JP2000189169A - L-glutamate productive bacterium and production of l- glutamic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an L-plutamate productive bacterium having high L- glutamate producibility and contribute to the development of the inexpensive and efficient production of L-glutamic acid. SOLUTION: This glutamate productive bacterium is a strain which belongs to the genus Enterobactor or Serratia and is enhanced in the enzyme activity for catalyzing the biosynthetic reaction of L-glutamic acid or a strain which is branched from the pathway of the biosynthesis of L-glutamic acid and decreased or lost in the enzyme activity for catalyzing the reaction of forming compounds other than L-glutamic acid and the production of L-glutamic acid is carried out by cultivating microorganisms which have L-glutamate producibility in a culture medium and separating the resultant L-glutamic acid from the medium.

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.

[0004]

An object of the present invention is to find a novel L-glutamic acid-producing bacterium having a high L-glutamic acid-producing ability and to develop an inexpensive and efficient method for producing L-glutamic acid. is there.

[0005]

Means for Solving the Problems In order to solve the above problems, the present inventors have extensively searched and studied microorganisms different from conventional microorganisms and capable of producing L-glutamic acid. As a result, they have found that a derivative derived from a microorganism belonging to the genus Enterobacter or Serratia has a high L-glutamic acid-producing ability, and have completed the present invention.

That is, the present invention is as follows.

(1) A microorganism belonging to the genus Enterobacter or the genus Serratia, having at least one of the following properties, and having an ability to produce L-glutamic acid: (a) an enzyme that catalyzes the biosynthesis reaction of L-glutamic acid (B) The activity of an enzyme that catalyzes a reaction that diverges from the L-glutamic acid biosynthetic pathway to produce a compound other than L-glutamic acid is reduced or defective.

(2) Citrate synthase (hereinafter abbreviated as “CS”), phosphoenolpyruvate carboxylase (hereinafter abbreviated as “PEPC”), and glutamate dehydrogenase (hereinafter abbreviated as “PEPC”) are enzymes that catalyze the biosynthesis reaction of L-glutamic acid. (Hereinafter abbreviated as "GDH").

(3) The microorganism according to (2), wherein the enzymes that catalyze the biosynthesis reaction of L-glutamic acid are all citrate synthase, phosphoenolpyruvate carboxylase and glutamate dehydrogenase.

(4) The enzyme which catalyzes a reaction for producing a compound other than L-glutamic acid by branching off from the L-glutamic acid biosynthetic pathway is α-ketoglutarate dehydrogenase (hereinafter abbreviated as “αKGDH”). ) ~
The microorganism according to any one of (3).

(5) The microorganism according to any one of (1) to (4), wherein the microorganism belongs to Enterobacter agglomerans or Serratia requifaciens.

(6) The microorganism according to any one of (1) to (5), wherein the microorganism 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.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The microorganism of the present invention is a microorganism belonging to the genus Enterobacter or the genus Serratia, and has at least one of the following properties. (A) The activity of an enzyme that catalyzes a biosynthesis reaction of L-glutamic acid is enhanced. (B) L-glutamic acid is diverged from the biosynthetic pathway to produce L-glutamic acid.
The activity of an enzyme that catalyzes a reaction for producing a compound other than glutamic acid is reduced or lacking.

Such a microorganism can be obtained by using a microorganism belonging to the genus Enterobacter or the genus Serratia as a parent strain and imparting the properties (a) and / or (b). The following microorganisms belong to the genus Enterobacter or Serratia used as parent strains.

[0015] Enterobacter agglomerans
robacter agglomerans 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) 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. Enterobacter agglomerans ATCC1228
7 Enterobacter agglomerans AJ13355 Serratia requesteaciens ATCC14460

Enterobacter agglomerans AJ
13355 is registered on February 19, 1998 by the Ministry of International Trade and Industry,
Deposit No. -16644, transferred to an international deposit based on the Budapest Treaty on January 11, 1999, and given the accession number FERM BP-6614. In addition, Enterobacter agglomerans ATCC1228
7, Serratia request faculty ATCC 1446
0 can be obtained from the ATCC.

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: negative (4) Oxidase: positive (5) Nitrate reducing ability: negative (6) Fogges-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 utilization: Arabinose: Positive Sucrose: Positive Lactose: Positive Xylose: Positive Sorbitol: Positive Inositol: Positive Trehalose: Positive Maltose: Positive Melibiose: Positive Adonitol: Negative Raffinose: 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) Pigmentation: Yellow (21) Gelatin liquefaction: Positive (22) Growth pH Poor growth, pH 4.5-7 growth Good (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.

In the examples described below, a strain in which the activity of an enzyme that catalyzes the biosynthesis reaction of L-glutamic acid is enhanced, or a reaction in which a compound other than L-glutamic acid is branched off from the biosynthetic pathway of L-glutamic acid to produce a compound other than L-glutamic acid Agromerans ATCC1 as a starting parent strain for obtaining a strain with reduced or deficient activity of an enzyme that catalyzes
2287, Enterobacter agglomerans AJ1
3355, or Serratia Riquefaciens A
TCC14460 was used. However, sugar metabolism by bacteria belonging to the genus Enterobacter or Serratia is performed via the Emden-Meierhof pathway, and pyruvate produced in the pathway is oxidized in the tricarboxylic acid cycle under aerobic conditions. 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. Thus, the L-glutamic acid biosynthesis pathway is common in these microorganisms, and in the present invention, microorganisms belonging to the genus Enterobacter and the genus Serratia form a single concept. Therefore, microorganisms belonging to the genus Enterobacter and the genus Serratia 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 genus Enterobacter or Serratia as described above,
It is a microorganism capable of producing L-glutamic acid. Here, “has the ability to produce L-glutamic acid” means that it has the ability to accumulate L-glutamic acid in the medium when cultured. In the present invention, L-glutamic acid-producing ability is imparted by imparting one or both of the following properties.

(A) The activity of an enzyme that catalyzes a biosynthesis reaction of L-glutamic acid is enhanced. (B) L-glutamic acid is diverged from the biosynthetic pathway to produce L-glutamic acid.
The activity of an enzyme that catalyzes a reaction for producing a compound other than glutamic acid is reduced or lacking.

Enzymes that catalyze the biosynthesis of L-glutamic acid by microorganisms of the genus Enterobacter or Serratia include GDH, glutamine synthetase, glutamate synthase, isocitrate dehydrogenase, aconitate hydratase, CS, PEPC, pyruvate dehydrogenase, and the like. Examples include pyruvate kinase, enolase, phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, fructosebisphosphate aldolase, phosphofructokinase, glucose phosphate isomerase and the like. Among these enzymes,
One, two or three of CS, PEPC and GDH are preferred. Further, in the microorganism of the present invention, it is preferable that the activities of all three enzymes, CS, PEPC and GDH, are enhanced. The activity of the target enzyme in the cell is increased, and the degree of the increase can be confirmed by measuring the enzyme activity of the microbial cell extract or purified fraction and comparing it with the wild-type or parent strain. .

The microorganism which belongs to the genus Enterobacter or the genus Serratia and has an enhanced activity of one or more enzymes that catalyze the biosynthesis reaction of L-glutamic acid includes, for example, Using the above microorganism as a starting parent strain, it can be obtained as a mutant strain in which a gene encoding these enzymes has been mutated, or as a genetically modified strain.

In order to enhance CS, PEPC or GDH activity, for example, a gene encoding CS, PEPC or GDH is cloned into a suitable plasmid, and the obtained parent strain is transformed into the host strain using the obtained plasmid. do it. Intracellular CS, PEPC in transformed cells
And genes encoding GDH (hereinafter referred to as "gltA gene", "ppc gene", "gdh
A gene) and the copy number of C gene
S, PEPC and GDH activities are increased.

The cloned gltA gene, pp
The c gene and the gdhA gene are introduced into the starting parent strain alone or in any combination of two or three. When introducing two or three types of genes, two or three types of genes may be cloned on one type of plasmid and introduced into a host, or two or three types of compatible plasmids may be introduced. Cloned separately and introduced into a host. The plasmid is not particularly limited as long as it is a plasmid that can autonomously replicate in cells of a microorganism belonging to the genus Enterobacter or Serratia.For example, pUC19, pUC18, pBR322, pHSG299, pHSG298,
pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW21
9, pMW218 and the like. In addition, phage DNA vectors can be used. Transformation can be performed, for example, by using DMMorris.
on (Methods in Enzymology 68, 326 (1979)) or a method in which recipient cells are treated with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A., J. Mol. Bio
l., 53, 159 (1970)).

[0030] Increasing CS, PEPC or GDH activity can be achieved by the gltA gene, the ppc gene or the gdhA gene.
The gene can also be achieved by allowing multiple copies of the gene to be present on the chromosomal DNA of the starting parent strain as a host. To introduce the gltA gene, the ppc gene, or the gdhA gene in multiple copies on the chromosomal DNA of a microorganism belonging to the genus Enterobacter or Serratia, repetitive DNA, inverted repeats present at the ends of transposable elements, and the like, Sequences present in multiple copies on chromosomal DNA can be used. Alternatively, it is also possible to mount the gltA gene, ppc gene, or gdhA gene on a transposon, transfer it, and introduce multiple copies into chromosomal DNA. The copy number of the gltA gene, the ppc gene, or the gdhA gene in the cells of the transformed strain is increased, and as a result, CS, PEPC or GDH activity is increased.

The gltA gene that increases copy number, p
As an organism that is a source of the pc gene and the gdhA gene, any organism having CS, PEPC and GDH activity may be used. Of these, bacteria that are prokaryotes, for example, bacteria belonging to the genera Enterobacter, Klebsiella, Erwinia, Pantoea, Serratia, Escherichia, Corynebacterium, Brevibacterium, and Bacillus are preferred. A specific example is Escherichia coli. The gltA gene, the ppc gene, and the gdhA gene can be obtained from chromosomal DNA of a microorganism as described above.

The gltA gene, ppc gene, and g
The dhA gene is CS, PEPC or GD, respectively.
It can be obtained by isolating a DNA fragment complementary to its auxotrophy from the chromosomal DNA of the microorganism using a mutant strain lacking H activity. In addition, the nucleotide sequences of these genes of the genus Escherichia and those of the genus Corynebacterium have already been determined (Bioc
hemistry, Vol. 22, pp. 5243-5249,
1983; Biochem. 95, 909
916, 1984; Gene, 27, 193.
199, 1984; Microbiology,
140, 1817-1828, 1994; Mo
l. Gen. Genet. 218, 330-33
9, 1989; Molecular Microb
iology, Vol. 6, pp. 317-326, 1992
Year) It is possible to synthesize primers based on each base sequence and obtain them by PCR using chromosomal DNA as a template.

The CS, PEPC or GDH activity can be enhanced by enhancing the expression of the gltA gene, the ppc gene or the gdhA gene in addition to the above gene amplification. For example, expression is enhanced by replacing the promoter of the gltA gene, ppc gene, or gdhA gene with another stronger promoter. For example, lac promoter, trp promoter, trc promoter, ta
c promoter, P _R promoter of lambda phage,
The P _L promoter and the like are known as strong promoters. The gltA gene with the promoter replaced, p
The pc gene or the gdhA gene is cloned into a plasmid and introduced into the host microorganism, or the chromosomal DNA of the host microorganism using repetitive DNA, inverted repeat, transposon, or the like.
Introduced above.

In order to enhance CS, PEPC or GDH activity, the promoter of the gltA gene, ppc gene or gdhA gene on the chromosome is replaced by a stronger promoter (WO 87/03006).
Or the insertion of a strong promoter upstream of the coding sequence for each gene (see Gene, 29, (1984) 231-241). it can. Specifically, a DN containing a gltA gene, a ppc gene or a gdhA gene or a part thereof replaced with a strong promoter
The homologous recombination may be caused between A and the corresponding gene on the chromosome.

Specific examples of microorganisms belonging to the genus Enterobacter or Serratia having enhanced CS, PEPC or GDH activity include Enterobacter agglomerans ATCC12287 / RSFCPG, Enterobacter agglomerans AJ13355 / RSFCPG,
And Serratia requesta ATCC 144
60 / RSFCPG.

Enzymes that catalyze the reaction that branches off from the L-glutamic acid biosynthetic pathway to produce a compound other than L-glutamic acid include αKGDH, isocitrate lyase, phosphate acetyltransferase, acetate kinase, acetohydroxy acid synthase, and the like. Examples include acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, and 1-pyrroline dehydrogenase. Of these enzymes, αKGDH is preferred.

In a microorganism belonging to the genus Enterobacter or the genus Serratia, the activity of the above enzyme can be reduced or deleted by a conventional mutation treatment method or a genetic engineering technique by adding the gene of the enzyme to
What is necessary is just to introduce | transduce the mutation which reduces or loses the activity of the said enzyme in a cell.

As the 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.

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 produce a gene that has lost its function (defective gene). Next, the defective 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 defective gene by using homologous recombination (gene disruption).

The activity of the target enzyme in the cell is reduced or deficient, and the degree of the decrease is determined by measuring the enzyme activity of the cell extract or purified fraction of the candidate strain, and comparing with the wild strain or the parent strain. It can be confirmed by comparing. For example, αKGDH activity can be determined by the method of Reed et al. (LJRe
ed and BBMukherjee, Methods in Enzymology 1969,1
3, p.55-61).

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 becomes possible by adding succinic acid or lysine, methionine, and diaminopimelic acid to a minimal medium containing glucose. Using these phenomena as indicators, it is possible to select mutant strains in which αKGDH 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. For other techniques such as gene cloning, DNA cleavage, ligation, and transformation methods, see Molecular Cloning, 2nd edition, Cold
Spring Harbor press (1989)).

Specific examples of the mutant strain obtained as described above, in which αKGDH activity is deficient or reduced, include Enterobacter agglomerans AJ13356. Enterobacter agglomerans AJ13
356 was issued by the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, Biotechnology and Industrial Technology Research Institute on February 19, 1998, under the accession number FERM P-1.
6645, transferred to the International Deposit under the Budapest Treaty on January 11, 1999, and received accession number FE.
RM BP-6615 is provided.

By culturing a microorganism belonging to the genus Enterobacter or the genus Serratia, having at least one of the properties (a) and (b), and capable of producing L-glutamic acid in a liquid medium, the L-glutamic acid can be expressed in the medium. Glutamic acid can be produced and accumulated.

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 can be 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 organic micronutrients, 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 completion of the cultivation, L-glutamic acid accumulated in the culture solution may be isolated 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.

[0051]

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

(1) Preparation of plasmid having gltA gene, ppc gene and gdhA gene The procedure for preparing a plasmid having gltA gene, ppc gene and gdhA gene will be described with reference to FIGS.

A plasmid pBRGDH having a gdhA gene derived from Escherichia coli (Japanese Patent Laid-Open No. 7-2039)
No. 80) was digested with HindIII and SphI, and T4DN
After blunting both ends by A polymerase treatment, gd
The DNA fragment having the hA gene was purified and recovered. on the other hand,
GltA gene and ppc from Escherichia coli
Plasmid pMWCP having the gene (WO97 / 08
294) was digested with XbaI, and both ends were made blunt with T4 DNA polymerase. To this, the g purified above
After mixing the DNA fragments having the dhA gene, they were ligated with T4 ligase to obtain a plasmid pMWCPG further carrying the gdhA gene in pMWCP (FIG. 1). Also,
After purifying and recovering a DNA fragment having the gdhA gene obtained by digesting pBRGDH with HindIII and SalI,
H of plasmid pSTV29 (purchased from Takara Shuzo Co., Ltd.)
Plasmid pSTVG was obtained by introducing into the indIII and SalI sites (FIG. 2).

At the same time, the broad host range plasmid RSF101
The plasmid pVIC40 having an origin of replication of 0 (Japanese Patent Laid-Open No. 08-047397) was digested with NotI, and T4 DNA was digested.
After digestion with PstI,
After BR322 was digested with EcoT141, treated with T4 DNA polymerase, and mixed with PstI digestion,
Plasmid R ligated by T4 ligase and having the RSF1010 origin of replication and the tetracycline resistance gene
SF-Tet was obtained (FIG. 3).

Next, pMWCPG was converted to EcoRI, Pst
I digestion and the gltA gene, ppc gene, and gd
The DNA fragment having the hA gene was purified and recovered, and RSF-
Tet was similarly digested with EcoRI and PstI, and RSF1 was digested.
After mixing with a purified and recovered DNA fragment having an origin of replication of 010, it was ligated with T4 ligase and RSF-Te
The gltA gene, ppc gene, and gdhA
The plasmid RSFCPG carrying the gene was obtained (FIG. 4). The resulting plasmid RSFCPG was used to replace the gltA gene, the ppc gene and the gdhA gene with pSTVG.
Expresses the gdhA gene according to the presence of the gltA gene, the ppc gene, or the gdA gene of Escherichia coli.
Complementation of the auxotrophy of the hA gene-deficient strain and confirmation of the activity of each enzyme were confirmed.

A plasmid having the gltA gene derived from Brevibacterium lactofermentum was constructed as follows. The nucleotide sequence of the gltA gene of Corynebacterium glutamicum (Microbiology, 1994,
140, 1817-1828), PCR was carried out using primer DNA having the nucleotide sequences shown in SEQ ID NOs: 6 and 7 and the chromosomal DNA of Brevibacterium lactofermentum ATCC 13869 as a template, and an approximately 3 kb gl was obtained.
A tA gene fragment was obtained. This fragment was inserted into SmaI-digested plasmid pHSG399 (purchased from Takara Shuzo Co., Ltd.) to obtain plasmid pHSGCB (FIG. 5). next,
pHSGCB was digested with HindIII and the cut out gltA gene fragment of about 3 kb was inserted into HindIII digested plasmid pMW218 (purchased from Nippon Gene Co., Ltd.) to obtain plasmid pMWCB (FIG. 5).
The expression of the gltA gene in the obtained plasmid pMWCB was confirmed by Enterobacter agglomerans A.
It was confirmed by measuring the enzyme activity in the J13355 strain.

(2) RSFCP to Enterobacter agglomerans or Serratia requifaciens
Introduction of G, pMWCB and pSTVG and evaluation of L-glutamic acid productivity Enterobacter agglomerans ATCC12287 using RSFCPG, pMWCB and pSTVG
Strain, Enterobacter agglomerans AJ1335
5 strains, or Serratia request faculty ATC
C14460 was prepared by electroporation (Miller J.
H., "A Short Course in Bacterial Genetics; Handboo
k "Cold Spring Harbor Laboratory Press, USA, 199
Transformation was performed according to 2), and a transformant showing tetracycline resistance was obtained.

The obtained transformant or each parent strain was subjected to 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. L, calcium chloride heptahydrate 0.25 g / L, ferrous sulfate heptahydrate 0.0
2 g / 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 0.72 mg / L, boric acid 0.4 mg / L, sodium molybdate dihydrate 1.2
mg / L, yeast extract 2g / L, calcium carbonate 30g
The medium was inoculated into a large 50 ml test tube into which 5 ml of the medium containing / L was injected, and cultured at 37 ° C. with shaking until the sugar in the medium was consumed. However, AJ13355 / pMWCB
The strain and the AJ13355 / pSTVG strain had a slower sugar consumption rate, and thus had the same culture time 1 as the parent strain AJ13355.
When about 10 g / L of sugar was consumed in 5 hours, the culture was stopped. For culture of the transformant, 25 mg / L of tetracycline was added. After the completion of the culture, the results of measurement of L-glutamic acid accumulated in the culture solution are shown in Table 1.

[0059]

Table 1 Table 1 L-glutamic acid accumulated amount 量 Strain L-glutamic acid accumulated amount ─ ────────────────────────────── ATCC12287 0.0 g / L ATCC12287 / RSFCPG 6.1 AJ13355 0.0 AJ13355 / RSFCPG 3 0.3 AJ13355 / pMWCB 0.8 AJ13355 / pSTVG 0.8 ATCC14460 0.0 ATCC14460 / RSFCPG 2.8 Medium only 0.2───────────────────── ───────────

[0060] Enterobacter agglomerans AT
The CC12287 strain, the Enterobacter agglomerans AJ13355 strain, or the Serratia requifaciens ATCC14460 strain did not accumulate L-glutamic acid, but the strains that amplified CS, PEPC, and GDH activities by introducing RSFCPG were:
6.1 g / L, 3.3 g / L and 2.8 g / L of L-glutamic acid were accumulated, respectively. In addition, 0.8 g / L of L-glutamic acid was accumulated by amplifying only the CS activity of AJ13355 strain, and 0.8 g / L of L-glutamic acid was also accumulated by amplifying only the GDH activity.

(3) The αKGDH gene of Enterobacter agglomerans AJ13355 (hereinafter referred to as “suc
AB ") Enterobacter agglomerans The sucAB gene of the AJ13355 strain is αKGD of Escherichia coli.
A DNA fragment complementary to the non-assimilation of acetate of a strain lacking the H-E1 subunit gene (hereinafter referred to as "sucA") was cloned by selecting from the chromosomal DNA of the Enterobacter agglomerans AJ13355 strain.

Enterobacter agglomerans AJ
The chromosomal DNA of the 13355 strain can be obtained by the same method as used for extracting chromosomal DNA in Escherichia coli (Bioengineering Experiments, Japan Biotechnology Society, 97
-98, Baifukan, 1992). As pTWV228 (ampicillin resistance) used as a vector, a commercial product manufactured by Takara Shuzo was used.

The chromosomal DNA of AJ13355 strain was
Digested with T221 and pTWV228 in Ps
The digested with tI was ligated with T4 ligase and su
cA-deficient Escherichia coli JRG465 strain (He
bert J. et al. Mol. Gen. Genetic
s 1969, 105, 182).
From the thus obtained transformants, a strain that grows in an acetic acid minimal medium was selected, and a plasmid was extracted therefrom to obtain pTWV
It was named EK101. The Escherichia coli JRG465 strain having pTWVEK101 restored the requirement for succinic acid or L-lysine and L-methionine in addition to the trait of non-assimilation of acetic acid. From this, pTWV
EK101 contains Enterobacter agglomerans
The ucA gene is considered to be included.

The Enterobacter pTWVEK101
FIG. 6 shows a restriction enzyme map of the DNA fragment derived from agglomerans. The result of determining the base sequence of the portion shown by the diagonal lines in FIG. 6 is shown in SEQ ID NO: 1. In this sequence, two full-length ORFs and a nucleotide sequence that was considered to be a partial sequence of the two ORFs were found. The amino acid sequences that can be encoded by these ORFs or partial sequences thereof are shown in SEQ ID NOs: 2 to 5 in order from the 5 'side. As a result of these homology searches, the base sequence determined was the partial sequence at the 3 'end of the succinate dehydrogenase iron-sulfur protein gene (sdhB), the full-length suc
A and αKGDH-E2 subunit gene (sucB gene) and succinyl-CoA synthetase β subunit gene (sucC gene) were found to contain partial sequences at the 5 ′ end. Amino acid sequences deduced from these nucleotide sequences were obtained from Escherichia coli (Eur. J. Biochem., 141, 351-359 (1984), Eur.
J. Biochem., 141, 361-374 (1984), Biochemistry, 24,
6245-6252 (1985)) are shown in FIGS. Thus, each amino acid sequence showed very high homology. In addition, Escherichia coli on the chromosome of Enterobacter agglomerans (Eur. J. Biochem.,
141, 351-359 (1984), Eur. J. Biochem., 141, 361-37.
4 (1984), Biochemistry, 24, 6245-6252 (1985)), s
It was found that dhB-sucA-sucB-sucC constituted a cluster.

(4) Acquisition of αKGDH-Deficient Strain Derived from Enterobacter agglomerans AJ13355 Using the sacAB gene of Enterobacter agglomerans obtained as described above, an αKGDH-deficient strain of Enterobacter agglomerans was obtained by homologous recombination. Acquisition was done.

First, pTWVEK101 was digested with BglII to remove the C-terminal region corresponding to about half of the sucA gene and the full length of the sucB gene. Subsequently, a chloramphenicol resistance gene fragment cut out by AccI from pHSG399 (manufactured by Takara Shuzo) was inserted therein. The resulting sdhB-ΔsucAB :: C
the area of m ^r -sucC AflII, was cut out by SacI. Using the obtained DNA fragment, the Enterobacter agglomerans AJ13355 strain was transformed by electroporation, to obtain the strains chloramphenicol resistance, sucAB gene on the chromosome is the sucAB :: Cm ^r S considered to be replaced
ucAB-deficient strain Enterobacter agglomerans A
J13356 strains were acquired.

AJ13356 obtained as described above
To confirm that the strain lacks αKGDH activity, the method of Reed et al. (LJ Reed and BBMukherjee, Meth.
odsin Enzymology 1969, 13, p.55-61). As a result, as shown in Table 2, AJ133
In 56 strains, αKGDH activity could not be detected.
It was confirmed that cAB was missing.

[0068]

Table 2 αKGDH activity {Strain αKGDH activity (ΔABS / min / mg protein)} Ａ AJ13355 0.481 AJ13356 <0.0001 ─────────

(5) Evaluation of L-glutamic acid productivity of Enterobacter agglomerans αKGDH-deficient strains AJ13355 and AJ13356 were isolated from glucose 4
0 g / L, ammonium sulfate 20 g / L, magnesium sulfate heptahydrate 0.5 g / L, potassium dihydrogen phosphate 2 g / L
L, sodium chloride 0.5 g / L, calcium chloride heptahydrate 0.25 g / L, ferrous sulfate heptahydrate 0.02 g / 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 0.72 mg / L, boric acid 0.4 mg
/ L, sodium molybdate dihydrate 1.2 mg / L,
Yeast extract 2 g / L, calcium carbonate 30 g / L, L-
Lysine monohydrochloride 200 mg / L, L-methionine 200
mg / L, DL-α, ε-diaminopimelic acid (DA
P) A 500 ml flask in which 20 ml of a medium containing 200 mg / L was injected was inoculated, and cultured at 37 ° C. with shaking until the sugar in the medium was consumed. After the culture, the results of measurement of L-glutamic acid and α-ketoglutaric acid (hereinafter abbreviated as “αKG”) accumulated in the culture solution are shown in Table 3.

[0070]

Table 3 L-glutamic acid and αKG accumulation ────────────────────────────── strain L-glutamic acid accumulation αKG Accumulated amount ────────────────────────────── AJ13355 0.0 g / L 0.0 g / L AJ13356 1.5 3.2 ──────────────────────────────

AJ13356 deficient in αKGDH activity
The strain accumulated 1.5 g / L of L-glutamic acid. At the same time, 3.2 g / L of αKG was accumulated.

(6) Introduction of RSFCP into Enterobacter agglomerans αKGDH-deficient strain and evaluation of L-glutamic acid productivity The AJ13356 strain was transformed with RSFCPG, and the resulting RSFCPG-introduced strain AJ13356 / RS
FCPG was prepared from 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, manganese sulfate tetrahydrate 0.02
g / L, zinc sulfate dihydrate 0.72 mg / L, copper sulfate pentahydrate 0.64 mg / L, cobalt chloride hexahydrate 0.72 mg
/ L, boric acid 0.4 mg / L, sodium molybdate dihydrate 1.2 mg / L, yeast extract 2 g / L, tetracycline 25 mg / L, calcium carbonate 30 g / L, L
-Lysine monohydrochloride 200 mg / L, L-methionine 20
The flask was inoculated into a 500 ml flask in which 20 ml of a medium containing 0 mg / L and 200 mg / L of DL-α, ε-DAP was injected, and cultured at 37 ° C. with shaking until the sugar in the medium was consumed. After completion of the culture, the results of measuring L-glutamic acid and αKG accumulated in the culture solution are shown in Table 4.

[0073]

Table 4 L-glutamic acid and αKG accumulation ──────────────────────────────────── strain L-glutamic acid accumulation amount αKG accumulation amount ──────────────────────────────────── AJ13356 1.4 g / L 2.9 g / L AJ13356 / RSFCPG 5.1 0.0 ────────────────────────────────────

By introducing RSFCPG, CS, PEPC,
And, in the strains in which GDH activity was amplified, the amount of accumulated αKG was decreased, and the accumulation of L-glutamic acid was further improved.

[0075]

EFFECT OF THE INVENTION Since the microorganism of the present invention has a high L-glutamic acid-producing ability, it is considered that a higher productivity can be imparted by using a conventionally known breeding technique for coryneform L-glutamic acid-producing bacteria. 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.

[0076]

[Sequence List] SEQUENCE LISTING <110> Ajinomoto Co., Inc. <120> L-glutamic acid producing bacteria and method for producing L-glutamic acid <130> P-6389 <150> JP 10-69068 <151 > 1998-03-18 <150> JP 10-297129 <151> 1998-10-19 <160> 7 <170> PatentIn Ver. 2.0

<210> 1 <211> 4556 <212> DNA <213> Enterobacter agglomerans <220> <221> CDS <222> (2) .. (121) <220> <221> CDS <222> (322 ) .. (3129) <220> <221> CDS <222> (3145) .. (4368) <220> <221> CDS <222> (4437) .. (4556) <400> 1 t gca ttc agc gtt ttc cgc tgt cac agc atc atg aac tgt gta agt gtt 49 Ala Phe Ser Val Phe Arg Cys His Ser Ile Met Asn Cys Val Ser Val 1 5 10 15 tgt cct aaa ggg cta aac ccg acg cgc gct atc ggc cac att aag tcg 97 Cys Pro Lys Gly Leu Asn Pro Thr Arg Ala Ile Gly His Ile Lys Ser 20 25 30 atg ctg ctg caa cgc agc gcg tagttatacc accgggaacc tcaggttccc 148 Met Leu Leu Gln Arg Ser Ala 35 ggtc gcatt gcattc gcattc gtaaacag gcatt gcattc gtaaacg gcattc gtaaacg ctcactacgg caaaccgcga 268 aagcggcaac aaatgaaacc tcaaaaaagc ataacattgc ttaagggatc aca atg 324 Met 1 cag aac agc gcg atg aag ccc tgg ctg gac tcc tcc tgg ctg gcc ggc 372 Gln Asg Ser Pg Tg Asp Serg Asp Ser Gl Asp Ser Pg aat cag tct tac ata gag caa ctc ta t gag gat ttc ctg acc gat 420 Ala Asn Gln Ser Tyr Ile Glu Gln Leu Tyr Glu Asp Phe Leu Thr Asp 20 25 30 cct gac tct gtg gat gca gtg tgg cgc tcg atg ttc caa cag tta cca 468 Pro Asp Ser Val Asp Ala Val Trp Arg Ser Met Phe Gln Gln Leu Pro 35 40 45 ggc acg gga gtg aaa cct gag cag ttc cac tcc gca act cgc gaa tat 516 Gly Thr Gly Val Lys Pro Glu Gln Phe His Ser Ala Thr Arg Glu Tyr 50 55 60 65 ttc cgt cgc ctg gcg aaa gac gca tct cgt tac acc tcc tca gtt acc 564 Phe Arg Arg Leu Ala Lys Asp Ala Ser Arg Tyr Thr Ser Ser Val Thr 70 75 80 gat ccg gca acc aac tcc aaa caa gtg aaa gtg ctg cag ctg att aac 612 Asp Pro Ala Thr Asn Ser Lys Gln Val Lys Val Leu Gln Leu Ile Asn 85 90 95 gcg ttt cgt ttc cgc gga cat cag gaa gca aat ctc gat ccg ctt ggc 660 Ala Phe Arg Phe Arg Gly His Gln Glu Ala Asn Leu Asp Pro Leu Gly 100 105 110 ctg tgg aaa cag gac cgc gtt gcc gat ctc gat cct gcc ttt cac gat 708 Leu Trp Lys Gln Asp Arg Val Ala Asp Leu Asp Pro Ala Phe His Asp 115 120 125 ctg acc gac gcc gat ttt cag gaa agc ttt aa c gta ggt tct ttt gcc 756 Leu Thr Asp Ala Asp Phe Gln Glu Ser Phe Asn Val Gly Ser Phe Ala 130 135 140 145 att ggc aaa gaa acc atg aag ctg gcc gat ctg ttc gac gcg ctg aag 804 Ile Gly Lys Glu Thr Met Lys Leu Ala Asp Leu Phe Asp Ala Leu Lys 150 155 160 cag acc tac tgt ggc tcg att ggt gca gag tat atg cac atc aat aac 852 Gln Thr Tyr Cys Gly Ser Ile Gly Ala Glu Tyr Met His Ile Asn Asn 165 170 175 acc gaa gag aaa cgc tgg atc cag cag cgt atc gaa tcc ggt gcg agc 900 Thr Glu Glu Lys Arg Trp Ile Gln Gln Arg Ile Glu Ser Gly Ala Ser 180 185 190 cag acg tca ttc agt ggc gaa gag aaa aaa ggt ttc ctg 948 Gln Thr Ser Phe Ser Gly Glu Glu Lys Lys Gly Phe Leu Lys Glu Leu 195 200 205 acc gcg gca gaa ggg ctg gaa aaa tat ctg ggc gcg aaa ttc ccg ggt 996 Thr Ala Ala Glu Gly Leu Glu Lys Tyr Lys Phe Pro Gly 210 215 220 225 gca aaa cgt ttc tcg ctg gaa ggc ggt gat gcg ctg gtg ccg atg ctg 1044 Ala Lys Arg Phe Ser Leu Glu Gly Gly Asp Ala Leu Val Pro Met Leu 230 235 240 cgc gag atg g cg ggc aaa agc ggc aca cgt gaa gtg gta 1092 Arg Glu Met Ile Arg His Ala Gly Lys Ser Gly Thr Arg Glu Val Val 245 250 255 ctg ggg atg gcg cac cgt ggc cgt ctt aac gta ctg att aac gta Gly Met G140 G140 Ala His Arg Gly Arg Leu Asn Val Leu Ile Asn Val Leu 260 265 270 ggt aaa aag cca cag gat ctg ttc gac gaa ttc tcc ggt aaa cac aaa 1188 Gly Lys Lys Pro Gln Asp Leu Phe Asp Glu Phe Ser Gly Lys His Lys 275 280 285 gag cat ctg ggc acc ggt gat gtg aag tat cac atg ggc ttc tct tcg 1236 Glu His Leu Gly Thr Gly Asp Val Lys Tyr His Met Gly Phe Ser Ser 290 295 300 305 gat att gaa acc gaa ggt ggt ctg gtg cat gcg ctg gcg ttt aac 1284 Asp Ile Glu Thr Glu Gly Gly Leu Val His Leu Ala Leu Ala Phe Asn 310 315 320 ccg tct cac ctg gaa att gtc agc ccg gtg gtc atg gga tcg gta cgt 1332 Pro Ser His Leu Glu Pro Val Val Met Gly Ser Val Arg 325 330 335 gca cgt ctc gat cgt ctg gcc gaa ccg gtc agc aat aaa gtg ttg cct 1380 Ala Arg Leu Asp Arg Leu Ala Glu Pro Val Ser Asn Lys Val Leu Pro 340 345 350 atc ac c att cac ggt gat gcg gcg gtg att ggt cag ggc gtg gtt cag 1428 Ile Thr Ile His Gly Asp Ala Ala Val Ile Gly Gln Gly Val Val Gln 355 360 365 gaa acc ctg aac atg tct cag gcg cgc ggc gc gg ggc acg 1476 Glu Thr Leu Asn Met Ser Gln Ala Arg Gly Tyr Glu Val Gly Gly Thr 370 375 380 385 gta cgt atc gtc att aac aac cag gtt ggt ttt acc acc tcc aac ccg 1524 Val Arg Ile Val Ile Asn Asn Gln Val Gly Phe Thr Thr Ser Asn Pro 390 395 400 aaa gat gcg cgt tca acc ccg tac tgt act gac atc ggc aag atg gtg 1572 Lys Asp Ala Arg Ser Thr Pro Tyr Cys Thr Asp Ile Gly Lys Met Val 405 410 415 ctg gca ccg att ttc cac gtc aat gct gac gat ccg gaa gcg gtg gcc 1620 Leu Ala Pro Ile Phe His Val Asn Ala Asp Asp Pro Glu Ala Val Ala 420 425 430 ttt gtt acc cgc ctg gcg ctg gac tat cgc aac acc ttc aaa cgc gat 1668 Arg Leu Ala Leu Asp Tyr Arg Asn Thr Phe Lys Arg Asp 435 440 445 gtg ttt atc gat ctg gtg tgc tat cgc cgt cat ggt cac aac gag gcg 1716 Val Phe Ile Asp Leu Val Cys Tyr Arg Arg His Gly His Asn Glu 450 455 460 465 gat gag cca agt gct acc cag ccg ttg atg tac cag aaa atc aaa aag 1764 Asp Glu Pro Ser Ala Thr Gln Pro Leu Met Tyr Gln Lys Ile Lys Lys 470 475 480 cat ccg acg ccg cgt aaa att tac gcc gat cgt ctg gaa ggc gaa ggt 1812 His Pro Thr Pro Arg Lys Ile Tyr Ala Asp Arg Leu Glu Gly Glu Gly 485 490 495 gtc gcg tcg cag gaa gat gcc acc gag atg gtg aac ctg tac cgc gat 1860 G Alu Ala Ser Gln Thr Glu Met Val Asn Leu Tyr Arg Asp 500 505 510 gcg ctc gat gcg ggc gaa tgc gtg gtg ccg gaa tgg cgt ccg atg agc 1908 Ala Leu Asp Ala Gly Glu Cys Val Val Pro Glu Trp Arg Pro Met Ser 515 c tcc ttc acg tgg tcg cct tat ctg aac cac gaa tgg gat gag 1956 Leu His Ser Phe Thr Trp Ser Pro Tyr Leu Asn His Glu Trp Asp Glu 530 535 540 540 545 cct tat ccg gca cag gtt gac atg aaa cgc ctg agga ttg 2004 Pro Tyr Pro Ala Gln Val Asp Met Lys Arg Leu Lys Glu Leu Ala Leu 550 555 560 cgt atc agc cag gtc cct gag cag att gaa gtg cag tcg cgc gtg gcc 2052 Arg Ile Ser Gln Val Pro Glu Gln Ilu Glu Val Gln Ser Arg Val Ala 565 570 575 aag atc tat aac gat cgc aag ctg atg gcc gaa ggc gag aaa gcg ttc 2100 Lys Ile Tyr Asn Asp Arg Lys Leu Met Ala Glu Gly Glu Lys Ala Phe 580 585 590 gac ggg gag aat ctg gcg tac gcc acg ctg gtg gat gaa 2148 Asp Trp Gly Gly Ala Glu Asn Leu Ala Tyr Ala Thr Leu Val Asp Glu 595 600 605 ggt att ccg gtt cgc ctc tcg ggt gaa gac tcc ggtcgt gacc Pro Val Arg Leu Ser Gly Glu Asp Ser Gly Arg Gly Thr Phe 610 615 620 625 ttc cat cgc cac gcg gtc gtg cac aac cag gct aac ggt tca acc tat 2244 Phe His Arg His Ala Val Val His Asn Gln Ala Asn Gly Ser Thr Tyr 630 635 640 acg ccg ctg cac cat att cat aac agc cag ggc gag ttc aaa gtc tgg 2292 Thr Pro Leu His His Ile His Asn Ser Gln Gly Glu Phe Lys Val Trp 645 650 655 gat tcg gtg ctg tct gaga gag gc g gcg ttt gaa tac ggt tac 2340 Asp Ser Val Leu Ser Glu Glu Ala Val Leu Ala Phe Glu Tyr Gly Tyr 660 665 670 gcc acg gct gag ccg cgc gtg ctg acc atc tgg gaa gcg cag ttt ggt 2388 Ala Thr Ala Glu A rg Val Leu Thr Ile Trp Glu Ala Gln Phe Gly 675 680 685 gac ttt gcc aac ggt gct cag gtg gtg att gac cag ttc atc agc tct 2436 Asp Phe Ala Asn Gly Ala Gln Val Val Ile Asp Gln Phe Ile Ser Ser 690 6700 705 ggc gaa cag aag tgg ggc cgt atg tgt ggc ctg gtg atg ttg ctg ccg 2484 Gly Glu Gln Lys Trp Gly Arg Met Cys Gly Leu Val Met Leu Leu Pro 710 715 720 cat ggc tac gaa ggt cg gcc gcc gcc gcc cgt ctg gaa 2532 His Gly Tyr Glu Gly Gln Gly Pro Glu His Ser Ser Ala Arg Leu Glu 725 730 735 cgc tat ctg caa ctt tgc gcc gag cag aac atg cag gtt tgc gtc ccg 2580 Arg Tyr Leu Gln Leu Cys AG Met Gln Val Cys Val Pro 740 745 750 tcg acg ccg gct cag gtg tat cac atg ctg cgc cgt cag gcg ctg cgc 2628 Ser Thr Pro Ala Gln Val Tyr His Met Leu Arg Arg Gln Ala Leu Arg 755 760 765 ggg atgcgc ctg gtg gtg atg tcg ccg aag tcg ctg tta cgc 2676 Gly Met Arg Arg Pro Leu Val Val Met Ser Pro Lys Ser Leu Leu Arg 770 775 780 785 cat cat cca ctg gcg atc tcg tcg ctg gat gag ctg gca ag 4 His Pro Leu Ala Ile Ser Ser Leu Asp Glu Leu Ala Asn Gly Ser Phe 790 795 800 cag ccg gcc att ggt gag atc gac gat ctg gat ccg cag ggc gtg aaa 2772 Gln Pro Ala Ile Gly Glu Ile Asp Asp Leu Asp Pro Gln Gly Val Lys 805 810 815 cgc gtc gtg ctg tgc tcc ggt aag gtt tac tac gat ctg ctg gaa cag 2820 Arg Val Val Leu Cys Ser Gly Lys Val Tyr Tyr Asp Leu Leu Glu Gln 820 825 830 cgt cgt aaa gac gag gtt gcc atc gtg cgc atc gaa cag 2868 Arg Arg Lys Asp Glu Lys Thr Asp Val Ala Ile Val Arg Ile Glu Gln 835 840 845 ctt tac ccg ttc ccg cat cag gcg gta cag gaa gca ttg aaa gcc Tat Pro 2916 Leu His Gln Ala Val Gln Glu Ala Leu Lys Ala Tyr 850 855 860 865 tct cac gta cag gac ttt gtc tgg tgc cag gaa gag cct ctg aac cag 2964 Ser His Val Gln Asp Phe Val Trp Cys Gln Glu Glu Pro Leu Asn Gln 870 875 880 ggc gcc tgg tac tgt agc cag cat cat ttc cgt gat gtc gtg ccg ttt 3012 Gly Ala Trp Tyr Cys Ser Gln His His Phe Arg Asp Val Val Pro Phe 885 890 895 895 ggt gcc acc ctg cgt tat gca ggt cgc ccg gca tc gct tct ccg gcc 3060 Gly Ala Thr Leu Arg Tyr Ala Gly Arg Pro Ala Ser Ala Ser Pro Ala 900 905 910 gtg ggt tat atg tcc gta cac caa caa cag cag caa gac ctg gtt aat 3108 Val Gly Tyr Met Ser Val His Gln Gln Gln Gln Gln Asp Leu Val Asn 915 920 925 gac gca ctg aac gtc aat taattaaaag gaaagata atg agt agc gta gat 3159 Asp Ala Leu Asn Val Asn Met Ser Ser Val Asp 930 935 1 5 att ctc gtt ccc gac ctg cct gcat gat gcc aca gta gca 3207 Ile Leu Val Pro Asp Leu Pro Glu Ser Val Ala Asp Ala Thr Val Ala 10 15 20 acc tgg cac aag aaa cca ggc gat gca gtc agc cgc gat gaa gtc atc 3255 Thr Trp His Lys Lys Pro Gly Asp Ala Val Ser Arg Asp Glu Val Ile 25 30 35 gtc gaa att gaa act gac aaa gtc gtg ctg gaa gtg ccg gca tct gcc 3303 Val Glu Ile Glu Thr Asp Lys Val Val Leu Glu Val Pro Ala Ser Ala 40 45 50 gat ggc gtg ctg gaa gcc gtg ctg gaa gac gaa ggg gca acc gtt acg 3351 Asp Gly Val Leu Glu Ala Val Leu Glu Asp Glu Gly Ala Thr Val Thr 55 60 65 tcc cgc cag atc ctg ggt cgc ctg aaa gaa ggc gac agt gc 399 Ser Arg Gln Ile Leu Gly Arg Leu Lys Glu Gly Asn Ser Ala Gly Lys 70 75 80 85 gaa agc agt gcc aaa gcg gaa agc aat gac acc acg cca gcc cag cgt 3447 Glu Ser Ser Ala Lys Ala Glu Ser Asn Asp Thr Thr Pro Ala Gln Arg 90 95 100 cag aca gcg tcg ctt gaa gaa gag agc agc gat gcg ctc agc ccg gcg 3495 Gln Thr Ala Ser Leu Glu Glu Glu Ser Ser Asp Ala Leu Ser Pro Ala 105 110 115 atc cgt cgc ctg att g cat aat ctt gac gct gcg cag atc aaa 3543 Ile Arg Arg Leu Ile Ala Glu His Asn Leu Asp Ala Ala Gln Ile Lys 120 125 130 ggc acc ggc gta ggc gga cgt tta acg cgt gaa gac gtt gaa aaa cat 3591 Gly Thr G Val Gly Gly Arg Leu Thr Arg Glu Asp Val Glu Lys His 135 140 145 ctg gcg aac aaa ccg cag gct gag aaa gcc gcc gcg cca gcg gcg ggt 3639 Leu Ala Asn Lys Pro Gln Ala Glu Lys Ala Ala Ala Pro Ala Ala Gly 150 155 160 165 gca gca acg gct cag cag cct gtt gcc aac cgc agc gaa aaa cgt gtt 3687 Ala Ala Thr Ala Gln Gln Pro Val Ala Asn Arg Ser Glu Lys Arg Val 170 175 180 ccg atg acg cgt tta cgt aag cgc gtc gc gagctg ctg gaa gcc 3735 Pro Met Thr Arg Leu Arg Lys Arg Val Ala Glu Arg Leu Leu Glu Ala 185 190 195 aag aac agc acc gcc atg ttg acg acc ttc aac gaa atc aac atg aag 3783 Lys Asn Ser Thr Ala Met Leu Thr Phe Asn Glu Ile Asn Met Lys 200 205 210 ccg att atg gat ctg cgt aag cag tac ggc gat gcg ttc gag aag cgt 3831 Pro Ile Met Asp Leu Arg Lys Gln Tyr Gly Asp Ala Phe Glu Lys Arg 215 220 225 cac ggt ctg ggc ttt atg tct ttc tac atc aag gcc gtg gtc 3879 His Gly Val Arg Leu Gly Phe Met Ser Phe Tyr Ile Lys Ala Val Val 230 235 240 245 gaa gcg ctg aag cgt tat cca gaa gtc aac gcc tct atc gat 3 Glu Ala Leu Lys Arg Tyr Pro Glu Val Asn Ala Ser Ile Asp Gly Glu 250 255 260 gac gtg gtg tac cac aac tat ttc gat gtg agt att gcc gtc tct acg 3975 Asp Val Val Tyr His Asn Tyr Phe Asp Val Ser Ile Ala Val Ser Thr 265 270 275 cca cgc gga ctg gtg acg cct gtc ctg cgt gac gtt gat gcg ctg agc 4023 Pro Arg Gly Leu Val Thr Pro Val Leu Arg Asp Val Asp Ala Leu Ser 280 285 290 atg gct gac atc gag aag aaa a tt aaa gaa ctg gca gtg aaa ggc cgt 4071 Met Ala Asp Ile Glu Lys Lys Ile Lys Glu Leu Ala Val Lys Gly Arg 295 300 305 gac ggc aag ctg acg gtt gac gat ctg acg ggc ggt aac ttt acc atc 4119 Thr Val Asp Asp Leu Thr Gly Gly Asn Phe Thr Ile 310 315 320 325 acc aac ggt ggt gtg ttc ggt tcg ctg atg tct acg cca atc atc aac 4167 Thr Asn Gly Gly Val Phe Gly Ser Leu Met Ser Thr Pro Ile Ile Asn 330 335 340 ccg cca cag agc gcg att ctg ggc atg cac gcc att aaa gat cgt cct 4215 Pro Pro Gln Ser Ala Ile Leu Gly Met His Ala Ile Lys Asp Arg Pro 345 350 355 atg gcg gtc aat ggt cag gtt gtg atc ct atg tac ctg gct 4263 Met Ala Val Asn Gly Gln Val Val Ile Leu Pro Met Met Tyr Leu Ala 360 365 370 ctc tcc tac gat cac cgt tta atc gat ggt cgt gaa tct gtc ggc tat 4311 Leu Ser Tyr Asp His Arg Leu Ile Asp Gly Arg Glu Ser Val Gly Tyr 375 380 385 ctg gtc gcg gtg aaa gag atg ctg gaa gat ccg gcg cgt ctg ctg ctg 4359 Leu Val Ala Val Lys Glu Met Leu Glu Asp Pro Ala Arg Leu Leu Leu 390 at 395 400 c tgattcatca ctgggcacgc gttgcgtgcc caatctcaat actcttttca 4415 Asp Val gatctgaatg gatagaacat catg aac tta cac gaa tac cag gct aaa cag 4466 Met Asn Leu His Glu Tyr Gln Ala Lys Gln 1 5 10 cg cg ccc gcc cg ccc gcc ccc gcc ccc gcc ccc gcc cg ccc gcc ccc gcc ccc gcc tgt act 4514 Leu Phe Ala Arg Tyr Gly Met Pro Ala Pro Thr Gly Tyr Ala Cys Thr 15 20 25 aca cca cgt gaa gca gaa gaa gcg gca tcg aaa atc ggt gca 4556 Thr Pro Arg Glu Ala Glu Glu Ala Ala Ser Lys Ile Gly Ala 30 35 40

<210> 2 <211> 39 <212> PRT <213> Enterobacter agglomerans <400> 2 Ala Phe Ser Val Phe Arg Cys His Ser Ile Met Asn Cys Val Ser Val 1 5 10 15 Cys Pro Lys Gly Leu Asn Pro Thr Arg Ala Ile Gly His Ile Lys Ser 20 25 30 Met Leu Leu Gln Arg Ser Ala 35

<210> 3 <211> 935 <212> PRT <213> Enterobacter agglomerans <400> 3 Met Gln Asn Ser Ala Met Lys Pro Trp Leu Asp Ser Ser Trp Leu Ala 1 5 10 15 Gly Ala Asn Gln Ser Tyr Ile Glu Gln Leu Tyr Glu Asp Phe Leu Thr 20 25 30 Asp Pro Asp Ser Val Asp Ala Val Trp Arg Ser Met Phe Gln Gln Leu 35 40 45 Pro Gly Thr Gly Val Lys Pro Glu Gln Phe His Ser Ala Thr Arg Glu 50 55 60 Tyr Phe Arg Arg Leu Ala Lys Asp Ala Ser Arg Tyr Thr Ser Ser Val 65 70 75 80 Thr Asp Pro Ala Thr Asn Ser Lys Gln Val Lys Val Leu Gln Leu Ile 85 90 95 Asn Ala Phe Arg Phe Arg Gly His Gln Glu Ala Asn Leu Asp Pro Leu 100 105 110 Gly Leu Trp Lys Gln Asp Arg Val Ala Asp Leu Asp Pro Ala Phe His 115 120 125 Asp Leu Thr Asp Ala Asp Phe Gln Glu Ser Phe Asn Val Gly Ser Phe 130 135 140 Ala Ile Gly Lys Glu Thr Met Lys Leu Ala Asp Leu Phe Asp Ala Leu 145 150 155 160 Lys Gln Thr Tyr Cys Gly Ser Ile Gly Ala Glu Tyr Met His Ile Asn 165 170 175 Asn Thr Glu Glu Lys Arg Trp Ile Gln Gln Arg Ile Glu Ser Gly Ala 180 185 190 Ser Gln Thr Ser Phe Ser Gly Glu Glu Lys Lys Gly Phe Leu Lys Glu 195 200 205 Leu Thr Ala Ala Glu Gly Leu Glu Lys Tyr Leu Gly Ala Lys Phe Pro 210 215 220 Gly Ala Lys Arg Phe Ser Leu Glu Gly Gly Asp Ala Leu Val Pro Met 225 230 235 240 Leu Arg Glu Met Ile Arg His Ala Gly Lys Ser Gly Thr Arg Glu Val 245 250 255 Val Leu Gly Met Ala His Arg Gly Arg Leu Asn Val Leu Ile Asn Val 260 265 270 Leu Gly Lys Lys Pro Gln Asp Leu Phe Asp Glu Phe Ser Gly Lys His 275 280 285 Lys Glu His Leu Gly Thr Gly Asp Val Lys Tyr His Met Gly Phe Ser 290 295 300 Ser Asp Ile Glu Thr Glu Gly Gly Leu Val His Leu Ala Leu Ala Phe 305 310 315 320 Asn Pro Ser His Leu Glu Ile Val Ser Pro Val Val Met Gly Ser Val 325 330 335 Arg Ala Arg Leu Asp Arg Leu Ala Glu Pro Val Ser Asn Lys Val Leu 340 345 350 Pro Ile Thrile Ile His Gly Asp Ala Ala Val Ile Gly Gln Gly Val Val 355 360 365 Gln Glu Thr Leu Asn Met Ser Gln Ala Arg Gly Tyr Glu Val Gly Gly 370 375 380 Thr Val Arg Ile Val Ile Asn Asn Gln Val Gly Phe Thr Thr Ser Asn 385 390 395 400 400 Pro Lys AspAla Arg Ser Thr Pro Tyr Cys Thr Asp Ile Gly Lys Met 405 410 415 Val Leu Ala Pro Ile Phe His Val Asn Ala Asp Asp Pro Glu Ala Val 420 425 430 Ala Phe Val Thr Arg Leu Ala Leu Asp Tyr Arg Asn Thr Phe Lys Arg 435 440 445 Asp Val Phe Ile Asp Leu Val Cys Tyr Arg Arg His Gly His Asn Glu 450 455 460 Ala Asp Glu Pro Ser Ala Thr Gln Pro Leu Met Tyr Gln Lys Ile Lys 465 470 475 480 480 Lys His Pro Thr Pro Arg Lys Ile Tyr Ala Asp Arg Leu Glu Gly Glu 485 490 495 Gly Val Ala Ser Gln Glu Asp Ala Thr Glu Met Val Asn Leu Tyr Arg 500 505 510 Asp Ala Leu Asp Ala Gly Glu Cys Val Val Pro Glu Trp Arg Pro Met 515 520 525 Ser Leu His Ser Phe Thr Trp Ser Pro Tyr Leu Asn His Glu Trp Asp 530 535 540 Glu Pro Tyr Pro Ala Gln Val Asp Met Lys Arg Leu Lys Glu Leu Ala 545 550 555 560 Leu Arg Ile Ser Gln Val Pro Glu Gln Ile Glu Val Gln Ser Arg Val 565 570 575 Ala Lys Ile Tyr Asn Asp Arg Lys Leu Met Ala Glu Gly Glu Lys Ala 580 585 590 Phe Asp Trp Gly Gly Ala Glu Asn Leu Ala Tyr Ala Thr Leu Val Asp 595 600 605 Glu Gly Ile ProVal Arg Leu Ser Gly Glu Asp Ser Gly Arg Gly Thr 610 615 620 Phe Phe His Arg His Ala Val Val His Asn Gln Ala Asn Gly Ser Thr 625 630 635 640 Tyr Thr Pro Leu His His Ile His Asn Ser Gln Gly Glu Phe Lys Val 645 650 655 Trp Asp Ser Val Leu Ser Glu Glu Ala Val Leu Ala Phe Glu Tyr Gly 660 665 670 Tyr Ala Thr Ala Glu Pro Arg Val Leu Thr Ile Trp Glu Ala Gln Phe 675 680 685 Gly Asp Phe Ala Asn Gly Ala Gln Val Val Ile Asp Gln Phe Ile Ser 690 695 700 Ser Gly Glu Gln Lys Trp Gly Arg Met Cys Gly Leu Val Met Leu Leu 705 710 715 715 720 Pro His Gly Tyr Glu Gly Gln Gly Pro Glu His Ser Ser Ala Arg Leu 725 730 735 Glu Arg Tyr Leu Gln Leu Cys Ala Glu Gln Asn Met Gln Val Cys Val 740 745 750 Pro Ser Thr Pro Ala Gln Val Tyr His Met Leu Arg Arg Gln Ala Leu 755 760 765 arg Arg Gly Met Arg Arg Pro Leu Val Val Met Ser Pro Lys Ser Leu Leu 770 775 780 Arg His Pro Leu Ala Ile Ser Ser Leu Asp Glu Leu Ala Asn Gly Ser 785 790 795 800 Phe Gln Pro Ala Ile Gly Glu Ile Asp Asp Leu Asp Pro Gln Gly Val 805 810 815 Lys Arg Val ValLeu Cys Ser Gly Lys Val Tyr Tyr Asp Leu Leu Glu 820 825 830 Gln Arg Arg Lys Asp Glu Lys Thr Asp Val Ala Ile Val Arg Ile Glu 835 840 845 Gln Leu Tyr Pro Phe Pro His Gln Ala Val Gln Glu Ala Leu Lys Ala 850 855 860 Tyr Ser His Val Gln Asp Phe Val Trp Cys Gln Glu Glu Pro Leu Asn 865 870 875 880 Gln Gly Ala Trp Tyr Cys Ser Gln His His Phe Arg Asp Val Val Pro 885 890 895 Phe Gly Ala Thr Leu Arg Tyr Ala Gly Arg Pro Ala Ser Ala Ser Pro 900 905 910 Ala Val Gly Tyr Met Ser Val His Gln Gln Gln Gln Gln Asp Leu Val 915 920 925 Asn Asp Ala Leu Asn Val Asn 930 935

<210> 4 <211> 407 <212> PRT <213> Enterobacter agglomerans <400> 4 Met Ser Ser Val Asp Ile Leu Val Pro Asp Leu Pro Glu Ser Val Ala 1 5 10 15 Asp Ala Thr Val Ala Thr Trp His Lys Lys Pro Gly Asp Ala Val Ser 20 25 30 Arg Asp Glu Val Ile Val Glu Ile Glu Thr Asp Lys Val Val Leu Glu 35 40 45 Val Pro Ala Ser Ala Asp Gly Val Leu Glu Ala Val Leu Glu Asp Glu 50 55 60 Gly Ala Thr Val Thr Ser Arg Gln Ile Leu Gly Arg Leu Lys Glu Gly 65 70 75 80 Asn Ser Ala Gly Lys Glu Ser Ser Ala Lys Ala Glu Ser Asn Asp Thr 85 90 95 Thr Pro Ala Gln Arg Gln Thr Ala Ser Leu Glu Glu Glu Ser Ser Asp 100 105 110 Ala Leu Ser Pro Ala Ile Arg Arg Leu Ile Ala Glu His Asn Leu Asp 115 120 125 Ala Ala Gln Ile Lys Gly Thr Gly Val Gly Gly Arg Leu Thr Arg Glu 130 135 140 Asp Val Glu Lys His Leu Ala Asn Lys Pro Gln Ala Glu Lys Ala Ala 145 150 155 160 Ala Pro Ala Ala Gly Ala Ala Thr Ala Gln Gln Pro Val Ala Asn Arg 165 170 175 Ser Glu Lys Arg Val Pro Met Thr Arg Leu Arg Lys Arg Val Ala Glu 180 185 190 Arg Leu Leu Glu Ala Lys Asn Ser Thr Ala Met Leu Thr Thr Phe Asn 195 200 205 Glu Ile Asn Met Lys Pro Ile Met Asp Leu Arg Lys Gln Tyr Gly Asp 210 215 220 Ala Phe Glu Lys Arg His Gly Val Arg Leu Gly Phe Met Ser Phe Tyr 225 230 235 240 Ile Lys Ala Val Val Glu Ala Leu Lys Arg Tyr Pro Glu Val Asn Ala 245 250 255 Ser Ile Asp Gly Glu Asp Val Val Tyr His Asn Tyr Phe Asp Val Ser 260 260 265 270 Ile Ala Val Ser Thr Pro Arg Gly Leu Val Thr Pro Val Leu Arg Asp 275 280 285 Val Asp Ala Leu Ser Met Ala Asp Ile Glu Lys Lys Ile Lys Glu Leu 290 295 300 Ala Val Lys Gly Arg Asp Gly Lys Leu Thr Val Asp Asp Leu Thr Gly 305 310 315 320 Gly Asn Phe Thr Ile Thr Asn Gly Gly Val Phe Gly Ser Leu Met Ser 325 330 335 Thr Pro Ile Ile Asn Pro Pro Gln Ser Ala Ile Leu Gly Met His Ala 340 345 350 Ile Lys Asp Arg Pro Met Ala Val Asn Gly Gln Val Val Ile Leu Pro 355 360 365 Met Met Tyr Leu Ala Leu Ser Tyr Asp His Arg Leu Ile Asp Gly Arg 370 375 380 Glu Ser Val Gly Tyr Leu Val Ala Val Lys Glu Met Leu Glu Asp Pro 385 390 395 400 Ala Arg LeuLeu Leu Asp Val 405

<210> 5 <211> 40 <212> PRT <213> Enterobacter agglomerans <400> 5 Met Asn Leu His Glu Tyr Gln Ala Lys Gln Leu Phe Ala Arg Tyr Gly 1 5 10 15 Met Pro Ala Pro Thr Gly Tyr Ala Cys Thr Thr Pro Arg Glu Ala Glu 20 25 30 Glu Ala Ala Ser Lys Ile Gly Ala 35 40

<210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 6 gtcgacaata gccygaatct gttctggtcg 30

<210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 7 aagcttatcg acgctcccct ccccaccgtt 30

[Brief description of the drawings]

FIG. 1. gltA gene, ppc gene and gdh
The figure which shows construction of the plasmid pMWCPG which has A gene.

FIG. 2 Plasmid pSTV having gdhA gene
The figure which shows construction of G.

FIG. 3. Plasmid RS containing the origin of replication of the broad host range plasmid RSF1010 and the tetracycline resistance gene
The figure which shows construction of F-Tet.

FIG. 4. Origin of replication of broad host range plasmid RSF1010, tetracycline resistance gene, gltA gene, p
Plasmid R having pc and gdhA genes
The figure which shows construction of SFCPG.

FIG. 5: Plasmid pMWC having gltA gene
The figure which shows construction of B.

FIG. 6 is a restriction map of a DNA fragment derived from Enterobacter agglomerans of pTWVEK101.

FIG. 7: s from Enterobacter agglomerans
The figure which shows the comparison of the amino acid sequence predicted from the base sequence of a ucA gene, and the thing derived from Escherichia coli.
Upper: Enterobacter agglomerans, Lower: Escherichia coli (hereinafter the same).

FIG. 8: s from Enterobacter agglomerans
The figure which shows the comparison of the amino acid sequence predicted from the base sequence of ucB gene, and the thing derived from Escherichia coli.

FIG. 9: s from Enterobacter agglomerans
The figure which shows the comparison of the amino acid sequence predicted from the base sequence of a dhB gene, and the thing derived from Escherichia coli.

FIG. 10 is a view showing a comparison between the amino acid sequence predicted from the nucleotide sequence of the sucC gene derived from Enterobacter agglomerans and that derived from Escherichia coli.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:01) (C12N 1/20 C12R 1: 425) (C12P 13/14 C12R 1:01) (C12P 13/14 C12R 1: 425) (72) Inventor Kazuhiko Matsui 1-1, Suzukicho, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Ajinomoto Co., Inc. Fermentation Research Laboratory (72) Mika Moriya Suzukicho, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture 1-1 Ajinomoto Co., Ltd. Fermentation Technology Research Laboratories (72) Inventor Hisao Ito 1-1 Ajinomoto Co., Ltd. Fermentation Technology Research Laboratories, Kawasaki City, Kawasaki City, Kanagawa Prefecture (72) Inventor Yoshihiko Hara Kawasaki City, Kanagawa Prefecture 1-1 Fuzume, Ajinomoto Co., Ltd. Fermentation Technology Research Laboratories, Ajinomoto Co., Ltd. 4B024 AA03 BA74 GA19 4B064 AE19 CA02 DA01 DA10 DA16 4B065 AA01X AA48X AC15 BA22 CA1 7 CA41 CA44

Claims (6)

[Claims] 1. A genus belonging to the genus Enterobacter or Serratia, having at least one of the following properties, and
A microorganism capable of producing glutamic acid: (a) an activity of an enzyme that catalyzes a biosynthesis reaction of L-glutamic acid is enhanced, and (b) a compound other than L-glutamic acid that is branched from the L-glutamic acid biosynthetic pathway. The activity of the enzyme that catalyzes the resulting reaction is reduced or missing. 2. The microorganism according to claim 1, wherein the enzyme that catalyzes the biosynthesis reaction of L-glutamic acid is selected from citrate synthase, phosphoenolpyruvate carboxylase, and glutamate dehydrogenase. 3. The microorganism according to claim 2, wherein the enzyme that catalyzes the biosynthesis reaction of L-glutamic acid is all of citrate synthase, phosphoenolpyruvate carboxylase, and glutamate dehydrogenase. 4. The enzyme according to claim 1, wherein the enzyme that catalyzes a reaction for producing a compound other than L-glutamic acid by branching off from the L-glutamic acid biosynthetic pathway is α-ketoglutarate dehydrogenase. Microorganisms. 5. The microorganism according to any one of claims 1 to 4, wherein the microorganism belongs to Enterobacter agglomerans or Serratia requifaciens. 6. 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.