JP2003159092A - Method for producing l-amino acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an L-amino acid such as L-lysine or L-glutamic acid by a fermentation process further improved compared to conventional ones, and to provide a strain for use in the method. <P>SOLUTION: A coryneform group of bacteria having the ability to produce the L-amino acid is transferred with a gene encoding glucose 6-phosphate isomerase to enhance the glucose 6-phosphate isomerase activity of the bacteria and thus improve the ability of the bacteria to produce the L-amino acid. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an L-based fermentation method.
The present invention relates to a method for producing an amino acid, particularly 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 raw material for seasonings and the like.

[0002]

2. Description of the Related Art Conventionally, L-amino acids such as L-lysine and L-glutamic acid have been fermented by a coryneform bacterium belonging to the genus Brevibacterium or Corynebacterium having the ability to produce these L-amino acids. It is manufactured industrially. For these coryneform bacteria, strains isolated from nature or artificial mutants of these strains are used in order to improve productivity.

Further, various techniques have been disclosed for increasing the L-amino acid-producing ability by enhancing the L-amino acid biosynthetic enzyme by recombinant DNA technology. For example, in coryneform bacteria having L-lysine-producing ability, a gene encoding aspartokinase in which feedback inhibition by L-lysine and L-threonine is released (mutant lysC), dihydrodipicolinate reductase gene (dapB), Dihydrodipicolinate synthase gene (dapA), diaminopimelate decarboxylase gene (lysA) gene, and diaminopimelate dehydrogenase gene (ddh) (WO96 / 40934), lysA and ddh
(JP-A-9-322774), lysC, lysA and phosphoenolpyruvate carboxylase gene (ppc) (JP-A-1-322774).
0-165180), mutant lysC, dapB, dapA, lysA and aspartate aminotransferase gene (aspC).
It is known that the introduction of (JP-A-10-215883) improves the L-lysine-producing ability of the bacterium.

In Escherichia bacteria, da
Sequential enhancement of pA, mutant lysC, dapB, diaminopimelate dehydrogenase gene (ddh) (or tetrahydrodipicolinate succinylase gene (dapD) and succinyldiaminopimelate deacylase gene (dapE)) improves L-lysine productivity Is known (WO 95/16042). In WO 95/16042, tetrahydrodipicolinate succinylase is erroneously described as succinyldiaminopimelate transaminase. On the other hand, in a bacterium of the genus Corynebacterium or genus Brevibacterium, the introduction of a gene encoding a citrate synthase derived from Escherichia coli or Corynebacterium glutamicum was effective for enhancing the L-glutamic acid-producing ability. Has been reported (Japanese Patent Publication No. 7-121228). Further, 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 Laid-Open No. 63-214189 discloses a technique for increasing the L-glutamic acid production ability by enhancing the glutamate dehydrogenase gene, the isocitrate dehydrogenase gene, the aconitate hydratase gene, and the citrate synthase gene. Has been done.

However, the structure of the gene encoding glucose 6-phosphate isomerase has not been reported in coryneform bacteria, and it is also known that the gene encoding glucose 6-phosphate isomerase is used for breeding coryneform bacteria. Has not been done.

DISCLOSURE OF THE INVENTION It is an object of the present invention to provide a method for producing L-amino acids such as L-lysine or L-glutamic acid by a fermentation method which is further improved than before, and a strain used therefor. To do.

[0006]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventors have found that glucose 6
It was found that the production amount of L-lysine or L-glutamic acid can be increased by introducing a gene encoding -phosphate isomerase into a coryneform bacterium to enhance glucose 6-phosphate isomerase activity, and the present invention. Has been completed. That is, the present invention is as follows.

(1) A coryneform bacterium having an enhanced glucose 6-phosphate isomerase activity in cells and an L-amino acid-producing ability. (2) The coryneform bacterium of (1), wherein the L-amino acid is selected from L-lysine and L-glutamic acid. (3) The coryneform bacterium of (1) above, wherein the enhancement of glucose 6-phosphate isomerase activity is due to an increase in the copy number of the gene encoding glucose 6-phosphate isomerase in the bacterial cell. (4) The coryneform bacterium according to (3), wherein the gene encoding glucose 6-phosphate isomerase is derived from a bacterium belonging to the genus Escherichia. (5) The method of culturing the coryneform bacterium according to any one of (1) to (4) above in a medium to produce and accumulate L-amino acid in the culture, and collecting the L-amino acid from the culture. And a method for producing an L-amino acid. (6) The method according to (5), wherein the L-amino acid is selected from L-lysine and L-glutamic acid.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.

<1> Coryneform bacterium of the present invention The coryneform bacterium of the present invention has L-amino acid-producing ability,
It is a coryneform bacterium with enhanced glucose 6-phosphate isomerase activity in cells. As an L-amino acid, L
-Lysine, L-glutamic acid, L-threonine, L-isoleucine, L-serine and the like can be mentioned. Among these, L-lysine and L-glutamic acid are preferable. Hereinafter, the embodiment of the present invention will be described mainly for coryneform bacteria having L-lysine-producing ability or L-glutamic acid-producing ability. The same can be applied to those located downstream of pyruvate carboxylase.

The coryneform bacteria referred to in the present invention include Bergey's Manual of Determinative Baciology.
teriology) 8th edition pp. 599 (1974), a group of microorganisms that are aerobic, gram-positive, non-acidic, non-sporulating rods, and are conventionally classified as Brevibacterium. However, it contains bacteria that have been integrated as Corynebacterium (Int. J. Syst. Bacterio
l., 41, 255 (1981)), and also Brevibacterium and Microbacterium which are closely related to the genus Corynebacterium. Examples of coryneform bacterial strains that are preferably used for the production of L-lysine or L-glutamic acid include:
For example, the following may be mentioned.

[0011]   Corynebacterium acetoside film ATCC13870   Corynebacterium acetoglutamicum ATCC15806   Corynebacterium carnae ATCC15991   Corynebacterium glutamicum ATCC13032   (Brevibacterium divaricatum) ATCC14020   (Brevibacterium lactofermentum) ATCC13869   [Corynebacterium Lilium] ATCC15990   (Brevibacterium flavum) ATCC14067   Corynebacterium meracecola ATCC17965   Brevibacterium saccharolyticum ATCC14066   Brevibacterium Inmario Film ATCC14068   Brevibacterium roseum ATCC13825   Brevibacterium thiogenitalis ATCC19240   Microbacterium Ammonia Philum ATCC15354   Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539)

In order to obtain these, for example, the American Type Culture Collection can be purchased. That is, a registration number corresponding to each microorganism is given, and the registration number can be quoted to receive the sale. The registration number corresponding to each microorganism is listed in the American Type Culture Collection catalog. The AJ12340 strain has been deposited at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, under the Budapest Treaty.

In addition to the above-mentioned 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 artificial mutant strains include the following. S- (2-
Aminoethyl) -cysteine (hereinafter abbreviated as "AEC") resistant mutant (for example, Brevibacterium lactofermentum AJ11082 (NRRL B-11470), JP-B-56-19
No. 14, JP-B 56-1915, JP-B 57-14157, JP-B 57-
14158, Japanese Examined Sho 57-30474, Japanese Examined Sho 58-10075, Examined Sho 59-4993, Examined Sho 61-35840, Examined Sho 62-24074,
Japanese Examined Patent Publication No. 62-36673, Japanese Examined Patent Publication 511958, Japanese Examined Publication 7-12437
No. 7-112438), and a mutant strain that requires an amino acid such as L-homoserine for its growth (Japanese Patent Publication No. 48-28078).
, JP-B-56-6499), AEC-resistant, L-leucine, L-homoserine, L-proline, L-serine,
Mutant strains requiring amino acids such as L-arginine, L-alanine and L-valine (US Pat. Nos. 3,708,395 and 38254).
72), DL-α-amino-ε-caprolactam, α-
Amino-lauryllactam, aspartic acid-analog, sulfa drugs, quinoids, L-lysine-producing mutants resistant to N-lauroylleucine, oxaloacetate decarboxylase (decarboxylase) or L resistant to respiratory enzyme inhibitors -Lysine-producing mutant (Japanese Patent Laid-Open No. 50-53588)
JP-A-50-31093, JP-A-52-102498, JP-A-53-9394
JP-A-53-86089, JP-A-55-9783, JP-A-55-97
59, JP-A-56-32995, JP-A-56-39778, JP-B-53
-43591, Japanese Examined Patent Publication No. 53-1833), L-lysine-producing mutants that require inositol or acetic acid (JP-A-55-9784).
No. JP-A-56-8692), fluoropyruvic acid or 34
L-lysine-producing mutant strains that are sensitive to temperatures above ° C (JP-A-55-9783 and JP-A-53-86090), Brevibacterium that is resistant to ethylene glycol and produces L-lysine Production mutant strains of the genus or Corynebacterium (US Pat. No. 4411997).

As a coryneform bacterium having L-threonine-producing ability, corynebacterium acetoacidophilum AJ12318 (FERM BP-1172) (US Pat. No. 5,188,94)
Brevibacterium flavum AJ121 is a coryneform bacterium capable of producing L-isoleucine.
49 (FERM BP-759) (see US Pat. No. 4,656,135) and the like.

<2> Enhancement of glucose 6-phosphate isomerase activity To enhance glucose 6-phosphate isomerase activity in coryneform bacterium cells, a gene fragment encoding glucose 6-phosphate isomerase is used in the bacterium. Recombinant DNA is prepared by ligating with a functional vector, preferably a multi-copy type vector, and the recombinant DNA is prepared by using L-lysine or L-lysine.
-It may be introduced into a coryneform bacterium capable of producing glutamic acid and transformed. As a result of the increase in the copy number of the gene encoding glucose 6-phosphate isomerase in the cells of the transformant, glucose 6-phosphate isomerase activity is enhanced. Glucose 6-phosphate isomerase is encoded by the pgi gene in Escherichia coli.

As the glucose 6-phosphate isomerase gene, either the gene of coryneform bacterium or the gene of other organisms such as Escherichia bacterium can be used. The nucleotide sequence of the pgi gene of Escherichia coli has already been clarified (Froman, BE et a
l., Mol.Gen.Genet. 217, 126-131 (1989), Genbank / EMB
L / DDBJ accetion No. X15196), a PCR method using Escherichia coli chromosomal DNA as a template (PCR: polymerase) using primers prepared based on the nucleotide sequence, for example, the primers shown in SEQ ID NOS: 1 and 2 of the Sequence Listing.
chain reaction; White, TJ et al; Trends Genet.
5,185 (1989)), the pgi gene can be obtained. Glucose from other microorganisms such as coryneform bacteria
The gene encoding 6-phosphate isomerase can be obtained in the same 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. Miur).
a Biochem.Biophys.Acta, 72,619, (1963), Biotechnology Experiment Book, Japan Society for Biotechnology, pp. 97-98, Baifukan, 19
1992) and the like.

Glucose 6 amplified by the PCR method
-The gene encoding phosphate isomerase is ligated to a vector DNA capable of autonomous replication in the cells of Escherichia coli and / or coryneform bacterium to prepare recombinant DNA, which is introduced into Escherichia coli cells. If you leave it, later operations will be easier. As a vector capable of autonomously replicating 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, pHSG2.
99, pHSG399, pHSG398, RSF1010 and the like.

Examples of the vector capable of autonomously replicating in the cells of coryneform bacteria include pAM330 (see JP-A-58-67699) and pHM1519 (see JP-A-58-77895). When a DNA fragment capable of autonomously replicating a plasmid in a coryneform bacterium is extracted from these vectors and inserted into the Escherichia coli vector, autonomous replication is possible in both Escherichia coli and a coryneform bacterium. Can be used as a so-called shuttle vector. Examples of such shuttle vectors include the following. The microorganisms carrying the respective vectors and the deposit numbers of the international depository institutions are shown in parentheses.

To prepare a recombinant DNA by ligating a gene encoding glucose 6-phosphate isomerase with a vector functioning in coryneform bacterium, glucose 6
-Cut the vector with a restriction enzyme that fits the ends of the gene encoding phosphate isomerase. Connection is T4D
It is common to use a ligase such as NA ligase.

The recombinant DNA prepared as described above can be introduced into a coryneform bacterium by any of the transformation methods previously reported. For example, a method of treating 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 of preparing competent cells from cells in the growth stage and introducing DNA as described for Bacillus subtilis (Duncan, CH, Wil.
son, GAand Young, FE, Gene, 1, 153 (1977)). Alternatively, cells of DNA-accepting bacteria, such as those known for Bacillus subtilis, actinomycetes and yeast, are brought into a protoplast or spheroplast in which the recombinant DNA is easily taken up, and the recombinant DNA is DNA.
Method of introducing into recipient bacteria (Chang, S. And Choen, SN, Mole
c. Gen. Genet., 168, 111 (1979); Bibb, MJ, Ward, J.
M. and Hopwood, OA, Nature, 274, 398 (1978); Hinnen,
A., Hicks, JBand Fink, GR, Proc. Natl. Acad. Sci.
USA, 75 1929 (1978)) can also be applied. The transformation method used in the examples of the present invention was the electric pulse method (Japanese Patent Laid-Open No.
207791).

The enhancement of the activity encoding glucose 6-phosphate isomerase can also be achieved by allowing multiple copies of the gene encoding glucose 6-phosphate isomerase to be present on the chromosomal DNA of the above host. Glucose on chromosomal DNA of microorganisms belonging to coryneform bacteria
In order to introduce a gene encoding 6-phosphate isomerase in multiple copies, homologous recombination is carried out using a sequence existing in multiple copies on the chromosomal DNA as a target. Chromosome DN
Sequences that exist in multiple copies on A include repetitive DNA, inverted at the end of transposable elements.
Repeat is available. Alternatively, JP-A 2-1099
As disclosed in Japanese Patent Publication No. 85-85, glucose 6-
It is also possible to incorporate a gene encoding phosphate isomerase into a transposon and transfer it to introduce multiple copies of the gene into chromosomal DNA. Either method increases the copy number of the gene encoding glucose 6-phosphate isomerase in the transformant, resulting in enhanced glucose 6-phosphate isomerase activity.

The enhancement of glucose 6-phosphate isomerase activity is not limited to the above-mentioned gene amplification, but is not limited to chromosome DN.
It can also be achieved by substituting a strong expression control sequence such as a promoter of a gene encoding glucose 6-phosphate isomerase on A or on a plasmid. For example, lac promoter, trp promoter, trc promoter, tac promoter, lambda phage P _R promoter, P _L promoter and the like are known as strong promoters. The substitution of these promoters enhances the expression of the gene encoding glucose 6-phosphate isomerase, thereby enhancing glucose 6-phosphate isomerase activity.

In addition, the coryneform bacterium of the present invention has enhanced glucose 6-phosphate isomerase activity, as well as other enzyme amino acid biosynthetic pathways or glycolytic pathways. May be done. For example, examples of genes that can be used for producing L-lysine include aspartokinase α subunit protein or β subunit protein in which synergistic feedback inhibition by L-lysine and L-threonine is substantially released. Gene encoding (WO94 / 25605 international publication pamphlet),
A wild-type phosphoenolpyruvate carboxylase gene derived from coryneform bacteria (Japanese Patent Laid-Open No. 60-87788),
A gene encoding a wild-type dihydrodipicolinate synthase derived from coryneform bacteria (Japanese Patent Publication No. 6-55149) is known.

Examples of genes that can be used for the production of L-glutamic acid include glycolytic phosphofructokinase (PFK, JP-A-63-102692), phosphoenolpyruvate in the anaplerotic pathway. Carboxylase (PEPC, JP-A-60-87788, JP-A-62-55089), citrate synthase in the TCA cycle (CS, JP-A-62-201585, JP-A-63-
119688), aconitate hydratase (AC
O, JP 62-294086), isocitrate dehydrogenase (ICDH, JP 62-166890).
, JP-A-63-214189), and glutamate dehydrogenase (GDH, which is a catalyst for amination reaction) (JP-A-61-268185).

Furthermore, the activity of an enzyme that catalyzes a reaction that branches from the L-amino acid biosynthesis pathway to produce a compound other than the same amino acid may be reduced or lacking. For example, homoserine dehydrogenase is an enzyme that catalyzes a reaction that branches from the L-lysine biosynthesis pathway to produce a compound other than L-lysine (see WO 95/23864). In addition, as an enzyme that catalyzes a reaction that branches from the L-glutamic acid biosynthetic pathway to produce a compound other than L-glutamic acid, α-ketoglutarate dehydrogenase (αKGDH), isocitrate lyase, phosphate acetyltransferase, acetate kinase, Acetohydroxy acid synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, 1-pyrroline dehydrogenase, and the like.

Furthermore, by imparting a temperature-sensitive mutation to a coryneform bacterium capable of producing L-glutamic acid, a temperature-sensitive mutation for a biotin-inhibiting substance such as a surfactant, the biotin-inhibiting effect in a medium containing an excess amount of biotin. L-glutamic acid can be produced in the absence of a substance (see WO96 / 06180). Examples of such coryneform bacteria include Brevibacterium lactofermentum AJ13029 described in WO96 / 06180. AJ130
Twenty-nine shares were deposited at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science with the deposit number FERM P-14501 on September 2, 1994,
It was transferred to an international deposit under the Budapest Treaty on August 1, 1995 and is given the deposit number FERM BP-5189.

In addition, a coryneform bacterium capable of producing L-lysine and L-glutamic acid is provided with a temperature-sensitive mutation to a biotin action-suppressing substance, whereby a biotin action-suppressing substance is contained in a medium containing an excess amount of biotin. L-lysine and L-glutamic acid can be co-produced in the absence of (see WO96 / 06180). Examples of such strains include Brevibacterium lactofermentum AJ12993 strain described in WO96 / 06180. The strain was deposited at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science with the deposit number FERM P-14348 as of June 3, 1994, and was transferred to the international deposit under the Budapest Treaty on August 1, 1995 and deposited. It is numbered FERM BP-5188.

<3> Production of L-amino acid Glucose 6-phosphate isomerase activity is enhanced,
When the coryneform bacterium having L-amino acid-producing ability is cultured in a suitable medium, the L-amino acid accumulates in the medium.
For example, when a coryneform bacterium having enhanced glucose 6-phosphate isomerase activity and L-lysine acid-producing ability is cultured in a suitable medium, L-lysine accumulates in the medium. Further, when coryneform bacteria having enhanced glucose 6-phosphate isomerase activity and L-glutamic acid-producing ability are cultured in a suitable medium, L-glutamic acid is accumulated in the medium.

Furthermore, when a coryneform bacterium having an enhanced glucose 6-phosphate isomerase activity and an ability to produce L-lysine and L-glutamic acid is cultured in a medium,
L-lysine and L-glutamic acid accumulate in the medium. L
-When lysine and L-glutamic acid are simultaneously fermented and produced, the L-lysine-producing bacterium may be cultured under L-glutamic acid-producing conditions, or a coryneform bacterium capable of producing L-lysine and L-lysine Coryneform bacteria capable of producing glutamic acid may be mixed and cultured (Japanese Patent Laid-Open No. 5-3793).
Issue).

The medium used for producing L-amino acid using the microorganism of the present invention is a usual medium containing a carbon source, a nitrogen source, inorganic ions and, if necessary, other organic micronutrients. As the carbon source, glucose, lactose, galactose, fructose, sucrose, molasses, carbohydrates such as starch hydrolysate, alcohols such as ethanol and inositol, organic acids such as acetic acid, fumaric acid, citric acid, and succinic acid. Can be used.

As the nitrogen source, ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, inorganic ammonium salts such as ammonium acetate, ammonia, peptone, meat extract, yeast extract, yeast extract,
Organic nitrogen such as corn steep liquor and soybean hydrolyzate, ammonia gas, and ammonia water can be used.

As the inorganic ions, potassium phosphate, magnesium sulfate, iron ions, manganese ions and the like are added in small amounts. As the organic micronutrients, it is desirable to contain required substances such as vitamin B ₁ or yeast extract in an appropriate amount as necessary.

The culture is preferably carried out for 16 to 72 hours under aerobic conditions such as shaking culture and aeration and stirring culture. The culture temperature is controlled at 30 ° C to 45 ° C and the pH during culture is controlled at 5-9. To do. Inorganic or organic acidic or alkaline substances, ammonia gas, etc. can be used for pH adjustment.

Collection of L-amino acids from the fermentation broth can be carried out in the same manner as in ordinary L-amino acid production methods. For example, L-lysine can be usually carried out by combining an ion exchange resin method, a precipitation method and other known methods. The method of collecting L-glutamic acid may be carried out by 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, it is not necessary to separate these amino acids from one another if they are used as a mixture.

[0036]

EXAMPLES The present invention will be described in more detail below with reference to examples.

<1> Cloning of pgi gene of Escherichia coli JM109 strain The nucleotide sequence of the pgi gene of Escherichia coli has already been clarified (Froman, BE et al., Mol.Gen.Gene.
t. 217, 126-131 (1989), Genbank / EMBL / DDBJ accetion
No. X15196). The primers shown in SEQ ID NOS: 1 and 2 of the Sequence Listing were synthesized based on the reported nucleotide sequence, and PC was prepared using the chromosomal DNA of Escherichia coli JM109 strain as a template.
The pyruvate dehydrogenase gene was amplified by the R method.

Among the synthesized primers, SEQ ID NO: 1 is
Froman, BE et al., Mol.Gen.Genet. 217, 126-131 (1
989) corresponding to the sequence from the 1st to the 24th base of the base sequence of the pgi gene, and SEQ ID NO: 2 is
It corresponds to the sequence extending from the 2573th base to the 2550th base.

Chromosomal DN of Escherichia coli JM109 strain
Preparation of A was carried out by a conventional method (Biotechnology Experiment Book, edited by Japan Society for Biotechnology, pp. 97-98, Baifukan, 1992). Also,
For the PCR reaction, the standard reaction conditions described on page 185 of the PCR method front line (Takeo Sekiya et al., Edited by Kyoritsu Shuppan, 1989) were used.

After purifying the generated PCR product by a conventional method,
Plasmid pHC4 cleaved with SmaI (JP-A-5-749)
No. 1) and a ligation kit (manufactured by Takara Shuzo), followed by transformation using Escherichia coli JM109 competent cells (manufactured by Takara Shuzo), containing 5 μg / ml of chloramphenicol L medium (Bacto tryptone 10g / L, Bacto yeast extract 5g / L, Na
Cl 5g / L, agar 15g / L, pH7.2), and after overnight culture,
The white colonies that appeared were picked up, and single colonies were separated to obtain a transformant. A plasmid was extracted from the obtained transformant to obtain a plasmid pHC4pgi in which the pgi gene was bound to the vector.

Escherichia coli which retains pHC4 is named as a private number AJ12617, 199.
On April 24, 1991, the Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Institute of Biotechnology, Institute of Biotechnology (Zip code 305-8566, 1-3-1, Higashi, Tsukuba, Ibaraki, Japan) with the contract number FERM BP-1221
No. 5, deposited on August 26, 1991, as an international deposit under the Budapest Treaty, with accession number FERM
BP-3532 is added.

Next, in order to confirm that the cloned DNA fragment encodes a protein having glucose 6-phosphate isomerase activity, JM109 was used.
Strain and glucose 6-phosphate isomerase activity of the JM109 strain carrying pHC4pgi were analyzed by Muramatsu,
N. and Nosoh, Y., Arch. Biochem. Biophys. 144, 245
-252 (1971). as a result,
The JM109 strain that retains pHC4pgi is
Since the glucose 6-phosphate isomerase activity of JM109 strain not holding gi was about 15 times higher, it was confirmed that the pgi gene was expressed.

<2> Introduction of pHC4pgi into L-glutamic acid-producing strain of coryneform bacterium and plasmid pHC4pgi of L-glutamic acid-producing Brevibacterium lactofermentum AJ13029 by electric pulse method (see Japanese Patent Laid-Open No. 2-207791). The resulting transformant was obtained. The resulting transformant AJ13029 / pHC4pgi was used to carry out culture for producing L-glutamic acid as follows. CM2B containing 5 μg / ml chloramphenicol
The cells of the AJ13029 / pHC4pgi strain obtained by culturing in a plate medium were inoculated into an L-glutamic acid production medium having the following composition containing 5 μg / ml of chloramphenicol, and cultured with shaking at 31.5 ° C. The culture was performed with shaking 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 shake-cultured at 37 ° C. until the sugar in the medium was consumed. Corynebacterium bacterium AJ as a control
The 13029 strain was transformed with the already obtained plasmid pHC4 capable of autonomously replicating in a bacterium belonging to the genus Corynebacterium by the electric pulse method, and cultured in the same manner as above.

[L-glutamic acid-producing medium] The following components (in 1 L) were dissolved, adjusted to pH 8.0 with KOH, and then at 115 ° C for 15
Sterilize for minutes. Glucose _{150g KH 2 PO 4 2g MgSO 4} · 7H 2 O 1.5g FeSO 4 · 7H 2 O 15mg MnSO 4 · 4H 2 O 15mg soy protein hydrolyzate 50ml biotin 2mg thiamine hydrochloride 3mg

After completion of the culturing, the accumulated amount of L-glutamic acid in the culture broth was measured by Asahi Kasei Kogyo Biotech Analyzer AS.
It was measured by -210. The results at this time are shown in Table 1.

[0046]

[Table 1] Table 1 ─────────────────────── Strain L-Glutamic acid production (g / L) ─────────────────────── AJ13029 / pHC4 21.0 AJ13029 / pHC4pgi 23.5 ───────────────────────

<3> Introduction of pHC4pgi into L-lysine-producing strain of coryneform bacterium and plasmid pHC4pgi of L-lysine-producing Brevibacterium lactofermentum AJ11082 by the electric pulse method (see Japanese Patent Laid-Open No. 2-207791). The resulting transformant was obtained. Using the obtained transformant AJ11082 / pHC4pgi, L-
Culture for lysine production was performed as follows. 5 μg / m
5 μg / ml of AJ11082 / pHC4pgi strain cells obtained by culturing in a CM2B plate medium containing 1 chloramphenicol
Was inoculated into the L-lysine production medium having the following composition containing chloramphenicol and cultured at 31.5 ° C. with shaking until the sugar in the medium was consumed. As a control, the strain AJ11082 of the genus Corynebacterium was transformed by the electric pulse method with the plasmid pHC4 capable of autonomous replication in the bacterium of the genus Corynebacterium already obtained, and cultured in the same manner as above.

Brevibacterium lactofermentum AJ11082 was prepared on the 18th of June, 1979.
Research Service Culture Collection (Agri
cultural research service culture collection) and has been deposited with the deposit number NRRL B-11470.

[L-lysine production medium] The following components (in 1 L) other than calcium carbonate were dissolved, adjusted to pH 8.0 with KOH, sterilized at 115 ° C for 15 minutes, and then dry heat-sterilized calcium carbonate was added. Add 50 g. Glucose 100 g (NH ₄ ) ₂ SO ₄ 55 g KH ₂ PO ₄ 1 g MgSO ₄ / 7H ₂ O 1 g biotin 500 μg thiamine 2000 μg FeSO ₄ / 7H ₂ O 0.01 g MnSO ₄ / 7H ₂ O 0.01 g nicotinamide 5 mg protein hydrolyzate (bean concentrate) 30 ml calcium carbonate 50 g

After completion of the culturing, the amount of L-glutamic acid accumulated in the culture broth was measured as Biotech Analyzer AS manufactured by Asahi Kasei Corporation.
It was measured by -210. The results at this time are shown in Table 2.

[0051]

[Table 2] Table 2 ─────────────────────── Strain L-lysine production (g / L) ─────────────────────── AJ11082 / pHC4 29.1 AJ11082 / pHC4pgi 33.4 ───────────────────────

<4> L-lysine and L- of coryneform bacterium
Introduction of pHC4pgi into a glutamic acid-producing strain and Brevibacterium lactofermentum AJ12993 co-producing L-lysine and L-glutamic acid were transformed with the plasmid pHC4pgi by the electric pulse method (see Japanese Patent Laid-Open No. 2-207791). A transformed strain was obtained. Using the obtained transformant AJ12993 / pHC4pgi, L-
Culturing for lysine and L-glutamic acid production was performed as follows. AJ12993 / pHC4pg obtained by culturing in CM2B plate medium containing 5 μg / ml chloramphenicol
The strain i cells were inoculated into the L-lysine production medium containing 5 μg / ml chloramphenicol and cultured at 31.5 ° C.
The culture temperature was shifted to 34 ° C. 12 hours after the culture was started, and the culture was performed with shaking until the sugar in the medium was consumed.
Corynebacterium bacterium AJ12993 as a control
A strain obtained by transforming the already obtained plasmid pHC4 capable of autonomous replication in a bacterium belonging to the genus Corynebacterium by the electric pulse method was cultured in the same manner as above.

After completion of the culture, L-lysine and L in the culture solution
-The amount of accumulated glutamic acid was measured by Biotech Analyzer AS-210 manufactured by Asahi Kasei. The results at this time are shown in Table 3.

[0054]

[Table 3]                                 Table 3 ───────────────────────────────────     Strain L-lysine production (g / L) L-glutamic acid production (g / L) ───────────────────────────────────   AJ12993 / pHC4 10.0 18.9   AJ12993 / pHC4pgi 12.1 20.8 ───────────────────────────────────

[0055]

Industrial Applicability According to the present invention, the ability of coryneform bacteria to produce L-amino acids such as L-lysine or L-glutamic acid can be improved.

[0056]

[Sequence list]                             Sequence Listing

[0057] <110> Ajinomoto Co., Inc. <120> Method for producing L-amino acid <130> P-6530 <141> 1999-07-02 <160> 2 <170> PatentIn Ver. 2.0

[0058] <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for       amplifying Esherichia coli pgi gene <400> 1 ggatccattt tcagccttgg caca 24

[0059] <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for       amplifying Esherichia coli pgi gene <400> 2 gcatgcgaat tacggcgcgg ggaa 24

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12P 13/06 C12P 13/06 D 13/14 A 13/14 13/08 // (C12P 13/08 C12R 1:15 C12R 1:15) C12P 13/06 (C12P 13/06 13/14 C12R 1:15) C12N 15/00 ZNAA (C12P 13/14 C12R 1:15) (72) Inventor Osamu Kurahashi Kawasaki, Kanagawa Prefecture 1-1, Suzuki-cho, Kawasaki-ku, Ajinomoto Co., Inc. F-term in the Fermentation Research Laboratory

Claims (6)

[Claims] 1. A coryneform bacterium having an enhanced glucose 6-phosphate isomerase activity in cells and an L-amino acid-producing ability. 2. The L-amino acid is L-lysine or L-
Glutamic acid, L-threonine, L-isoleucine, L
-The coryneform bacterium of claim 1, selected from serine. 3. The coryneform bacterium according to claim 1, wherein the glucose 6-phosphate isomerase activity is enhanced by increasing the copy number of the gene encoding glucose 6-phosphate isomerase in the bacterial cell. . 4. The coryneform bacterium according to claim 3, wherein the gene encoding glucose 6-phosphate isomerase is derived from a bacterium belonging to the genus Escherichia. 5. The coryneform bacterium according to any one of claims 1 to 4 is cultured in a medium, L-amino acids are produced and accumulated in the culture, and the L-amino acids are collected from the culture. A method for producing an L-amino acid, which comprises: 6. The L-amino acid is L-lysine or L-
Glutamic acid, L-threonine, L-isoleucine, L
-The method of claim 5 selected from serine.