JP2004313202A - Process for constructing amino acid-producing bacterium and process for producing amino acid by fermentation method using the constructed amino acid-producing bacterium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fermentation method for producing glutamic acid at a lower cost by using a coryneform glutamic acid-producing microorganism and increasing glutamic acid yield. <P>SOLUTION: The fermentation method for producing L-glutamic acid or L-arginine comprises cultivating a coryneform bacterium in a medium, producing and accumulating L-glutamic acid or L-arginine in the medium and collecting the L-glutamic acid or L-arginine from the medium, wherein the coryneform bacterium is introduced, at -35 region of a promoter sequence of amino acid-biosynthesizing genes on a chromosome of a coryneform bacterium, with at least one kind of DNA sequence selected from the group consisting of TTGTCA, TTGACA, TTGCTA and TGCCA, and/or introduced, at -10 region, with TATAAT sequence or a sequence substituted its base of ATAAT with another base. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for constructing a mutant having the ability to produce an amino acid at a high yield and a method for producing an L-amino acid by a fermentation method using the mutant.

There are roughly two methods for constructing mutant strains used for the production of amino acids by fermentation. One is a method of introducing mutations into DNA randomly using a chemical mutagen, and the other is a method of constructing a gene. This is a method using replacement. In the method using genetic recombination, strains with improved target substance productivity are obtained by enhancing genes in metabolic pathways involved in the biosynthesis of the target substance or weakening genes of enzymes involved in degradation. Can be developed. At that time, a plasmid capable of autonomously replicating independently of a chromosome in a cell has been mainly used as a method for enhancing a target gene.
However, there is a problem in a method for enhancing a target gene using a plasmid. Specifically, the degree of enhancement of the target gene is determined by the copy number of the plasmid itself. Depending on the type of the target gene, the copy number is too high and the expression level is too high, so that the growth is significantly suppressed or the reverse. In many cases, the ability to produce the target substance is reduced. In such a case, the degree of enhancement of the target gene can be reduced by using a plasmid having a low copy number, but the type of plasmid is often limited, and the expression level of the target gene is reduced. It is impossible to adjust freely.

Another problem is that the replication of the plasmid is often unstable and the plasmid is lost.
For example, Patent Literature 1 discloses a DNA fragment containing a glutamate dehydrogenase (GDH) -producing gene (glutamic acid dehydrogenase gene) derived from a glutamate-producing coryneform bacterium and a DNA fragment containing a gene required for autonomous replication in a cell ( And a recombinant DNA containing the plasmid (plasmid). By introducing the recombinant DNA into cells, a GDH-enriched strain can be bred and the production of substances (amino acids, proteins, etc.) by microorganisms is improved. It is disclosed that it is possible.
On the other hand, in Patent Document 2, the above recombinant DNA was transferred into Corynebacterium to prepare a strain with enhanced enzyme activity, and L-glutamic acid was produced by fermentation using this strain. However, the production amount and yield are not yet satisfactory, and it is said that it is desired to further improve the productivity of L-glutamic acid. This demand was met by transferring a recombinant DNA containing two genes, a glutamate dehydrogenase producing gene and an isocitrate dehydrogenase gene (ICDH), from a glutamate-producing coryneform bacterium to a glutamate-producing coryneform bacterium. It has been achieved.

  On the other hand, Patent Document 3 discloses a Corynebacterium strain, a first functional DNA sequence for expression in the strain, a second DNA sequence encoding an amino acid, a polypeptide and / or a protein, and A secretion cassette containing a third DNA sequence inserted between the two DNA sequences, wherein said third DNA sequence ensures secretion of amino acids, polypeptides and / or proteins by said Corynebacterium strain. And a corynebacterium expression and secretion system characterized by encoding a protein element selected from the group consisting of: Specifically, polypeptide secretion is disclosed, and NTG mutagenesis is performed on a Corynebacterium strain to select a mutant strain that confers resistance to a glutamate analog, 4-fluoroglutamic acid 4-fluoroglutamate (4FG), This is subjected to transformation with pCGL141, and it is disclosed that a strain with enhanced GDH expression can be obtained from the above-mentioned analog-resistant bacteria. Here, it is shown that a mutation occurs in the nucleotide sequence 251 to 266 of the GDH promoter.

JP-A-61-268185 Japanese Patent No. 2520895 Japanese Patent Publication No. Hei 6-502548

The present invention relates to a method for constructing a mutant strain capable of appropriately enhancing and regulating the expression level of a target gene without using a plasmid and having the ability to produce amino acids in high yield by genetic recombination or mutation. The purpose is to provide.
An object of the present invention is to provide a promoter for GDH capable of conferring the ability to produce glutamic acid in a high yield on a Corynebacterium strain without causing a significant increase in by-product aspartic acid and alanine.
Another object of the present invention is to provide a GDH gene having the above-mentioned GDH promoter sequence.
Another object of the present invention is to provide an L-glutamic acid-producing corynebacterium strain having the above gene.
An object of the present invention is to provide a method for producing an amino acid by a fermentation method using a constructed amino acid-producing bacterium.
An object of the present invention is to provide a glutamate fermentation method that uses a coryneform glutamate-producing bacterium, improves the yield of glutamic acid, and produces glutamic acid at lower cost.

The present invention has been made based on the finding that the above problem can be efficiently solved by variously modifying the promoter of the amino acid biosynthesis system gene on the chromosome and adjusting the expression level of the gene of interest. is there. In particular, the present invention has been made based on the finding that the above problem can be efficiently solved by introducing a specific mutation into the -35 region and / or the -10 region which is a specific region of the promoter.
That is, the present invention provides a mutation in a coryneform bacterium by causing a mutation approaching a consensus sequence or by introducing a gene into a promoter sequence of an amino acid on the chromosome of a coryneform bacterium or a nucleic acid biosynthesis gene, or introducing the mutation by genetic recombination. The present invention provides a method for preparing a coryneform bacterium having improved ability to produce amino acids or nucleic acids, comprising preparing a body, culturing the mutant, and collecting a mutant having a high production amount of the desired amino acid or nucleic acid. .

The present invention also provides a method wherein at least one DNA sequence selected from the group consisting of CGGTCA, TTGTCA, TTGACA and TTGCCA is substituted in the -35 region and / or the TATAAT sequence or the ATAAT base in the sequence is substituted with another base in the -10 region. And a promoter for a glutamate dehydrogenase (GDH) -producing gene, which has a sequence that does not inhibit the promoter function.
The present invention also provides a glutamate dehydrogenase producing gene having the above promoter.
The present invention also provides a coryneform L-glutamic acid producing bacterium having the above gene.

The present invention also provides a coryneform bacterium having an improved ability to produce amino acids or nucleic acids constructed by the above-described method, by culturing in a medium, producing and accumulating a target amino acid or nucleic acid in the medium, and collecting the same from the medium. A method for producing the amino acid or nucleic acid by a fermentation method is provided.
The present invention also provides a method for culturing a coryneform L-glutamic acid-producing bacterium having resistance to 4-fluoroglutamic acid in a liquid medium, producing and accumulating L-glutamic acid in the medium, and collecting the L-glutamic acid from the medium. And a method for producing L-glutamic acid by a fermentation method.

  The coryneform glutamate-producing bacterium referred to in the present invention includes a bacterium that has been conventionally classified into the genus Brevibacterium, but is now integrated as a bacterium belonging to the genus Corynebacterium (Int.J. Syst.Bacteriol., 41, 255 ( 1981)), and also includes bacteria of the genus Brevibacterium, which is closely related to the genus Corynebacterium. Therefore, the mutant strain used in the present invention can be derived from the following coryneform glutamate-producing bacteria belonging to the genus Brevibacterium or Corynebacterium. In the present specification, when not referring to glutamic acid productivity, the genus Corynebacterium and the genus Brevibacterium may be simply referred to as coryneform bacterium.

Corynebacterium acetoacid film ATCC13870
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium carnae ATCC15991
Corynebacterium glutamicum ATCC13032
Brevibacterium divaricatum ATCC14020
Brevibacterium lactofermentum ATCC13869
Corynebacterium lilium ATCC15990
Brevibacterium flavum ATCC14067
Corynebacterium meracecola ATCC17965
Brevibacterium saccharolyticum ATCC14066
Brevibacterium in Mario film ATCC14068
Brevibacterium roseum ATCC13825
Brevibacterium thiogenitalis ATCC19240
Microbacterium ammonia phyllum ATCC15354
Corynebacterium thermoaminogenes AJ12310 (FERM 9246)

The amino acid as the target substance may be any amino acid as long as the gene involved in biosynthesis and its promoter are known. Specific examples of enzymes involved in biosynthesis include GDH, citrate synthase (CS), isocitrate dehydrogenase (ICDH), pyruvate dehydrogenase (PDH), and aconitase (ACO) in the case of glutamic acid fermentation. Is valid.
In lysine fermentation, biosynthetic enzymes such as aspartate kinase (AK), dihydrodipicolinate synthase, dihydrodipiconate reductase, diaminopimelate dehydrogenase, and diaminopimelate decarboxylase are added. Lysine excretion protein (lysE gene) involved in membrane excretion is also effective.
In arginine fermentation, N-acetylglutamate synthase, N-acetylglutamate kinase, N-acetylglutamylphosphate reductase, acetylornithine aminotransferase, N-acetylornitinase, ornithine carbamyltransferase, argininosuccinate synthase and argininosuccinase Produced in catalyzed reactions. And these enzymes are effective. Further, these enzymes are encoded by the respective genes of argA, argB, argC, argD, argE, argF, argG, and argH in order.

In serine fermentation, enzymes such as 3-phosphoglycerate dehydrogenase, phosphoserine transaminase, and phosphoserine phosphatase are effective.
In the phenylalanine fermentation, deoxyarabinoheptulonate phosphate synthase, 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, shikimate dehydrogenase, shikimate kinase, 5-enolpyruvylshikimate-3-phosphate synthase Biosynthetic enzymes such as chorismate synthase, chorismate mutase and prephenate dehydratase are effective. In addition, sugar metabolism enzymes such as transketolase, transaldolase, and phosphoenol pyruvate synthase are also effective.
In tryptophan fermentation, enzymes belonging to the tryptophan operon are effective in addition to the enzymes considered to be effective in the phenylalanine fermentation and the enzymes considered to be effective in the serine fermentation.
In proline fermentation, γ-glutamyl kinase, γ-glutamyl semialdehyde dehydrogenase, pyrroline-5-carboxylate reductase, and the like are effective in addition to the enzymes considered to be effective in glutamic acid fermentation.
In glutamine fermentation, glutamine synthetase is effective in addition to the enzymes considered to be effective in glutamic acid fermentation.

In inosine production, enhanced expression of enzymes such as 5-phosphoribosyl 1-diphosphate synthase, 5-phosphoribosyl 1-2-phosphate aminotransferase, and phosphoribosylaminoimidazolecarboxamide formyltransferase is considered to be effective.
In guanosine production, in addition to 5-phosphoribosyl 1-2-phosphate synthase, 5-phosphoribosyl 1-2-phosphate aminotransferase, and phosphoribosylaminoimidazolecarboxamide formyltransferase, 5'-inosinic acid dehydrogenase and 5'-xanthylic acid aminase Is thought to be effective.
In adenosine production, in addition to 5-phosphoribosyl 1-2-phosphate synthase, 5-phosphoribosyl 1-2-phosphate aminotransferase, and phosphoribosylaminoimidazolecarboxamide formyltransferase, enhanced expression of adenylosuccinate synthetase is effective. it is conceivable that.
In nucleotide production, enhanced expression of phosphoribosyltransferase, inosine kinase, guanosine kinase, and adenosine kinase is considered to be effective.

In the present invention, a promoter sequence of a desired amino acid biosynthesis system gene on the chromosome of a coryneform amino acid-producing bacterium, for example, a promoter sequence such as the above-mentioned GDH promoter is mutated using a chemical or the like to approach a consensus sequence. A mutant of a coryneform amino acid-producing bacterium is prepared by causing the mutation or introducing the mutation by genetic recombination.
Here, the consensus sequence is a sequence in which the most frequently occurring bases are arranged by comparing many promoter sequences. Such consensus sequences include consensus sequences for E. coli, Bacillus subtilis, and the like. The consensus sequence of E. coli is described in Diane K. Hawley and William R. McClure Nuc. Acid. Res. Genet 186: 339-346 (1982).
The above mutation may be caused only in one promoter sequence, for example, a GDH promoter alone, but two or more promoter sequences, for example, a GDH promoter, citrate synthase (CS) or isocitrate dehydrogenase (ICDH) May be woken up.
In the present invention, the mutant thus obtained is cultured to collect a mutant having a high production amount of the target amino acid.

In the case of glutamate fermentation, GDH of coryneform glutamate producing bacteria has been shown to have its own promoter sequence in its upstream region (Sahm et al. Molecular Microbiology (1992), 6, 317-326).
For example, the GDH promoter of the present invention, the GDH gene having the GDH promoter sequence, and the L-glutamic acid-producing corynebacterium strain having the gene can be obtained, for example, as follows.
That is, the above strains are subjected to mutation treatment such as ultraviolet irradiation, X-ray irradiation, radiation irradiation, and treatment with a mutagenic agent, and are resistant to 4-fluoroglutamic acid on an agar plate medium containing 4-fluoroglutamic acid. Is obtained. That is, the mutated strain may be applied to an agar plate medium containing 4-fluoroglutamic acid at a concentration that suppresses the growth of the parent strain, and the grown mutant strain may be isolated.
In addition, a large number of GDH gene promoter sequences were prepared by substituting various mutation-introduced sequences using a site-directed mutagenesis method, and the relationship between each sequence and GDH activity was examined to determine L-glutamic acid productivity. Can be selected.

In the present invention, in particular, the DNA sequence of the -35 region of the promoter of the GDH gene is at least one DNA sequence selected from the group consisting of CGGTCA, TTGTCA, TTGACA and TTGCCA, and / or the -10 region of the promoter. It is preferable that the DNA sequence is TATAAT or that the -10 region has the ATAAT base in the TATAAT sequence replaced with another base and does not inhibit the promoter function. The ATAAT base of the -10 sequence is replaced with another base in the ATAAT base, and a sequence that does not inhibit the promoter function can be selected. This is because a dramatic increase in GDH specific activity was observed only by replacing “C” with “T” (see Table 1, p6-4), and it is considered that other bases may be used.
The promoter sequence of the GDH gene is described, for example, in Sahm et al., Molecular Microbiology (1992), 6, 317-326, supra, and is described in SEQ ID NO: 1. The sequence of the GDH gene itself is described in, for example, Sahm et al. Molecular Microbiology (1992), 6, 317-326, and is also described in SEQ ID NO: 1.
Similarly, mutations can be made in the promoters of citrate synthase (CS) and isocitrate dehydrogenase (ICDH).
Thus, the promoter for GDH includes at least one DNA sequence selected from the group consisting of CGGTCA, TTGTCA, TTGACA and TTGCCA in the −35 region and / or the TATAAT sequence or the ATAAT base of the sequence in the −10 region. Those having a sequence substituted with another base and not inhibiting the promoter function can be mentioned. Also, there is provided a glutamate dehydrogenase producing gene having the above promoter.

Examples of the CS promoter include those having a TTGACA sequence in the -35 region and / or a TATAAT sequence in the -10 region, and having a sequence that does not inhibit the promoter function. Further, the present invention provides a CS gene having the above promoter.
The promoter for ICDH has either the TTGCCA sequence or the TTGACA sequence in the first or second promoter of the -35 region and / or the TATAAT sequence in the first or second promoter of the -10 region. And those having a sequence that does not inhibit the promoter function. Also provided is an icd gene having the above promoter.
Examples of the PDH promoter include those having a TTGCCA sequence in the -35 region and / or a TATAAT sequence in the -10 region, and which do not inhibit the promoter function. Also provided is a PDH gene having the above promoter.
The present invention also provides a coryneform L-glutamic acid producing bacterium having the above gene.

As the promoter for argininosuccinate synthase, at least one kind of DNA sequence selected from the group consisting of TTGCCA, TTGCTA and TTGTCA in the -35 region and / or the TATAAT sequence or the ATAAT base of the sequence substituted with another base in the -10 region. And has a sequence that does not inhibit the promoter function. Also provided is an argininosuccinate synthase gene having the above promoter.
The present invention also provides a coryneform arginine-producing bacterium having the above gene.
The coryneform amino acid of the present invention, preferably an L-glutamic acid-producing bacterium, is cultured in a liquid medium to produce and accumulate a desired amino acid, preferably L-glutamic acid, in the medium. Obtainable.
In the present invention, a normal nutrient medium containing a carbon source, a nitrogen source, inorganic salts, growth factors and the like is used as a liquid medium for culturing the above strain.
As the carbon source, glucose, fructose, sucrose, molasses, carbohydrates such as starch hydrolysate, alcohols such as ethanol and glycerol, and organic acids such as acetic acid are used. As the nitrogen source, ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, ammonium acetate, ammonia, peptone, meat extract, yeast extract, corn steep liquor and the like are used. When auxotrophic mutants are used, it is preferable to add those required substances as a sample or a natural product containing the same.

Coryneform bacteria generally produce L-glutamic acid under biotin restriction. Therefore, the amount of biotin in the medium is restricted, or a biotin action inhibitor such as a surfactant or penicillin is added.
The fermentation is preferably performed for 2 to 7 days under aerobic conditions such as shaking culture or aeration and stirring culture while maintaining the pH of the culture solution between 5 and 9. It is preferable to use urea, calcium carbonate, ammonia gas, aqueous ammonia or the like for adjusting the pH. The culture temperature is preferably from 24 to 37 ° C.
The L-glutamic acid produced and accumulated in the culture solution may be collected by a conventional method, for example, an ion exchange resin method, a crystallization method, or the like. Specifically, L-glutamic acid may be adsorbed and separated by an anion exchange resin, or may be neutralized and crystallized.
According to the present invention, a target amino acid can be obtained in high yield by introducing a mutation into the promoter region of the amino acid biosynthetic gene of a coryneform amino acid-producing bacterium and controlling the expression level of the target gene. As described above, the desired amino acid can be stably obtained at a high yield without dropout, which is a great industrial advantage.

Further, according to the present invention, various promoters capable of conferring the ability to produce amino acids, particularly glutamic acid, in high yields on Corynebacterium strains without causing an increase in by-product aspartic acid and alanine, especially promoters for GDH Can be provided.
Further, according to the present invention, a coryneform L-glutamic acid-producing bacterium is subjected to a mutation treatment, and a strain in which a mutation has occurred in the promoter region of the GDH gene and which is resistant to 4-fluoroglutamic acid is collected. Glutamic acid can be obtained in high yield by culturing, which is industrially advantageous.

Next, the present invention will be described with reference to examples.
Example 1 Preparation of Mutant GDH Promoter Using a site-directed mutagenesis method, a mutant GDH promoter was prepared by the following method.
(1) Preparation of GDH Gene Having Various Mutant Promoters The wild-type sequence of the -35 region and the -10 region of the promoter of the GDH gene of coryneform bacterium is shown in Sequence 1. However, the wild-type promoter sequence has already been reported (Molecular Microbiolgy (1992), 6, 317-326).
A method for preparing a plasmid carrying a GDH gene having a mutant promoter is as follows. As shown in FIG. 1, the chromosomal gene of the coryneform bacterium ATCC13869 strain prepared based on the “Bacterial Genome DNA purification kit” (Advanced Genetic Technologies Corp.) was used as a template, and PCR was performed upstream and downstream of the GDH gene by PCR. After amplifying the gene and blunting both ends, it was inserted into the SmaI site of plasmid pHSG399 (Takara Shuzo). Next, a plasmid pGDH was prepared by introducing a replication origin obtained from the plasmid pSAK4 having a replication origin capable of replicating in coryneform bacteria into the SalI site of this plasmid. In this method, the GDH gene having each of the above promoter sequences can be prepared by using a primer having a sequence shown in Sequence Listings 1 to 6 as an upstream primer of the GDH gene. In addition, it was confirmed by base sequence determination that no mutation was introduced into the PCR amplified fragment used here except for the mutation in the introduced promoter sequence. In order to construct pSAK4, a plasmid pHK4 having a replication origin derived from a plasmid pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)) capable of autonomously replicating in Corynebacterium bacteria already obtained. (Japanese Unexamined Patent Publication (Kokai) No. 5-7491) was digested with restriction enzymes BamHI and KpnI to obtain a DNA fragment containing an origin of replication, and the obtained fragment was blunted using a DNA blunt-end kit (Blunting kit, manufactured by Takara Shuzo Co., Ltd.). After termination, a SalI linker (manufactured by Takara Shuzo Co., Ltd.) was ligated, and this was inserted into the SalI site of pHSG299. The resulting plasmid is pSAK4.

(2) Comparison of the expression level of GDH having each promoter sequence The plasmids prepared as described above were introduced into a coryneform bacterium ATCC13869 wild type strain, respectively. An electroporation method was used for the introduction (see Japanese Patent Application Laid-Open No. 2-2077791). In order to compare the expression levels of GDH in these produced strains, the specific activity of GDH was examined. The activity was measured according to the method of Sahm et al. Table 1 shows the results.

Table 1

Strain Promoter sequence GDH specific activity Relative value
−35 −10
ATCC 13869 TGGTCA CATAAT 7.7 0.1
/ pGDH TGGTCA CATAAT 82.7 1.0
/ p6-2 CGGTCA CATAAT 33.1 0.4
/ p6-4 TGGTCA TATAAT 225.9 2.7
/ p6-3 TTGACA TATAAT 327.2 4.0
/ p6-7 TTGCCA TATAAT 407.0 4.9
/ p6-8 TTGTCA TATAAT 401.3 4.9

The above ATCC 13869 / p6-2 to ATCC 13869 / p6-8 correspond to SEQ ID NOs: 2 to 6, and these sequences are based on the sequence described in SEQ ID NO: 1 (wild type) and are underlined as follows: It has been changed.
SEQ ID NO: 1 5'-TTAATTCTTTG TGGTCA TATCTGCGACACTGC CATAAT TTGAACGT-3 '
Sequence number 2 CGGTCA CATAAT
SEQ ID NO: 3 TGGTCA TATAAT
SEQ ID NO: 4 TTGACA TATAAT
SEQ ID NO: 5 TTGCCA TATAAT
SEQ ID NO: 6 TTGTCA TATAAT
These sequences are linear, double-stranded synthetic DNA.

Example 2: Acquisition of mutant strain
(1) Induction of a mutant strain having resistance to 4-fluoroglutamic acid
The AJ13029 strain is a glutamic acid producing strain described in WO96 / 06180, and does not produce glutamic acid at a culture temperature of 31.5 ° C, but produces glutamic acid even in the absence of a biotin action inhibitor when the culture temperature is shifted to 37 ° C. Mutant strain. In this example, Brevibacterium lactofermentum AJ13029 was used as a parent strain for mutant induction. Of course, a glutamate-producing strain other than the AJ13029 strain can be a parent strain for induction of a mutant having resistance to 4-fluoroglutamic acid.
The AJ13029 strain was cultured on a CM2B agar medium (Table 2) at 31.5 ° C. for 24 hours to obtain bacterial cells. The obtained cells were treated with an aqueous solution of 250 μg / ml N-methyl-N′-nitro-N-nitrosoguanidine at 30 ° C. for 30 minutes. The cells were inoculated on an agar plate medium (Table 3) containing fluoroglutamic acid (4FG) and cultured at 31.5 ° C for 20 to 30 hours to form colonies. At this time, first, a medium containing 1 mg / ml of 4FG was prepared with an inclination, and the same medium without 4FG was horizontally layered thereon. Thus, a concentration gradient of 4FG is created on the surface of the agar medium. When the above-mentioned mutation-treated cells are seeded on this plate, a boundary line is formed around the growth limit region of the strain. A strain that formed a colony in a region where 4FG was present at a higher concentration than this boundary was collected. Thus, about 50 4FG-resistant strains were obtained from about 10,000 mutant-treated strains.

Table 2 CM2B agar medium

Component concentration
Polypeptone (Nippon Pharmaceutical) 1.0%
Yeast extract (Difco) 1.0%
NaCl 0.5%
d-biotin 10 μg / l
Agar 1.5%
(Adjusted to pH 7.2 KOH)

Table 3 Agar medium

Component Amount added per liter of water
10 g of glucose
MgSO ₄ · 7H ₂ O 1 g
FeSO ₄ · 7H ₂ O 0.01 g
MnSO ₄ · 4-6H ₂ O 0.01 g
Siamine hydrochloride 0.2 mg
d-biotin 0.05 mg
(NH ₄ ) ₂ SO ₄ 5 g
Na ₂ HPO ₄・ 12H ₂ O 7.1 g
KH ₂ PO ₄ 1.36 g
Agar 15 g

(2) Confirmation of L-glutamic acid-producing ability of mutant strain showing resistance to 4FG The glutamate-producing ability of the about 50 mutant strains obtained in (1) above and the parent strain AJ13029 was determined as follows. Confirmed as follows.
Cells obtained by culturing AJ13029 and each mutant strain on a CM2B agar medium at 31.5 ° C. for 20 to 30 hours were inoculated into a liquid medium having the composition shown in Table A medium A at 31.5 ° C. Shaking culture was started. After about 22 hours, a new medium was added so that the final concentration became the concentration shown in Table B medium, the temperature was shifted to 37 ° C., and culture was performed for about 24 hours. After completion of the culture, the presence or absence of L-glutamic acid was examined using a Biotech Analyzer manufactured by Asahi Kasei Corporation. As a result, about 50 strains were cultured, and two strains having higher glutamic acid yield and higher GDH activity than the parent strain were separated (strains A and B). When the respective GDH activities were measured, the specific activity of GDH was increased in both strains (Table 5). The measurement of GDH activity followed the method of ER Bormann et al. (Molecular Microbiol., 6, 317-326 (1996)). Therefore, when the nucleotide sequence of the GDH gene was analyzed, mutation points were present only in the promoter region of GDH (Table 6).

Table 4

Component A medium B medium
Glucose 3 g / dl 5 g / dl
KH ₂ PO ₄ 0.14 g / dl 0.14 g / dl
_{MgSO 4 · 7H 2 O 0.04 g} / dl 0.04 g / dl
_{FeSO 4 · 7H 2 O 0.001 g} / dl 0.001 g / dl
_{MnSO 4 · 4H 2 O 0.001 g} / dl 0.001 g / dl
(NH ₄ ) ₂ SO ₄ 1.5 g / dl 2.5 g / dl
Soy protein hydrolyzate 1.5 ml / dl 0.38 ml / dl
Thiamine hydrochloride 0.2 mg / l 0.2 mg / l
Biotin 0.3 mg / l 0.3 mg / l
Defoamer 0.05 ml / l 0.05 ml / l
CaCO ₃ 5 g / dl 5 g / dl
pH 7.0 (adjusted with KOH)

Table 5 Glutamate production and GDH activity of mutants

Strain Glu (g / dl) GDH specific activity Relative value
AJ13029 2.6 7.7 1.0
FGR1 2.9 23.1 3.0
FGR2 3.0 25.9 3.4

Table 6 Nucleotide sequence of GDH promoter region of mutant strain

Strain GDH promoter sequence
−35 −10
AJ13029 TGGTCA TTCTGTGCGACACTGC CATAAT
FGR1 TGGTCA TTCTGTGCGACACTGC TATAAT
FGR2 TTGTCA T-CTGTGCGACACTGC TATAAT

Example 3 Introduction of Mutations into the CS Gene Promoter Region of Coryneform Glutamate-Producing Bacteria This example shows an example in which a promoter-enhanced strain of a gene encoding glutamate dehydrogenase (GDH) and citrate synthase (CS) was prepared.
(1) Cloning of gltA gene The nucleotide sequence of the gene gltA encoding citrate synthase of coryneform bacterium has already been elucidated (Microbiol. 140 1817-1828 (1994)). Based on this sequence, primers shown in SEQ ID NOs: 7 and 8 were synthesized. On the other hand, chromosomal DNA of Brevibacterium lactofermentum ATCC13869 was prepared using the Bacterial Genome DNA Purification Kit (Advanced Genetic Technologies Corp.). 0.5 μg of this chromosomal DNA, 10 pmol of each of the oligonucleotides, 8 μl of dNTP mixture (2.5 mM each), 5 μl of 10 × LA Taq Buffer (Takara Shuzo), 2 U of LA Taq (Takara Shuzo) A liquid was prepared. This reaction solution was subjected to 30 cycles of PCR using a thermal cycler TP240 (Takara Shuzo) under the conditions of denaturation at 94 ° C for 30 seconds, association at 55 ° C for 15 seconds, and extension reaction at 72 ° C for 3 minutes. A 3 Kbp DNA fragment was amplified. The obtained amplified fragment was purified by SUPREC02 manufactured by Takara Shuzo and blunt-ended. Blunting was performed using a Blunting Kit manufactured by Takara Shuzo. This and pHSG399 (Takara Shuzo) completely degraded with SmaI were mixed and ligated. The ligation reaction was performed using DNA ligation kit ver2 manufactured by Takara Shuzo. After ligation, transformation was performed using competent cells of Escherichia coli JM109 (manufactured by Takara Shuzo Co., Ltd.), and IPTG (isopropyl-β-D-thiogalactopyranoside) 10 μg / ml and X-Gal (5-bromo L medium containing 40 μg / ml of 4-chloro-3-indolyl-β-D-galactoside) and 40 μg / ml of chloramphenicol (10 g / l of bactotryptone, 5 g / l of bactoeast extract, 5 g / l of NaCl, After coating overnight on agar (15 g / l, pH 7.2) and culturing overnight, the appeared white colonies were picked and separated into single colonies to obtain transformed strains.
A plasmid was prepared from the transformant using the alkaline method (Biotechnical Experiments, edited by The Biotechnology Society of Japan, p. 105, Baifukan, 1992), and a restriction enzyme map was prepared, which was equivalent to the restriction map shown in FIG. Was named pHSG399CS.

(2) Mutation into the gltA promoter
Mutan-Super Express Km manufactured by Takara Shuzo was used to introduce a mutation into the gltA promoter region. A specific method will be described below. pHSG399CS was completely digested with EcoRI and SalI to prepare an EcoRI-SalI fragment containing the gltA gene, and this fragment was ligated to a fragment obtained by completely digesting pKF19kM (Takara Shuzo) with EcoRI and SalI. After ligation, transformation was performed using competent cells of Escherichia coli JM109 (Takara Shuzo), applied to an L medium containing 10 μg / ml of IPTG, 40 μg / ml of X-Gal and 25 μg / ml of kanamycin, and cultured overnight. Appeared white colonies were picked and separated into single colonies to obtain transformed strains. A plasmid was prepared from the transformed strain, and one containing the gltA gene was named pKF19CS.

  Using pKF19CS as a template, PCR was performed using 5′-terminal phosphorylated synthetic DNA shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 and selection primer attached to Mutan super Express Km. This PCR product was used to transform competent cells of Escherichia coli MV1184 (Takara Shuzo), applied to an L medium containing 25 μg / ml of kanamycin, cultured overnight, and the emerged colonies were picked and separated into single colonies. Thus, a transformant was obtained. Plasmid DNA was prepared from the transformant, and the nucleotide sequence of the gltA promoter was determined using the synthetic DNA shown in SEQ ID NO: 12 by the method of Sanger (J. Mol. Biol., 143, 161, (1980)). Specifically, the nucleotide sequence was analyzed by Genetic Analyzer ABI310 (Applied Biosystems) using the Dye terminator sequencing kit (Applied Biosystems). Those in which the gltA promoter region was replaced with the sequence shown in Table 7 were named pKF19CS1, pKF19CS2, and pKF19CS4, respectively.

Table 7

-35 area -10 area
pKF19CS ATGGCT TATAGC
pKF19CS1 ATGGCT TATAAC
pKF19CS2 ATGGCT TATAAT
pKF19CS4 TTGACA TATAAT

(3) Construction of mutant gltA plasmid pKF19CS, pKF19CS1, pKF19CS2, and pKF19CS4 constructed in (2) were completely digested with SalI and EcoRI (Takara Shuzo), respectively. On the other hand, plasmid pSFK6 (Japanese Patent Application No. 11-69896) having a replication origin derived from plasmid pAM330 (Japanese Patent Application Laid-Open No. 58-67699) capable of autonomous replication in coryneform bacteria was completely digested with EcoRI and SalI, and this and gltA were digested. The about 2.5 kb fragment was ligated. After ligation, transformation was performed using competent cells of Escherichia coli JM109, and applied to an L medium containing 10 μg / ml of IPTG, 40 μg / ml of X-Gal, and 25 μg / ml of kanamycin. Was picked up and a single colony was separated to obtain a transformed strain. Plasmids were prepared from the transformants, and plasmids containing the gltA gene were designated as pSFKC, pSFKC1, pSFKC2, and pSFKC4, respectively.

(4) Measurement of CS expression level of mutant gltA plasmid in coryneform bacteria The plasmid constructed in (3) above was introduced into Brevibacterium lactofermentum ATCC13869. Specifically, using an electric pulse method (Japanese Patent Laid-Open No. 2-07791), the selection of transformants was performed on a CM2B plate medium containing 25 μg / ml kanamycin (Bactotryptone 10 g / l, Bacto yeast extract 10 g / l, NaCl 5 g / l, biotin 10 μg / L, agar 15 g / l, pH 7.0) at 31 ° C. After culturing for two nights, the emerged colonies were picked up, single colonies were separated, and transformants carrying pSFKC, pSFKC1, pSFKC2, and pSFKC4 were named BLCS, BLCS1, BLCS2, and BLCS4, respectively. The transformant was inoculated into the medium shown in Table 8, culture was continued at 31 ° C., and the culture was terminated before glucose was completely consumed. The culture was centrifuged to separate the cells. The cells were washed with 50 mM Tris buffer (pH 7.5) containing 200 mM sodium glutamate, suspended in the same buffer, and sonicated. Ultrasonic crushing was performed by UD-201 of TOMY. After sonication, the mixture was centrifuged at 10,000 g for 20 minutes to remove unbroken cells to obtain a crude enzyme solution. The activity of citrate synthase may be measured according to (Methods Enzymol. 13, 3-11 (1969)). Specifically, a crude enzyme solution was added to a reaction solution containing TisHCl 100 mM (pH 8), DTNB 0.1 mM, sodium glutamate 200 mM, and acetyl-CoA 0.3 mM, and the increase in absorbance at 412 nm at 30 ° C. was measured by Hitachi Spectrophotometer U-3210. And determined as the background. Further, oxaloacetic acid was added to a final concentration of 0.5 mM, the increase in absorbance at 412 nm was measured, and the value obtained by subtracting the background value was defined as the activity of citrate synthase. Further, Protein Assay (BIO-RAD) was used for measuring the protein concentration of the crude enzyme solution. Bovine serum albumin was used as a standard protein. Table 9 shows the measurement results. It was confirmed that the citrate synthase activity was increased in the gltA promoter mutant strain as compared with the wild-type gltA promoter.

Table 8
Ingredient concentration
Glucose 50 g / l
KH ₂ PO ₄ 1 g / l
_{MnSO 4 · 7H 2 O 0.4 mg} / l
_{FeSO 4 · 7H 2 10 mg /} l
Soy protein hydrolyzate 20 ml / l
Biotin 0.5 mg / l
Siamine hydrochloride 2 mg / l

Table 9

Strain dABS / min / mg Relative activity Relative activity
Wild strain 6.8 1.0
BLCS00 38.8 5.7 1.0
BLCS01 57.1 8.4 1.2
BLCSO2 92.5 13.6 1.9
BLCS04 239.4 35.2 4.8

(5) Introduction of Mutant gltA Gene into Temperature-Sensitive Plasmid As a method for integrating a mutant gltA promoter sequence into a chromosome, a method using a plasmid whose replication is temperature-sensitive in coryneform bacteria is known ( JP-A-5-7491. Here, pSFKT2 (Japanese Patent Application No. 11-81693) was used as a plasmid vector whose replication is temperature-sensitive in coryneform bacteria. As mutant gltA promoter sequences, pKFCS1, pKFCS2, and pKFCS4 were completely degraded with SalI and BstPI and blunt-ended, and those obtained by completely degrading pSFKT2 with SmaI were ligated. After ligation, transformation was performed using competent cells of Escherichia coli JM109 (manufactured by Takara Shuzo Co., Ltd.), applied to an L medium containing 10 μg / ml of IPTG, 40 μg / ml of X-Gal and 25 μg / ml of kanamycin, and cultured overnight. Thereafter, the emerged white colonies were picked and separated into single colonies to obtain transformed strains. Plasmids were prepared from the transformants, and the temperature-sensitive shuttle vectors containing the gltA gene were named pSFKTC1, pSFKTC2, and pSFKTC4, respectively.

(6) Introduction of mutant gltA promoter into chromosome
pSFKTC1, pSFKTC2, and pSFKTC4 were each introduced into the strain Brevibacterium lactofermentum FGR2 by the electric pulse method. Transformants were selected at 25 ° C. on a CM2B plate medium containing 25 μg / ml kanamycin. After the introduction, the obtained strain was cultured in a CM2B liquid medium, and diluted with CM2B plate containing 25 μg / ml kanamycin so as to have a density of 10 ³ to 10 ⁵ cfu per plate, followed by application at 34 ° C. Cultured. Strains harboring the temperature-sensitive plasmid become kanamycin-sensitive and cannot form colonies due to inhibition of plasmid replication at this temperature, but strains with plasmid DNA integrated into the chromosome form colonies, and should be selected. Can be. Emerging colonies were picked up and single colonies were separated. A chromosomal DNA was extracted from this strain, and PCR was performed using this as a template and the primers shown in SEQ ID NO: 8 and SEQ ID NO: 13, and an amplified fragment of about 3 kb was confirmed. Therefore, this strain was shown to have a mutant gltA gene derived from a temperature-sensitive plasmid integrated near the gltA gene on the host chromosome by homologous recombination. Strains derived from pSFKTC1, 2, and 4 were named BLCS11, BLCS12, and BLCS14, respectively.

(7) Acquisition of gltA promoter-substituted strain By homologous recombination, first, a kanamycin-sensitive strain was obtained from the BLCS11, BLCS12, and BLCS14 strains incorporating the mutant gltA gene. The plasmid-incorporated strain is diluted and applied to a CM2B plate, and cultured at 34 ° C. After colony formation, the cells are replicated on a CM2B plate containing 25 μg / ml kanamycin and cultured at 34 ° C. At this time, a kanamycin-sensitive strain was obtained.
A chromosome was extracted from the kanamycin-sensitive strain, and PCR was performed using the primers shown in SEQ ID NOs: 7 and 8 to prepare a gltA gene fragment. The obtained amplified fragment was purified by SUPREC02 manufactured by Takara Shuzo Co., Ltd., and then subjected to a sequence reaction using the primer shown in SEQ ID NO: 13, to determine the sequence of the promoter region. As a result, a strain having the same promoter sequence as pKF19CS1 in Table 7 was named GB01, a strain having the same promoter sequence as pKF19CS2 was named GB02, and a strain having the same promoter sequence as pKF19CS4 was named GB03. In these strains, when the plasmid and the overlapping gltA gene are eliminated from the chromosome, the mutant gltA gene introduced by the plasmid remains on the chromosome, and the wild-type gltA gene originally on the chromosome, together with the vector plasmid Dropped out, meaning that gene replacement has occurred.

(8) Measurement of citrate synthase activity of mutant mutant of gltA promoter FGR2, GB01, GB02, GB03 and FGR2 / pSFKC obtained in (7) were subjected to citrate synthase in the same manner as described in (4). Was measured for activity. Table 10 shows the measurement results. It was confirmed that the citrate synthase activity was increased in the gltA promoter-substituted strain as compared to the parent strain.

Table 10

Strain dABS / min / mg Relative activity
FGR2 7.9 1.0
GB01 9.5 1.2
GB02 15.0 1.9
GB03 31.6 4.0
FGR2 / pSFKC 61.6 7.8

(9) Culture performance of gltA promoter-substituted strain Each strain obtained in (7) above was inoculated into a seed culture medium having the composition shown in Table 11, and cultured at 31.5 ° C for 24 hours with shaking to obtain a seed culture. After 300 ml of the main culture medium having the composition shown in Table 11 was dispensed into a 500-ml glass jar fermenter and sterilized by heating, 40 ml of the above seed culture was inoculated. The cultivation was started at a culture temperature of 31.5 ° C. at a stirring speed of 800 to 1300 rpm and an aeration rate of 1/2 to 1/1 vvm. The pH of the culture solution was maintained at 7.5 with ammonia gas. Eight hours after the start of the culture, the temperature was shifted to 37 ° C. In each case, the culture was terminated when glucose was completely consumed in 20 to 40 hours, and the amount of L-glutamic acid produced and accumulated in the culture solution was measured.
As a result, as shown in Table 12, a remarkable improvement in the yield of L-glutamic acid was observed in the GB02 and GB02 strains rather than in the GB01 and FGR2 / pSFKC strains. From the above, it was shown that in improving the glutamic acid yield of these strains, a good result could be obtained by introducing a mutation into the promoter and enhancing the CS activity by 2 to 4 times.

Table 11

Component concentration
Seed culture Main culture
Glucose 50 g / l 150 g / l
KH ₂ PO ₄ 1 g / l 2 g / l
_{MgSO 4 · 7H 2 O 0.4 g} / l 1.5 g / l
_{FeSO 4 · 7H 2 O 10 mg} / l 15 mg / l
_{MnSO 4 · 4H 2 O 10 mg} / l 15 mg / l
Soy protein hydrolysis 20 ml / l 50 ml / l
Biotin 0.5 mg / l 2 mg / l
Thiamine hydrochloride 2 mg / l 3 mg / l

Table 12

Strain L-glutamic acid (g / l)
FGR2 8.9
GB01 9.1
GB02 9.4
GB03 9.4
FGR2 / pSFKC 9.1

Example 4 Introduction of Mutation into ICDH Gene Promoter Region of Coryneform Glutamate-Producing Bacteria This example shows an example in which a promoter-enhanced strain of a gene encoding glutamate dehydrogenase, citrate synthase and isocitrate dehydrogenase was prepared.
(1) Cloning of icd gene The nucleotide sequence of the gene icd encoding isocitrate dehydrogenase of coryneform bacterium has already been elucidated (J. Bacteriol. 177 774-782 (1995)). Based on this sequence, primers represented by SEQ ID NOs: 14 and 15 were synthesized, and PCR was performed using the chromosomal DNA of Brevibacterium lactofermentum ATCC13869 as a template to amplify an approximately 3 Kbp DNA fragment containing the icd gene and its promoter. did. The obtained amplified fragment was completely digested with EcoRI, and a fragment obtained by completely digesting pHSG399 (Takara Shuzo) with EcoRI was mixed and ligated. After ligation, transformation was performed using competent cells of Escherichia coli JM109, applied to an L medium containing IPTG 10 μg / ml, X-Gal 40 μg / ml and chloramphenicol 40 μg / ml, and cultured overnight. Appeared white colonies were picked and separated into single colonies to obtain transformed strains.
The plasmid having the icd gene was named pHSG399icd.

(2) Mutation introduction to icd promoter
The exact promoter location of the icd gene has not been determined. Therefore, the possibility of increasing the mRNA transcription amount of the icd gene by artificially modifying the upstream sequence of the gene encoding ICDH to a promoter-like sequence was examined. Specifically, a mutation was introduced into a -10-like region present in a DNA sequence of about 190 bp (first promoter) and about 70 bp (second promoter) upstream of the first ATG of the ICDH protein.
Mutan-Super Express Km manufactured by Takara Shuzo was used to introduce a mutation into the upstream region of the icd gene. A specific method will be described below. pHSG399icd was completely digested with PstI to prepare a PstI fragment containing the promoter of the icd gene, and this was ligated to a fragment obtained by completely digesting pKF18kM (Takara Shuzo) with PstI. After ligation, transformation was performed using competent cells of Escherichia coli JM109 (manufactured by Takara Shuzo Co., Ltd.), applied to an L medium containing 10 μg / ml of IPTG, 40 μg / ml of X-Gal and 25 μg / ml of kanamycin, and cultured overnight. Thereafter, the emerged white colonies were picked and separated into single colonies to obtain transformed strains. A plasmid was prepared from the transformant, and one containing the icd gene promoter was designated as pKF18icd.

  Using pKF18icd as a template, PCR was performed using 5'-terminal phosphorylated synthetic DNA shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21 and selection primer. Using this PCR product, the competent cells of Escherichia coli JM109 were transformed, applied to an L medium containing 25 μg / ml of kanamycin, cultured overnight, and the colonies that appeared were picked, separated into single colonies, and transformed. Got the strain. Plasmid DNA was prepared from the transformant, and the nucleotide sequence of the icd promoter was determined by the method of Sanger (J. Mol. Biol., 143, 161, (1980)) using the synthetic DNA shown in SEQ ID NO: 22. Specifically, the nucleotide sequence was analyzed by Genetic Analyzer ABI310 (Applied Biosystems) using the Dye terminator sequencing kit (Applied Biosystems). Those in which the icd promoter region was replaced with the sequences shown in Table 7 were named pKF18ICD1, pKF18ICD2, pKF18ICD3, pKF18ICD4, pKF18ICD5, and pKF18ICD6, respectively. Of these, pKF18ICD2 was completely digested with PstI to prepare a PstI fragment containing the promoter of the icd gene, and this was ligated to a fragment of pKF18kM (Takara Shuzo) completely digested with PstI. After ligation, transformation was performed using competent cells of Escherichia coli JM109 (manufactured by Takara Shuzo), applied to an L medium containing 10 μg / ml of IPTG, 40 μg / ml of X-Gal and 25 μg / ml of kanamycin, and cultured overnight. Thereafter, the emerged white colonies were picked and separated into single colonies to obtain transformed strains. A plasmid was prepared from the transformant, and one containing the promoter of the icd gene was named pKF18ICDM2. Using pKF18ICDM2 as a template, PCR was performed using 5'-terminal phosphorylated synthetic DNA shown in SEQ ID NO: 20 and SEQ ID NO: 21 and selection primer. Using this PCR product, the competent cells of Escherichia coli JM109 were transformed, applied to an L medium containing 25 μg / ml of kanamycin, cultured overnight, and the colonies that appeared were picked, separated into single colonies, and transformed. Got the strain. Plasmid DNA was prepared from the transformant, and the nucleotide sequence of the icd promoter was determined using the synthetic DNA shown in SEQ ID NO: 22. Those in which the icd promoter region was replaced with the sequence shown in Table 13 were named pKF18ICD25 and pKF18ICD26, respectively.

Table 13
Plasmid Promoter 1 Promoter 2
-35 -10 -35 -10
pKF18ICD GCGACT GAAAGT TTTCCA CACCAT
pKF18ICD01 GCGACT TATAAT TTTCCA CACCAT
pKF18ICD02 TTGACA TATAAT TTTCCA CACCAT
pKF18ICD03 TTGACT TAAAGT TTTCCA CACCAT
pKF18ICD04 GCGACT GAAAGT TTTCCA TATAAT
pKF18ICD05 GCGACT GAAAGT TTGCCA TATAAT
pKF18ICD06 GCGACT GAAAGT TTGACA TATAAT
pKF18ICD25 TTGACA TATAAT TTGCCA TATAAT
pKF18ICD26 TTGACA TATAAT TTGACA TATAAT

(3) Construction of Plasmid for Measuring Promoter Activity In order to easily measure promoter activity, a method of indirectly measuring promoter activity using a reporter gene is considered. Desirable properties of the reporter gene include simple activity measurement, no significant decrease in activity even when amino acids are not available at the N-terminal side, no background reaction, and appropriate restrictions for genetic manipulation. The presence of an enzyme cleavage site. Β-galactosidase (LacZ) of Escherichia coli is widely used as a reporter gene, and bacteria of the genus Corynebacterium have no ability to assimilate lactose (J. Gen. Appl. Microbiol., 18, 399-416 (1972 )), It was determined that using LacZ as the reporter gene was optimal. Thus, a plasmid pNEOL carrying LacZ as a reporter gene was constructed (FIG. 3). The process is described in detail below. PCR was performed using the synthetic DNAs shown in SEQ ID NOS: 23 and 24 as primers and chromosomal DNA obtained from E. coli ME8459 (ME8459 was deposited at the National Institute of Genetics in Japan) as a template. The PCR product was completely digested with SmaI and BamHI, and then ligated with pKF3 (Takara Shuzo) digested with HindIII and blunt-ended. After ligation, transformation was performed using competent cells of Escherichia coli JM109 (manufactured by Takara Shuzo Co., Ltd.), applied to an L medium containing 25 μg / ml of kanamycin, and cultured overnight. Separated to obtain a transformed strain. A plasmid was prepared from the transformant and named pKF3nptII. Next, this was digested with SalI, while pSAK4 was completely digested with SmaI and SalI and blunt-ended in the same manner as in Example 1, (1), and ligated to form a shuttle capable of replicating in coryneform bacteria. The vector pNEO was constructed. This plasmid confers chloramphenicol resistance and kanamycin resistance to the host. Furthermore, a coryneform bacterium having LacZ, in which pNEO was completely degraded with SmaI and Sse8387I, and this was completely digested with pMC1871 (Pharmacia Biotech) with PstI and SmaI, and the N-terminal 8 amino acids were deleted as a reporter gene, was obtained. A shuttle vector pNEOL capable of replication in E. coli was constructed (FIG. 3).

(4) Measurement of mutant icd promoter activity Plasmids pKF18ICD1, pKF18ICD2, pKF18ICD3, pKF18ICD4, pKF18ICD5, pKF18ICD6, pKF18ICD25, pKF18ICD26, and pKF18ICD, which were completely digested with the plasmids pKF18ICD1, pKF18ICD2, pKF18ICD3, pKF18ICD4, and pKF18ICD26, which were constructed in (2). It was blunt-ended and ligated with pNEOL cut with SmaI. After ligation, transformation was performed using competent cells of Escherichia coli JM109. The colonies were picked and separated into single colonies to obtain a transformant.
Plasmids were prepared from the transformants and those having a structure capable of producing a fusion protein of ICDH and LacZ were designated as pNEOICD1, pNEOICD2, pNEOICD3, pNEOICD4, pNEOICD5, pNEOICD6, pNEOICD25, pNEOICD26, and pNEOLICD. These plasmids and pNEOL were introduced into Brevibacterium lactofermentum ATCC13869 by the electric pulse method. Transformants were selected using a CM2B plate medium containing 25 μg / ml kanamycin and X-Gal 40 μg / ml (Bactotryptone 10 g / l, Bacto yeast extract 10 g / l, NaCl 5 g / l, biotin 10 μg / L, agar 15 g). / l, pH 7.0) at 31 ° C. After cultivation for two nights, the emerged colonies were picked, and single colonies were separated and transformed into pNEOICD1, pNEOICD2, pNEOICD3, pNEOICD4, pNEOICD5, pNEOICD6, pNEOICD25, pNEOICD26, and pNEOLICD. , Named BNEOL. All the transformants except BNEO formed blue colonies. A crude enzyme solution was prepared from these transformants by the method described in Example 3 (4). However, Z-Buffer (KCl 10 mM, MgSO4 1 mM, 2-ME 270 μl / 100 mM NaPi (pH 7.5)) was used as a buffer for washing and suspending the cells. The activity of LacZ was measured according to the following procedure. Mix Z-Buffer with the crude enzyme solution, add ONPG dissolved in Z-Buffer to a final concentration of 0.8 mg / ml, and measure the increase in absorbance at 420 nm at 30 ° C with Hitachi spectrophotometer U-3210 The value obtained was defined as LacZ activity. In addition, Protein Assay (BIO-RAD) was used for measuring the protein concentration of the crude enzyme solution. Bovine serum albumin was used as a standard protein. Table 14 shows the measurement results. It was confirmed that the strain expressing the ICDH-LacZ fusion protein having a mutation in the icd promoter had higher LacZ elementary activity than the strain expressing the wild-type ICDH-LacZ fusion protein.

Table 14
Strain dABS / min / mg Relative activity
BNEOL Not detected 0.0
PNEOLI 42 1.0
BNEOLI-1 84 2.0
BNEOLI-2 168 4.0
BNEOLI-3 80 1.9
BNEOLI-4 126 3.0
BNEOLI-5 139 3.3
BNEOLI-6 84 2.0
BNEOLI-25 168 4.0
BNEOLI-26 170 4.0

(5) Introduction of Mutant icd Gene into Temperature-Sensitive Plasmid A plasmid vector pSFKT2 (Japanese Patent Application No. 11-81693) whose replication is temperature-sensitive in coryneform bacteria was used. The mutant icd promoter sequence (pKF18ICD1, pKF18ICD2, pKF18ICD3, pKF18ICD4, pKF18ICD5, pKF18ICD6, pKFICD25, and pKFICD26 was completely digested with PstI, and this fragment was ligated. Transformation was performed using competent cells of JM109 (Takara Shuzo Co., Ltd.), applied to an L medium containing 10 μg / ml of IPTG, 40 μg / ml of X-Gal and 25 μg / ml of kanamycin, and cultured overnight, followed by white colonies that appeared. A plasmid was prepared from the transformant, and the temperature-sensitive shuttle vectors containing the icd promoter were replaced with pSFKTI1, pSFKTI2,, pSFKTI3, pSFKTI4, pSFKTI5, pSFKTI6, pSFKTI25, pSFKTI26, respectively. I named it.

(6) Introduction of Mutant icd Promoter into Chromosome The plasmid constructed in (5) was introduced into Brevibacterium lactofermentum GB02 strain by the electric pulse method. Selection of transformants was performed using a CM2B plate medium containing 25 μg / ml kanamycin (Bactotryptone 10 g / L, Bacto yeast extract 10 g / L, NaCl 5 g / L, biotin 10 μg / L, agar 15 g / L, pH 7.0). ) At 25 ° C. After the introduction, the obtained strain was cultured in a CM2B liquid medium, and diluted with CM2B plate containing 25 μg / ml kanamycin so as to have a density of 10 ³ to 10 ⁵ cfu per plate, followed by application at 34 ° C. Cultured. Strains harboring the temperature-sensitive plasmid become kanamycin-sensitive and cannot form colonies due to inhibition of plasmid replication at this temperature, but strains with plasmid DNA integrated into the chromosome form colonies, and should be selected. Can be. The appeared colonies were picked up and separated into single colonies. A chromosomal DNA was extracted from this strain, and PCR was carried out using the chromosomal DNA as a template and the primers shown in SEQ ID NOs: 13 and 15 to confirm an amplified fragment of about 3 kb. Therefore, this strain was shown to have a mutant icd gene derived from a temperature-sensitive plasmid integrated near the icd gene in the host chromosome by homologous recombination.

(7) Acquisition of icd promoter-substituted strain A kanamycin-sensitive strain was firstly obtained from the strain described in (6) in which the mutant icd gene was incorporated by homologous recombination. The plasmid-incorporated strain is diluted and applied to a CM2B plate, and cultured at 34 ° C. After colony formation, the cells are replicated on a CM2B plate containing 25 μg / ml kanamycin and cultured at 34 ° C. At this time, a kanamycin-sensitive strain was obtained.
A chromosome was extracted from the kanamycin-sensitive strain and subjected to PCR using primers shown in SEQ ID NO: 14 and SEQ ID NO: 15 to prepare an icd gene fragment. The obtained amplified fragment was purified with SUPREC02 manufactured by Takara Shuzo Co., Ltd., and then subjected to a sequence reaction using the primer shown in SEQ ID NO: 22 to determine the sequence of the promoter region. As a result, strains having an icd promoter sequence derived from pSFKTI1, pSFKTI2, pSFKTI3, pSFKTI4, pSFKTI5, pSFKTI6, pSFKTI25, and pSFKTI26 were named GC01, GC02, GC03, GC04, GC05, Gc06, Gc25, and GC26, respectively. In these strains, when the plasmid and the overlapping icd gene are eliminated from the chromosome, the mutated icd gene introduced by the plasmid remains on the chromosome, and the wild-type icd gene originally on the chromosome is transformed into a vector plasmid. With it.

(8) Measurement of isocitrate dehydrogenase activity of mutant of icd promoter An ICDH crude enzyme solution was prepared from 8 strains and GB02 strain obtained in (7) in the same manner as in Example 3 (7). The measurement of ICDH activity was performed according to the following procedure. Add the crude enzyme solution to a reaction solution containing 35 mM TisHCl (pH 7.5), 1.5 mM MnSO4, 0.1 mM NADP, 1.3 mM isocitrate, and measure the increase in absorbance at 340 nm at 30 ° C with the Hitachi U-3210 spectrophotometer. The value obtained was defined as the activity of ICDH. In addition, Protein Assay (BIO-RAD) was used for measuring the protein concentration of the crude enzyme solution. Bovine serum albumin was used as a standard protein. Table 15 shows the measurement results. It was confirmed that the icd promoter-substituted strain had increased isocitrate dehydrogenase activity compared to its parent strain.

Table 15
Strain dABS / min / mg Relative activity
GB02 3.9 1.0
GC01 8.2 2.1
GC02 19.1 4.9
GC03 7.0 1.8
GC04 12.5 3.2
GC05 19.1 4.9
GC06 10.5 2.7
GC25 30.4 7.8
GC26 24.2 6.2

(9) Culture results of icd promoter-substituted strains Eight strains obtained in (7) were cultured in the same manner as in Example 3 (9). As a result, as shown in Table 16, the GC02, GC04, GC05, GC25 and GC26 strains showed an improvement in the yield of L-glutamic acid. It was shown that a good result could be obtained by introducing a mutation into the icd promoter and increasing the ICDH activity three-fold or more.

Table 16

Strain L-glutamic acid (g / l)
GB02 9.2
GC01 9.0
GC02 9.5
GC03 9.1
GC04 9.4
GC05 9.6
GC06 9.2
GC25 9.9
GC26 9.8

Example 5 Introduction of Mutation into PDH Gene Promoter Region of Coryneform Glutamate-Producing Bacteria
(1) Cloning of pdhA gene from coryneform bacterium A region having high homology between the E1 subunits of pyruvate dehydrogenase (PDH) of Escherichia coli, Pseudomonas aeruginosa and Mycobacterium tuberculosis was selected and shown in SEQ ID NOS: 25 and 26. Primers were synthesized, and using the chromosome of Brevibacterium lactofermentum ATCC 13869 prepared with the Bacterial Genomic DNA Purification Kit manufactured by Advanced Genetic Technologies Corp. as a template, PCR technology (Henry Erich, Stockton Press, 1989), p. PCR was performed under the standard reaction conditions described, and the reaction solution was subjected to agarose gel electrophoresis. As a result, it was found that a DNA fragment of about 1.3 kilobases had been amplified. The nucleotide sequence of both ends of the obtained DNA was determined using the synthetic DNAs of SEQ ID NO: 25 and SEQ ID NO: 26. The nucleotide sequence was determined using the DNA Sequencing Kit (manufactured by Applied Biosystems) according to the method of Sanger (J. Mol. Biol., 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 homology was high, so the DNA fragment amplified by PCR was Brevibacterium Lactofermentum brevibacterium lactofermentum It was determined that it was part of the pdhA gene encoding the E1 subunit of pyruvate dehydrogenase of ATCC 13869, and the upstream and downstream portions of the gene were cloned. The cloning method is to clone the upstream portion from a DNA fragment obtained by digesting Brevibacterium lactofermentum ATCC 13869 chromosome with restriction enzymes EcoRI, BamHI, HindIII, PstI, SalI and XbaI (Takara Shuzo). Using the primers shown in SEQ ID Nos. 27 and 28 in the Sequence Listing, and using the primers shown in SEQ ID Nos. 29 and 30 to clone the downstream portion, using the LA PCR in vitro cloning Kit (Takara Shuzo) To perform cloning. As a result of performing PCR with this kit, the upstream portion was amplified with EcoRI, Hind III, Pst I, Sal I, and Xba I digested fragments of about 0.5, 2.5, 3.0, 1.5, and 1.8 kilobase DNA fragments, respectively. The downstream portion was a fragment digested with BamHI, Hind III, and Pst I, which amplified DNA fragments of about 1.5, 3.5, and 1.0 kilobases, respectively.These DNA fragments were sequenced in the same manner as above. Was. 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 existed further upstream. The amino acid sequence of the product deduced from the nucleotide sequence of this open reading frame is highly homologous to the known E1 subunit of pyruvate dehydrogenase such as Escherichia coli. Fermentum ATCC13869 was found to be a pdhA gene encoding the E1 subunit of pyruvate dehydrogenase. The nucleotide sequence of this open reading frame is as shown in SEQ ID NO: 31 of the Sequence Listing. The sequence shown in SEQ ID NO: 31 also shows the amino acid sequence of the product deduced from the nucleotide sequence. The methionine residue at the N-terminus of the protein is often independent of the original function of the protein because it is derived from the initiation codon ATG, and it is well known that it is removed by the action of peptidase after translation. Also, in the case of the above protein, the methionine residue on the N-terminal side may be removed. However, there is a GTG sequence 6 bases upstream of the ATG shown in SEQ ID NO: 31 in the Sequence Listing, and amino acids may be translated therefrom. In addition, 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. There was no open reading frame in the approximately 3 kilobases downstream of the gene, which could be the E2 and E3 subunits of pyruvate dehydrogenase. Instead, it has been clarified that a putative terminator sequence is present downstream of this open reading frame, and thus E2 and P2 of pyruvate dehydrogenase of Brevibacterium lactofermentum ATCC 13869 are known. The E3 subunit was thought to be located elsewhere on the chromosome.

(2) Construction of plasmid for amplifying pdhA A strain obtained by introducing a gene encoding three subunits constituting E. coli PDH into Brevibacterium lactofermentum ATCC 13869 has an improved glutamic acid yield. Has already been clarified (Japanese Patent Application No. 10-360619). However, only the pdhA gene encoding the E1 subunit has been cloned from the PDH of Brevibacterium lactofermentum ATCC 13869, and it has not yet been investigated whether amplification of this gene alone has an effect on glutamate yield. Therefore, here, it was determined whether amplification of the pdhA gene alone had an effect on glutamate yield.
Primers shown in SEQ ID NOs: 33 and 34 were synthesized based on the nucleotide sequences already cloned, and the chromosome of Brevibacterium lactofermentum ATCC 13869 prepared by Bacterial Genomic DNA Purification Kit manufactured by Advanced Genetic Technologies Corp. was used as a template, PCR was performed under standard reaction conditions described on page 8 of PCR technology (Henry Erich, edited by Stockton Press, 1989) to amplify the pdhA gene. Of the synthesized primers, SEQ ID NO: 33 corresponds to the sequence from the 1397th base to the 1416th base in the nucleotide sequence diagram of the pdhA gene described in SEQ ID NO: 32 in the Sequence Listing, and SEQ ID NO: 34 The reverse strand of the base sequence corresponding to the sequence from the 5535th base to the 5374th base of SEQ ID NO: 32 is shown from the 5 'side.

After purifying the generated PCR product by a conventional method, a restriction enzyme SalI and EcoT22I were reacted, and pSFK (Japanese Patent Application No. 11-69896) cut with restriction enzymes SalI and PstI and a ligation kit (Takara Shuzo) were used. After ligation, transformation was performed using competent cells of Escherichia coli JM109 (manufactured by Takara Shuzo Co., Ltd.), and IPTG (isopropyl-β-D-thiogalactopyranoside) 10 μg / ml and X-Gal (5- L medium containing 40 μg / ml of bromo-4-chloro-3-indolyl-β-D-galactoside 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 and separated into single colonies to obtain transformed strains.
After preparing a plasmid from the transformant using the alkaline method (Biotechnology Experiments, edited by Biotechnology Society of Japan, p. 105, Baifukan, 1992), a restriction enzyme map of the DNA fragment inserted into the vector was prepared. Compared with the restriction enzyme map of the pdhA gene reported in SEQ ID NO: 32 in the Sequence Listing, a plasmid into which a DNA fragment having the same restriction enzyme map was inserted was named pSFKBPDHA.

(3) Introduction of pASFKBPDHA into Brevibacterium lactofermentum ATCC13869 and CG25 and evaluation of fermentation culture Brevibacterium lactofermentum ATCC13869 and GC25 were transformed with plasmid pSFKBPDHA by the electric pulse method (see JP-A-2-207791). After transformation, a transformant was obtained. Culture for L-glutamic acid production was performed as follows using the transformants ATCC13869 / pSFKBPDHA and GC25 / pSFKBPDHA obtained by introducing the plasmid pSFKBPDHA into Brevibacterium lactofermentum ATCC13869 and GC25. The cells of the ATCC13869 / pSFKBPDHA and GC25 / pSFKBPDHA strains obtained by culturing in a CM2B plate medium containing 25 μg / mL kanamycin were obtained by adding 80 g of glucose, 1 g of KH ₂ PO ₄ , 0.4 g of MgSO ₄ .7H ₂ O, ( 30 g of NH ₄ ) ₂ SO ₄ , 0.01 g of FeSO ₄ .7H ₂ O, 0.01 g of MnSO ₄ .7H ₂ O, 15 ml of soybean hydrolysate, 200 μg of thiamine hydrochloride, 60 μg of biotin, 25 mg of kanamycin and 50 g of CaCO ₃ (PH adjusted to 8.0 with KOH in a medium contained in 1 L of water), and cultured with shaking at 31.5 ° C. until the sugar in the medium was consumed. The obtained culture was added to a medium having the same composition as described above for GC25 / pSFK6 and GC25 / pSFKBPDHA, and to a culture medium containing biotin for ATCC13869 / pSFK6 and ATCC13869 / pSFKBPDHA in the same manner as described above, except for 5%. The cells were inoculated and cultured with shaking at 37 ° C. until the sugar in the medium was consumed. As a control, a strain obtained by transforming Brevibacterium lactofermentum ATCC13869 and GC25 with an already obtained plasmid pSFK6 capable of autonomously replicating by Corynebacterium bacteria by an electric pulse method (see JP-A-2-207791). Was cultured in the same manner as described above. After the completion of the culture, the amount of L-glutamic acid accumulated in the culture solution was measured using a Biotech Analyzer AS-210 manufactured by Asahi Kasei Corporation. Table 17 shows the results.

Table 17

Strain L-glutamic acid yield (%)
ATCC13869 / pSFK 3.6
ATCC13869 / pSFKBPDHA 3.8
GC25 / pSFK6 5.1
GC25 / pSFKBPDHA 5.3

From these results, it was clarified that in the Brevibacterium lactofermentum ATCC13869 and GC25, even the single amplification of the pdhA gene was effective in improving the Glu yield.
(4) Construction of a Plasmid for Measuring Mutated pdhA Promoter Activity Promoter region of the pdhA gene of Brevibacterium lactofermentum ATCC13869 which has been cloned in order to prepare a promoter mutant of pyruvate dehydrogenase (PDH). The determination of the expression level and the measurement of the difference in the expression level due to the modification of the promoter region were performed by measuring the activity of β-galactosidase.
When the promoter site of the pdhA gene was estimated from the nucleotide sequence that had already been revealed by cloning, the 2252th to 2257th and the 2279th to 2284th of SEQ ID NO: 32 in the Sequence Listing were -35 region and -10 region, respectively. It was presumed that this was likely. Therefore, primers shown in SEQ ID NOs: 35 and 36 in the Sequence Listing were synthesized, and a DNA fragment containing the promoter region of the pdhA gene was amplified by PCR using the chromosomal DNA of Brevibacterium lactofermentum ATCC13869 as a template. Among the synthesized primers, SEQ ID NO: 35 corresponds to the sequence extending from the 2194th to 2221st bases in the nucleotide sequence of SEQ ID NO: 32 in Sequence Listing, but base 2198th in C, 2200th and 2202nd in bases. Is changed to G, and a recognition sequence for the restriction enzyme SmaI is inserted. SEQ ID NO: 36 corresponds to the sequence extending from the 2372nd to the 2398th base in the nucleotide sequence of SEQ ID NO: 32 in Sequence Listing, but changing the 2393rd and 2394th bases to G and inserting a recognition sequence for restriction enzyme SmaI The reverse strand of the base sequence is shown from the 5 'side. Using the chromosome of Brevibacterium lactofermentum ATCC 13869 prepared with the Bacterial Genomic DNA Purification Kit manufactured by Advanced Genetic Technologies Corp. as a template, a standard described on page 8 of PCR technology (Henry Erich, Stockton Press, 1989), page 8 PCR was performed under the reaction conditions to amplify the promoter region of the pdhA gene. After purifying the generated PCR product by a conventional method, it is reacted with a restriction enzyme SmaI, and pNEOL capable of replication in a coryneform bacterium lacking the promoter region of the lacZ gene is cleaved with the restriction enzyme SmaI ((3) in Example 4). After ligation was performed using a ligation kit (Takara Shuzo), transformation was performed using competent cells of Escherichia coli JM109 (Takara Shuzo), and X-Gal (5-bromo-4-chloro-) was transformed. L medium containing 10 μg / ml of 3-indolyl-β-D-galactoside and 25 μg / ml of kanamycin (10 g / l of bactotryptone, 5 g / l of bactoeast extract, 5 g / l of NaCl, 15 g / l of agar, pH 7.2) ), And after overnight culture, emerged blue colonies were picked and separated into single colonies to obtain transformed strains. After preparing a plasmid from the transformant using the alkaline method (Biotechnical Experiments, edited by Biotechnology Society of Japan, p. 105, Baifukan, 1992), the DNA fragment inserted into the vector is sequenced by a standard method, and the DNA fragment is prepared. The plasmid into which was inserted was named pNEOLBPDHApro1.

  Furthermore, in order to construct a plasmid in which a region presumed to be a promoter site was changed to a common sequence of a coryneform bacterium promoter, primers shown in SEQ ID NOs: 37, 38, and 39 in the Sequence Listing were synthesized, and these primers were combined with the respective primers. A DNA fragment in which the promoter region of the pdhA gene was changed to a common sequence was amplified by a PCR method using the chromosomal DNA of Brevibacterium lactofermentum ATCC13869 as a template using the primers shown in SEQ ID NO: 36 in the sequence listing. Of the synthesized primers, SEQ ID NO: 37 corresponds to the sequence extending from the 2244th base to the 2273th base in the nucleotide sequence of SEQ ID NO: 32, and changing the 2255th base to C and the 2257th base to A. , -35 region corresponds to a common sequence of coryneform bacteria. In addition, SEQ ID NO: 38 corresponds to the sequence extending from the nucleotide sequence 2249 to the nucleotide 2288 in the nucleotide sequence of SEQ ID NO: 32 in the sequence listing, the nucleotides 2279 and 2281 are changed to T, and only the -10 region is coryneform. This corresponds to the change to a common sequence of bacteria. SEQ ID NO: 39 corresponds to the sequence extending from the 2249th base to the 2288th base in the nucleotide sequence of Sequence Listing SEQ ID NO: 32, the 2255th base to C, the 2257th base to A, the 2279th and 2281 This corresponds to a case where the -th base is changed to T and both the -35 region and the -10 region are changed to a common sequence of coryneform bacteria. Using the chromosome of Brevibacterium lactofermentum ATCC 13869 prepared with the Bacterial Genomic DNA Purification Kit manufactured by Advanced Genetic Technologies Corp. as a template, a standard described on page 8 of PCR technology (Henry Erich, Stockton Press, 1989), page 8 PCR was performed under the reaction conditions, and using these primers, the promoter region of the pdhA gene in which the promoter region was changed to a common sequence was amplified. After purifying the generated PCR product by a conventional method, the restriction enzyme SmaI was reacted, and pNEOL capable of replicating in a coryneform bacterium lacking the promoter region of the lacZ gene was cut with the restriction enzyme SmaI and a ligation kit (Takara Shuzo Co., Ltd.). ), And transformed using competent cells of Escherichia coli JM109 (manufactured by Takara Shuzo), followed by X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). ) Spread on an L medium containing 40 μg / ml and kanamycin 25 μg / ml (Bactotryptone 10 g / l, Bacto yeast extract 5 g / l, NaCl 5 g / l, agar 15 g / l, pH 7.2), and culture overnight The emerged blue colonies were picked up and separated into single colonies to obtain transformed strains. After preparing a plasmid from the transformant using the alkaline method (Biotechnical Experiments, edited by The Biotechnology Society of Japan, p. 105, Baifukan, 1992), the DNA fragment inserted into the vector was sequenced by a standard method, and- A plasmid in which a DNA fragment in which only the 35 region was changed to a common sequence was named pNEOLBPDHApro35, and a plasmid in which a DNA fragment in which only the -10 region was changed to a common sequence was named pNEOLBPDHApro35, the -35 region and- A plasmid into which a DNA fragment in which ten regions were changed to a common sequence was inserted was named pNEOLBPDHApro3510.

(5) Evaluation of Mutated pdhA Promoter Activity The plasmids named pNEOLBPDHApro1, pNEOLBPDHApro10 and pNEOLBPDHApro3510 constructed here were used to transform Brevibacterium lactofermentum ATCC13869 by the electric pulse method (see JP-A-2-207791). As a result, a transformant was obtained. Β-galactosidase activity of the obtained transformant was measured by the method described in Example 4 (4). The β-galactosidase activity when the promoter region was changed to a consensus sequence was as shown in Table 18, where the enzyme activity of β-galactosidase having the promoter region of the pdhA gene was 1.

Table 18
Strain β-galactosidase activity (relative value)
ATCC13869 / pNEOLBPDHApro1 1
ATCC13869 / pNEOLBPDHApro10 6
ATCC13869 / pNEOLBPDHApro3510 7.5

This result implies that the putative promoter site is the promoter of the pdhA gene, and shows that the expression level of PdhA can be changed (amplified) by changing this region to a common sequence. . This indicates that the expression level can be changed without using a plasmid by changing the promoter region of the pdhA gene.
(6) Construction of a Plasmid for Producing a Mutant Promoter Since it was revealed that the expression level of PdhA can be changed by causing a mutation in the promoter, a plasmid for producing a promoter mutant of the pdhA gene was constructed. Was. Three types of plasmids for constructing the promoter mutant strain were constructed by changing the -35 region and the -10 region to a common sequence and by changing both of them to a common sequence.

4-1) Construction of Plasmid for Producing Mutant Promoter Primers shown in SEQ ID NOS: 40 and 41 were newly synthesized based on the nucleotide sequence already cloned. Among the synthesized primers, SEQ ID NO: 40 is the reverse strand of the nucleotide sequence corresponding to the sequence from the 2491st to the 2521st nucleotides in SEQ ID NO: 32 in the Sequence Listing, which is represented by 5 'from the 5' end. An array in which four Ts are connected after the connection is obtained. In addition, SEQ ID NO: 33 shows the reverse strand of the base sequence from the 5020th to the 5039th base in the base sequence diagram of the pdhA gene described in SEQ ID NO: 32 in the Sequence Listing from the 5 'side. PCR technology (Henry Erich, Stockton Press) using the chromosome of Brevibacterium lactofermentum ATCC 13869 prepared by Bacterial Genomic DNA Purification Kit manufactured by Advanced Genetic Technologies Corp. as a template and SEQ ID NOS: 33 and 40 as primers PCR was performed under the standard reaction conditions described on page 8. PCR was also performed using the chromosome of Brevibacterium lactofermentum ATCC 13869 as a template and SEQ ID NOS: 15 and 17 in the sequence listing. After purifying the generated PCR product by a conventional method, a PCR product using SEQ ID NOS: 9 and 16 as a primer and a PCR product using SEQ ID NOs: 39 and 41 as primers and SEQ ID NOS: 33 and 41 are used. Was used to perform PCR. The conditions at this time were such that these four DNAs were added to the reaction solution so as to have a concentration of 10 μM, and PCR was performed using LA taq (Takara Shuzo) without adding a template. The resulting PCR product was purified by a conventional method, and then reacted with restriction enzymes SalI and XhoI. A temperature-sensitive plasmid pSFKT2 capable of replicating in coryneform bacteria was cut with restriction enzyme SalI, and a ligation kit (Takara Shuzo) was used. After ligation using E. coli JM109, transformation was performed using competent cells (manufactured by Takara Shuzo), IPTG (isopropyl-β-D-thiogalactopyranoside) 10 μg / ml, X-Gal (5 -Bromo-4-chloro-3-indolyl-β-D-galactoside) L medium containing 40 μg / ml and kanamycin 25 μg / ml (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 and separated into single colonies. To obtain a quality transformed strains. After preparing a plasmid from the transformant using the alkaline method (Biotechnology Experiments, edited by Biotechnology Society of Japan, p. 105, Baifukan, 1992), the DNA fragment inserted into the vector was sequenced, and the sequence listing was performed. In comparison with the nucleotide sequence of the pdhA gene reported in No. 32, a plasmid into which a DNA fragment in which only the -35 region and the -10 region of the promoter were changed to a common sequence of coryneform bacterium was named pSFKTPDHApro3510. .

A plasmid in which the -35 region of the promoter of the pdhA gene was changed to a common sequence of coryneform bacteria and the pdhA gene A plasmid was constructed in which the -10 region of the promoter was changed to a common sequence of coryneform bacteria. These plasmids were named pSFKTDHApro35 and pSFKTPDHApro10, respectively.
(7) Preparation of Promoter Mutant A promoter mutant of the pdhA gene was prepared by homologous recombination using the plasmid for preparing a promoter mutant constructed in (6).
First, GC25 was transformed by an electric pulse method (see JP-A-2-207791) using a plasmid pSFKTPDHApro3510 for preparing a mutant promoter, and a CM2B plate medium (polypeptone 10 g / l, Bacto yeast extract 10 g / l, NaCl 5 g / l, biotin 10 microg / ml, agar 15 g / l, pH 7.2) was applied to obtain a transformant at a culture temperature of 25 ° C. The resulting transformant was cultured in a CM2B liquid medium overnight in a test tube, then spread on a CM2B plate medium containing 25 μg / ml of kanamycin, cultured at a culture temperature of 34 ° C., and transformed into a plasmid on a chromosome by homologous recombination. A single recombinant strain into which pSFKTPDHpro3510 was inserted was obtained. After single colony separation of the obtained single recombinant strain, it is placed on a CM2B liquid medium, cultivated at a culture temperature of 31.5 ° C., and when a colony appears, replica to a CM2B plate medium containing 25 μg / ml of kanamycin. Thus, a kanamycin-sensitive strain was obtained. As the promoter site of the pdhA gene of the obtained strain, two types of strains can be obtained: a wild-type sequence and a mutation-introduced strain. The introduced promoter mutant strain was obtained. This strain was a strain in which the -35 region and the -10 region of the promoter of the pdhA gene were changed to a common sequence of coryneform bacteria, and this strain was named GD3510.

In addition, the plasmid pSFKTPDHApro3510 for producing the promoter mutant strain was replaced with the plasmids pSFKTPDHApro35 and pSFKTPDHApro10 for preparing the promoter mutant strain, and in exactly the same manner as described above, the -35 region and the -10 region of the pdhA gene promoter were coryneform bacteria, respectively. Strains with the consensus sequence were obtained and named GD35 and GD10, respectively.
(8) Evaluation of Flask Culture of Mutant of pdhA Gene Promoter Flask culture for production of L-glutamic acid was performed as follows using the three types of mutant mutants of pdhA gene promoter obtained, GD3510, GD35, GD10 and GC25. Cells of the promoter mutant strains GD3510, GD35, GD10 and GC25 obtained by culturing on a CM2B plate medium were subjected to 30 g of glucose, 1 g of KH ₂ PO ₄ , 0.4 g of MgSO ₄ .7H ₂ O, 0.4 g of (NH ₄ ) ₂ SO _{4 30g, FeSO 4 · 7H 2} O 0.01g, MnSO 4 · 7H 2 O 0.01g, soybean hydrolyzate solution 15 ml, thiamin hydrochloride 200 [mu] g, KOH biotin 60μg and CaCO ₃ 50 g in a medium containing in 1L of pure water And adjusted to pH 8.0 using the above method) and cultured at 31.5 ° C. with shaking until the sugar in the medium was consumed. The obtained product was inoculated at a concentration of 5% into a medium having the same composition as described above, and cultured with shaking at 37 ° C. until the sugar in the medium was consumed. After the completion of the culture, the amount of L-glutamic acid accumulated in the culture solution was measured using a Biotech Analyzer AS-210 manufactured by Asahi Kasei Corporation. Table 19 shows the results.

Table 19

Strain L-glutamic acid (g / dl)
GC25 1.9
GD35 2.0
GD10 2.0
GD3510 2.1
From these results, it was revealed that the obtained mutant promoter had an improved Glu yield.

Example 6 Introduction of Mutation into Promoter Region of Arginosuccinate Synthase Gene into Corynetype Arginine-Producing Bacterium
1) Determination of the nucleotide sequence of the upstream region of the argG gene In order to amplify the argG gene of Brevibacterium flavum by PCR, the nucleotide sequence of the upstream and downstream regions of the ORF was determined. The nucleotide sequence was determined by synthesizing primers based on the known ORF nucleotide sequence of the argG gene of Corynebacterium glutamicum (GenBank accession AF030520) and using an in vitro LA PCR cloning kit (Takara Shuzo). Was performed according to the instructions attached to the kit. Specifically, oligonucleotides (primers 1 and 2) having the nucleotide sequences shown in SEQ ID NOs: 42 and 43 for the upstream region of the ORF, and nucleotide sequences shown in SEQ ID NOs: 44 and 45 for the downstream region are used for the primer. (Primers 3, 4) were used, respectively. Complete digestion of the chromosomal DNA of Brevibacterium flavum strain 2247 (ATCC14067) with the restriction enzyme EcoRI, primary PCR with primers 2 or 3, and secondary PCR with primers 1 or 4. As a result, the nucleotide sequences upstream and downstream of argG were determined.

2) Prediction of promoter site From the above sequences, a promoter-like sequence upstream of the ORF of the argG gene was searched using commercially available software (GENETYX). A mutation was introduced at the site with the highest score (about 120 bp upstream from the first ATG), and then the activity of the promoter was evaluated.
3) Mutation into the promoter sequence and measurement of the activity of the mutant promoter In the site with the highest score, the primer for mutation introduction primer9 or primer10 or primer11 or primer12 or primer13 and primer7 (SEQ ID NOS: 50, 51, 52, 53, respectively) 54, 48), the first PCR was performed using the chromosomal DNA of the AJ12092 strain as a template, and the PCR product was used as the 3′-primer, and primer8 (SEQ ID NO: 49) was used as the 5′-primer again, and Was used as a template to obtain a DNA fragment in which a mutation was introduced into the target promoter portion. Next, in order to measure the activity of this mutant promoter, these DNA fragments were inserted into the SmaI site of the promoter probe vector pNEOL so as to be in the forward direction with the reporter gene lacZ, plasmid pNEOL-1, pNEOL-2, pNEOL -3, pNEOL-4 and pNEOL-7 were obtained. As a control of the activity, plasmid pNEOL-0 was constructed by similarly inserting a DNA fragment obtained by performing PCR using chromosomal DNA of AJ12092 strain as a template using primer7 and primer8 upstream of the lacZ gene of pNEOL.

pNEOL-0, pNEOL-1, pNEOL-2, pNEOL-3, pNEOL-4 and pNEOL-7 were introduced into AJ12092 strain. The plasmid was introduced by the electric pulse method (JP-A-2-207791). The transformant was resistant to chloramphenicol on a CM2G plate medium (10 g of polypeptone, 10 g of yeast extract, 5 g of glucose, 5 g of NaCl, and 15 g of agar in 1 liter of pure water, pH 7.2) containing 4 μg / ml of chloramphenicol. Selected as strain.
These strains were obtained by culturing at 31.5 ° C for 20 hours on agar medium containing 0.5 g / dl of glucose, 1 g / dl of polypeptone, 1 g / dl of yeast extract, 0.5 g / dl of NaCl, and 5 μg / l of chloramphenicol. 1 g of the bacterial cell was purified from glucose 3 g / dl, ammonium sulfate 1.5 g / dl, KH2PO4 0.1 g / dl, MgSO4 0.04 g / dl, FeSO4 0.001 g / dl, MnSO4 0.01 g / dl, VB1 5 μg / dl, biotin 5 μg / dl dl, soybean hydrolyzate β-galactosidase activity was measured using cells obtained by inoculating a medium containing 45 mg / dl as an N content and culturing at 31.5 ° C for 18 hours.
As shown in Table 20, β-galactosidase activity was detected in AJ12092 / pNEOL-0, indicating that the DNA fragment inserted upstream of the lacZ structural gene functions as a promtoer. In addition, β-galactosidase activity was higher in each plasmid-introduced strain as compared to AJ12092 / pNEOL-0, and it was found that transcriptional activity was increased as shown in Table 20 by introducing a mutation into this promoter-like sequence. .

Table 20

4) Construction of plasmid for mutagenesis
A DNA fragment obtained by performing PCR using the chromosomal DNA of the AJ12092 strain as a template using primers 14 and 15 (SEQ ID NOs: 55 and 56) was inserted into the SmalI site of a multicloning site made by TaKaRa to construct plasmid p0. Next, p0 was digested with restriction enzymes EcoRV and BspHI, and a DNA fragment obtained by similarly digesting pNEOL-3 and pNEOL-7 with restriction enzymes EcoRV and BspHI was ligated to obtain a plasmid p3 for mutagenesis. Mutation derived from primer 11 for mutagenesis) and p7 (mutation derived from primer 13 for mutagenesis) were obtained.
5) Introduction of plasmid for mutation introduction into Arg-producing bacteria The above-mentioned plasmid was introduced into Arg-producing bacteria Bevribacterium lactofermentum AJ12092 strain. The plasmid was introduced by the electric pulse method (JP-A-2-207791). Since these plasmids cannot be replicated autonomously in Bevribacterium, only strains in which this plasmid has been integrated into the chromosome by homologous recombination can be selected as Cm-resistant strains. The strain in which the mutation-introducing plasmid has been integrated into the chromosome contains a CM2G plate medium containing 5 μg / ml of chloramphenicol (10 g of polypeptone, 10 g of yeast extract, 5 g of glucose, 5 g of NaCl, and 15 g of agar in 1 liter of pure water, pH 7.2). ) Was selected as a chloramphenicol resistant strain.
Next, a strain in which the promoter portion of the argG gene was replaced with the target mutant sequence was selected from the strains that had undergone homologous recombination and became Cm-sensitive.
As a result, those substituted with the sequence of P3 (AJ12092-P3) and those substituted with the sequence of P7 (AJ12092-P7) were obtained.

6) Cloning of argG gene Based on the nucleotide sequence determined as in 1), oligonucleotides (primers 5, 6) having the nucleotide sequences shown in SEQ ID NOs: 46 and 47 were synthesized, and Brevibacterium flavum 2247 was synthesized. PCR was performed using the chromosomal DNA of No. 1 as a template. In the PCR reaction, 25 cycles of 94 ° C. for 30 seconds, 55 ° C. for 1 second, and 72 ° C. for 2 minutes and 30 seconds were performed. The obtained DNA fragment was cloned into the SmaI site in a multiple cloning site of a cloning vector pSTV29 (manufactured by Takara Shuzo Co., Ltd.) to prepare pSTVarG. Further, pargG was prepared in which a fragment containing a replication origin obtained by treating pSAK4 described in Example 1 (1) with SalI was inserted into the SalI site of pSTVargG.
7) Introduction of pargG to Brev.
pargG was introduced into Bevribacterium lactofermentum AJ12092 strain. The plasmid was introduced by the electric pulse method (JP-A-2-207791). The transformant was resistant to chloramphenicol on a CM2G plate medium (10 g of polypeptone, 10 g of yeast extract, 5 g of glucose, 5 g of NaCl, and 15 g of agar in 1 liter of pure water, pH 7.2) containing 4 μg / ml of chloramphenicol. Selected as strain.

8) ArgG activity of promoter mutant strain The ArgG activity of the above two types of ArgG promoter mutant strain and the strain (AJ12092 / pargG) in which argG was amplified by plasmid was measured. These strains were obtained by culturing at 31.5 ° C for 20 hours on agar medium containing 0.5 g / dl of glucose, 1 g / dl of polypeptone, 1 g / dl of yeast extract, 0.5 g / dl of NaCl, and 5 μg / l of chloramphenicol. 1 g of the bacterial cell was purified from glucose 3 g / dl, ammonium sulfate 1.5 g / dl, KH2PO4 0.1 g / dl, MgSO4 0.04 g / dl, FeSO4 0.001 g / dl, MnSO4 0.01 g / dl, VB1 5 μg / dl, biotin 5 μg / dl dl, soybean hydrolyzate Inoculated in a medium containing 45 mg / dl as an N amount, and using cells obtained by culturing at 31.5 ° C for 18 hours, reported previously (Journal of General Microbiology (1990), 136, 1177-1183 )), The ArgG activity was measured. Table 21 shows the ArgG activity of the two types of ArgG promoter mutants and the strain (AJ12092 / pargG) in which argG was amplified by plasmid. As shown in Table 21, by introducing a mutation into the promoter, ArgG activity was increased about twice as much as that of the parent strain in AJ12092-P3 and about three times in AJ12092-P7. In addition, ArgG activity of AJ12092 / pargG was about 4.5 times that of the parent strain.

Table 21

9) Arg production by promoter mutant
ArgG promoter mutant strains were cultured in flasks. As controls, parent strains AJ12092 and AJ12092 / pargG were similarly cultured. KH2PO4 0.1 g / dl, MgSO4 0.04 g / dl, FeSO4 0.001 g / dl, MnSO4 0.01 g / dl, VB1 5 μg / dl, biotin 5 μg / dl, soybean hydrolyzate Inoculated in a medium containing 45 mg / dl as N content A bacterium obtained by culturing at 31.5 ° C for 20 hours, painting on an agar medium containing 0.5 g / dl of glucose, 1 g / dl of polypeptone, 1 g / dl of yeast extract, 0.5 g / dl of NaCl, and 5 μg / l of chloramphenicol Body 1 Ese was cultured in a flask with glucose 4 g / dl, ammonium sulfate 6.5 g / dl at 31.5 ° C. until glucose was completely consumed.
The absorbance at 620 nm (OD620), the amount of arginine produced (concentration (g / dl) and the culture time) of a liquid obtained by diluting the culture solution with 0.2N HCl 51-fold are shown.
As shown in Table 22, the Arg yield was improved in the argG promoter mutant, and the yield was equivalent to that of the argG amplified strain in the plasmid. In addition, while the culture time was delayed in the plasmid amplified strain, the culture time of both the AJ12092-P3 and AJ12092-P7 in the promoter mutant strain was equivalent to that of the parent strain, indicating that Arg productivity was improved compared to the plasmid amplified strain. Was.

Table 22

Example 7: Introduction of Mutation into GDH Gene Promoter Region of Coryneform Glutamate-Producing Bacterium (1) Construction of Mutant gdh Plasmid It was constructed by a specific mutation technique. To obtain the GDH promoter sequence of the FGR1 strain, PCR was performed using the synthetic DNA shown in SEQ ID NO: 57 and the synthetic DNA shown in SEQ ID NO: 60 as a primer and the chromosomal DNA of ATCC13869 as a template, while the synthesis shown in SEQ ID NO: 58 PCR was performed using the DNA and the synthetic DNA shown in SEQ ID NO: 59 as primers and the chromosomal DNA of ATCC13869 as a template. Furthermore, PCR was performed using the mixture of the PCR products as a template and the synthetic DNAs shown in SEQ ID NOs: 57 and 58 as primers. The thus obtained PCR product was inserted into the Smal site of pSFKT2 (Japanese Patent Application No. 11-69896) to construct pSFKTG11. To obtain the GDH promoter sequence of the FGR2 strain, PCR was performed using the synthetic DNA shown in SEQ ID NO: 57 and the synthetic DNA shown in SEQ ID NO: 62 as a primer and the chromosomal DNA of ATCC13869 as a template, while the synthesis shown in SEQ ID NO: 58 PCR was performed using the DNA and the synthetic DNA shown in SEQ ID NO: 61 as primers and the chromosomal DNA of ATCC13869 as a template. Furthermore, PCR was performed using the mixture of the PCR products as a template and the synthetic DNAs shown in SEQ ID NOs: 57 and 58 as primers. The thus obtained PCR product was inserted into the Smal site of pSFKT2 (Japanese Patent Application No. 11-69896) to construct pSFKTG07. In addition, the nucleotide sequence of the DNA fragment inserted into the Smal site of pSFKTG11 and pSFKTG07 was determined, and it was confirmed that no mutation was introduced except for the promoter region of GDH.

(2) Construction of gdh Promoter Mutant Next, pSFKTG11 and pSFKTG07 were introduced into the AJ13029 strain by an electric pulse method, and transformants growing at 25 ° C. on a CM2B plate containing 25 μg / ml kanamycin were selected. The transformant was cultured at 34 ° C, and a strain showing kanamycin resistance at 34 ° C was selected. Showing kanamycin resistance at 34 ° C. means that pSFKTG11 or pSFKTG07 was integrated on the chromosome of AJ13029 strain. A kanamycin-sensitive strain was obtained from such a strain in which the plasmid was integrated on the chromosome. The GDH promoter sequences of these strains were determined, and strains having the same gdh promoter sequence as pSFKTG11 and pSFKTG07 were designated GA01 and GA02, respectively.
(3) Confirmation of L-glutamic acid producing ability of mutant gdh promoter
The glutamic acid-producing ability of the GA01 strain, the GA02 strain and its parent strain AJ13029 was confirmed by the same method as in Example 2 (2) above. As a result, remarkable improvement in glutamic acid accumulation was observed in GA01 and GA02 (Table 23).

Table 23
Strain Glu (g / dl) GDH specific activity Relative value
AJ13029 2.6 7.7 1.0
GA01 3.0 22.3 2.9
GA02 2.9 27.0 3.5

(4) Construction of self-cloning type gdh plasmid First, a self-cloning vector pAJ220 was constructed. pAJ226 (JP-A-61-152289) was treated with EcoRV, Pstl to prepare a fragment containing a region capable of autonomous replication in coryneform bacteria, and pAJ224 (JP-A-61-152289) was digested with EcoRV, Pstl. The plasmid ligated to a DNA fragment of about 0.7 kb generated by the treatment is pAJ220. This plasmid is capable of autonomous replication in coryneform fungi and confers trimethoprim resistance to the host.
A gdh gene fragment was prepared by performing a PCR reaction using the chromosomal DNA of the ATCC13869 strain of the coryneform bacterium ATCC13869 as a template as a primer to prepare a gdh gene fragment, and this was inserted into the Ball site of pAJ220 to form pAJ220G It was constructed. A promoter is present near the Ball site of pAJ220, and the expression level of the gene inserted into the Ball site is enhanced depending on the direction of insertion. A strain in which pAJ220G and pGDH were introduced into the ATCC13869 strain by the electric pulse method was constructed, and the GDH activity was measured by the method described in (1) above. As a result, the GDH activity of the pAJ220G-introduced strain was found to be about 1.5 times that of the pGDH-introduced strain. (Table 24)

Table 24
Strain GDH specific activity Relative value
ATCC13869 7.7 1.0
ATCC13869 / pGDH 82.7 10.7
ATCC13869 / pAJ220G 120.l 15.6

(5) Effect of gdh activity on yield and by-product Asp
pGDH and pAJ220G were introduced into AJ13029 by the electric pulse method. These strains and each of the strains obtained in the above (2) were inoculated into a seed culture medium having the composition shown in Table 25, and cultured with shaking at 31.5 ° C for 24 hours to obtain a seed culture. 300 ml of the main culture medium having the composition shown in Table 25 was dispensed into a 500-ml glass jar fermenter, sterilized by heating, and then inoculated with 40 ml of the above seed culture. The cultivation was started at a culture temperature of 31.5 ° C. at a stirring speed of 800 to 1300 rpm and an aeration rate of 1/2 to 1/1 vvm. The pH of the culture solution was maintained at 7.5 with ammonia gas. Eight hours after the start of the culture, the temperature was shifted to 37 ° C. In all cases, the culture was terminated when glucose was completely consumed in 20 to 40 hours, and the amount of L-glutamic acid produced and accumulated in the culture solution was measured (Table 26). The GDH activity for maximizing the yield was about three-fold. When the GDH activity increased, the extent of improvement in the yield decreased, and when the GDH activity was enhanced to about 16-fold, the yield decreased rather. When the by-product amino acids were analyzed using Hitachi's amino acid analyzer L-8500, it was found that the accumulation of aspartic acid and alanine increased with increasing GDH activity. From these results, in order to improve the glutamate yield, it is necessary to appropriately enhance GDH activity so as not to cause a significant increase in aspartic acid and alanine. It has been shown that the effective means is to regulate GDH activity to about three times that of the parent strain by introducing E. coli.

Table 25
concentration
component
Seed culture Main culture
Glucose 50 g / l 150 g / l
KH ₂ PO ₄ 1 g / l 2 g / l
_{MgSO 4 · 7H 2 O 0.4 g} / l 1.5 g / l
_{FeSO 4 · 7H 2 O 10 mg} / l 15 mg / l
_{MnSO 4 · 4H 2 O 10 mg} / l 15 mg / l
Soy protein hydrolysis 20 ml / l 50 ml / l
Biotin 0.5 mg / l 2 mg / l
Thiamine hydrochloride 2 mg / l 3 mg / l

Table 26
Strain Glu (g / dl) Asp (mg / dl) Ala (mg / dl) GDH specific activity Relative value
AJ 13029 8.3 49 60 7.7 1.0
GA01 9.0 145 152 22.3 2.9
GA02 8.9 153 155 27.0 3.5
AJ13029 / pGDH 8.6 201 190 82.7 10.7
AJ13029 / pAJ220G 7.5 290 590 120.12 15.6

1 shows a construction flow of a GDH gene having a mutant promoter. 1 shows a construction flow of a CS gene having a mutant promoter. 1 shows a construction flow of a shuttle vector having lacZ as a reporter gene.

Claims (10)

  At least one DNA sequence selected from the group consisting of TTGTCA, TTGACA, TTGCTA and TTGCCA, and / or a TATAAT sequence or a -10 region at the −35 region of the promoter sequence of the amino acid biosynthesis gene on the chromosome of the coryneform bacterium. A coryneform bacterium in which a sequence in which the base of ATAAT in the sequence is replaced by another base is cultured in a medium, L-glutamic acid or L-arginine is produced and accumulated in the medium, and the L-glutamic acid or L-arginine is collected from the medium. A method for producing L-glutamic acid or L-arginine by a fermentation method.   The amino acid is L-glutamic acid, and the promoter is selected from the group consisting of a promoter for glutamate dehydrogenase (GDH), citrate synthase (CS), isocitrate dehydrogenase (ICDH), and a pyruvate dehydrogenase (PDH) gene. Item 7. The method according to Item 1.   The promoter for a glutamate dehydrogenase (GDH) -producing gene has a −35 region in which at least one DNA sequence selected from the group consisting of TTGTCA, TTGACA and TTGCCA and / or a −10 region in which the TATAAT sequence or the ATAAT base of the sequence is different. 3. The method according to claim 2, wherein the method has a sequence substituted with the following bases.   The method according to claim 3, wherein the GDH promoter has a TTGACA sequence or a TTGCCA sequence in the -35 region and / or has TATAAT as the -10 region.   The method according to claim 2, wherein the CS promoter has a TTGACA sequence in the -35 region and / or a TATAAT sequence or a TATAAC sequence in the -10 region. The ICDH promoter may have a TTGCCA sequence or a TTGACA sequence in the first or second promoter in the -35 region and / or a TATAAT sequence in the first or second promoter in the -10 region. Item 3. The method according to Item 2.   The method according to claim 2, wherein the PDH promoter has a TTGCCA sequence in a -35 region and / or a TATAAT sequence in a -10 region.   The method according to claim 1, wherein the amino acid is L-arginine, and the promoter is a promoter for argininosuccinate synthase. The promoter for argininosuccinate synthase has a −35 region in which at least one DNA sequence selected from the group consisting of TTGCCA, TTGCTA and TTGTCA and / or a −10 region in which the TATAAT sequence or the base of ATAAT in the sequence is replaced by another base. 9. The method according to claim 8, wherein the method has an aligned sequence.   The method according to claim 9, wherein the promoter for argininosuccinate synthase has a TTGCTA sequence in a -35 region and / or a TATAAT sequence in a -10 region.

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