K0H, and sterilize at 115 ° C for 15 minutes. Glucose 150 g

Soy protein hydrolyzate 50 ml

Piotin 2 mg

Thiamine hydrochloride 3 mg

After completion of the culture, the accumulated amount of L-glucamic acid in the culture solution was measured with a biotech analyzer AS-210 manufactured by Asahi Kasei Corporation. Table 1 shows the results. Bacterial L-glutamic acid production (g / L)

AJ13029 / pHC4 2 0.1

AJ13029 / pHC4pps 23.5

3> Introduction of 4pps of pHC into L-lysine-producing strain of coryneform bacterium and L-lysine production Brevibacterium 'Lactofamentum AJ11082 (Electrical pulse method using NRRL B- (see JP-A-2-207791)) The resulting transformant AJ11082 / pHC4pps was used to culture L-lysine as follows: 5 μg / ml The AJ11082 / pHC4pps strain obtained by culturing in a CM2B plate medium containing chloramphenicol was inoculated into an L-lysine production medium having the following composition containing 5 g / ml chloramphenic acid: And cultured with shaking until the sugar in the medium was consumed at 31.5 ° C. As a control, Corynebacterium sp.AJ11082 was used as a control with a plasmid pHC4 that can replicate autonomously with already obtained Corynebacterium sp. Transformation by electric pulse method The transformed strain was cultured as described above.

(L-lysine production medium)

Dissolve the following components (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 sterilized calcium carbonate.

100 g glucose

MgS〇4 7 H ₂ 0 1

Piotin 500 PL too

Thiamine 2000 z

F e S_rei_4 · 7 H ₂ 0 0.01 g

_{MnS 04 · 7 H 2 0 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 with a Biotech Analyzer AS 210 manufactured by Asahi Kasei Corporation. Table 2 shows the results. Table 2

-Lysine production (g / L)

AJ11082 / pHC4 30.2

AJ11082 / pHC4pps 33.5

Introduction of 4pps of pHC into L-lysine and L-glucamic acid producing strains of coryneform bacteria and simultaneous production of L-lysine and L-glucamic acid

Brevibacterium lactofermentum AJ12993 was transformed with plasmid pHC4pps by the electric pulse method (see Japanese Patent Application Laid-Open No. 2-207791) to obtain the resulting transformant. Using the obtained transformant AJ12993 / pHC4pps, culture for producing L-lysine and L-glucamic acid was performed as follows. The AJ12993 / pHC4pps strain obtained by culturing on a CM2B plate medium containing 5 g / ml chloramphenicol was inoculated into the L-lysine production medium containing Sg / 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 of Corynebacterium sp. AJ12993 was transformed by an electropulse method with a plasmid PHC4 capable of autonomous replication with a previously obtained Corynebacterium sp., And 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

¾i strain L-lysine production l (g / L) L-glutamic acid production (g / L)

AJ12993 / pHC4 1 0.2 19.9

AJ12993 / pHC4pps 1 2.5 22.3

Industrial applicability

According to the present invention, the ability of coryneform bacteria to produce L-amino acid such as L-lysine or L-glutamic acid can be improved. Also, an efficient method for producing an L-amino acid such as L-lysine or L-glutamic acid is provided.

Claims

The scope of the claims

1. Coryneform bacterium having enhanced phosphoenorubyruvate synthase activity in cells and capable of producing amino acids.

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 according to claim 1, wherein the enhancement of phosphoenolpyruvate synthase activity is due to an increase in the number of coby genes encoding a phosphoenolpyruvate synthase in the bacterial cell. Bacteria.

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

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5. culturing the coryneform bacterium according to any one of claims 1 to 4 in a medium, producing and accumulating L-amino acids in the culture, and collecting the L-amino acid from the culture. Characteristic method for producing L-amino acids.

6. The method according to claim 5, wherein said L-amino acid is selected from L-lysine, L-glutamic acid, L-threonine, L-isoleucine and L-serine.