JP2000189175A - L-glutamic acid-producing bacterium and production of l-glutamic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new bacterial strain for production of L-glutamic acid comprising a bacterium, into which a citric acid synthase gene derived from a coryneform bacterium is introduced, belonging to an enteric bacterium group having L-glutamic acid-producing ability and useful for foods, medicines, etc. SOLUTION: This new bacterium is obtained by introducing citric acid synthase gene derived from a coryneform bacterium (e.g. Brevibacterium lactofermentum) thereinto and has L-glutamic acid-producing ability and belongs to an enteric bacterium group of the genus Enterobacter or the genus Klebsiella, etc., and is used for inexpensive and efficient production, etc., of L-glutamic acid useful for medicines, etc. The glutamic acid-producing bacterium is obtained by carrying out PCR by using chromosome DNA of Brevibacterium lactofermentum as a template to afford gltA gene, inserting the gene into SimaI- digested plasmid pHSG399, cutting out gene with HindIII, inserting the gltA gene into Hind III-digested plasmid pMW218 and inserting the gene into an enteric bacterium.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel L-glutamic acid-producing bacterium and a method for producing L-glutamic acid by a fermentation method using the same. L-glutamic acid is a food,
It is an important amino acid for pharmaceuticals.

[0002]

2. Description of the Related Art Conventionally, L-glutamic acid is mainly produced by a fermentation method using a so-called coryneform L-glutamic acid-producing bacterium belonging to the genus Brevibacterium, Corynebacterium or Microbacterium, or a mutant thereof. (Amino Acid Fermentation, Academic Press, 19
5-215, 1986). As a method for producing L-glutamic acid by fermentation using other strains, methods using microorganisms such as Bacillus, Streptomyces, Penicillium (US Pat. No. 3,220,929), Pseudomonas, Arthro A method using microorganisms such as Bacter, Serratia, Candida (US Pat.
563,857), Bacillus, Pseudomonas,
Methods using microorganisms such as Serratia and Aerobacterium aerogenes (now Enterobacter aerogenes) are known (Japanese Patent Publication No. 32-9393), and a method using a mutant strain of Escherichia coli (JP-A-5-244970) is known. ing.

[0003] Although the productivity of L-glutamic acid has been considerably increased due to the improvement of the breeding and production methods of the microorganisms as described above, in order to respond to a further increase in demand in the future, a more inexpensive and efficient method is required. There is a need for the development of a method for producing L-glutamic acid.

In view of the above, the present inventor has extensively searched and studied microorganisms having L-glutamic acid-producing ability. As a result, the activity of enzymes (citrate synthase, phosphoenolpyruvate carboxylase, and glutamate dehydrogenase) that catalyze the biosynthesis reaction of L-glutamic acid in microorganisms belonging to the genus Enterobacter, Serratia, Klebsiella or Erwinia is increased. Thus, it has been found that microorganisms having high L-glutamic acid-producing ability can be obtained (Japanese Patent Application Nos. 10-224909 and 10-297129).

Further, a gene encoding citrate synthase (hereinafter, also referred to as “CS”) and phosphoenol pyruvate carboxylase derived from a bacterium belonging to the genus Escherichia is introduced into a valine-sensitive strain belonging to the genus Escherichia, and the enzymatic activities of these enzymes are introduced. To increase the L
-It has been found that a microorganism having glutamic acid-producing ability can be obtained (WO 97/08294).

On the other hand, in a bacterium of the genus Corynebacterium or Brevibacterium, introduction of a gene (CS gene) encoding a citrate synthase derived from Escherichia coli or Corynebacterium glutamicum is performed by L
-It has been reported that it was effective in enhancing glutamate-producing ability (Japanese Patent Publication No. 7-112228). When these coryneform bacteria were used as hosts, the introduction of CS gene derived from Corynebacterium glutamicum of the same species as the host showed a slightly higher effect than the introduction of CS gene derived from Escherichia coli,
No noticeable difference was seen.

As described above, it is known that the CS gene is introduced into various microorganisms to enhance L-glutamic acid-producing ability. However, microorganisms belonging to the group of intestinal bacteria including Escherichia bacteria are coryneform bacteria. There is no known example of introducing a derived CS gene.

[0008]

The problem to be solved by the present invention is that L-
It is an object of the present invention to find a novel L-glutamic acid-producing bacterium having a glutamic acid-producing ability and to develop an inexpensive and efficient method for producing L-glutamic acid.

[0009]

Means for Solving the Problems The present inventors conducted breeding by gene transfer in order to improve L-glutamic acid-producing ability of enterobacteria. Generally, in breeding microorganisms by gene amplification, when the host has a target gene, using a host-specific gene or a gene derived from a microorganism closely related to the host is preferable to introducing a heterologous gene. It is believed that a better effect can be obtained. However, the present inventors have found that, in the intestinal bacterial group, the introduction of a CS gene derived from a coryneform bacterium is extremely effective in enhancing L-glutamic acid-producing ability, as compared to a CS gene derived from a homologous microorganism. As a result, the present invention has been completed. That is, the present invention is as follows.

(1) A microorganism belonging to the group of enteric bacteria having an ability to produce L-glutamic acid, into which a citrate synthase gene derived from a coryneform bacterium has been introduced.

(2) The microorganism according to (1), wherein the coryneform bacterium is Brevibacterium lactofermentum.

(3) The microorganism according to (1) or (2), wherein the microorganism belonging to the enterobacterial group is a bacterium belonging to the genus Enterobacter or the genus Klebsiella.

(4) The microorganism according to (3), wherein the bacterium belongs to Enterobacter agglomerans or Klebsiella planticola.

(5) culturing the microorganism according to any one of the above (1) to (4) in a liquid medium, producing and accumulating L-glutamic acid in the medium, and collecting the L-glutamic acid from the medium. A method for producing L-glutamic acid, which is characterized in that:

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

<1> Microorganism of the Present Invention The microorganism belonging to the enterobacterial group of the present invention belongs to the enteric bacterial group, and is imparted with L-glutamic acid-producing ability by introducing a CS gene derived from a coryneform bacterium. There is no particular limitation as long as the ability to produce L-glutamic acid is improved, and examples include bacteria belonging to the genus Enterobacter, the genus Klebsiella, the genus Serratia, the genus Erwinia, the genus Pantoea, or the genus Escherichia. Among these, bacteria belonging to the genus Enterobacter or the genus Klebsiella are preferred. Hereinafter, specific examples of these bacteria will be shown, but the microorganism of the present invention is not limited thereto.

The following microorganisms belong to the genus Enterobacter used in the present invention. Enterobacter agg
lomerans) Enterobacter aerog
enes) Enterobacter amnig
enus) Enterobacter asburia
e) Enterobacter cloaca
e) Enterobacter dissolvens
solvens) Enterobacter gergovia
viae) Enterobacter horma
echei) Enterobacter intermedius
intermedius) Enterobacter nimipresalis (Enterobacter n)
imipressuralis) Enterobacter sakazaki
i) Enterobacter taylora
e)

More preferably, the following strains can be mentioned. Enterobacter agglomerans ATCC1228
7 Enterobacter agglomerans AJ13355 Enterobacter agglomerans AJ13355
On February 19, 1998, the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, Institute of Biotechnology and Industrial Technology, has accession number FERM P-1664.
No. 4 and transferred to an international deposit under the Budapest Treaty on January 11, 1999, with accession number FERM.
BP-6614 is given. Enterobacter agglomerans ATCC 12287 is also a registered trademark of ATC
You can get a sale from C.

Enterobacter agglomerans AJ
13355 strain is a strain isolated from soil in Iwata City, Shizuoka Prefecture. The physiological properties of AJ13355 are described.

(1) Gram stainability: negative (2) Behavior to oxygen: facultative anaerobic (3) Catalase: positive (4) Oxidase: negative (5) Nitrate reducing ability: negative (6) Foges-Proscauer test : Positive (7) Methyl red test: Negative (8) Urease: Negative (9) Indole production: Positive (10) Motility: Yes (11) Hydrogen sulfide production in TSI medium: Weak activity (12) β- Galactosidase: positive

(13) Sugar assimilation: arabinose: positive sucrose: positive lactose: positive xylose: positive sorbitol: positive inositol: positive trehalose: positive maltose: positive melibiose: positive adonitol: negative raffinose: negative salicin: negative melibio positive

(14) Glycerose assimilation: positive (15) Organic acid assimilation: citric acid: positive tartaric acid: negative gluconic acid: positive acetic acid: positive malonic acid: negative (16) arginine dehydratase: negative (17) ol Tin decarboxylase: Negative (18) Lysine decarboxylase: Negative (19) Phenylalanine deaminase: Negative (20) Pigment formation Yellow (21) Gelatin liquefaction: Positive (22) Growth pH poor growth at pH 4, good growth at pH 4.5-7 (23) Growth temperature 25 ° C good growth, 30 ° C good growth,
37 ° C good, 42 ° C, 45 ° C

From these mycological properties, AJ13355 was determined to be Enterobacter agglomerans.

The following microorganisms belong to the genus Klebsiella used in the present invention. Klebsiella plantic
ola) K. terrigena, more preferably Klebsiella planticola
AJ13399. Klebsiella planticola AJ13399 was deposited on February 19, 1998 with the Ministry of International Trade and Industry's Institute of Biotechnology and Industrial Technology, under the accession number FERMP-16646.
Transferred to the International Deposit under the Budapest Treaty on January 11, 2001, and given the accession number FERM BP-6616.

Klebsierra Planticola AJ1
The 3399 strain is a strain isolated from the soil in Sapporo, Hokkaido. The physiological properties of AJ13399 are described below.

(1) Cell shape: Bacillus (2) Motility: None (3) Spores: None (4) Colony morphology on LabM nutrient agar: circular
The surface is smooth, creamy, smooth, raised and shiny. (5) Glucose OF test: Fermentation ability positive (6) Gram staining: negative (7) Behavior against oxygen: facultative anaerobic (8) Catalase: positive (9) Oxidase: negative (10) Urease: positive

(11) cytochrome oxidase: negative (12) β-galactosidase: positive (13) arginine dehydratase: negative (14) ornithine decarboxylase: negative (15) lysine decarboxylase: positive (16) tryptophan deaminase: negative (17) ) Foges-Proscowell test: Positive (18) Indole production: Positive (19) Hydrogen sulfide production in TSI medium: Negative (20) Citrate utilization: Positive

(21) M-hydroxybenzene acid assimilation: negative (22) Gelatin liquefaction: negative (23) Production of acid from sugar Glucose: positive Mannitol: positive Rhamnose: positive arabinose: positive sucrose: positive sorbitol : Positive inositol: Positive melibiose: Positive Amygdalin: Positive Adonitol-Peptone-Water: Positive Cellobiose-Peptone-Water: Positive Durucitol-Peptone-Water: Negative Raffinose-Peptone-Water: Positive (24) ℃ cannot grow

From these mycological properties, AJ13399
Was determined to be Klebsiella Planticola.

The microorganisms belonging to the genus Serratia used in the present invention include the following. Serratia liquefacien
ce) Serratia entomophila (Serratia entomophila) Serratia ficaria (Serratia fonticola) Serratia grimesi (Serratia grimesii) Serratia protea maculaans (Serratia proteamac)
ulans) Serratia odorifera Serratia plymuthica Serratia rubidaea

More preferably, the following strains can be mentioned. Serratia requesta ATCC14460 Serratia requesta ATCC14460
Can be obtained from the ATCC.

Microorganisms belonging to the genus Erwinia used in the present invention include the following. Erwinia herbicola (now Pantoea agglomeran)
s)) E. ananas E. cacticida E. chrysanthemi E. mallotivora E. mallotivora E. persicinus e. Psidi (E. psidii) E. quercina (E. quercina) E. rhapontici (E. rhapontici) E. rubrifaciens (E. salicis) E. salicis (E. uredovora)

More preferably, Ervinia herbicola IAM1595 (Pantair agglomerans)
AJ2666). Ervinia herbicola IAM 1595 can be ordered from the Institute for Molecular and Cellular Biology, The University of Tokyo. In addition, Bergey's Manual of Determinative Bacteriology (Bergey's Manual of Determinative Bacteriology)
The ninth edition does not describe Erwinia herbicola, and microorganisms classified as Erwinia herbicola are classified as Pantoea agglomerans. Thus, the microorganism belonging to the genus Erwinia and the microorganism belonging to the genus Pantoea are closely related to each other. Therefore, microorganisms belonging to the genus Pantoea can be used in the same manner as microorganisms belonging to the genus Erwinia. Microorganisms belonging to the genus Pantoea include Pantoea agglomerans and Pantoea dispersers (Pantoea
dispersa). Ervinia Herbicola
IAM1595 is Pantoea Agglomerans AJ
2666, and on February 25, 1999, the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology,
Deposited internationally as MBP-6660 under the Budapest Treaty.

The microorganism belonging to the genus Escherichia used in the present invention includes Escherichia coli
i).

More preferably, Escherichia coli having valine resistance, specifically, the following strains can be mentioned. Escherichia coli B11 Escherichia coli K-12 (ATCC10798) Escherichia coli B (ATCC11303) Escherichia coli W (ATCC9637) Escherichia coli K-12 (ATCC10798);
Escherichia coli B (ATCC 11303) and Escherichia coli W (ATCC 9637) can be obtained from the ATCC.

The genus Enterobacter as described above,
Metabolism of sugars by enterobacteria such as bacteria belonging to the genus Klebsiella, Serratia, Erwinia, Pantoea or Escherichia is performed via the Emden-Meyerhof pathway, and pyruvate produced in the pathway is under aerobic conditions. Oxidized in the tricarboxylic acid cycle. L-glutamic acid is biosynthesized by GDH or glutamine synthetase / glutamic acid synthase from α-ketoglutarate, which is an intermediate in the tricarboxylic acid cycle. As described above, the L-glutamic acid biosynthesis pathway is common in these microorganisms, and in the present invention, the microorganisms as described above form a single concept. Therefore, microorganisms belonging to the group of intestinal bacteria other than the above-described species or strains are also included in the present invention.

The microorganism of the present invention is a microorganism belonging to the intestinal bacteria group as described above, and is a microorganism into which a CS gene derived from a coryneform bacterium has been introduced and which has an ability to produce L-glutamic acid. Here, "has the ability to produce L-glutamic acid"
"It means having the ability to accumulate L-glutamic acid in the medium when cultured. The L-glutamic acid-producing ability may be a property of a wild-type strain, or may be a property imparted or enhanced by breeding. Alternatively, a microorganism to which L-glutamic acid-producing ability is imparted by introducing a CS gene derived from a coryneform bacterium may be used. Examples of the microorganisms belonging to the intestinal bacterial group to which L-glutamic acid-producing ability has been imparted or enhanced include, for example, a microorganism having an enhanced activity of an enzyme that catalyzes a biosynthesis reaction of L-glutamic acid, or a microorganism having an increased activity of L-glutamic acid. Microorganisms in which the activity of an enzyme that catalyzes a reaction that produces a compound other than L-glutamic acid by branching off from the synthesis pathway is reduced or deleted are exemplified. In addition, the activity of the enzyme that catalyzes the biosynthesis reaction of L-glutamic acid is increased, and the activity of the enzyme that catalyzes the reaction that produces a compound other than L-glutamic acid by branching off from the biosynthetic pathway of L-glutamic acid is decreased. Alternatively, a defective microorganism is also included.

Gl to be introduced into a microorganism belonging to the group of intestinal bacteria
Coryneform bacteria that serve as a source of the tA gene include bacteria that were previously classified into the genus Brevibacterium, but are now integrated into the genus Corynebacterium (Int. J. Syst. Bacter).
iol., 41, 255 (1981)), and also includes bacteria of the genus Brevibacterium which is closely related to the genus Corynebacterium. Examples of such coryneform bacteria include the following.

Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium carnae Corynebacterium glutamicum Corynebacterium lilium
Glutamicum) Corynebacterium meracecora Corynebacterium thermoaminogenes Corynebacterium herculis Brevibacterium dibaricatum (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium)
(Glutamicum) Brevibacterium inmariophyllum Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharisticacum Brevibacterium thiogenitalis Brevibacterium album Brevibacterium -Selinum microbacterium ammonia phyllum Specifically, the following strains can be exemplified.

Corynebacterium acetoacidophilum ATCC 13870 Corynebacterium acetoglutamicum ATCC1
5806 Corynebacterium alkanolicum ATCC2
1511 Corynebacterium karnamie ATCC15991 Corynebacterium glutamicum ATCC1302
0, 13032, 13060 Corynebacterium lilium (Corynebacterium
Glutamicum) ATCC15990 Corynebacterium meracecora ATCC1796
5 Corynebacterium thermoaminogenes AJ123
40 (FERM BP-1539) Corynebacterium herculis ATCC1386
8 Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC14020 Brevibacterium flavum (Corynebacterium
Glutamicum) ATCC13826, ATCC140
67 Brevibacterium inmariophilum ATCC1
4068 Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC 13665, A
TCC13869, Brevibacterium roseum ATCC13825 Brevibacterium saccharolyticum ATCC1
4066 Brevibacterium thiogenitalis ATCC 192
40 Brevibacterium album ATCC15111 Brevibacterium serinum ATCC15112 Microbacterium ammonia phyllum ATCC1
5354

The gltA gene derived from a coryneform bacterium is
It can be obtained by using a microorganism lacking S activity, for example, a mutant strain of a coryneform bacterium, and isolating a DNA fragment complementary to its auxotrophy from the chromosomal DNA of the coryneform bacterium. Since the nucleotide sequence of the gltA gene of coryneform bacteria has been elucidated (Microbiology, 1994, 14
0, 1817-1828), a primer can be synthesized based on the nucleotide sequence, and it can be obtained by a PCR method using chromosomal DNA as a template. Examples of the primer include oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 1 and 2.

In order to introduce a CS gene derived from a coryneform bacterium into a microorganism belonging to the group of intestinal bacteria, for example, the CS gene is cloned on a suitable plasmid, and the resulting recombinant plasmid is used as a host. The above-mentioned starting parent strain may be transformed. The copy number of the CS gene (hereinafter abbreviated as "gltA gene") in the cells of the transformed strain is increased, and as a result, CS activity is increased.

The plasmid is not particularly limited as long as it is a plasmid autonomously replicable in cells of a microorganism belonging to the enterobacteria group. For example, pUC19, pUC18, pBR322,
pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW11
9, pMW118, pMW219, pMW218 and the like. In addition, phage DNA vectors can be used.

The introduction of the gltA gene can also be achieved by allowing the gltA gene to be present, preferably in multiple copies, on the chromosomal DNA of the starting parent strain as a host. Gl on the chromosomal DNA of a microorganism belonging to the intestinal bacterial group
To introduce the tA gene in multiple copies, repetitive D
Sequences present in multiple copies on chromosomal DNA, such as NA and inverted repeats present at the end of transposable elements, can be used. Alternatively, it is also possible to mount the gltA gene on a transposon, transfer it, and introduce multiple copies on chromosomal DNA. G in the cells of the transformed strain
The copy number of the ltA gene is increased, resulting in increased CS activity.

Transformation can be performed, for example, by the method of DA Morrison (Methods in Enzymology 68, 326 (1979)) or a method of treating recipient cells with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A ., J. Mol. Biol., 53, 159
(1970)).

The gltA gene to be introduced is replaced with a promoter suitable for cells of a microorganism belonging to the group of intestinal bacteria, for example, lac, t, instead of the promoter specific to the gltA gene.
rp, it may be replaced with a promoter such as P _L.

Techniques such as gene cloning, DNA cleavage, ligation, and transformation methods are described in Molecular Clonin.
g, 2nd edition, Cold Spring Harbor press (1989))
In detail.

[0048] The microorganism of the present invention comprises g of coryneform bacterium.
In addition to the introduction of the ltA gene, the activity of enzymes other than CS that catalyze L-glutamic acid biosynthesis may be enhanced. Such enzymes that catalyze L-glutamic acid biosynthesis include glutamate dehydrogenase (GDH),
Glutamine synthase, glutamate synthase, isocitrate dehydrogenase, aconitate hydratase, phosphoenolpyruvate carboxylase (PE
PC), pyruvate dehydrogenase, pyruvate kinase, enolase, phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, fructosebisphosphate aldolase, phosphofructokinase, glucose phosphate And isomerase. Furthermore, the activity of an enzyme that catalyzes a reaction that branches off from the L-glutamic acid biosynthetic pathway to produce a compound other than L-glutamic acid may be reduced or lacking. Examples of an enzyme that catalyzes a reaction that produces a compound other than L-glutamic acid by branching off from the L-glutamic acid biosynthetic pathway include α-ketoglutarate dehydrogenase (αKGDH), isocitrate lyase, phosphate acetyltransferase,
There are acetate kinase, acetohydroxy acid synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, 1-pyrroline dehydrogenase, and the like. Of these enzymes, αKGDH is preferred.

Gene encoding PEPC, and GD
The gene encoding H is PEPC or GD, respectively.
Using a mutant strain lacking H activity, a DNA fragment that complements the auxotrophy can be obtained by isolating it from the chromosomal DNA of the microorganism. The nucleotide sequences of these genes of Escherichia bacteria and those of Corynebacterium bacteria have already been determined (B
iochemistry, Vol. 22, 5243-524
9, 1983; Biochem. , Volume 95,
909-916, 1984; Gene, 27,
193-199, 1984; Mol. Gen. Ge
net. 218, 330-339, 1989.
Year; Molecular Microbiology,
6, 317-326, 1992), primers were synthesized based on the respective nucleotide sequences, and chromosomal DNA was synthesized.
Can be obtained as a template by PCR.

In microorganisms belonging to the group of intestinal bacteria, the activity of the above-mentioned enzyme can be reduced or deleted by a conventional mutagenesis method or a genetic engineering technique by adding the gene of the enzyme to the cells in the cells. A mutation may be introduced so that the activity of the enzyme is reduced or deleted.

As a mutation treatment method, for example, a method of irradiating X-rays or ultraviolet rays, or N-methyl-N'-nitro-
There is a method of treating with a mutagen such as N-nitrosoguanidine. The site where the mutation is introduced into the gene may be a coding region encoding an enzyme protein or an expression control region such as a promoter.

Examples of the genetic engineering techniques include, for example, methods using gene recombination, transduction, cell fusion and the like. For example, a drug resistance gene is inserted into the cloned target gene to prepare a gene that has lost its function (deletion type gene). Next, the deletion gene is introduced into cells of a microorganism belonging to the genus Enterobacter or Serratia, and the target gene on the chromosome is replaced with the deletion gene using homologous recombination (gene disruption).

The activity of the target enzyme in the cell is reduced or deficient, and the degree of the reduction is determined by measuring the enzyme activity of the cell extract or purified fraction of the candidate strain and comparing it with the wild strain. Can be confirmed by For example,
αKGDH activity was determined by the method of Reed et al. (LJReed and BBM).
ukherjee, Methods in Enzymology 1969, 13, p.55-6
The enzyme activity can be measured according to 1).

Depending on the target enzyme, the target mutant can be selected depending on the phenotype of the mutant.
For example, a mutant strain in which αKGDH activity is deficient or reduced cannot grow on a minimal medium containing glucose or a minimal medium containing acetic acid or L-glutamic acid as a sole carbon source under aerobic culture conditions, or has a growth rate of less. It decreases significantly. However, even under the same conditions, normal growth can be achieved by adding succinic acid or L-lysine, L-methionine, and diaminopimelic acid to a minimal medium containing glucose. Using these phenomena as indices, αK
It is possible to select a mutant strain in which GDH activity is deficient or reduced.

A method for producing an αKGDH gene-deficient strain of Brevibacterium lactofermentum utilizing homologous recombination is described in detail in WO95 / 34672, and the same applies to microorganisms belonging to the genera Enterobacter and Serratia. The method can be applied.

Specific examples of the mutant strains having αKGDH activity deficient or reduced as described above include Enterobacter agglomerans AJ13356 and Klebsiella planticola AJ13410. Enterobacter agglomerans AJ1
3356 and Klebsiella Planticola AJ1
3410, respectively, on February 19, 1998, the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry,
MP-16645 and FERM P-16647, transferred to the International Deposit under the Budapest Treaty on January 11, 1999, and deposited under accession number FERM BP-
6615 and FERM BP-6617. L-glutamic acid can be produced and accumulated in a culture medium by culturing a microorganism belonging to the intestinal bacteria group and having a gltA gene derived from a coryneform bacterium introduced therein in a liquid medium.

As the medium, a normal nutrient medium containing a carbon source, a nitrogen source, inorganic salts, and, if necessary, organic trace nutrients such as amino acids and vitamins can be used. Either a synthetic medium or a natural medium can be used. The carbon source and nitrogen source used in the medium may be those available for the strain to be cultured.

As the carbon source, sugars such as glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolyzate, molasses and the like are used. In addition, organic acids such as acetic acid, citric acid and the like are used alone or in addition. It is used in combination with a carbon source.

As the nitrogen source, ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate and ammonium acetate, nitrates and the like are used.

As the organic trace nutrients, amino acids, vitamins, fatty acids, nucleic acids, and peptones, casamino acids, yeast extracts, soybean protein decomposed products containing these, and the like are used. When a sex mutant is used, it is necessary to supplement the required nutrients.

As the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like are used.
As the culture method, aeration culture is performed while controlling the fermentation temperature to 20 to 42 ° C. and the pH to 4 to 8. By culturing for about 10 hours to 4 days, a remarkable amount of L
-Glutamic acid is accumulated.

After the completion of the culture, a method for isolating L-glutamic acid accumulated in the culture solution may be performed according to a known method. For example, it can be isolated by a method of removing the cells from the culture and then concentrating and crystallization, or by ion exchange chromatography.

[0063]

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

<1> Preparation of plasmid having gltA gene derived from Brevibacterium lactofermentum gl derived from Brevibacterium lactofermentum
The plasmid having the tA gene was constructed as follows. GltA of Corynebacterium glutamicum
Gene sequence (Microbiology, 1994, 140, 1817-18)
Based on 28), PCR was performed using primer DNAs having the nucleotide sequences shown in SEQ ID NOS: 1 and 2 and chromosomal DNA of Brevibacterium lactofermentum ATCC 13869 as a template to obtain a gltA gene fragment of about 3 kb. . This fragment was digested with SmaI.
This was inserted into SG399 (purchased from Takara Shuzo Co., Ltd.) to obtain a plasmid pHSGCB (FIG. 1). Next, pHSGCB
Was digested with HindIII and excised about 3 kb of gl.
Plasmid p obtained by digesting the tA gene fragment with HindIII
It was inserted into MW218 (purchased from Nippon Gene Co., Ltd.) to obtain plasmid pMWCB (FIG. 1). The expression of the gltA gene in the obtained plasmid pMWCB was confirmed by Enterobacter agglomerans AJ1335.
It was confirmed by measuring the enzyme activity in 5 strains.

<2> gltA derived from Escherichia coli
Preparation of plasmid having gene As a target, a plasmid having a gltA gene derived from Escherichia coli was constructed as follows. g
Plasmid pTWVC (WO97) having the ltA gene
No. 08294) was digested with HindIII and EcoRI, and a DNA fragment containing the gltA gene was purified and recovered, followed by plasmid pMW219 (Nippon Gene Co., Ltd.).
The plasmid pMWC was obtained by introducing the plasmid into HindIII and EcoRI sites (purchased from No. 3) (FIG. 2). The expression of the gltA gene in the obtained plasmid pMWC was confirmed by measurement of complementation of auxotrophy and enzyme activity of the gltA gene-deficient strain of Escherichia coli.

<3> Introduction of gltA gene into Enterobacter agglomerans and Klebsiella planticola, and production of L-glutamic acid Enterobacter agglomerans AJ13355 and Klebsiella planticola AJ13399 were used by pMWC or pMWCB. And transformed. The resulting transformant, AJ13355 / pMWC, AJ13355 / pMWC
B, AJ13399 / pMWC and AJ13399 / pMWCB were each prepared by adding glucose 40 g / L, ammonium sulfate 20 g / L, magnesium sulfate heptahydrate 0.5 g / L, potassium dihydrogen phosphate 2
g / L, sodium chloride 0.5 g / L, calcium chloride heptahydrate 0.25 g / L, ferrous sulfate heptahydrate 0.02 g / L
L, manganese sulfate tetrahydrate 0.02 g / L, zinc sulfate dihydrate 0.72 mg / L, copper sulfate pentahydrate 0.64 mg / L,
Cobalt chloride hexahydrate 0.72mg / L, boric acid 0.4m
g / L, sodium molybdate dihydrate 1.2 mg /
L, 2 g / L of yeast extract, and 20 g of a medium containing 30 g / L of calcium carbonate were inoculated into a 500-ml flask, and cultured with shaking at 37 ° C. for 15 hours. After the cultivation, the results of measuring L-glutamic acid and residual glucose accumulated in the culture solution are shown in Table 1.

[0067]

[Table 1] 蓄積 Strain L-Cultural acid accumulation Amount of glucose remaining (g / L) (g / L) ─ ───────────────────────── AJ13355 0 0 AJ13355 / pMWC 0.01 6.0 AJ13355 / pMWCB 0.78 28.5 AJ13399 0 0 AJ13399 / pMWC 2.85 0 AJ13399 / pMWCB 4.71 0 ──────────────────────────

Enterobacter agglomerans AJ1
3355 strains and Klebsiella planticola AJ1
In all 3399 strains, L-glutamic acid-producing ability was confirmed by introduction of the gltA gene. The accumulation of L-glutamic acid was more remarkable when the gltA gene derived from Brevibacterium lactofermentum was introduced than the gltA gene derived from Escherichia coli. AJ13
Under the above conditions, 355 / pMWCB did not consume a considerable amount of glucose and remained, but if cultured until all glucose was consumed, it would accumulate about 1.5 to 2 g / L of L-glutamic acid. Is expected.

AJ13355 / pMWC, AJ13355 / pMWCB, and AJ13355 / pMWCB
J13399 / pMWC and AJ13399 / pMWCB did not show a significant difference in the copy number of the respective plasmids.

[0070]

According to the present invention, it is possible to efficiently impart L-glutamic acid-producing ability to microorganisms belonging to the intestinal bacteria group. Therefore, a breeding method conventionally known for coryneform L-glutamic acid-producing bacteria is used. It is considered that higher productivity can be imparted by using such methods. In addition, it is expected that studies on culture conditions and the like will lead to the development of an inexpensive and efficient method for producing L-glutamic acid.

[0071]

[Sequence List] SEQUENCE LISTING <110> Ajinomoto Co., Inc. <120> L-glutamic acid producing bacteria and method for producing L-glutamic acid <130> P-6906 <141> 1999-10-15 < 150> JP 10-297350 <151> 1998-10-19 <160> 2 <170> PatentIn Ver. 2.0

<210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 1 gtcgacaata gccygaatct gttctggtcg 30

<210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 2 aagcttatcg acgctcccct ccccaccgtt 30

[Brief description of the drawings]

FIG. 1. Plasmid pMWC having gltA gene
The figure which shows construction of B.

FIG. 2 Plasmid pMWC having gltA gene
FIG.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:13) (C12N 15/09 ZNA C12R 1:19) (C12N 1/21 C12R 1:01) ( (C12N 1/21 C12R 1:22) (C12N 1/21 C12R 1:13) (C12P 13/14 C12R 1:22) (C12P 13/14 C12R 1:01) (72) Inventor Yoshihiko Hara Kawasaki-shi, Kanagawa 1-1, Suzuki-cho, Kawasaki-ku Ajinomoto Co., Ltd. Fermentation Technology Research Laboratories (72) Inventor Hisao Ito 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-ku, Kanagawa Prefecture Ajinomoto Co., Ltd.

Claims (5)

[Claims] 1. A microorganism belonging to the intestinal bacterium group having a citrate synthase gene derived from a coryneform bacterium and capable of producing L-glutamic acid. 2. The microorganism according to claim 1, wherein the coryneform bacterium is Brevibacterium lactofermentum. 3. The microorganism according to claim 1, wherein the microorganism belonging to the group of intestinal bacteria is a bacterium belonging to the genus Enterobacter or the genus Klebsiella. 4. The microorganism according to claim 3, wherein the bacterium belongs to Enterobacter agglomerans or Klebsiella planticola. 5. The method according to claim 1, wherein the microorganism according to claim 1 is cultured in a liquid medium, L-glutamic acid is produced and accumulated in the medium, and the L-glutamic acid is collected from the medium. -A method for producing glutamic acid.