JP2000232890A - Production of l-glutamic acid through fermentation process - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an inexpensive and efficient production process for glutamic acid by growing a strain that has high ability to produce L-glutamic acid. SOLUTION: A coryneform bacterium that is increased its activity of the pyruvate dehydrogenase by increasing the copying number of the gene encoding the intracellular pyruvate dehydrogenase and has the capability of producing L-glutamic acid is cultured in a culture medium, preferably including >=20 μg/L of vitamin B1 to accumulate L-glutamic acid in the culture mixture. Finally L-glutamic acid is collected from the culture mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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

[0002]

[Prior art]

Hitherto, L-glutamic acid has been industrially produced by fermentation using a coryneform bacterium belonging to the genus Brevibacterium or Corynebacterium having the ability to produce L-glutamic acid (amino acid fermentation, Gakkai Shuppan Center, 195-215, 1986). As these coryneform bacteria, strains isolated from nature or artificial mutants of the strains are used in order to improve productivity.

[0004] Also, various techniques have been disclosed for increasing the ability to produce L-glutamic acid by enhancing the gene of an L-glutamic acid biosynthetic enzyme by recombinant DNA technology. For example, JP-A-63-214189 discloses a technique for increasing the ability to produce L-glutamic acid by enhancing the glutamate dehydrogenase gene, the isocitrate dehydrogenase gene, the aconitate hydratase gene, and the citrate synthase gene. Have been.

However, it has not been known that the pyruvate dehydrogenase gene is used for breeding coryneform L-glutamic acid producing bacteria.

[0006]

SUMMARY OF THE INVENTION The object of the present invention is to provide an L-
In order to respond to a further increase in demand for glutamic acid, it is an object of the present invention to breed a strain having a high L-glutamic acid-producing ability and to provide a more inexpensive and efficient method for producing L-glutamic acid.

[0007]

Means for Solving the Problems The present inventor aims to obtain a coryneform bacterium having an improved L-glutamic acid producing ability by enhancing the pyruvate dehydrogenase activity of a coryneform bacterium capable of producing L-glutamic acid. And succeeded in completing the present invention. That is, the present invention
It is as follows.

(1) Coryneform bacterium having enhanced pyruvate dehydrogenase activity in cells and having L-glutamic acid producing ability. (2) The enhancement of pyruvate dehydrogenase activity is as follows:
The coryneform bacterium according to (1), which is obtained by increasing the copy number of a gene encoding pyruvate dehydrogenase in the bacterial cell.

(3) The coryneform bacterium according to (2), wherein the gene encoding pyruvate dehydrogenase is derived from a bacterium belonging to the genus Escherichia. (4) The coryneform bacterium according to any one of (1) to (3) is cultured in a medium, L-glutamic acid is produced and accumulated in the culture, and L-glutamic acid is collected from the culture. A process for producing L-glutamic acid.

[0010] (5) The media is a vitamin B ₁ 20μg
/ L or more. (6) DNA encoding a protein shown in the following (A) or (B). (A) a protein having the amino acid sequence of SEQ ID NO: 10; (B) In the amino acid sequence of SEQ ID NO: 10, 1
Alternatively, a protein comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of several amino acids, and having pyruvate dehydrogenase activity. (7) a DNA represented by the following (a) or (b): (6)
DNA. (A) a DNA comprising a base sequence consisting of base numbers 2360 to 5125 of SEQ ID NO: 9; (B) a DNA which hybridizes with a base sequence consisting of base numbers 2360 to 5125 of SEQ ID NO: 9 or a probe prepared from the base sequence under stringent conditions and encodes a protein having pyruvate dehydrogenase activity. (8) The DNA according to (7), wherein the stringent condition is that washing is performed at 60 ° C. at a salt concentration corresponding to 1 × SSC and 0.1% SDS.

In the present invention, "L-glutamic acid-producing ability" refers to the ability to accumulate L-glutamic acid in a culture medium when the coryneform bacterium of the present invention is cultured in the medium.
The L-glutamic acid-producing ability may be a property of a wild type coryneform bacterium, or may be a property imparted or enhanced by breeding.

[0012]

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

<1> Coryneform bacterium of the present invention The coryneform bacterium of the present invention is a coryneform bacterium having enhanced pyruvate dehydrogenase activity in cells and having an ability to produce L-glutamic acid.

[0014] Coryneform bacteria include bacteria that were previously classified into the genus Brevibacterium, but are now integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 25).
5 (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 carnaye Corynebacterium glutamicum Corynebacterium lilium
Glutamicum) Corynebacterium meracecora Corynebacterium thermoaminogenes Corynebacterium herculis Brevibacterium dibaricatum (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium)
(Glutamicum) Brevibacterium inmaliophyllum Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharisticacum Brevibacterium thiogenitalis Brevibacterium ammoniagenes (coryne) Bacterium ammoniagenes) Brevibacterium album Brevibacterium serinum Microbacterium ammoniafilum 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 ammoniagenes (Corynebacterium ammoniagenes) ATCC6871 Brevibacterium album ATCC15111 Brevibacterium serinum ATCC15112 Microbacterium ammoniaphilum ATCC1
5354

In order to obtain these, for example, they can be obtained from the American Type Culture Collection. That is, a corresponding registration number is assigned to each strain, and the strain can be ordered by referring to this registration number. The registration number corresponding to each strain is described in the catalog of the American Type Culture Collection. The AJ12340 strain has been deposited with the Ministry of International Trade and Industry of the National Institute of Advanced Industrial Science and Technology under the accession number FERM BP-1539 under the Budapest Treaty.

<2> Amplification of pyruvate dehydrogenase activity In order to amplify pyruvate dehydrogenase activity in coryneform bacterium cells, a gene fragment encoding pyruvate dehydrogenase is converted into a vector which functions in the bacterium, preferably a multicopy type. Recombinant DN linked to vector
A may be prepared, introduced into a host bacterium having L-glutamic acid-producing ability, and transformed. As a result of an increase in the copy number of the gene encoding pyruvate dehydrogenase in the cells of the transformed strain, pyruvate dehydrogenase activity is amplified.

In Escherichia coli, pyruvate dehydrogenase is a hetero-oligomer composed of three subunits of E1, E2 and E3, each of which is aceE gene, ac
Encoded by the eF and lpd genes, these genes form an operon. The gene encoding pyruvate dehydrogenase or the pyruvate dehydrogenase gene according to the present invention refers to an operon encoding such a pyruvate dehydrogenase complex or a gene encoding a monomer having pyruvate dehydrogenase activity. Examples of the monomer having pyruvate dehydrogenase activity include an E1 subunit encoded by the pdhA gene of Brevibacterium lactofermentum.

As the pyruvate dehydrogenase gene, any of a gene derived from a coryneform bacterium and a gene derived from another organism such as a bacterium belonging to the genus Escherichia can be used.

The nucleotide sequence of the pyruvate dehydrogenase complex gene of Escherichia coli K-12 has already been elucidated (FEMS Microbiology Letters, Vol. 44, No. 4).
17 (1987), using a primer prepared based on the nucleotide sequence, for example, the primers shown in SEQ ID NOs: 1 and 2 in the Sequence Listing, using a chromosomal DNA of Escherichia coli as a template (PCR: polymerase chain reactio).
n; White, TJ et al; Trends Genet. 5,185 (1989)) to obtain a pyruvate dehydrogenase gene. Pyruvate dehydrogenase genes from other microorganisms can be obtained in a similar manner. Genes derived from coryneform bacteria will be described later.

Chromosomal DNA can be obtained from a DNA donor bacterium, for example, by the method of Saito and Miura (H. Saito and K. Miur).
a Biochem. Biophys. Acta, 72, 619, (1963)) and the like.

The pyruvate dehydrogenase gene amplified by the PCR method is connected to a vector DNA capable of autonomous replication in the cells of Escherichia coli and / or coryneform bacteria to prepare a recombinant DNA, which is then ligated to Escherichia coli. When introduced into cells, subsequent operations become easier. As the vector capable of autonomous replication in Escherichia coli cells, a plasmid vector capable of autonomous replication in host cells is preferable, for example, pUC19, pUC1
8, pBR322, pHSG299, pHSG399, pHSG398, RSF1010 and the like.

A vector that functions in a coryneform bacterium is, for example, a plasmid that can autonomously replicate in a coryneform bacterium.
Specific examples include the following.

PAM330 See JP-A-58-67699. PHM 1519 See JP-A-58-77895. PAJ655 See JP-A-58-192900. PAJ 611 Same as above pAJ 1844 Same as above pCG 1 P134 See JP-A-58-35197. PCG4 See JP-A-57-183799. PCG11 Same as above. PHK4 See JP-A-5-7591.

Recombinant DNA by linking the pyruvate dehydrogenase gene with a vector that functions in coryneform bacteria
Is prepared by cutting the vector with a restriction enzyme that matches the end of the pyruvate dehydrogenase gene. Ligation is generally performed using a ligase such as T4 DNA ligase.

The recombinant DNA prepared as described above can be introduced into a coryneform bacterium according to the transformation method reported so far. For example, a method of increasing the permeability of DNA by treating recipient cells with calcium chloride, as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol. , 53,159
(1970)) and a method for preparing competent cells from cells in the growth stage and introducing DNA as described in Bacillus subtilis (Duncan, CH, Wil).
son, GAand Young, FE, Gene, 1, 153 (1977)). Alternatively, the DNA of a DNA-receptor bacterium, as is known for Bacillus subtilis, actinomycetes and yeast, is placed into a protoplast or spheroplast that readily incorporates the recombinant DNA, and the recombinant DNA is converted to DNA.
Method for introduction into recipient bacteria (Chang, S. and Choen, SN, Mole
c. Gen. Genet., 168, 111 (1979); Bibb, MJ, Ward, J.
M. and Hopwood, OA, Nature, 274, 398 (1978); Hinnen,
A., Hicks, JBand Fink, GR, Proc. Natl. Acad. Sci.
USA, 75 1929 (1978)). The transformation method used in the examples of the present invention is an electric pulse method (Japanese Unexamined Patent Publication No.
No. 207791).

Amplification of the pyruvate dehydrogenase activity can also be achieved by the presence of multiple copies of the pyruvate dehydrogenase gene on the chromosomal DNA of a coryneform bacterium. Chromosome DN of microorganism belonging to coryneform bacterium
In order to introduce the pyruvate dehydrogenase gene into A in multiple copies, homologous recombination is performed using a sequence present in multiple copies on chromosomal DNA as a target. Chromosomal DNA
As a sequence having multiple copies above, repetitive DN
A, Inverted repeats at the end of the transposable element can be used. Alternatively, Japanese Unexamined Patent Application Publication No.
As disclosed in Japanese Patent Application Laid-Open Publication No. H10-107, it is also possible to mount a pyruvate dehydrogenase gene on a transposon, transfer it, and introduce multiple copies on chromosomal DNA. Either method increases the copy number of the pyruvate dehydrogenase gene in the transformant, resulting in amplification of pyruvate dehydrogenase activity.

The amplification of pyruvate dehydrogenase activity can be achieved by replacing the expression control sequence such as the promoter of the pyruvate dehydrogenase gene on the chromosomal DNA or plasmid with a strong one, in addition to the above gene amplification. (See Japanese Patent Application Laid-Open No. 1-215280). For example, lac promoter, trp promoter, trc promoter, tac promoter, P _R promoter of lambda phage, the P _L promoter, etc. are known as strong promoters. By substitution with these promoters, pyruvate dehydrogenase activity is amplified by enhancing the expression of the pyruvate dehydrogenase gene. The enhancement of expression control sequences may be combined with increasing the copy number of the pyruvate dehydrogenase gene.

The coryneform bacterium of the present invention may have enhanced activity of an enzyme that catalyzes L-glutamic acid biosynthesis other than pyruvate dehydrogenase. Such enzymes that catalyze L-glutamic acid biosynthesis include:
Glutamate dehydrogenase, glutamine synthetase, glutamate synthase, isocitrate dehydrogenase, aconitate hydratase, citrate synthase, phosphoenolpyruvate carboxylase, pyruvate kinase, enolase, phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde-3-phosphate Dehydrogenase, triosephosphate isomerase, fructosebisphosphate aldolase, phosphofructokinase, glucose phosphate isomerase and the like.

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. As an enzyme that catalyzes a reaction that produces a compound other than L-glutamic acid by branching off from the biosynthetic pathway of L-glutamic acid, α-ketoglutarate dehydrogenase (αK
GDH), isocitrate lyase, phosphate acetyltransferase, acetate kinase, acetohydroxyacid synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, glutamate decarboxylase, 1-pyrroline dehydrogenase, and the like. Of these enzymes, αKGDH is preferred.

Further, by imparting a temperature-sensitive mutation to a biotin action inhibitor such as a surfactant to a coryneform bacterium having an ability to produce L-glutamic acid, the biotin action is inhibited in a medium containing an excessive amount of biotin. L-glutamic acid can be produced in the absence of a substance (see WO96 / 06180). An example of such a coryneform bacterium is Brevibacterium lactofermentum AJ13029 described in WO96 / 06180. AJ130
29 strains were deposited with the National Institute of Advanced Industrial Science and Technology on September 2, 1994 under the accession number FERM P-14501,
It was transferred to an international deposit under the Budapest Treaty on August 1, 1995 and has been assigned the accession number FERM BP-5189.

<3> Pyruvate dehydrogenase gene of Brevibacterium lactofermentum The pyruvate dehydrogenase gene (pdhA) of Brevibacterium lactofermentum is, for example, Brevibacterium lactofermentum ATCC 13869.
It can be obtained from the chromosomal DNA of the strain by PCR using the chromosomal DNA as a template and a primer having the nucleotide sequence shown in SEQ ID NO: 11 and a primer having the nucleotide sequence shown in SEQ ID NO: 12 in the sequence listing.

The pdhA gene has been obtained by PCR as described above, and is hybridized with an oligonucleotide prepared based on the nucleotide sequence of the DNA of the present invention as a probe to obtain Brevibacterium lactofera. It can also be obtained from Mentam's chromosomal DNA library.

Preparation of genomic DNA library, hybridization, PCR, preparation of plasmid DNA,
Methods such as DNA cleavage and ligation and transformation are described in Sambroo
k, J., Fritsch, EF, Maniatis, T., Molecular Cloning, C
Old Spring Harbor Laboratory Press, 1.21 (1989).

The DNA of the present invention may comprise one or more amino acid substitutions at one or more positions, as long as the activity of the encoded pyruvate dehydrogenase is not impaired.
It may encode a pyruvate dehydrogenase containing deletions, insertions, additions, or inversions. here,
"Several" differs depending on the position and type of the amino acid residue in the three-dimensional structure of the protein. Specifically, the "several" is 2 to 400, preferably 2 to 1
00, more preferably 2 to 20.

DNA encoding a protein substantially identical to pyruvate dehydrogenase as described above can be obtained by substituting, deleting, inserting, adding, or reversing amino acid residues at specific sites by, for example, site-directed mutagenesis. It is obtained by modifying the nucleotide sequence to include the position. The modified DNA as described above can also be obtained by a conventionally known mutation treatment. As the mutation treatment, a method of in vitro treating DNA encoding pyruvate dehydrogenase with hydroxylamine or the like, and a microorganism having a DNA encoding pyruvate dehydrogenase, such as a bacterium belonging to the genus Escherichia, irradiated with ultraviolet light or N
-Methyl-N'-nitro-N-nitrosoguanidine (NTG)
Alternatively, a method of treating with a mutagen commonly used in mutagenesis treatment, such as nitrous acid, may be mentioned.

The substitution, deletion, insertion, addition, or inversion of a base as described above may occur naturally due to individual differences of microorganisms having pyruvate dehydrogenase and differences based on species and genera. The resulting mutation (mutant or variant) is also included.

The DNA having the above mutation is expressed in a suitable cell, and the expression product is examined for pyruvate dehydrogenase activity, whereby DNA encoding a protein substantially identical to pyruvate dehydrogenase can be obtained. In addition, DNA encoding pyruvate dehydrogenase having a mutation or a cell carrying the same can be obtained, for example, from base numbers 2360 to 5 of SEQ ID NO: 9 in the sequence listing.
DNA hybridizing with a DNA having a nucleotide sequence consisting of 125 or a probe prepared from a DNA having the same nucleotide sequence under stringent conditions, and encoding a protein having pyruvate dehydrogenase
Can also be obtained by isolating a DNA encoding a protein substantially identical to pyruvate dehydrogenase. The probe was prepared by converting the DNA of SEQ ID NO: 9
It can be obtained by amplifying an appropriate region with R. The term "stringent conditions" as used herein refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Although it is difficult to quantify these conditions clearly, as an example, DNAs having high homology, for example, DNAs having homology of 40% or more hybridize to each other, and DNAs having lower homology A salt corresponding to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS at 60 ° C., which is a condition under which they do not hybridize with each other, or a washing condition for ordinary Southern hybridization. Conditions for hybridizing at a concentration are listed.

Some of the genes that hybridize under such conditions include those in which a stop codon has been generated in the middle and those in which the activity has been lost due to mutation of the active center. Pyruvate dehydrogenase activity was determined by a known method (Methods
in Enzymology, 9, 248-249, 1966).

<4> L-type using the coryneform bacterium of the present invention
Glutamate Production A strain belonging to a coryneform bacterium, in which pyruvate dehydrogenase activity is amplified, and in which L-glutamic acid is produced using a strain having L-glutamic acid producing ability, a carbon source,
It can be carried out by a conventional method using a usual nutrient medium containing a nitrogen source, inorganic salts, and other organic trace nutrients such as amino acids and vitamins as necessary. Either a synthetic medium or a natural medium can be used. As the carbon source and the nitrogen source used in the medium, any type can be used as long as the strain to be cultured can be used.

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 and citric acid are used alone or in combination. Used in combination with other carbon sources.

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.
The coryneform bacterium of the present invention can be prepared by the conventional method even in the presence of a smaller amount of vitamin B ₁ than that required for a medium used for culturing a normal L-glutamic acid producing bacterium, for example, in the presence of 20 μg / L or less of vitamin B _1. L-glutamic acid at the same level as that of L-glutamic acid can be produced. Also, the medium used for culturing coryneform bacterium of the present invention, vitamin B ₁ 20 [mu] g / L or more, by preferably be contained more than 500 [mu] g / L, L
Glutamic acid production can be increased.

The cultivation method is as follows: fermentation temperature 20-45 ° C., pH
Aeration culture is performed while controlling the pH to 5-9. If the pH drops during the culture, calcium carbonate is added or neutralized with an alkali such as ammonia gas. Thus 10 hours to 4
By culturing for about one day, a remarkable amount of L-glutamic acid is accumulated in the culture solution.

The method of collecting L-glutamic acid from the culture solution after completion of the culture may be performed according to a known recovery method. For example, it is collected by a method in which cells are removed from the culture solution and then concentrated and crystallized, or by ion exchange chromatography.

[0048]

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

[0049]

Example 1 <1> Cloning of pyruvate dehydrogenase gene of Escherichia coli K-12 The nucleotide sequence of the pyruvate dehydrogenase gene of Escherichia coli K-12 has already been elucidated (FEMS).
Microbiology Letters, Vol. 44, p. 417, 1987). Based on the reported base sequence, the primers shown in SEQ ID NOs: 1 and 2 in the Sequence Listing were synthesized, and Escherichia coli K-
The pyruvate dehydrogenase gene was amplified by PCR using chromosomal DNA of JM109 strain 12 (manufactured by Takara Shuzo) as a template.

Among the synthesized primers, SEQ ID NO: 1
FEMS Microbiology Letters, vol. 44, p. 417, which corresponds to the sequence extending from base 1039 to base 1071 in the base sequence diagram of the pyruvate dehydrogenase gene described in 1987, but at positions 1039, 1042, and 1046 Base of A
In addition, the bases at positions 1040 and 1043 are changed to T, and the base at position 1044 is changed to G, and a recognition sequence for the restriction enzyme XhoI is inserted. SEQ ID NO: 2 is from FEMS Microbiology Letters, No. 44
Volume, page 417, which corresponds to the sequence from the 7700th base to the 7732th base in the nucleotide sequence diagram of the pyruvate dehydrogenase gene described in 1987, but with the 7726th and 7727th positions
The 7730th base is changed to A, the 7729th base is changed to C, the 7724th base is changed to T, and the reverse strand of the base sequence into which the recognition sequence of the restriction enzyme XhoI is inserted is shown from the 5 'side. .

The preparation of chromosomal DNA of Escherichia coli JM109 strain was carried out by a conventional method (Biological Engineering Experiment, edited by The Society of Biotechnology, Japan, pages 97-98, Baifukan, 1992). In addition, the PCR reaction, the forefront of the PCR method (Takeo Sekiya et al.,
Standard reaction conditions described on page 185 of Kyoritsu Shuppan Co., Ltd. (1989) were used.

After purifying the generated PCR product by an ordinary method,
After the restriction enzymes XbaI and XhoI were reacted and ligated using pHSG396 (manufactured by Takara Shuzo) cut with restriction enzymes XbaI and XhoI using a ligation kit (manufactured by Takara Shuzo), Escherichia
Transformation was performed using competent cells of E. coli JM109 (Takara Shuzo), and IPTG (isopropyl-β-D
-Thiogalactopyranoside) 10 μg / ml, X-Ga
l (5-bromo-4-chloro-3-indolyl-β-D
-Galactoside) and 20 μg / ml chloramphenicol in L medium (bactotryptone 10
g / L, Bacto yeast extract 5 g / L, Na
Cl 5 g / L, agar 15 g / L, pH 7.2) and cultured overnight. Thereafter, the emerged white colonies were picked and separated into single colonies to obtain transformed strains.

From the transformed strain, the alkaline method (Biological Engineering Experiment, edited by The Society of Biotechnology, Japan, p. 105, Baifukan, 1992)
Year), a restriction map of the DNA fragment inserted into the vector was prepared, and the reported restriction map of the pyruvate dehydrogenase gene (FEMS Microbiology Letters, vol. 44, p. 417, 1987
Year), a plasmid into which a DNA fragment having the same restriction enzyme map was inserted was named pHSGEPDH.

Further, the prepared plasmid pHSGEPDH is
The restriction enzymes XbaI and SacI are reacted, and after ligation using pMW219 (manufactured by Nippon Gene) and a ligation kit (manufactured by Takara Shuzo) cut with the restriction enzymes XbaI and SacI, competent cells of Escherichia coli JM109 (manufactured by Takara Shuzo)
And IPTG (isopropyl-β
-D-thiogalactopyranoside) 10 μg / ml, X-
Gal (5-bromo-4-chloro-3-indolyl-β
-D-galactoside) 40 μg / ml and kanamycin
L medium containing 25 μg / ml (Bactotryptone 10 g /
L, Bacto yeast extract 5g / L, NaCl
5 g / L, agar 15 g / L, pH 7.2), and after overnight culture, appeared white colonies were picked up and separated into single colonies to obtain transformed strains. From the transformed strain, use the alkaline method (Biological Engineering Experiment, edited by the Society of Biotechnology, Japan, p. 105,
After preparing a plasmid using Baifukan, 1992), a restriction map of the DNA fragment inserted into the vector was prepared and compared with the reported restriction map of the pyruvate dehydrogenase gene. The plasmid into which the DNA fragment was inserted was named pMWEPDH.

Further, in order to confirm that the cloned pyruvate dehydrogenase gene is expressed, the pyruvate dehydrogenase activity of the JM109 strain and the JM109 strain carrying pMWEPDH was determined by the method of Willms et al.
in Enzymology, Vol. 9, pp. 248-249, 1966). As a result, as shown in Table 1, since the JM109 strain carrying pMWEPDH exhibited about four times the pyruvate dehydrogenase activity of the JM109 strain, it was confirmed that the pyruvate dehydrogenase gene was expressed.

[0056]

[Table 1] Table 1 PD Strain PDH activity (units / mg protein) ─────────── ──────────── JM109 0.03 JM109 / pMWEPDH 0.12 ───────────────────────

<2> Preparation of plasmid having pyruvate dehydrogenase gene of JM109 strain derived from Escherichia coli K-12 strain and origin of replication of coryneform bacterium Replication of origin of pyruvate dehydrogenase gene of Escherichia coli JM109 strain and coryneform bacterium In order to construct a plasmid having a plasmid pHM1519 (Agric.
Biol. Chem., 48, 2901-2903 (1984)) was digested with the restriction enzymes BamHI and KpnI to obtain a gene fragment containing the replication origin. After the obtained fragment was blunt-ended using a DNA blunting kit (manufactured by Takara Shuzo), the above-mentioned <1> was used using an XbaI linker (manufactured by Takara Shuzo).
The plasmid pMWEPDH carrying the pyruvate dehydrogenase gene of Escherichia coli JM109 cloned in the above was inserted into the XbaI site. Transfer this plasmid to pEP
Named DH.

(3) Introduction of pEPDH into Coryneform Bacteria and Evaluation of Culture It was obtained by transforming Brevibacterium lactofermentum AJ13029 with the plasmid pEPDH by the electric pulse method (see JP-A-2-207791). A transformant was obtained. Using the obtained transformant AJ13029 / pEPDH, culture for producing L-glutamic acid was performed as follows. The cells of the AJ13029 / pEPDH strain obtained by culturing on a CM2B plate medium containing 25 μg / mL kanamycin,
Glucose _{30g, KH 2 PO 4 1g,} MgSO 4 · 7H 2 O 0.4g, (NH 4) 2
30 g of SO ₄ , 0.01 g of FeSO ₄ .7H ₂ O, 0.01 g of MnSO ₄ .7H ₂ O, 15 ml of soybean hydrolyzate, 200 μg of thiamine hydrochloride, 60 μg of biotin, 25 mg of kanamycin and 50 g of CaCO ₃ in pure water 1
Medium contained in L (adjusted to pH 8.0 using KOH)
And cultured with shaking at 31.5 ° C. until the sugar in the medium was consumed. The obtained culture was inoculated into a medium having the same composition as described above in an amount of 5%, and cultured at 37 ° C. with shaking until the sugar in the medium was consumed. As a control, a strain obtained by transforming Brevibacterium lactofermentum AJ13029 with pHK4 by the electric pulse method was cultured in the same manner as described above.

After completion of the culture, the amount of L-glutamic acid accumulated in the culture solution was measured using a Biotech Analyzer AS manufactured by Asahi Kasei Corporation.
Measured by -210. Table 2 shows the results. In addition, Brevibacterium lactofermentum
AJ13029 was approved by the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, Biotechnology and Industrial Technology Research Institute on September 2, 1994, under the accession number FERMP-1.
Deposit No. 4501 and transferred to an international deposit under the Budapest Treaty on August 1, 1995, with accession number FER
MBP-5189 has been assigned.

[0060]

Table 2 {Yield of strain L-glutamic acid (%)} ──────────── AJ13029 / pHK4 55.1 AJ13029 / pEPDH 57.3 ───────────────────────

[0061] (4) To investigate the effect of addition of vitamin B ₁ (thiamine), which is a precursor of coenzyme thiamine diphosphate (TPP) Evaluation pyruvate dehydrogenase vitamin B ₁ addition effect against L- glutamic acid productivity A pyruvate dehydrogenase activity-enhancing strain AJ13029 / pEPDH strain,
The culture for L- glutamic acid production in the medium changed the concentration of vitamin B ₁ was carried out as follows.

CM containing 25 μg / mL kanamycin
The cells of the AJ13029 / pEPDH strain obtained by culturing on a 2B plate medium were cultured in 30 g of glucose, 1 g of KH ₂ PO ₄ , MgSO ₄ .7H ₂ O 0.4
_{g, (NH 4) 2 SO} 4 30g, FeSO 4 · 7H 2 O 0.01g, MnSO 4 · 7H 2 O 0.0
1 g of a soy hydrolyzate, 15 ml of soy hydrolyzate, 60 μg of biotin, 25 mg of kanamycin and 50 g of CaCO ₃ in 1 liter of pure water (adjusted to pH 8.0 using KOH) in 1 liter of pure water. Until the sugars were consumed. The resulting cultures were treated with 0, 10 μg, 20
A medium having the same composition as described above except that the medium contained μg, 200 μg, 2 mg or 20 mg was inoculated in an amount of 5%, and cultured at 37 ° C. with shaking until the sugar in the medium was consumed. As a control, Brevibacterium lactofermentum AJ13029 strain was subjected to the above-mentioned pHK4 by the electric pulse method (JP-A-2-207791).
The strain transformed by the above method was cultured in the same manner as described above. After completion of the culture, the amount of L-glutamic acid accumulated in the culture solution was measured using a Biotech Analyzer A manufactured by Asahi Kasei Corporation.
It was measured by S-210. The results at this time are shown in Table 3 and FIG.

[0063]

Table 3 ─────────────────────────────── Bacterial strain Thiamin hydrochloride concentration (μg / L) L-Cultamic acid Yield (%) ─────────────────────────────── AJ13029 / pHK4 0 53.5 10 53.6 20 54.3 200 55.1 2000 55.5 20000 55.6 ─────────────────────────────── AJ13029 / pEPDH 0 53.6 10 53.9 20 55.0 200 57.3 2000 57.7 20000 57.8 ─── ────────────────────────────

From the above results, it was found that L-glutamic acid producing bacteria belonging to the coryneform bacterium in which the pyruvate dehydrogenase activity was enhanced by introducing the pyruvate dehydrogenase gene had an improved L-glutamic acid yield.

The L-glutamic acid-producing bacteria belonging to the coryneform bacterium and the L-glutamic acid-producing bacteria belonging to the coryneform bacterium in which the pyruvate dehydrogenase gene is introduced to enhance the pyruvate dehydrogenase activity are all contained in the medium. vitamin B ₁ concentration was found that the yield is improved by culturing in medium or 20 [mu] g / L.

[0066]

Example 2 <1> Cloning of pdhA gene of Brevibacterium lactofermentum ATCC 13869 A region having high homology between the E1 subunits of pyruvate dehydrogenase of Escherichia coli, Pseudomonas aeruginosa and Mycobacterium tuberculosis was selected. And the primer shown in SEQ ID NO: 4 was synthesized,
Bacterial Gen manufactured by Advanced Genetic Technologies Corp.
Brevibacterium lactofermentum ATCC1386 prepared by omic DNA Purification Kit
Using the chromosome 9 as a template, PCR was performed under standard reaction conditions described on page 8 of PCR technology (Henry Erich, edited by Stockton Press, 1989), and the reaction solution was subjected to agarose gel electrophoresis. It was found that a kilobase DNA fragment was amplified.

The base sequences at both ends of the obtained DNA were determined using the synthetic DNAs of SEQ ID NO: 3 and SEQ ID NO: 4. The nucleotide sequence can be determined using the DNA Sequencing Kit (Applie
d Biosystems) using the method of Sanger (J. Mol. Bi
143, 161 (1980)). When the determined nucleotide sequence was translated into amino acids and compared with the E1 subunit of pyruvate dehydrogenase of Escherichia coli, Pseudomonas aeruginosa and Mycobacterium tuberculosis, the DNA fragment amplified by PCR showed high homology because of the high homology. -It was determined that it was part of the pdhA gene encoding the E1 subunit of pyruvate dehydrogenase of lactofermentum ATCC 13869, and the upstream and downstream portions of the gene were cloned as described below.

Brevibacterium lactofermentum ATCC 13869 chromosome was ligated with restriction enzymes EcoRI, BamHI,
D digested with HindIII, PstI, SalI, XbaI (Takara Shuzo)
From the NA fragment, LA PCR was performed using the primers shown in SEQ ID NO: 5 and SEQ ID NO: 6 to clone the upstream portion, and the primers shown in SEQ ID NO: 7 and SEQ ID NO: 8 to clone the downstream portion. in vi
Each clone was performed using tro cloning Kit (Takara Shuzo). As a result of PCR with this kit, the upstream part was digested with EcoRI, HindIII, PstI, SalI, and XbaI, and the DNA fragments of about 0.5, 2.5, 3.0, 1.5, and 1.8 kilobases were amplified, and the downstream part was amplified. BamHI, HindII
DNA fragments of about 1.5, 1.0, and 3.5 kilobases were amplified by the fragments digested with I and PstI, respectively, and the base sequence of this DNA fragment was determined in the same manner as described above.

As a result, it was revealed that the amplified DNA fragment further contained an open reading frame of about 920 amino acids, and that a region presumed to be a promoter region was present further upstream thereof. . The amino acid sequence of the product deduced from the nucleotide sequence of this open reading frame is highly homologous to the known pyruvate dehydrogenase E1 subunit of Escherichia coli. Mentum ATCC138
It was estimated to be a pdhA gene encoding the E1 subunit of 69 pyruvate dehydrogenase. The nucleotide sequence containing the open reading frame, the promoter region, and the region presumed to be a terminator is shown in SEQ ID NO: 9 in the Sequence Listing. In addition, this open
The amino acid sequence of the product deduced from the reading frame is shown in SEQ ID NOs: 9 and 10. The methionine residue at the N-terminus of the protein is the start codon AT
Since it is derived from G, it is often unrelated to the original function of the protein, and it is well known that it is removed by the action of post-translation peptidase. In the case of the above protein, the N-terminal methionine residue Removal may have occurred. However, there is a GTG sequence 6 bases upstream of the ATG shown in SEQ ID NO: 7, from which amino acids may be translated.

The pyruvate dehydrogenase of other microorganisms such as Escherichia coli is composed of three subunits, E1, E2 and E3, and the gene encoding these is often an operon. Became pd
Approximately 3 kilobases downstream of the hA gene did not have an open reading frame that could be the E2 and E3 subunits of pyruvate dehydrogenase. Instead, it has been found that a sequence that is a putative terminator is present downstream of this open reading frame, so that pyruvate dehydrogenases E2 and E3 of Brevibacterium lactofermentum ATCC 13869 are known. The subunit was thought to be located elsewhere on the chromosome.

<2> Evaluation of Glutamate Yield Improvement Effect of pdhA Amplified Strain (1) Construction of pdhA Amplification Plasmid Based on the base sequence of the pdhA gene, SEQ ID NO: 11
And 12 were synthesized, and the Advanced Genetic
Technologies Corp. Bacterial Genomic DNAPurifica
PCR was performed using the chromosome of Brevibacterium lactofermentum ATCC 13869 prepared by the Option Kit as a template under the standard reaction conditions described on page 8 of PCR technology (Henry Erich, Stockton Press, 1989). Was amplified. Among the synthesized primers, SEQ ID NO: 11 corresponds to the sequence extending from the 1397th base to the 1416th base of the base sequence of the pdhA gene shown in SEQ ID NO: 9 in Sequence Listing, and SEQ ID NO: 12 is
It shows the reverse strand of the base sequence corresponding to the sequence from the 5355th base to the 5374th base in SEQ ID NO: 9 from the 5 'side. The resulting PCR product was purified by a conventional method, and then digested with restriction enzymes SalI and EcoT22I.

On the other hand, plasmid vectors capable of autonomous replication in both Escherichia coli and coryneform bacteria were prepared. First, a vector having a drug resistance gene of Streptococcus faecalis was constructed. The kanamycin resistance gene of Streptococcus faecalis was amplified by PCR from a known plasmid containing the gene. The nucleotide sequence of the kanamycin resistance gene of Streptococcus faecalis has already been elucidated (Trieu-
Cuot, P. and Courvalin, P .: Gene 23 (3), 331-341 (19
83)). Based on this sequence, primers represented by SEQ ID NOs: 13 and 14 were synthesized, and pDG783 (Anne-Marie Guerout-Fl
PCR was performed using eury et al., Gene, 167, 335-337 (1995)) as a template to amplify a DNA fragment containing the kanamycin resistance gene and its promoter.

After purifying the above DNA fragment with SUPREC02 manufactured by Takara Shuzo, it was completely digested with restriction enzymes HindIII and HincII and blunt-ended. Blunting Kit manufactured by Takara Shuzo Co., Ltd.
Performed by Using this DNA fragment and the primers shown in SEQ ID NOS: 15 and 16, pHSG399 (S. Takeshita et
al: Gene 61, 63-74 (1987)), a PCR product was used as a template, and the obtained amplification product was purified and blunt-ended, and mixed and ligated. The ligation reaction was DNA lig from Takara Shuzo.
ation kit ver2. Using the ligated DNA,
Escherichia coli JM109 competent cells (Takara Shuzo) were transformed, and IPTG (isopropyl-β-D-thiogalactopyranoside) 10 μg / ml and X-Gal (5-bromo-4-chloro-3-) L medium (10 g of bactotryptone) containing 40 μg / ml of indolyl-β-D-galactoside) and 25 μg / ml of kanamycin
/ L, Bacto yeast extract 5g / L, NaCl 5g / L,
After coating overnight on agar (15 g / L, pH 7.2) and culturing overnight, the emerged blue colonies were picked and separated into single colonies to obtain transformed strains.

Plasmids were prepared from the transformants using the alkaline method (Biotechnical Experiments, edited by Biotechnology Society of Japan, p. 105, Baifukan, 1992), and a restriction enzyme map was prepared.
The one equivalent to the restriction enzyme map shown in the above was named pK1. This plasmid is stably maintained in Escherichia coli and confers kanamycin resistance on the host. Also,
Since it contains the lacZ 'gene, it is suitable for cloning vectors. Brevibacterium lactofermentum AT
Plasmid pAM330 extracted from CC13869 (JP-A-58-67699)
Was digested completely with HindIII and then blunt-ended, and this was digested with pK1 and completely digested with BsaAI. Brevibacterium lactofermentum ATCC13869 was transformed using the ligated DNA. The transformation method is the electric pulse method (Japanese Patent Laid-Open No. 2-20
7791) was used. Transformants were selected on an M-CM2B plate containing 25 μg / ml kanamycin (10 g / polypeptone /
L, yeast extract 10 g / L, NaCl 5 g / L, biotin 10 μg / L, agar 15 g / L, pH 7.2). After culturing for two nights, the colonies were picked and separated into single colonies to obtain transformants. Plasmid DNA was prepared from the transformant, a restriction map was prepared, and one having the same restriction map as the restriction map shown in FIG. 3 was named pSFK6. This plasmid is capable of autonomous replication in Escherichia coli and coryneform bacteria and confer kanamycin resistance to the host.

The plasmid pSFK6 was replaced with the restriction enzymes SalI and Pst
Cut with I. This was ligated to the SalI-EcoT22I fragment of the PCR product using a ligation kit (Takara Shuzo), and then transformed using Escherichia coli JM109 competent cells (Takara Shuzo) to perform IPTG.
(Isopropyl-β-D-thiogalactopyranoside) 1
0 μg / ml, X-Gal (5-bromo-4-chloro-
3-indolyl-β-D-galactoside) 40 μg / m
medium containing 25 g / ml of kanamycin and 25 μg / ml of kanamycin (Bactotryptone 10 g / l, Bacto yeast extract 5 g / l, NaCl 5 g / l, agar 15 g / l, pH
7.2), and after overnight culture, appeared white colonies were picked up and single colonies were separated to obtain transformed strains.

From the transformed strain, the alkaline method (Biotechnology Experiments, edited by the Society of Biotechnology, Japan, p. 105, Baifukan, 1992)
Year), a restriction map of the DNA fragment inserted into the vector was prepared, and SEQ ID NO: 9
A plasmid into which a DNA fragment having the same restriction enzyme map was inserted as compared with the restriction enzyme map of the pdhA gene shown in (1) was named pSFKBPDHA.

(2) Introduction of pSFKBPDHA into Brevibacterium lactofermentum ATCC 13869 and Brevibacterium lactofermentum AJ13029 and evaluation of flask culture Brevibacterium lactofermentum ATCC1
3869 and Brevibacterium lactofermentum AJ13029 were subjected to an electric pulse method (Japanese Patent Laid-Open No. 2-2077791).
With the plasmid pSFKBPDHA to obtain a transformed strain. The obtained transformant ATCC1
Using 3869 / pSFKBPDHA and AJ13029 / pSFKBPDHA, culture for L-glutamic acid production was performed as follows. CM2B containing 25 μg / mL kanamycin
The cells of the AJ13029 / pSFKBPDHA strain obtained by culturing on a plate medium were mixed with 30 g of glucose, 1 g of KH ₂ PO ₄ ,
_{O 4 · 7H 2 O0.4g, (} NH 4) 2 SO 4 30g, Fe
_{SO 4 · 7H 2 O0.01g, MnSO} 4 · 7H 2 O0.0
1 g, soy hydrolyzate 15 ml, thyamine hydrochloride 20
0 μg, 60 μg of biotin, 25 mg of kanamycin and 50 g of CaCO ₃ in 1 L of pure water (adjusted to pH 8.0 using KOH) were inoculated to 31.5 μl.
Shaking culture was performed at ℃ until the sugar in the medium was consumed.

The obtained culture was inoculated into a medium having the same composition except that biotin was not added in an amount of 5%.
C13869 was cultured at 31.5 ° C and AJ13029 at 37 ° C with shaking until the sugar in the medium was consumed. As a control, Brevibacterium lactofermentum A
TCC13869 and AJ13029 strains were transformed with pSFK6 by the electric pulse method and cultured in the same manner as described above. After completion of the culture, the accumulated amount of L-glutamic acid in the culture solution was measured using a Biotech Analyzer AS-210 manufactured by Asahi Kasei Corporation.
Was measured by The results are shown in Table 4.

[0079]

Table 4 {Strain L-glutamic acid yield (%)} ──────────── ATCC13869 / pSFK6 48.3 ATCC13869 / pSFKBPDHA 53.2 AJ13029 / pSFK6 52.3 AJ13029 / pSFKBPDHA 59.2 ────────────────────── ─

From these results, it was found that Brevibacterium
In lactofermentum ATCC 13869 and Brevibacterium lactofermentum AJ13029, it was revealed that amplification of the pdhA gene alone was also effective in improving L-glutamic acid yield.

[0081]

Industrial Applicability The L-glutamic acid-producing bacterium of the present invention belonging to the coryneform bacterium having enhanced pyruvate dehydrogenase activity can produce L-glutamic acid more efficiently than before.

[0082] Further, by vitamin B ₁ is added 20 [mu] g / L in the medium, the L- glutamic acid yield can be further improved. Further, the present invention provides a novel pdhA gene.

[0083]

[Sequence List] Sequence Listing <110> Ajinomoto Co., Inc. <120> Method for producing L-glutamic acid by fermentation (130) P-7027 <141 > 1999-12-15 <150> JP 10-360619 <151> 1998-12-18 <160> 16 <170> PatentIn Ver. 2.0

<210> 1 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying Esherichia coli pyruvate dehydrogenase gene <400> 1 ggctctagag cgagagttca atgggacagg ttc 33

<210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying Esherichia coli pyruvate dehydrogenase gene <400> 2 ggcctcgagg gaaaaagtac agcagcgccc act 33

<210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying Brevibacterium lactofermentum pdhA <220> <221> misc_feature <222> (3, (6,8,15,18,20) <223> n = inosine <400> 3 acngtntcna tgggnctngg ncc 23

<210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying Brevibacterium lactofermentum pdhA <220> <221> misc_feature <222> (6, 12,15,18,20) <223> n = inosine <400> 4 ccttcnccgt tnagngtngt ncg 23

<210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for LA in vitro cloning of Brevibacterium lactofermentum pdhA gene <400> 5 ttgcagttaa ccacgaaggt caggttgtcc 30

<210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for LA in vitro cloning of Brevibacterium lactofermentum pdhA gene <400> 6 tggatgagac cacgtgattc tggctcgtcc 30

<210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for LA in vitro cloning of Brevibacterium lactofermentum pdhA gene <400> 7 acagatcctg cacgaaggca tcaacgaggc 30

<210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for LA in vitro cloning of Brevibacterium lactofermentum pdhA gene <400> 8 tcatcgctgc gggtacctcc tacgccaccc 30

<210> 9 <211> 5370 <212> DNA <213> Brevibacterium lactofermentum <220> <221> CDS <222> (2360) .. (5125) <400> 9 tcacgttacg gcgatcaaca ccgcaaccac tacgagaaga tctccaaacg agaccaagac 60agt cccgtctcat tttgcacctg ccattctgtg aggatatggc aggtgctttt 120 tcatgccact atcttggggt tctcggtatt agatcttctg ataaaaaccc gatagttttc 180 ttgcgctaga cactaattac ggcaccgctt aagcatggtc gtgacacgta aaacctgact 240 taggccattt tgatgtggtg tagatcatat tgacgtcaat gaatgaagtg actaactccg 300 ccgaatccac atcgtctaaa aggcctgggc gaccacgtaa agacgggcac gacgagaaga 360 tcatcgacgc aactttacgg ctcatcgaca gcaatcgtcc cgtcacggtc aatgcagttg 420 tcaaagaaag cggagtggca cgtgcagcgg tttatcgacg ctggcccagg ctagtggatc 480 tagtagcgga agctttagat gccgggcgag ctccagttga aatagatacc ccaggggaca 540 tcaaagagac cttgattgat gggctgttta caaatcaggc gaaaaccact ggagtctcct 600 atcctcgtca gcgatttcgc aaacggctcg agttggtgat gtcagatcaa gaattacagc 660 tcgcctaatg gaattcacat gtgaagagac gtcgagaagc aaatattcgc gcgctgcaag 720 tcgcgcaaga aaaaggccaa atccgggcgg atctagacat cgaggcgtgc ctcgatgcaa 780 tccttggggt gttttattac caatcggtcg cgcgtggagt aaatttcacc gaccaaggta 840 caacgcaacg atgcagagaa gccttggagg tgatctggca tggaatggaa ccttaaattc 900 aggttctgac gaggtgcgaa gcaagttgtc gcgcgccgca cctcagtatc cggatcaact 960 taatttcgaa gtgctgggtt ttctcgcgca tacccaatgc gtaccgatgt gcccatgagc 1020 gaaaaacagg ccacgataag tttcttaaaa cttatcgtgg cctgcttcta tatttgtgcg 1080 ccctgacggg ctcgaaccgc cgacctgctg ggtgtaaacc agctgctctt ccagctgagc 1140 taaaggcgcg cacgtgcttt tctagaacca ccttggtggc ctcgaaagca acgagtgaaa 1200 tactaacaca caatctccac agacctaaaa tcgctgctca ggccgtggaa attagcgatt 1260 gttaaggctt cttgtttcca cgctggacga ggcaagaacc ttgccaatta ccgagacgtt 1320 ccgccttggt ctgcacgaga cctgccagtt gtgctgattc agagataact ccaggagcca 1380 gggctccttc tttaccaatg ccaggagtca acacccagat acgaccattc tcagcgaggg 1440 agcggatgga atccacaagt ccgtcgacga gatcgccgtc atcctcgcgc caccagagca 1500 gcacgacatc gcacagctcg tcggtttctt catcgagtag ttcctcaccg attgcatctt 1560 cgatggactc gctgatcagc gtgtcggaat cttcatccc a tccaatttct tgaacgatat 1620 gacccgattg aatgccgagt agttgagcat aatcctgggc accttgcttg actgcgcccg 1680 gagcgtcggc cactttaata atcctcctcg tgtgggcccc gatgtgtttt tcgattacat 1740 ggattcaaca tgaaaccgcg gggctattga tatatccgaa ttgcacatta ccgtccaacc 1800 ggtactttga accacctttc cctggaattt tttccttttc ctcccccttt acgctcaaga 1860 atcaatgaat tcaatcactg gccagcgatt aacttttcga gttttcagtc ttggatttcc 1920 acaattctct tcaaaataat ggtggctaga tttttcatca aaccctcacc aaaaggacat 1980 cagacctgta gttttatgcg attcgcgtca aacgtgagag aaacatcaca tctcacggga 2040 aactacccga taattctttg caaaactttg caaagggtaa tgaacatgca gctagtttcc 2100 gtagaaatgt tctttaaaaa atccacaaca attgccagga agcacaccga ttgatggata 2160 cctgaaatcc cagtgagcgc accactcccc ttacgtcaca gtctgtaaaa caaatcttcg 2220 gtgttgcgta tccttgttaa taacttatgc gttgacccat tcgtgcactt cggtgtgcca 2280 caattaggta cgaccaagaa tgggaccggg aaaccgggac gtataaacga aataaaacat 2340 tccaacagga ggtgtggaa atg gcc gat caa gca aaa ctt ggt ggt aag ccc 2392 Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro 1 5 1 0 tcg gat gac tct aac ttc gcg atg atc cgc gat ggc gtg gca tct tat 2440 Ser Asp Asp Ser Asn Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr 15 20 25 ttg aac gac tca gat ccg gag gag acc aac gag atgg gat tca ctc 2488 Leu Asn Asp Ser Asp Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu 30 35 40 gac gga tta ctc cag gag tct tct cca gaa cgt gct cgt tac ctc atg 2536 Asp Gly Leu Leu Gln Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met 45 50 55 ctt cgt ttg ctt gag cgt gca tct gca aag cgc gta tct ctt ccc cca 2584 Leu Arg Leu Leu Glu Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro 60 65 70 75 atg acg tca acc gac tac gtc aac acc att cca acc tct atg gaa cct 2632 Met Thr Ser Thr Asp Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro 80 85 90 gaa ttc cca ggc gat gag gaa atg gag aag cgt tac cgt cgt tgg att 2680 Glu Phe Pro Gly Asp Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile 95 100 105 cgc tgg aac gca gcc atc atg gtt cac cgc gct cag cga cca ggc atc 2728 Arg Trp Asn Ala Ala Ile Met Val His Arg Ala Gln Arg Pro Gly Ile 110 115 120 ggc gtc ggc gga cac att tcc act tac gca ggc gca gcc cct ctg tac 2776 Gly Val Gly Gly His Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr 125 130 135 gaa gtt ggc ttc aac cac ttc ttc cgc gac agag ag cca ggc ggc 2824 Glu Val Gly Phe Asn His Phe Phe Arg Gly Lys Asp His Pro Gly Gly 140 145 150 155 ggc gac cag atc ttc ttc cag ggc cac gca tca cca ggt atg tac gca 2872 Gly Asp Gln Ile Phe Phe Gln Gly Ala Ser Pro Gly Met Tyr Ala 160 165 170 cgt gca ttc atg gag ggt cgc ctt tct gaa gac gat ctc gat ggc ttc 2920 Arg Ala Phe Met Glu Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe 175 180 185 cgt cag tcc cgt gag cag ggt ggc att ccg tcc tac cct cac 2968 Arg Gln Glu Val Ser Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His 190 195 200 cca cac ggt atg aag gac ttc tgg gag ttc cca act gtg tcc atg ggt 3016 Pro His Gly Met Lys Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly 205 210 215 ctt ggc cca atg gat gcc att tac cag gca cgt ttc aac cgc tac ctc 3064 Leu Gly Pro Met Asp Ala Ile Tyr Gln Ala Arg Phe Asn A rg Tyr Leu 220 225 230 235 gaa aac cgt ggc atc aag gac acc tct gac cag cac gtc tgg gcc ttc 3112 Glu Asn Arg Gly Ile Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe 240 245 250 ctt ggc gac ggc gaa atg gag cca gaa tca cgt ggt ctc atc cag 3160 Leu Gly Asp Gly Glu Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln 255 260 265 cag gct gca ctg aac aac ctg gac aac ctg acc ttc gtg gtt aac tgc A208 LaGln Asn Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys 270 275 280 aac ctg cag cgt ctc gac gga cct gtc cgc ggt aac acc aag atc atc 3256 Asn Leu Gln Arg Leu Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile 285 290 295 cag gaa ctc gag tcc ttc ttc cgt ggc gca ggc tgg tct gtg atc aag 3304 Gln Glu Leu Glu Ser Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys 300 305 310 315 gtt gtt tgg ggt cgc gag tgg gatg aag gac cag gat 3352 Val Val Trp Gly Arg Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp 320 325 330 ggt gca ctt gtt gag atc atg aac aac acc tcc gat ggt gac tac cag 3400 Gly Ala Leu Val Glu Ile Met As n Asn Thr Ser Asp Gly Asp Tyr Gln 335 340 345 acc ttc aag gct aac gac ggc gca tat gtt cgt gag cac ttc ttc gga 3448 Thr Phe Lys Ala Asn Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly 350 355 360 cgt gac cca cgc acc gca aag ctc gtt gag aac atg acc gac gaa gaa 3496 Arg Asp Pro Arg Thr Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu 365 370 375 atc tgg aag ctg cca cgt ggc ggc cac gat tac cgc aag gtt 3544 Ile Trp Lys Leu Pro Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala 380 385 390 395 gcc tac aag cga gct ctt gag acc aag gat cgc cca acc gtc atc ctt 3592 Ala Tyr Lys Arg Ala Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu 400 405 410 gct cac acc att aag ggc tac gga ctc ggc cac aac ttc gaa ggc cgt 3640 Ala His Thr Ile Lys Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg 415 420 425 aac gca acc cac cag atg aag aag ctg acg ctt gat gat ctg aag ttg 3688 Asn Ala Thr His Gln Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu 430 435 440 ttc cgc gac aag cag ggc atc cca atc acc gat gag cag ctg gag Asp 3736 Phe Lys Gln Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys 445 450 455 gat cct tac ctt cct cct tac tac cac cca ggt gaa gac gct cct gaa 3784 Asp Pro Tyr Leu Pro Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu 460 465 470 475 atc aag tac atg aag gaa cgt cgc gca gcg ctc ggt ggc tac ctg cca 3832 Ile Lys Tyr Met Lys Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro 480 485 490 gag cgt cgt gat cat gat cat gat cca cca ctg gat aag 3880 Glu Arg Arg Glu Asn Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys 495 500 505 ctt cgc tct gtc cgt aag ggc tcc ggc aag cag cag atc gct acc act 3928 Leu Arg Ser Val Arg Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr 510 515 520 atg gcg act gtt cgt acc ttc aag gaa ctg atg cgc gat aag ggc ttg 3976 Met Ala Thr Val Arg Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu 525 530 cg gat ctt gtc cca atc att cct gat gag gca cgt acc ttc ggt 4024 Ala Asp Arg Leu Val Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly 540 545 550 555 555 ctt gac tct tgg ttc cca acc ttg aag atc tac aac ccg cac g gt cag 4072 Leu Asp Ser Trp Phe Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln 560 565 570 aac tac gtg cct gtt gac cac gac ctg atg ctc tcc tac cgt gag gca 4120 Asn Tyr Val Pro Val Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala 575 580 585 cct gaa gga cag atc ctg cac gaa ggc atc aac gag gct ggt tcc gtg 4168 Pro Glu Gly Gln Ile Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val 590 595 600 gca tcg ttc atc ggt acc tcc tac gcc acc cac ggc aag gcc 4216 Ala Ser Phe Ile Ala Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala 605 610 615 atg att ccg ctg tac atc ttc tac tcg atg ttc gga ttc cag cgc acc 4264 Met Iet Leu Tyr Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr 620 625 630 635 ggt gac tcc atc tgg gca gca gcc gat cag atg gca cgt ggc ttc ctc 4312 Gly Asp Ser Ile Trp Ala Ala Ala Asp Gln Met Ala Arg Gly 640 645 650 ttg ggc gct acc gca ggt cgc acc acc ctg acc ggt gaa ggc ctc cag 4360 Leu Gly Ala Thr Ala Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln 655 660 665 cac atg gat gga cac tcc cct gtc ttg t tcc acc aac gag ggt gtc 4408 His Met Asp Gly His Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val 670 675 680 gag acc tac gac cca tcc ttt gcg tac gag atc gca cac ctg gtt cac 4456 Glu Thr Tyr Asp Pro Ser Phe Ala Tyr Glu Ile Ala His Leu Val His 685 690 695 cgt ggc atc gac cgc atg tac ggc cca ggc aag ggt gaa gat gtt atc 4504 Arg Gly Ile Asp Arg Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile 700 705 710 tac tac atc acc atc tac aac gag cca acc cca cag cca gct gag cca 4552 Tyr Tyr Ile Thr Ile Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro 720 725 730 gaa gga ctg gac gta gaa ggc ctg cac aag ggc atc tac ct tac tcc 4600 Glu Gly Leu Asp Val Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser 735 740 745 cgc ggt gaa ggc acc ggc cat gag gca aac atc ttg gct tcc ggt gtt 4648 Arg Gly Glu Gly Thr Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val 750 755 760 ggt atg cag tgg gct ctc aag gct gca tcc atc ctt gag gct gac tac 4696 Gly Met Gln Trp Ala Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr 765 770 775 gga gtt cgt gcc aac att tac tcc gct act tct tgg gtt aac ttg gct 4744 Gly Val Arg Ala Asn Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala 780 785 790 795 cgc gat ggc gct gct cgt aac aag gca cag ctg cgc aac cca ggt gca 4792 Asp Gly Ala Ala Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala 800 805 810 gat gct ggc gag gca ttc gta acc acc cag ctg aag cag acc tcc ggc 4840 Asp Ala Gly Glu Ala Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly 815 820 825 cca tac gtt gca gtg tct gac ttc tcc act gat ctg cca aac cag atc 4888 Pro Tyr Val Ala Val Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile 830 835 840 cgt gaa tgg gtc cca ggc gac tac acct ctc ggt gca gat ggc ttc 4936 Arg Glu Trp Val Pro Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe 845 850 855 ggt ttc tct gat acc cgc cca gct gct cgt cgc ttc ttc aac atc gac 4984 Gly Phe Ser Asp Thr Ala Ala Arg Arg Phe Phe Asn Ile Asp 860 865 870 875 gct gag tcc att gtt gtt gca gtg ctg aac tcc ctg gca cgc gaa ggc 5032 Ala Glu Ser Ile Val Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly 880 885 890 aag atc gac gtc tcc gtt gct gct cag gct gct gag aag ttc aag ttg 5080 Lys Ile Asp Val Ser Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu 895 900 905 gat gat cct acg agt gtt tcc gta gat cca aac gct gag gaa 5125 Asp Asp Pro Thr Ser Val Ser Val Asp Pro Asn Ala Pro Glu Glu 910 915 920 taaatcacct caagggacag ataaatcccg ccgccagacg ttagtctggc ggcgggattc 5185 gtcgtaaagc aagctctttt tagccgagaa acgccttgtc agacaatgtt gcgcccttga 5245 tattggcgaa ctcctgcagc aaatcgcgca cagtcaactt cgacttggta gcctgatctg 5305 cctggtagac aatctggcct tcatgcatca tgatcaggcg attgcccagg cgaattgcct 5365 gttcc 5370

<210> 10 <211> 922 <212> PRT <213> Brevibacterium lactofermentum <400> 10 Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn 1 5 10 15 Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp 20 25 30 Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45 Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60 Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp 65 70 75 80 Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp 85 90 95 Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala 100 105 110 Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His 115 120 125 Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn 130 135 140 His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe 145 150 155 160 Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu 165 170 175 Gly Arg Leu Ser Glu Asp Asp Lepsp Asp Gly Val Ser 180 185 190 Arg G lu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys 195 200 205 Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220 Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile 225 230 235 240 Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu 245 250 255 Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn 260 265 270 Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285 Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser 290 295 300 Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg 305 310 315 320 Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335 Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn 340 345 350 Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365 Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro 370 375 380 Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala 385 390 395 400 Leu G lu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys 405 410 415 Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln 420 425 430 Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445 Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460 Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys 465 470 475 480 Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495 Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510 Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg 515 520 525 Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 530 535 540 Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe 545 550 555 560 Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575 Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile 580 585 590 Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala 595 600 605 Ala Gly T hr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr 610 615 620 Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp 625 630 635 640 Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala 645 650 655 Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670 Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685 Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg 690 695 700 Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile 705 710 710 720 Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val 725 730 735 Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750 Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765 Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780 Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala 785 790 795 800 Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala 805 810 815 815 Phe Val T hr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830 Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro 835 840 845 Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860 Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val 865 870 875 880 Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser 885 890 895 Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910 Val Ser Val Asp Pro Asn Ala Pro Glu Glu 915 920

<210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for constructing plasmid for amplifying pdhA gene of Brevibacterium lactofermentum <400> 11 aatgccagga gtcaacaccc 20

<210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for constructing plasmid for amplifying pdhA gene of Brevibacterium lactofermentum <400> 12 acatggaaca ggcaattcgc 20

<210> 13 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying kanamycin resistant gene of Streptococcus faecalis <400> 13 cccgttaact gcttgaaacc caggacaata ac 32

<210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying kanamycin resistant gene of Streptococcus faecalis <400> 14 cccgttaaca tgtacttcag aaaagattag 30

<210> 15 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying Escherichia coli cloning vector pHSG399 <400> 15 gatatctacg tgccgatcaa cgtctc 26

<210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying Escherichia coli cloning vector pHSG399 <400> 16 aggccttttt ttaaggcagt tattg 25

[Brief description of the drawings]

FIG. 1 shows the effect of vitamin B ₁ for L- glutamic acid productivity of coryneform bacteria.

FIG. 2 is a diagram showing a construction process of a plasmid pK1.

FIG. 3 is a view showing a construction process of a plasmid pSFK6.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) C12R 1:13) (C12N 1/21 C12R 1:13) (C12P 13/14 C12R 1:13) (72 ) Inventor Kazuhiko Matsui 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture, Ajinomoto Co., Inc. (72) Inventor Osamu Kurahashi 1-1-Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Pref. ) Inventor Kazunari Horino 1-1 Ajinomoto Co., Ltd., Ajinomoto Fermentation Technology Co., Ltd. 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki City, Kanagawa Prefecture (72) Inventor Wataru Nakamatsu 1-1, Ajinomoto Co., Ltd.

Claims (8)

[Claims] 1. A coryneform bacterium having enhanced pyruvate dehydrogenase activity in a cell and having an ability to produce L-glutamic acid. 2. The coryneform bacterium according to claim 1, wherein the enhancement of pyruvate dehydrogenase activity is caused by increasing the copy number of a gene encoding pyruvate dehydrogenase in the bacterial cell. 3. The coryneform bacterium according to claim 2, wherein the gene encoding pyruvate dehydrogenase is derived from a bacterium belonging to the genus Escherichia or a coryneform bacterium. 4. The coryneform bacterium according to claim 1, which is cultured in a medium, L-glutamic acid is produced and accumulated in the culture, and L-glutamic acid is collected from the culture. A method for producing L-glutamic acid, comprising: Wherein said medium is vitamin B ₁ 20 [mu] g / L
5. The method according to claim 4, wherein the content is above. 6. A DNA encoding a protein represented by the following (A) or (B): (A) a protein having the amino acid sequence of SEQ ID NO: 10; (B) In the amino acid sequence of SEQ ID NO: 10, 1
Alternatively, a protein comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of several amino acids, and having pyruvate dehydrogenase activity. 7. The DNA according to claim 2, which is a DNA shown in (a) or (b) below. (A) a DNA comprising a base sequence consisting of base numbers 2360 to 5125 of SEQ ID NO: 9; (B) a DNA which hybridizes with a base sequence consisting of base numbers 2360 to 5125 of SEQ ID NO: 9 or a probe prepared from the base sequence under stringent conditions and encodes a protein having pyruvate dehydrogenase activity. 8. The method according to claim 1, wherein the stringent condition is 1 × S
The DNA according to claim 7, wherein the washing is performed at 60 ° C at a salt concentration corresponding to SC and 0.1% SDS.