AU737421B2 - Process for constructing amino acid-producing bacterium and process for producing amino acid by fermentation method with the use of the thus constructed amino acid-producing bacterium - Google Patents

Description

SPECIFICATION

Method of constructing amino acid producing bacterial strains, and method of preparing amino acids by fermentation with the constructed amino acid producing bacterial strains Background of the Invention The present invention relates to a method of constructing a mutant strain capable of producing amino acids in a high yield, and a method of producing L-amino acids by the fermentation with the mutant.

Methods of constructing mutant strains usable for the production of amino acids by the fermentation can be roughly classified into two methods. One of them comprises introducing random mutations into DNA with a chemical mutagen, and the other comprises the genetic recombination. In the latter method, a strain having an improved capacity of producing an intended substance can be developed by enhancing a gene on a metabolic pathway relating to the biosynthesis of an intended substance, or by weakening a gene of an enzyme relating to-the destruction. In this connection, for enhancing an intended gene, a plasmid capable of autonomously replicating independently from the chromosome in a cell has been mainly used.

However, the method of enhancing the intended gene with a plasmid has problems. In particular, the degree of enrichment of the intended gene is variable depending on the number of copies of the plasmid itself. Therefore, for some kinds of intended genes, the copies are often too many in number and, as a result, the expression becomes excessive, the growth is seriously inhibited or the capacity of producing the intended substance is lowered. In such a case, although the degree of the enhancement of the intended gene can be lowered by using a plasmid of a small number of the copies, the variety of the plasmid is limited in many cases, and the intended control of the expression level of the intended gene is impossible.

Another problem is that since the replication of the plasmid is often unstable, the plasmid is eliminated.

For example, Japanese Patent Unexamined Published Application (hereinafter referred to as P. KOKAI") No:61-268185 discloses a recombinant DNA comprising a DNA fragment containing a glutamate dehydrogenase (GDH)-producing gene (glutamate dehydrogenase gene) derived from a glutamate-producing coryneform bacterium, and a DNA fragment (plasmid) containing a gene necessary for the autonomous replication in the cell. It is also disclosed therein that by introducing the recombinant DNA into a cell, a GDH-enriching strain can be grown to improve the production of substances (such as amino acids and proteins) with microorganisms.

On the other hand, in Japanese Patent No. 2,520,895, the above described recombinant DNA is introduced into Corynebacterium to obtain a strain having the improved enzymatic activity, and L-glutamic acid is produced by the fermentation with the strain. However, the production and yield of L-glutamic acid were yet unsatisfactory. Thus, it is demanded to further improve the productivity of L-glutamic acid. It is reported that the demand had been attained by introducing a recombinant DNA comprising two kinds of genes, i.e. a glutamate dehydrogenase-producing gene derived from a glutamate-producing coryneform bacterium, and an isocitrate dehydrogenase (ICDH)gene, into a glutamate-producing coryneform bacterium.

Further, JP Kokai No.6-502548 discloses an expression system and a secretion system of Corynebacterium comprising a Corynebacterium strain and a secretory cassette comprising the first functional DNA sequence for the expression in the strain, the second DNA sequence encoding for amino acids, polypeptides and/or proteins and the third DNA sequence inserted between the first DNA sequence and the second DNA sequence, wherein the third DNA sequence encodes the protein element selected from PS1 and PS2 which guarantee the secretion of the amino acids, polypeptides and/or proteins. Specifically, the secretion of polypeptides is disclosed therein and in particular, NTG mutagtenesis was conducted with Corynebacterium ._and a mutant resistant to 4-fluoroglutamate (4FG) which is an analogue to glutamate is selected and subjected to the transformation with PCGL141. It is described therein that a strain having an enhanced expression of GDH can be obtained from the analogue resistant bacteria. It is also described therein that a mutation was observed in nucleotide sequence No.251 to No.266 of GDH promoter.

Disclosure of the Invention The present invention relates to a method of constructing a mutant capable of suitably enhancing or controlling the expression of an intended gene without using a plasmid and also capable of producing amino acids in a high yield, by gene recombination or mutation.

The present invention also relates to a promoter for GDH capable of imparting a capability of producing glutamic acid in a high yield to a Corynebacterium strain without seriously increasing the amount of by-produced aspartic acid and alanine.

;t The present invention also relates to a GDH gene having a sequence of the above-described promoter for GDH.

The present invention also relates to a Corynebacterium strain having the above-described gene and capable of producing I-glutamic acid.

The present invention also relates to a method of producing amino acids by fermentation wherein amino acid-producing microorganism thus constructed is used.

The present invention also relates to a fermentation method of producing glutamic acid at a low cost by increasing the yield of glutamic acid by using a glutamic acid-producing coryneform bacterium.

The present invention has been completed on the basis of a finding that the above-described problems can be efficiently solved by variously modifying the promoter of amino acid-biosynthesizing genes on a chromosome to control the amount of the expression of the intended genes. Particularly, the invention has been completed on the basis of a finding that the above-described problem can be 3 efficiently solved by introducing a specific mutation into -35 region or -10 region which is a specific region of the promoter.

Namely, the present invention provides a method of producing coryneform bacteria having an improved amino acid- or nucleic acid-productivity, which comprises the steps of introducing a mutations in a promoter sequence of amino acid- or nucleic acid-biosynthesizing genes on a chromosome of a coryneform bacteria to make it C close to a consensus sequence or introducing a change in a promoter sequence of amino acid- or nucleic acid-biosynthesizing genes on a chromosome of Coryneform bacteria by gene recombination to make it close to a consensus sequence, to obtain mutants of the coryneform amino acid- or nucleic acid-producing microorganism, culturing the mutants, and selecting a mutant capable of producing the intended amino acid or nucleic acid in a large amount.

The present invention also provides a promoter for glutamate dehydrogenase (GDH)-producing gene, which has the sequence of at least one DNA sequence selected from the group consisting of CGGTCA, TTGTCA, TTGACA and TTGCCA in region, (ii) TATAAT sequence or the same TATAAT sequence but in which the base of ATAAT is replaced with another base in -10 region, or (iii) a combination of (i) and wherein the sequence does not inhibit the promoter function.

The present invention also provides a glutamate dehydrogenase-producing gene having the above-described promoter.

The present invention also provides a coryneform L-glutamate-producing microorganism having the above-described gene.

The present invention also provides a process for producing an amino acid by the fermentation, which comprises the steps of culturing a coryneform bacterium constructed by the above-described method and having an improved amino acidproducing capacity in a medium to form and also to accumulate the intended amino acid in the medium, and collecting the amino acid from the medium.

The present invention also provides a process for producing L-glutamic acid by thefermentation, which comprises the steps of culturing a coryneform L-glutamic acid-producing microorganism resistant to 4-fluoroglutamic acid in a liquid medium to form and also to accumulate L-glutamic acid in the medium, and collecting L-glutamic acid from the medium.

Brief Description of the Drawings Fig. 1 show a flow of construction of GDH gene having a mutant promoter.

Fig.2 show a flow of construction of CS gene having a mutant promoter.

Fig.3 show a flow of construction of shuttle vector carrying lacZ as a reporter gene.

Best Mode for Carrying out the Invention The term "coryneform glutamic acid producing microorganism" as used herein includes also bacteria which were classified to be the genus Brevibacterium before but now integrated into the genus Corynebacterium [Int. J. Syst. Bacteriol., 41, 255 (1981)] and also bacteria of the genus Brevibacterium which are very close to those of the genus Corynebacterium. Therefore, the mutants used in the present invention can be derived from the coryneform glutamic acid-producing bacteria of the genus Brevibacterium or Corynebacterium shown below. Bacteria of the genus Corynebacterium and those of the genus Brevibacterium will be collectively referred to as "coryneform bacteria" so far as they do not concern the glutamic acid productivity.

Corynebacterium acetoacidophilum ATCC13870 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium callunae ATCC15991 Corynebacterium glutamicum ATCC13032 Brevibacterium divaricatum ATCC14020 Brevibacterium lactofermentum ATCC13869 Corynebacterium lilium ATCC 15990 Brevibacterium flavum ATCC14067 Corynebacterium melassecola ATCC17965

NOFF\

Brevibacterium saccharolyticum. ATCC14066 Brevibacterium immariophilum ATCC14068 Brevibacterium roseum ATCC13825 Brevibacterium thiogenitalis ATCC19240 Microbacterium ammoniaphilum ATCC15354 Corynebacterium thermoaminogenes AJ12310(FERM 9246) The amino acids to be produced are not particularly limited so far as the genes concerning the biosynthesis and promoters thereof have been elucidated.

Examples of effective enzymes concerning the biosynthesis include GDH, citrate S synthase isocitrate synthase (ICDH), pyruvate dehydrogenase (PDH) and aconitase (ACO) for glutamic acid fermentation.

The term "glutamic acid-synthesizing gene" as used herein includes a gene encoding an enzyme which is involved in the bosynthesis of glutamic acid.

Especially, "glutamic acid-synthesizing gene" includes glutamate dehydrogenase (GDH) gene, citrate synthase (CS) gene, isocitrate synthase (ICDH) gene, pyruvate dehydrogenase (PDH) gene and aconitase (ACO) gene.

E'nzymes for lysine fermentation including biosynthesis enzymes such as aspartate kinase dihydrodipicolinate synthase, dihydrodipicolinate reductase, diaminopimelate dehydrogenase and diaminopimelate decarboxylase are also effective. Lysine eccrisis protein (lysE gene) concerning the membrane eccrisis of lysine is also effective.

The term "arginine-synthesizing gene" as used herein includes Nacetylglutamate synthase gene, N-acetylglutamate kinase gene, N-acetylglutamyl phosphate reductase gene, acetylornithine aminotransferase gene, Nacetylornithinase gene, ornithine carbamyltransferase gene, argininosuccinate synthase gene, and arginosuccinase gene.

Effective enzymes for arginine fermentation include N-acetylglutamate synthase, N-acetylglutamate kinase, N-acetylglutamyl phosphate reductase, acetylornithine aminotransferase, N-acetylornithinase, ornithine carbamyltransferase, argininosuccinate synthase, and arginosuccinase. arginine is formed by the reaction catalyzed by these enzymes. These enzymes are effective. These enzymes are coded by enzymes argA, argB, argC, argD, aegE, argF, argG and argH, relatively.

Effective enzymes for serine fermentation includes 3-phosphoglyceric acid dehydrogenase, phosphoserine trans-amylase, phosphoserine phosphatase and the like.

Effective enzymes for phenylalanine fermentation include bio-synthesizing enzymes such as deoxyarabinohepturonic phosphate synthetase, 3-dehydrokinate synthetase, 3-dehydrokinic acid dehydroratase, shikimic acid dehydrogenase, 6A shikimic kinase, 5-enol pyrvilshikimic acid-3-phosphate synthetase, chorismic acid synthetic enzyme, chorismate synthetase, chorismate mutase, prephenate dehydroratase, and the like. Sugar metabolic enzymes such as transketorase, transaldolase, phosphoenolpyrvic acid synthetic enzyme are also effective.

Effective enzymes for tryptophan fermentation include enzymes belonging to tryptophan operon, in addition to various enzymes effective in the above-mentioned phenylalanine fermentation and various enzymes effective in the above-mentioned serine fermentation.

Effective enzymes for proline fermentation include y -glutamylkinase, glutamylcemialdehyde dehydrogenase, pyrroline-5-carboxylate reductase, in addition to various enzymes effective in the above-mentioned glutamic acid fermentation.

Effective enzymes for glutamine fermentation include glutamine synthetase, in addition to various enzymes effective in the above-mentioned glutamic acid fermentation.

In the inosine production, it is considered to be useful to enhance the expression of 5-phosphoribosyl 1-diphosphate synthetase, 5-phosphoribosyl 1-diphosphate aminotransferase, phosphoribosylaminoimidazolecarboxamide formyltransferase and the like.

In the guanosine production, it is considered to be useful to enhance the expression of 5'-inosinic acid dehydrogenase and 5'-xanthylic acid aminase, addition to 5-phospholibosyl 1-diphosphate synthetase, 5-phospholibosyl 1-diphosphate aminotransferase, phosphoribosylaminoimidazolecarboxamide formyltransferase and the like.

In the adenosine production, it is considered to be useful to enhance the expression of adenirosuccinate synthase, in addition to 5-phosphoribosyl 1diphosphoric acid synthetic enzyme, 5-phosphoribosyl 1-diphosphoric acid aminotransferase, phosphoribosylaminoimidazole-carboxamide formyltransferase and the like.

In the nucleotide production, it is considered to be useful to enhance the expression of phosphoribosyl transferase, inosine kinase, guanosine kinase and adenosine kinase.

In the present invention, a mutant of a coryneform amino acid-producing bacterium is obtained by, introducing a mutation in a promoter sequence of desired amino acid-biosynthesizing genes on a chromosome of a coryneform amino acidproducing bacterium, such as the above-described promoter sequence for GDH, to make it close to a consensus sequence with a chemical or by introducing the mutation by the genetic recombination to obtain a mutant of the coryneform amino acidproducing microorganism.

The term "consensus sequence" is a sequence which appears most frequently in various promoter sequences. Such consensus sequences include, for example, those of E. coli and Bacillus subtilis. The consensus sequence of E. coli is described in Diane K. Hawley and William R. McClure Nuc. Acid. Res. 11:2237- 2255(1983), and that of B. subtilis is described in Charles et al. Mol. Gen. Genet 186:339-346(1982).

The mutation may be caused in either only one promoter sequence such as that for GDH or two or more promoter sequences such as those for GDH, citrate synthase (citrate-synthesizing enzyme) (CS) and isocitrate synthase (isocitratesynthesizing enzyme) (ICDH).

In the present invention, the mutant thus obtained is cultured to obtain the mutant capable of producing a large amount of an intended amino acid.

It was already elucidated that in the fermentation of glutamic acid, GDH derived from a coryneform glutamate-producing microorganism has its own promoter sequence in upstream region thereof [Sahm et al., Molecular Microbiology (1992), 6, 317-326].

For example, the promoter for GDH of the present invention, GDH gene having the promoter sequence for GDH and L-glutamate-producing Corynebacterium strain having this gene can be obtained by, for example, the following methods: ,uo Namely, the strain is subjected to a mutagenesis treatment such as the irradiation with UV, X-rays or radiation, or treatment with a mutagen to obtain a strain resistant to 4-fluoroglutamic acid on an agar plate culture medium containing 4fluoroglutamic acid. Namely, the mutagenized cells are spread on agar plates culture medium containing 4-fluoroglutamic acid in such a concentration that it inhibits the growth of the parent, and the mutant thus grown is separated.

Further, the promoter sequence of GDH genes can be replaced with variously modified sequences by site directed mutagenesis, and the relationship between the respective sequences and GDH activity is examined so as to select the ones having a high L-glutamate-productivity.

It is particularly preferred in the present invention that the DNA sequence in region of the prompter for GDH-producing gene is at least one DNA sequence selected from the group consisting of CGGTCA, TTGTCA, TTGACA and TTGCCA and/or the DNA sequence in -10 region of the promoter is TATAAT, or the bases of ATAAT in TATTAT sequence in -10 region is replaced with another base, while they do not inhibit the promoter function. The reason why the strain in which the bases of ATAAT in TATAAT sequence in -10 region is replaced with another base and the promoter function is not inhibited can be selected is as follows: Because a remarkable increase in the specific activity of GDH was observed by merely replacing the first "C" of CATAAT with in wild type -10 sequence (refer to p6-4 in Table it was considered that such a replacement with another base is possible.

The promoter sequence of GDH gene is described in, for example, the above-described Sahm et al., Molecular Microbiology (1992), 6, 317-326. It is described therein as Seq ID No. 1. The sequence of GDH gene itself is also described in Sahm et al., Molecular Microbiology (1992), 6, 317-326 to be Seq ID No.

1.

Similarly, the mutation can be introduced in the promoter for citratesynthesizing enzyme (CS) or isocitrate-synthesizing enzyme (ICDH).

Thus, the promoters for GDH are those having at least one DNA sequence in region selected from the group consisting of CGGTCA, TTGTCA, TTGACA and

I,

TTGCCA in -35 region and/or or TATAAT sequence or the TATAAT sequence but in which the base of ATAAT is replaced with another base, wherein they do not inhibit the promoter function. Genes for producing glutamate dehydrogenase, which have the above-described promoter, are also provided.

The promoters for CS are those having TTGACA sequence in -35 region and/or TATAAT sequence in -10 region, which do not inhibit the promoter function.

CS genes having the above-described promoter are also provided.

Promoters for ICDH are those having TTGCCA or TTGACA sequence in the first or the second promoter in -35 region and/or TATAAT sequence in the first or the second promoter in -10 region which do not inhibit the function of the promoter. The icd genes having the above-described promoter are also provided.

Promoters for PDH are those having TTGCCA sequence in -35 region and/or TATAAT sequence in -10 region, which do not inhibit the function of the promoter.

PDH genes having the above-described promoter are also provided.

The present invention also provides coryneform L-glutamate-producing bacterium having the above-described genes.

The promoters for algininosuccinate synthase are those having at least one DNA sequence selected from the group consisting of TTGCCA, TTGCTA, and TTGTCA in -35 region and/or TATAAT sequence in -10 region, or the base of ATAAT in TATTAT sequence is replaced with another base, which do not inhibit the function of the promoter. Argininosuccinate synthase gene having the above-described promoter are also provided.

The present invention also provides coryneform arginine-producing bacterium having the above-described genes.

Amino acids can be obtained by culturing a coryneform bacterium of the present invention, which produces an amino acid, preferably L-glutamic acid, in a liquid culture medium to form and thereby to accumulate the intended amino acid, preferably L-glutamic acid, and collecting the amino acid from the culture medium.

The liquid culture medium used for cultivating the above-described strain of F

OPFFOC

the bacterium in the present invention is an ordinary nutrition medium containing carbon sources, nitrogen sources, inorganic salts, growth factors, etc.

The carbon sources include carbohydrates such as glucose, fructose, sucrose, molasses and starch hydrolyzates; alcohols such as ethanol and glycerol; and organic acids such as acetic acid. The nitrogen sources include ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, ammonium acetate, ammonia, peptone, meat extract, yeast extract and corn steep liquor. When an auxotrophic mutant is used, the required substances are added to the medium as the reagents or natural substances containing them.

The coryneform bacteria usually produce L-glutamic acid under reduced biotin condition. Therefore, the amount of biotin in the medium is restricted or a substance inhibiting the effect of biotin such as a surfactant or penicillin is added.

The fermentation is preferably conducted by shaking the culture or agitating the culture with aeration while the pH of the culture liquid is kept in the range of 5 to 9 for 2 to 7 days. The pH is preferably controlled with urea, calcium carbonate, gaseous ammonia, ammonia water or the like. The culture temperature is preferably 24 to 37°C.

L-glutamic acid thus produced and accumulated in the culture liquid is collected by an ordinary method such as ion-exchange resin method or crystallization method. Specifrrfically, L-glutamic acid is separated by the adsorption on an anionexchange resin or by the neutralization crystallization.

According to the present invention, the intended amino acid can be obtained in a high yield by introducing a mutation into a promoter region of amino acidbiosynthesizing genes of a coryneform amino acid-producing bacterium to control the expression of the intended genes. In addition, since any elimination of the intended gene does not occur in the bacteria according to the present invention, contrary to the cases using plasmid, the intended amino acid can be stably obtained in a high yield.

Thus, the industrial merit of the invention is great.

The present invention provides various promoters, particularly, promoters for I(B ~11 11 GDH, capable of imparting a power of producing amino acids, particularly glutamic acid, in a high yield to Corynebacterium strains without increasing the amount of byproduced aspartic acid and alanine.

In the present invention, a coryneform L-glutamate-producing bacterium is mutagenized, a strain in which the mutation introduced in a promoter region of GDH gene and which is resistant to 4-fluoroglutamic acid is collected, and the strain is cultured to obtain glutamic acid in a high yield. Thus, the present invention is industrially very advantageous.

The following Examples will further illustrate the present invention.

Example 1: Production of mutant GDH promoter: A mutant GDH promoter was prepared by site-directed mutagenesis method as follows: Preparation of GDH genes having various mutant promoters: The wild type sequence in -35 region and -10 region of a promoter of GDH gene of a coryneform bacteria is shown in sequence 1. The promoter sequence of wild type has already been reported [Molecular Microbiology (1992), 6, 317-326].

The method of preparing a plasmid carrying GDH gene having a mutant promoter is as follows: As shown in Fig. 1, a chromosomal gene of a wild type strain of a coryneform bacterium ATCC13869 prepared with "Bacterial Genome DNA purification kit" (Advanced Genetic Technologies Corp.) was used as the template for PCR. The gene amplification was conducted by PCR using upstream and downstream sequences of GDH gene. Both ends were blunt-ended. The product thus obtained was inserted in Smal site of plasmid pHSG399 (a product of Takara Shuzo Co., Ltd.).

Then a replication origin taken from plasmid pSAK4 having the replication origin capable of replicating in a coryneform bacterium was introduced into Sal I site of the plasmid to obtain plasmid pGDH. By this method, GDH genes having each abovedescribed promoter sequence can be obtained by using a primer having each of the sequence of Seq ID No. 1 to Seq ID No. 6 shown in the Sequence Listing as the upstream primer for GDH gene, respectively. It was confirmed by sequencing the PCR amplified fragment that any mutation, other than the introduced mutation in the promoter sequence, was not occured in the amplified fragment. pSAK4 is constructed as follows: previously obtained plasmid pHK4 P. KOKAI No.5-7491] having an autonomous replication origin derived from plasmid pHM1519 [Agric. Biol.

Chem., 48, 2901-1903 (1984)] ,which is capable of autonomously replicating in Corynebacterium microorganism, is digested with restriction enzymes BamHI and Kpnl to obtain a DNA fragment having the replication origin. Then the fragment thus obtained is blunt-ended with DNA-Blunting Kit (Blunting kit of Takara Shuzo Co., Ltd.). After the ligation with Sail linker, the product thus obtained was inserted into Sal I site of pHSG299 (a product of Takara Shuzo Co., Ltd.) to obtain plasmid pSAK4.

Comparison of the degrees of expression of GDH having each promoter sequence: Each plasmid prepared as described above was introduced into wild type strain of coryneform bacterium ATCC13869 by electroporation method (refer to J. P.

KOKAI No. 2-207791. For comparing the degrees of expression of GDH for these strains, the specific activity of GDH was determined by the above-described method of Sahm et al. The results are shown in Table 1.

Table 1 Strain Promoter sequence Specific activity of GDH -10 ATCC13869 TGGTCA CATAAT 7.7 /pGDH TGGTCA CATAAT 82.7 /p6-2 CGGTCA CATAAT 33.1 /p6-4 TGGTCA TATAA.T 225.9 Relative value 0.1 0.4 2.7 ATCC 13869/p6-2 through ATCC 13869p6-8/ corresponded to the sequences of Seq ID No. 2 through Seq ID No.6, respectively. These sequences were the same as the sequence No.1 (wild type) except that the underlined parts were changed as follows: Sequence No. 1 5'-TTAATTCTTTGTGGTCATATCTGCGACACTGC 2

CGGTCA

3.

TGGTCA

4.

TTGACA

TTGCCA

6.

TTGTCA

CATAATTTGAACGT-3'

CATAAT

TATAAT

TATAAT

TATAAT

TATATT

These were those of synthetic linear doubled stranded DNA.

Example 2: Preparation of mutant strains: Preparation of mutant strains resistant to 4 -fluoroglutamic acid: AJ13029 is a mutant strain producing glutamic acid and disclosed in W096/06180. Although it does not produce glutamic acid at a culture temperature of 31,5 0 C, it produces glutamic acid even in the absence of a biotin-inhibitor when the culture temperature is shifted to 37 °C In this Example, Brevibacterium lactofermentum AJI3029 strain was used as the parent strain for preparing the mutant strains. As a matter of course, any of glutamic acid-producing strains other than AJ13029 can be used as a parent strain for preparing mutant strains resistant to 4fluoroglutamic acid.

AJ13029 were cultured on a CM2B agar medium (Table 2) at 31.5 0 C for 24 hours to obtain the bacterial cells. The cells were treated with 250pg/ml aqueous solution of N-methyl-N'-nitro-N-nitrosoguanidine at 30°C for 30 minutes. Then a suspension of the cells having a survival rate of 1 was spread on agar plates culture medium (Table 3) containing 4-fluoroglutamic acid (4FG). Colonies were formed after incubating the plate at 31.5°C for 20 to 30 hours. In this experiment, a slant medium containing 1 mg/ml of 4FG was prepared at first, and then a layer of the same medium without 4FG was formed thereon horizontally. Thus, 4FG concentration gradient was obtained on the surface of the agar medium. When the plate was inoculated with the mutant cells obtained as described, a boundary line was formed at a border of the growing limit of the strain. Bacterial trains which formed colonies in a area containing 4FG of a concentration higher than that of the boundary line were taken. Thus, about 50 strains resistant to 4FG were obtained from about 10,000 mutagenized cells.

Table 2 CM2B agar medium Ingredient Concentration Polypeptone (Nippon Seiyaku Co.) 1.0 Yeast extract (Difco Co.) 1.0 NaCI 0.5 d-Biotin 10 jg/ Agar (pH 7.2 adjusted with KOH) Table 3 Agar medium Component Amount in one liter of wate Glucose MgSO 4 *7H 2 0 1g S7z FeSO 4 .7H 2 0 0.01g MnS04-4-6H 2 0 0.01 g Thiamine hydrochloride 0.2 mg d-Biotin 0.05 mg

(NH

4 2 S0 4 5 g Na 2

HPO

4 -12H 2 0 7.1g

KH

2

PO

4 1.36g Aaar Confirmation of capability of L-glutamic acid-production of 4FG-resistant mutant strains: The capability of glutamic acid-production of about 50 mutant strains obtained in above and parent AJ13029 strain were confirmed as described below.

AJ13029 and mutant strains were each cultured on CM2B agar medium at 31.5°C for 20 to 30 hours. A liquid medium having a composition shown as "medium A" in Table 4 was inoculated with the cells thus obtained, and the shaking culture was started at 31.5°C. About 22 hours after, the fresh medium-was added so that the final concentration would be that of medium B shown in Table 4. The temperature was shifted to 37°C and then the culture was continued further for about 24 hours.

After the completion of the culture, the culture was examined with a Biotic Analyzer (a product of Asahi Chemical Industry Co., Ltd.) to determine whether L-glutamic acid was produced or not. It was thus found that when the 50 strains were cultured, two strains having a yield of glutamic acid higher than that obtained from the parent strains and a high GDH activity were separated (strain A and strain GDH activity of each of them was determined to find that the specific GDH activity of both of them was increased (Table The GDH activity was determined by the method of E. R.

Bormann et al. [Molecular Microbiol., 6, 317-326 (1996)]. By sequencing the GDH genes, it was identified that the mutation points were found only in the promoter region of GDH (Table 6).

A

Table 4 Ingredient Mdium.* A.e.u Glucose 3 g/dl 5 g/di

KH

2

PO

4 0. 14 g/dl 0. 14 g/dI MgSO 4 7H 2 0 0.04 g/dl 0.04 g/dl FeSO 4 7 2 0 0.001 g/dl 0.001 g/dl MnSO 4 -4H 2 0 0.001 g/dl 0.001 g/dl

(NH

4 2 S50 4 1.5 g/dl 2.5 g/dl Soybean protein hydrolyzate solution 1.5 mi/dI 0.38 mI/dIl Thiamine hydrochloride 0.2 mg/I 0. 2 mg/I Biotin 0.3 mg/I 0.3 mg/I Antifoaming agent 0.05ml/l 0.05ml/l CaCO 3 5 gd/I 5 gd/I pH 7.0(adiusted with KOH) Table 5. Glutamic acid formation and GDH activity of mutant §trains Strain Glu(g/dl) GDH specific activity -Relative value AJ 13029 2.6 7.7 FGR1 2.9 23.1 FGR2 3.0 25.9 3.4 Table 6. DNA sequences in GDH promoter region of mutant strains Strain GDH promoter sequence -351r AJ13029 TGGTCA TTCTGTGCGACACTGC

CATAAT

FGR1 TGGTCA TTCTGTGCGACACTGC

TATAAT

17 FGR2 Tr rr A TATAA r Example 3 Introduction of mutation into CS gene promoter region of coryneform glutamate-producing bacterium: In this Example, a strain having an enhanced promoter for the genes which codes glutamate dehydrogenase (GDH) and citrate-synthesizing enzyme (CS) was produced.

Cloning of gItA gene: The sequence of gItA gene of a coryneform bacterium, which codes citratesynthesizing enzyme, has already been elucidated [Microbial. 140, 1817-1828 (1994)].

On the basis of this sequence, primers shown in Seq ID No. 7 and Seq ID No. 8 were synthesized. On the other hand, chromosomal DNA from Brevibacterium lactofermentum ATCC13869 was prepared using Bacterial Genome DNA Purification Kit (Advanced Genetic Technologies Corp.). Sterilized water was added to a mixture of 0.5 tg of the chromosomal DNA, 10 pmol of each of the oligonucleotides, 8Pl of dNTP mixture (2.5 mM each), 5jp of 10xLa Taq Buffer (Takara Shuzo Co., Ltd.) and 2 U of La Taq (Takara Shuzo Co., Ltd.) to obtain 50p. of PCR-reaction cocktail. The reaction cocktail was subjected to PCR. The PCR conditions were 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 15 seconds and extention at 720C for 3 seconds using Thermal Cycler TP240 (Takara Shuzo Co., Ltd.) to amplify about 3 Kbp of DNA fragments containing gItA gene and promoter thereof. The amplified fragements thus obtained were purified with SUPRECO2 (Takara Shuzo Co., Ltd.) and then blunt-ended. The blunting was conducted with Blunting Kit of Takara Shuzo Co., Ltd. The blunt-ended fragment was mixed with pHSG399 (Takara Shuzo Co., Ltd.) completely digested with Smal to conduct the ligation. The ligation reaction was conducted with DNA Ligation Kit ver 2 (Takara Shuzo Co., Ltd.). After the completion of the ligation, the transformation was conducted with competent cells of E. coli JM109 (Takara Shuzo Co., Ltd.). The cells were spread on an L medium plates (comprising 10 g/1 of bactotryptone, 5 g/l of bactoyeast extract, 5 g/I of NaCI and 15 g/I of agar; pH 7.2) containing 10 pg/ml of IPTG (isopropyl-P-D-thiogalactopyranoside), 40p g/ml of X-Gal (5-bromo-4-chloro-3indolyl- P -D-galactoside) and 40 pg/ml of chloramphenicol. After culturing them overnight, white colonies were taken to obtain the transformed strains after single colony isolation.

From the transformed strains, plasmids were prepared by the alkali method (Seibutsu Kogaku Jikken-sho edited by Nippon Seibutsu Kogaku-kai and published by Baifukan, p. 105, 1992). Restriction enzyme maps were prepared, and the plasmid which has the same restriction map as the map shown in Fig. 2 was named "pHSG399CS".

Introduction of mutations into gItA promoter: Mutan-Super Express Km (Takara Shuzo Co., Ltd.) was used for the introduction of mutation into gItA promoter region. The method is speciffically described below. PHSG399CS was completely digestied with EcoRI and Sail to obtain EcoRI-Sall fragment containing gltA genes, which were ligated to the fragment obtained by complete digestion of pKF19kM (Takara Shuzo Co., Ltd.) with EcoRI and Sail. After the completion of the ligation, the transformation was conducted with competent cells of E. coli JM109 (Takara Shuzo Co., Ltd.). The cells was spread on L medium plates containing 10 pg/ml of IPTG, 40pg/ml of X-Gal and 25 pg/ml of kanamycin. After overnight incubation, white colonies were taken and transformants were obtained by single colony isolation. From the transformants, plasmids were prepared and the plasmid containing gltA gene was named pKF19CS.

PCR was conducted by using pKF19CS as the template and phosphorylated synthetic DNA shown in sequence of Seq ID No. 9, Seq ID No.10 and Seq ID No.11 together with the selection primer from Mutan super Express Km. The transformation was conducted with competent cells of E. coli MV1184 (Takara Shuzo Co., Ltd.) by using the PCR product. The cells were spread on L-medium plates containing 25 pg/ml of kanamycin. After overnight incubation, colonies were taken and the transformants were obtained after single colony isolation. From the 19

-A

2' OF 411 transformants, plasmid DNA was prepared. The sequence of gltA promoter region was determined by Sanger method Mol. Biol., 143,161 (1980)] using synthetic DNA having the sequence of Seq ID No. 12. Specifically, the sequence was determined with a Dye Terminator Sequencing Kit (Applied Biosystems) and analyzed by Genetic Analyzer ABI310 (Applied Biosystems). The plasmids in which gltA promoter region was replaced with the sequence shown in Table 7 were named pKF19CS1, pKF19CS2 and pKF19CS4, respectively.

Table 7 region_-10 region pKF19CS ATGGCT

TATAGC

pKF19CS1 ATGGCT

TATAAC

pKF19CS2 ATGGCT TATAAT oKF19CS4 TTGACA

TATAAT

Construction of mutant gltA plasmid: pKF19CS, pKF19CS1, pKF19CS2 and pKF19CS4 constructed in step (2) were completely digested with Sail and EcoRI (Takara Shuzo Co., Ltd.). On the other hand, plasmid pSFK6 (Japanese Patent Application No.11-69896) having a replication origin derived from plasmid pAM330 which can autonomously replicate in a coryneform bacterium [Japanese Patent Publication for Opposition Purpose (hereinafter referred to as P. KOKAI") No. 58-67699] was completely digested with EcoRI and Sail. The obtained fragment was ligated with about the 2.5 kb fragment containing gItA. After the completion of the ligation, transformation was conducted with competent cells of E. coli JMI09. The cells was spread on the L-medium plates containing 10 pg/ml of IPTG, 40 g/ml of X-Gal and 25 pg/ml of kanamycin. After overnight incubation, colonies were taken and the transformants were obtained after single colony isolation. From the transformants, plasmids were prepared. The plasmids containing gItA gene were named pSFKC, pSFKC1, pSFKC2 and pSFKC4, respectively.

Determination of CS expression from mutant gltA plasmid in coryneform bacterium: The plasmid constructed in above step was introduced into Brevibacterium lactofermentum ATCC13869. Specifically, this treatment was conducted by electrical pulse method P. KOKAI No. 2-07791). The transformants were selected at 31°C with CM2B medium plate (comprising 10 g/1 of bactotryptone, g/1 of bactoyeast extract, 5 g/1 of NaCI, 10pg/l of biotin and 15 g/1 of agar; pH containing 25 jpg/ml of kanamycin. After incubating for two days, colonies were taken and the transformants containing pSFKC, pSFKC1, pSFKC2 and pSFKC4 were named BLCS, BLCS1, BLCS2 and BLCS4, respectively, after single colony isolation.

A medium having a composition shown in Table 8 was inoculated with the transformant. The culture was continued at 31°C and terminated before the glucose had been completely consumed. The culture liquid was centrifuged to separate the cells. The cells were washed with 50 mM tris buffer solution (pH 7.5) containing 200m of sodium glutamate and then suspended in the same buffer solution. After the sonication with UD-201 (TOMY) followed by the centrifugation (10,000g), the cells remaining unbroken were removed to obtain a crude enzyme solution. The activity of citrate synthase can be determined according to Methods Enzymol. 13, 3-11 (1969).

Specifically, the crude enzyme solution was added to a reaction mixture containing 100 mM of TisHCI. (pH 0.1 mM, of DTNB, 200 mM of sodium glutamate and 0.3 mM of acetyl CoA, and the background was determined as the increase in the absorbance at 412 nm at 30°C determined by Hitachi spectrophotometer U-3210.

Further, oxaloacetic acid was added in such an amount that the final concentration thereof would be 0.5 mM. The increase in the absorbance at 412 nm was determined, from which the background value was deducted to determine the activity of the citrate synthase. The protein concentration in the crude enzyme solution was determined by Protein Assay (BIO-RAD.). Bovine serum albumin was used as the (Asr& I standard protein. The results are shown in Table 9. It was confirmed that the citrate synthase activity of mutant gItA promoters was increased compared to wildtype gItA promoter.

Table 8 Ingredient Glucose

KH

2

PO

4 MnSO 4 -7H20 FeSO 4 -7H 2 0 Soybean protein hydrolysates Biotin Thiamine hvdrochloride 2 ma/I Concentration 50 g/l 1 g/I 0.4 mg/I 10 mg/I 20 ml/I 0.5 mg/I 2 ma/ll 2 mci/I Strain Wild type4 BLCS00 BLCS01 BLCS02 BLCS04 Table 9 dABS/min/mg Relative activity Relative activity 6.8 38.8 5.7 57.1 8.4 1.21 92.5 13.6 1.9 239.5 35.2 4.8 Introduction of mutant gltA gene into temperature-sensitive plasmid: For integrating mutant gltA promoter sequences into a chromosome, a method is known wherein a plasmid of which replication in a coryneform bacterium is temperature sensitive is used(J. P. KOKAI No. 5- 7491). PSFKT2 (Japanese Patent Application No.11-81693) was used as the plasmid vector, the replication of which in a coryneform bacterium is temperature sensitive. pKFCS1, pKFCS2 and pKFCS3 22 s

C)

I 0 completely digested with Sail and BstPI and blunt-ended were used as the mutant gltA promoter sequences. They were ligated to pSFKT2 completely digested with Smal. After the completion of the ligation, the transformation was conducted with competent cells of E. coli JM109 (Takara Shuzo Co., Ltd.). The cells were spread on the L-medium plates containing 10 p g/ml of IPTG, 40 p g/ml of X-Gal and 25 p g/ml of kanamycin. After overnight incubation, white colonies were taken and the transformants were obtained after single colony isolation. From the transformants, plasmids were prepared. Temperature-sensitive shuttle vectors containing gltA gene were named pSFKTC1, pSFKTC2 and pSFKTC4, respectively.

Introduction of mutant gItA promoter into chromosome: pSFKTC1, pSFKTC2 and pSFKTC4 were each introduced into Brevibacterium lactofermentum FGR2 strain by electrical pulse method. The transformants were selected on CM2B medium plates containing 25 p g/ml of kanamycin at 25°C. After introduction of each plasmid, each obtained strain was cultured in CM2B liquid medium, spread on CM2B plates containing 25p g/ml of kanamycin, after the dilution to a concentration of 103 to 10 5 cfu per plate and cultured at 34 0 C. The strain having the temperature-sensitive plasmid became sensitive to kanamycin because the replication of the plasmid was inhibited at this temperature and, therefore, it could not form colonies. On the other hand, the strain having plasmid DNA integrated into the chromosome could be selected because it formed the colonies. Colonies thus obtained were taken and separated into respective colonies. Chromosomal DNA was extracted from the strain. PCR was conducted by using the chromosomal DNA as the template and primers of sequence shown in Seq ID No. 8 and Seq ID No. 13. About 3 kb of amplified fragments were confirmed.

It was thus proved that in this strain, mutant gItA gene derived from the temperaturesensitive plasmid was integrated near gItA gene in the host chromosome by homologous recombination. Strains derived from pSFKTC1, 2 and 4 were named BLCS11, BLCS12 and BLCS14, respectively.

Preparation of substituted gItA promoters: First, kanamycin-sensitive strains were obtained from the strains BLCS11, BLCS 12 and BLCS14 having mutant gltA gene integrated therein by homologous recombination. The strains having plasmid integrated therein were diluted and spread on CMM2B plates and then cultured at 34°C. After the formation of colonies, the replicas of the plates were made using CM2B plates containing 25 p g/ml of kanamycin and were cultured at 34°C. Thus, kanamycin-sensitized strains were obtained.

The chromosome was extracted from the kanamycin sensitive strain, and PCR was conducted with primers having the sequence shown in Seq ID No. 7 and Seq ID No.8 to prepare gItA gene fragments. The amplified fragments thus obtained were purified with SUPRECO2 (Takara shuzo Co., Ltd.) and then subjected to the sequencing reaction using a primer of Seq ID No. 13 to determine the sequence in the promoter region thereof. As a result, the strain having the same promoter sequence as that of pKF19CS1 in Table 7 was named GB01, the strain having the same promoter sequence as that of pKF19CS2 was named GB02 and the strain having the same promoter sequence as that of pKF19CS4 was named GB03. It was indicated that In these strains, the gItA gene of wild type originally located on the chromosome was excised together with the vector plasmid while the mutant gItA gene introduced by the plasmid was remained on the chromosome when the plasmid and duplicated gItA gene were excised from the chromosome.

Determination of activity of citrate synthase of mutant gltA promoter strains: The activities of the citrate synthase were determined by treating FGR2, GB01, GB02, GB03 and FGR2/pSFKC strains obtained in step in the same manner as that of step The results are shown in Table 10. It was confirmed that the citrate synthase activity of the substitited gItA promoter strain was higher than that of the parent strains thereof.

Table Strain dABS/min/ma Relative activity FGR2 7.9 GB01 9.5 1.2 GB02 15.0 1.9 GB03 31.6 FGR2/pSFKC 61.6 7.8 Results of culture of substituted gltA promoter strains: Each of the strains obtained in above-described step was inoculated into a seed culture medium having a composition shown in Table 11, and the culture was shaked at 31.5°C for 24 hours. 300 ml of a main culture medium having a composition shown in Table 11 was placed into 500 ml glass jar fermenters and then sterilized by heating and was inoculated by 40 ml of the seeds cultured as described above. The culture was started at a culture temperature of 31.5°C while the stirring rate and the aeration rate were controlled at 800 to 1300 rpm and 1/2 to 1/1 vvm, respectively. The pH of the culture liquid was kept at 7.5 with gaseous ammonia.

The temperature was shifted to 37°C 8 hours after the initiation of the culture. The culture was terminated when glucose had been completely consumed in 20 to hours, and the quantity of L-glutamic acid formed and accumulated in the culture liquid was determined.

As a result, the larger improvement in the yield of L-glutamic acid was confirmed when each of the strains GB02 and GB03 rather than GB01 and FGR2/pSFKC was used as shown in Table 12. From these facts, it was found that good results were obtained by introducing the mutation into the gttA promoter to increase the CS activity to 2 to 4 times for the improvement in the yield of glutamic acid produced by those strains.

Table 11 Concentration Seed culture Main culture Ingredient Glucose

KH

2

PO

4 MgSO 4 7H 2 0 FeSO 4 7H 2 0 MnSO 4 4H 2 0 Soybean protein hydrolyzate Biotin Thiamine hvdrochloride 50 g/ 1 g/l 0.4 g/l 10 mg/I 10 mg/l 20 mi/I 0.5 mg/I 2 ma/I 150 g/l 2 g/l 1.5 g/l 15 mg/I 15 mg/I 50 ml/I 2 mg/I 3 ma/l j 3 ma/I Table12 Strain L-glutamic acid (g/1) 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 bacterium: In this Example, strains having enhanced promoters for genes which codes glutamate dehydrogenase, citrate synthase and isocitrate dehydrogenase were produced.

Cloning of icd gene: The DNA sequence of icd gene of coryneform bacterium, which codes citrate synthase, has already been elucidated Bacteriol. 177, 774-782 (1995)]. On the 17' ST7 a 26 bases of this sequence, primers shown in Seq ID No. 14 and Seq ID No.15 were synthesized. PCR was conducted by using chromosomal DNA of Brevibacterium lactofermentum ATCC13869 as the template to amplify about 3 Kbp of DNA fragment containing icd gene and promoter thereof. The amplified fragment thus obtained was completely digested with EcoRI, and mixed with that obtained by complete digestion of pHSG399 (Takara Shuzo Co., Ltd.) with EcoRI to conduct the ligation.

After the completion of the ligation, the transformation was conducted using competent cells of E. coli JM109. The cells were spread on the L-medium plates containing 10 y g/ml of IPTG, 40 g/ml of X-Gal and 40p g/ml of chloramphenicol After overnight incubation, white colonies were taken and the transformants was obtained after single colony isolation.

The plasmid carrying icd gene was named pHSG399icd.

Introduction of mutations into icd promoter: The accurate location of the promoter of icd gene has not yet been determined. The possibility of increasing mRNA transcription level of icd gene was investigated by artificially modifying the upstream sequence of the gene which codes ICDH into a promoter-like sequence. Specifically, mutations were introduced into the like region existing in the DNA sequence about 190 bp upstream (the first promoter) and about 70 bp (the second promoter) upstream from the first ATG of ICDH protein.

Mutan-Super Express Km (Takara Shuzo Co., Ltd.) was used for the introduction of mutation into an upstream region of icd gene. The method is specifically described below. pHSG399icd was completely digested with Pstl to obtain Pstl fragment containing the promoter of icd gene. The fragments were ligated with the fragment obtained by complete digestion of pKF18kM (Takara Shuzo Co., Ltd.) with Pstl. After the completion of the ligation, the transformation was conducted with competent cells of E. coli JM109 (Takara Shuzo Co., Ltd.). The cells was spread on the L-medium containing 10 p g/ml of IPTG, 40 p g/ml of X-Gal and 'c 27

A

p g/ml of kanamycin. After overnight incubation, white colonies were taken and transformants were obtained after single colony isolation. From the transformants, plasmids were prepared, and the plasmid containing the promoter of icd gene was named pKF18icd.

PCR was conducted by using pKF18icd as the template and 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 the selection primer. These PCR products were used for transforming competent cells of E. coli JM109. The cells were spread on the L-medium plates containing 25 g/ml of kanamycin. After overnight incubation, formed colonies were taken and the transformants were obtained after single colony isolation. From the transformants, plasmid DNA was prepared, and the sequence of icd promoter region was determined using synthetic DNA shown in Seq ID No. 22 by Sanger's method Mol. Biol., 143, 161 (1980)].

Specifically, the DNA sequence was determined with Dye Terminator Sequencing Kit (Applied Biosystems), and analyzed with Genetic Analyzer ABI310 (Applied Biosystems). Those obtained by replacing icd promoter region with a sequence shown in Table 7 were named pKF181CD1, pKF181CD2, pKF181CD3, pKF181CD4, pKF181CD5 and pKF181CD6m respectively. Among them, pKF181CD2 was completely digested with Pstl to obtain Pstl fragment containing the promoter of icd gene. The fragment was ligated with the fragment obtained by complete Pstl digestion of pKF18kM (Takara Shuzo Co., Ltd.). After the completion of the ligation, the transformation was conducted with competent cells of E. coli JM109 (Takara Shuzo Co., Ltd.). The cells were spread on the L-medium plates containing 10 p g/ml of IPTG, 40 1 g/ml of X-Gal and 25 p g/ml of kanamycin. After overnight incubaton, white colonies were taken and the transformed strains were obatined after single colony isolation. From the transformed strains, plasmids were prepared, and the plasmid containing the promoter of icd gene was named pKF181CDM2. PCR was conducted using pKF181CDM2 as the template and 5'-phosphorilated synthetic DNA shown in Seq ID No. 20 and Seq ID No.21 and the selection primer. The v.i transformation of competent cells of E. coli JM109 was conducted with the PCR product. The cells were spread on the L-medium plates containing 25p g/ml of kanamycin. After overnight incubation, colonies thus formed were taken and transformants were obatined after single colony isolation. From the transformants, plasmids DNA were prepared, and the sequence of icd promoter region was determined using synthetic DNA shown in Seq ID No. 22. Those obtained by replacing icd promoter region with the sequence shown in Table 13 were named pKF181CD25 and pKF181CD26, repectively.

Table 13 Plasmid 1st Promoter 2nd Promoter -10 -35 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 pKF18ICDO6 GCGACT GAAAGT TTGACA TATAAT pKF18ICD25 TTGACA TATAAT TTGCCA TATAAT pKF18ICD26 TTGACA TATAAT TTGACA TATAAT Plasmid construction for determination of promoter activity: For easily determining the promoter activity, a possible method is the indirect determination of the promoter activity using a reporter gene. Desirable properties required of the reporter gene are that the activity can be easily determined, that even when an amino acid is added to an N-terminal, the activity is not seriously lowered, that the background reaction does not occur and that it has a restriction enzyme s 29 pusF\ 0pnc cleavage site suitable for the gene manipulation. Because 3 galactosidase (LacZ) of E. coli is widely used as a reporter gene and bacteria of the genus Corynebacterium do not have lactose assimilability Gen. Appl. Microbiol., 18, 399- 416 (1972)], LacZ was determined to be the optimum reporter gene. Then, plasmid pNEOL carrying LacZ as the reporter gene was constructed (see Fig.3). The process for the construction is described in detail below. PCR was conducted by using a chromosomal DNA obtained from E coli ME8459(ME8459 was deposited with National Institute of Genetics (Japan)) as the template with synthetic DNA shown in Seq ID No. 23 and Seq ID No.24 as the primer. The PCR product was completely digested with Smal and BamHI and then ligated with fragments obtained by digesting pKF3 (Takara Shuzo Co., Ltd.) with Hindlll and blunt-ended. After the completion of the ligation, the transformation was conducted with competent cells of E. coli JM109 (Takara Shuzo Co., Ltd.). The cells were spread on the L-medium plates containing g/ml of kanamycin. After overnight incubation, colonies thus formed were taken and separated into respective colonies to obtain the transformed strain. The plasmid obtained from the transformed strain was named pKF3nptll. Then, this plasmid was digested with Sail. On the other hand, pSAK4 described in Example 1(1) was completely digested with Smal and Sail and blunt-ended. These fragments were ligated together to construct a shuttle vector pNEO which can replicate in a coryneform bacterium. This plasmid was capable of imparting a resistance to chloramphenicol and resistance to kanamycin to the hosts. Further, pNEO was completely digested with Smal and Sse83871. The resultant fragments were ligated to those obtained by complete digestion of pMC1871 (Farmacia Biotech.) with Pstl and Smal. Thus, shuttle vector pNEOL which can be replicated in a coryneform bacterium and having LacZ lacking 8 amino acid on N-terminal as the reporter gene was constructed (see Fig.3).

Determination of activity of mutant icd promoter: Plasmids having mutant icd promoter constructed in above-described step -w I;" -i r i. e. pKF181CD1, pKF181CD2, pKF181CD3, pKF181CD4, pKF181CD5, pKF181CD6, pKF181CD25, pKF18ICD26 and pKF181CD, were completely digestede with Sacll and Pstl and then blunt-ended. They were ligated with fragment obtained by digesting pNEOL with Smal. After the completion of the ligation, the transformation was conducted with competent cells of E. coli JM109. The cells were spread on the Lmedium plates containing IPTG, X-Gal and 40 p g/ml of chloramphenicol. After overnight incubation, blue colonies were taken and the transformed strains were obtained after single colony isolation.

From the transformed strains, plasmids were prepared. Plasmids having a structure capable of producing a fused protein of ICDH and LacZ were named pNEOICD1, pNEOICD2, pNEOICD3, pNEOICD4, pNEOICDS, pNEOICD6, pNEOICD26 and pNEOLICD, respectively. Each of These plasmids or pNEOL was introduced into Brevibacterium lactofermentum ATCC13869 by electrical pulse method. The transformants were selected by using CM2B medium plates (comprising 10 g/l of bactotryptone, 10 g/l of bactoyeast extract, 5 g/l of NaCI, 10 1 g/l of biotin and 15 g/l of agar and having pH 7.0) containing 25 p g/ml of kanamycin and p g/ml of X-Gal at 31 C for two days. After the completion of the introduction, colonies thus formed were taken and isolated as single colonies. The transformants containing pNEOICD1, pNEOICD2, pNEOICD3, pNEOICD4, pNEOICD5, pNEOICD6, pNEOICD25, pNEOICD26 and pNEOLICD were named BLAC1, BLAC2, BLAC3, BLAC4, BLAC5, BLAC6, BLAC25, BLAC26, BLAC and BNEOL, respectively. All the transformants other than BNEO formed blue colonies. Crude enzyme solutions were prepared from the transformants in the same manner as that of step in Example 3 except that "Z-Buffer" (comprising 10 mM of KCI, 1 mM of MgSO 4 270 p g/100 mM of 2-ME and NaPi and having pH 7.5) was used as a washing and suspension buffer.

The activity of LacZ was determined as follows: Z-Buffer was mixed with the crude enzyme solution, ONPG in Z-Buffer having the final concentration of 0.8 mg/ml was added to the resultant mixture, and the increase in the absorbance at 420 nm at was determined with Hitachi spectrophotometer U-3210 as the activity of LacZ. The f _31 te protein concentration in the crude enzyme solution was determined by Protein Assay (BIO-RAD). Bovine serum albumin was used as the standard protein. The results are shown in Table 14. It was confirmed that the LacZ activity of the strain having a mutation in icd promoter and expressing ICDH-LacZ fused protein was higher than that expressing the wild type ICDH-LacZ fused protein.

Table 14 Strain dABS/min/mg Relative activity BNEOL Not detected 0.0 BNEOLI 42 BNEOLI-1 84 BNEOLI-2 168 BNEOLI-3 80 1.9 BNEOLI-4 126 139 3.3 BNEOLI-6 84 168 BNEOLI-26 170 Introduction of mutant icd gene into temperature-sensitive plasmid: Plasmid vector pSFKT2 (Japanese Patent Application No. 11-81693) the replication of which in a coryneform bacterium was temperature-sensitive was used.

pKF181CD1, pKF181CD2, pKF181CD3, pKF181CD4, pKF181CD5, pKF181CD6, pKFICD25 and pKFICD26 were completely digested with Pstl and the obtained fragments were used as the mutant icd promoter sequences. The fragments thus obtained were ligated with pSFKT2 completely digested with Pstl. After the completion of the ligation, the transformation was conducted with competent cells of E.

coli JM109 (Takara Shuzo Co., Ltd.). The cells were spread on the L-medium plates 32 (rn containing 10 u g/ml of IPTG, 40 p g/ml of X-Gal and 25 p g/ml of kanamycin. After overnight incubation, white colonies were taken and transformed strains were obtained after single colony isloation. From the transformed strains, plasmids were prepared. Temperature-sensitive shuttle vectors containing icd promoter were named pSFKTI1, pSFKTI2, pSFKTI3, pSFKTI4, pSFKTI5, pSFKTI6, and pSFKTI26, respectively.

Integreation of mutant icd promoter into chromosome: The plasmids constructed in above-described step were each introduced into Brevibacterium lactofermentum GB02 strain by electrical pulse method. The transformants were selected with CM2B medium plates(comprising 10 g/l of g/l of bactoyeast extract, 5 g/l of NaCI, 10 A g/l of biotin and 15 g/l of agar and having pH 7.0) containing 25 p g/ml of kanamycin at 25°C. After the completion of the introduction, the obtained strains were cultured in CM2B liquid medium, spread on CM2B plates containing 25# g/ml of kanamycin after the dilution to a concentration of 103 to 10 5 cfu per plate and cultured at 34°C. The strain having the temperature-sensitive plasmid became sensitive to kanamycin because the replication of the plasmid was inhibited at this temperature and, therefore, it could not form colonies. On the other hand, the strain having plasmid DNA integrated into the chromosome could be selected because it could form colonies. Colonies thus obtained were taken and separated into isolated colonies. Chromosomal DNA was extracted from the strain and PCR was conducted by using the chromosomal DNA as the template with primers shown in Seq ID No. 13 and Seq ID No. 15. About 3 kb of amplified fragments were confirmed. It was thus proved that in this strain, mutant icd gene derived from the temperature-sensitive plasmid was integrated near icd gene in the host chromosome by homologous recombination.

Preparation of strains having substituted icd promoter: First, kanamycin-sensitive strain was obtained from the strains having mutant icd _gene integrated therein by the homologous recombination as described in step The strains having the plasmid integrated therein were diluted and spread on CM2B plates and then cultured at 34°C. After the formation of colonies, replicas were made on CM2B plates containing 25 p g/ml of kanamycin, and they were incubated at 34°C.

Thus, kanamycin-sensitive strains were obtained.

The chromosome was extracted from the kanamycin resistant strain, and PCR was conducted using primers shown in Seq ID No.14 and Seq ID No.15 to prepare icd gene fragments. The amplified fragments thus obtained were purified with SUPRECO2 (Takara shuzo Co., Ltd.) and then subjected to the.sequencing reaction using a primer shown in Seq ID No. 22 to determine the sequence of the promoter region thereof. As a result, strains having icd promoter sequences derived from pSFKTI1, pSFKTI2, pSFKTI3, pSFKTI4, pSFKT15, pSFKTI6, pSFKTI25 and pSFKTI26 were named GC01, GC02, GC03, GC04, GC05, GC06, GC25 and GC26, respectively. In these strains, when the plasmid and duplicate icd gene were excised from the chromosome, the icd gene of wild type originally located on the chromosome was excised together with the vector plasmid, while the mutant icd gene introduced by the plasmid remained on the chromosome.

Determination of isocitrate-dehydrogenase activity of the mutant strains having mutant icd promoter: ICDH crude enzyme solution was prepared by using each of the 8 strains obtained in above-described step and GB02 strain in the same manner as that of step in Example 3. The ICDH activities were determined as follows: The crude enzyme solution was added to a reaction solution containing 35 mM of TisHCI (pH mM of MnSO 4 0.1 mM of NADP and 1.3 mM of isocitric acid, and the increase in the absorbance at 340 nm at 30°C was determined with Hitachi spectrophotometer U- 3210 as the activity of ICDH. The protein concentration in the crude enzyme solution was determined by Protein Assay (BIO-RAD). Bovine serum albumin was used as the standard protein. The results are shown in Table 15. It was confirmed that the :isocitrate dehydrogenase activity of substituted icd promoter strains was higher than that of the parent strain.

Table Strain GB02 GC01 GC02 GC03 GC04 GC06 GC26 dABS/min/mg 3.9 8.2 19.1 7.0 12.5 19.1 10.5 30.4 94 9 Relative activity 2.1 4.9 1.8 3.2 4.9 2.7 7.8 6.2 Results of culturing the strains containing substituted icd promoter: Each of the 8 strains obtained in above-described step was cultured in the same manner as that in step in Example 3. As a result, the improvement in the yield of L-glutamic acid was confirmed when any one of the strains GC02, GC04, GC25 and GC26 was used as shown in Table 16. It was found that good results were obtained by introducing the mutation into icd promoter to increase the ICDH activity to at least 3 times.

Strain GB02 GC01 GC02 Table16 L-glutamic acid (g/dl) 9.2 9.1 GC04 9.4 9.6 GC06 9.2 9.9 GC26 9.8 Example 5 Introduction of mutation into PDH gene promoter region of coryneform glutamate-producing bacterium: Cloning of pdhA gene from coryneform bacteria Primers shown in Seq ID No.25 and Seq ID No.26 were synthesized by selecting regions having a high homology among El subunits of pyruvate dehydrogenase (PDH) of Escherichia coli, Pseudomonas aeruginosa and Mycobacterium tuberculosis.

PCR was conducted by using chromosome of Brevibacterium lactofermentum ATCC13869, prepared with a bacterial genomic DNA purification kit (Advanced Genetic Technologies Corp.), as the template under standard reaction conditions described on page 8 of PCR Technology (edited by H. Erlich and published by Stockton Press, 1989). The reaction solution was subjected to the electrophoresis in an agarose gel to find that about 1.3 kilobases of DNA fragment was amplified. The sequence of both end of the obtained DNA was determined with synthetic DNA shown in Seq ID No. 25 and Seq ID No.26. The sequence was determined by Sanger's method Mol. Biol., 143, 161 (1980)] with DNA Sequencing Kit (Applied Biosystems The determined sequence was deduced to amino acids, and compared with El subunits of pyruvate dehydrogenase derived from each of Escherichia coli, Pseudomonas aeruginosa and Mycobacterium tuberculosis to find a high homology among them. Consequently, it was dtermined that the DNA fragment amplified by PCR was a part of pdhA gene which codes El subunit of pyruvate dehydrogenase of Brevibacterium lactofermentum ATCC13869. The cloning of the upstream and downstream region of the gene was conducted. The cloning method was as f fjloWy, A chromosome of Brevibacterium lactofermentum ATCC13869 was digested with restriction enzymes EcoRI, BamHI, Hind III, Pst I, Sal I and Xba I (Takara Shuzo Co., ltd.) to obtain DNA fragments. LA PCR in vitro cloning Kit (Takara Shuzo Co., Ltd.) was used for the cloning, using the sequences shown in Seq ID No. 27 and Seq ID No.28 in the Sequence Listing as primers for cloning the upstream region, and sequences shown in Seq ID No. 29 and Seq ID No.30 as primers for cloning the downstream region. After PCR using the kit, DNA fragments of about 0.5, 2.5, 3.0, 1.5 and 1.8 kilobases were amplified for the upstream region from the fragments obtained by digestion with EcoRI, Hind III, Pst I, Sal I and Xba I, respectrively; and DNA fragments of about 1.5, 3.5 and 1.0 kilobase were amplified for the downstream region from the fragements obtained by digestion with BamHI, Hind III and Pst I, respectively. The sequences of these DNA fragments were determined in the same manner as that described above. It was found that the amplified DNA fragments further contained an open reading frame of about 920 amino acids and also that a region supposed to be a promoter region was present in the upstream region. Because the deduced amino acid sequence from the DNA sequence of the open reading frame is highly homologous to known El subunit of pyruvate dehydrogenase such as that of E. coli, it was apparent that the open reading frame was the pdhA gene which codes El subunit of pyruvate dehydrogenase of Brevibacterium lactofermentum ATCC13869. The DNA sequence of the open reading frame was shown in Seq ID No. 31 in the Sequence Listing. In Seq ID No.

31 in the Sequence Listing, deduced amino acid sequence from the DNA sequence is also shown. Since methionine residue at N-terminal of the protein is derived from ATG which is an initiation codon, it usually does not concern the essential function of protein, and it is well known that the methionine residue is removed by the effect of peptidase after the translation. Therefore, in the above-described protein, it is possible that methionine residue at the N-terminal has been removed. However, the GTG sequence is present in 6 bases upstream of ATG shown in Seq ID No. 31 in the Sequence Listing, and it is also possible that amino acids is translated from this point.

,.Pyruvate dehydrogenase of other microorganisms such as E. coli are composed of S37 %n V three subunits of El, E2 and E3, and genes which encode them constitute an operon in many cases. However, there was no open reading frame considered to be E2 and E3 subunit of pyruvate dehydrogenase in about 3 kilobases downstream of pdhA gene. Instead, it was shown that a sequence supposed to be a terminator was present in the downstream of the open reading frame. From these facts, it was supposed that E2 and E3 subunits of pyruvate dehydrogenase of Brevibacterium lactofermentum ATCC13869 were present in another regon on the chromosome.

Construction of a plasmid for amplifying pdhA: It was already apparent that a strain obtained by introducing a gene which codes three subunits constituting PDH of E. coli into Brevibacterium lactofermentum ATCC13869 gives an improved glutamic acid yield (JP No.10-360619). However, in PDH of Brevibacterium lactofermentum ATCC13869, only pdhA gene which codes El subunit had been cloned, and no examination had not been made to know whether the amplification of the gene alone is effective in improving the yield of glutamic acid.

Under these circumstances, examination was made to know whether the amplification of pdhA gene alone is effective in improving the yield of glutamic acid or not.

Primers shown in Seq ID No. 33 and Seq ID No.34 were synthesized on the basis of the previously cloned DNA sequences. PCR was conducted by using chromosome of Brevibacterium lactofermentum ATCC13869, prepared with a Bacterial Genomic DNA Purification kit (Advanced Genetic Technologies Corp.), as the template under standard reaction conditions described on page 8 of PCR Technology (edited by H. Erlich and published by Stockton Press, 1989) to amplify pdhA gene. Among the primers thus synthesized, Seq ID No. 33 corresponded to a sequence of base No. 1397 to No.1416 in pdhA gene described in Seq ID No. 32 in the Sequence Listing. Seq ID No. 34 was the complementary strand of the DNA sequence corresponding to the sequence of base No. 5355 to No.5374 in Seq ID No.

32 in the Sequence Listing, which was represented from the 5' side.

PCR product thus obtained was purified by an ordinary method and reacted with restriction enzyme Sal I and EcoT221. The fragment was ligated with pSFK(Patent Application No.11-69896 cleaved with restriction enzymes Sal I and Pst I, with a ligation kit (Takara Shuzo Co., Ltd.). After the transformation with competent cells (Takara Shuzo Co., Ltd.) of E. coli JM109, the cells were spread to the L-medium medium plates(comprising 10 g/l of bactotryptone, 5 g/l of bactoyeast extract, 5 g/l of NaCI and 15 g/l of agar and having pH 7.2) containing 101 g/ml of IPTG (isopropyl- -D-thiogalactopyranoside), 40 p g/ml of X-Gal (5-bromo-4-chloro-3indolyl- 3 -D-galactoside) and 25 g g/ml of kanamycin. After overnight incubation, white colonies were taken and the transformed strains were obtained after single colony isolation.

From the transformned strains, plasmids were prepared by alkali method (Seibutsu Kogaku Jikken-sho edited by Nippon Seibutsu Kogaku kai and published by Baifukan, p. 105, 1992). Restriction enzyme maps of DNA fragments inserted into the vectors were prepared and compared with the restriction enzyme map of pdhA gene reported in sequence No. 32 of the Sequence Listing. A plasmid containing DNA fragments inserted therein having the same restriction enzyme map as that of pdhA gene was named pSFKBPDHA.

Introduction of pASFKBPDHA into Brevibacterium lactofermentum ATCC13869 and GC25 and evaluation of the fermentation experiments: Brevibacterium lactofermentum ATCC13869 and GC25 were transformed with plasmid pSFKBPDHA by electrical pulse method P. KOKAI No.2-207791) to obtain the transformed strains. The culture for producing L-glutamic acid was conducted with transformed strain ATCC13869/pSFKBPDHA and GC25/pSFKBPDHA obtained by introducing plasmid pSFKBPDHA into Brevibacterium lactofermentum ATCC13869 and GC25 as follows: Cells of ATCC13869/pSFKBPDHA and obtained by the culture on CM2B medium plates containing 25 p g/ml of kanamycin were inoculated into a medium (comprising 1 liter of pure water containing 80 g of glucose, 1 g of KH 2

PO

4 0.4 g of MgSO 4 7H20, 30 g of (NH 4 2 SO4, 0.01 g of FeSO 4 7H 2 0, 0.01 g of MnSO 7H 2 0, 15 ml of soybean protein hydrolyzate, 200 p g of thiamine hydrochloride, 60 L g of biotin, 25 mg of kanamycin and 50 g of CaCO 3 and having a pH adjusted to 8.0 with KOH). Then the culture was shaked at 31.5°C until sugar in the medium had been consumed. The obtained products were inoculated into the medium of the same composition as that described above (for GC25/pSFK6 and GC25/pSFKBDHA) or the medium eliminated Biotin from the composition as that described above(for ATCC13869/pSFK6 and ATCC13869/pSFKBPDHA) in an amount of 5 and the shaking culture was conducted at 37°C until sugar in the medium had been consumed. As a control, strains obtained by transforming Brevibacterium lactofermentum ATCC13869 and GC25 with previously obtained palsmid pSFK6 capable of autonomously replicating in coryneform bacterium by electrical pulse method P. KOKAI No. 2-207791), were cultured in the same manner as that described above. After the completion of the culture, the amount of L-glutamic acid accumulated in the culture medium was determined with Biotic Analyzer AS-210 (a product of Asahi Chemical Industry Co., Ltd.). The results are shown in Table 17.

Table 17 Strain Yield of L-glutamic acid (g/dl) ATCC13869/Psfk 3.6 ATCC13869/pSFKBPDHA 3.8 6 5.1 5.3 From these results, it was apparent that even the amplification of pdhA gene alone is sufficiently effective in improving the yield of Glu in Brevibacterium lactofermentum ATCC13869 and Construction of plasmids for determination of the activity of mutated pdhA promoter: To produce promoter mutant of pyruvate dehydrogenase (PDH), the determination of the previously cloned promoter region of pdhA gene of Brevibacterium lactofermentum ATCC13869 was conducted and also the determination of difference in the expression caused by the modification of the promoter region were conducted by determining the activity of -galactosidase.

The promoter region of pdhA gene was presumed from the DNA sequence which had been already elucidated by cloning. As a result, it was supposed to be possible that base No. 2252 to No.2257 and No. 2279 to No. 2284 in Seq ID No. 32 in the Sequence Listing were -35 region and -10 region, respectively. Therefore, primers shown as Seq ID No. 35 and Seq ID No.36 in the Sequence Listing were synthesized, and DNA fragments containing promoter region of pdhA gene were amplified by PCR method by using chromosomal DNA of Brevibacterium lactofermentum ATCC13869 as a template. Among the synthesized primers, Seq ID No. 35 corresponded to the sequence ranging from base No. 2194 to base No. 2221 in Seq ID No. 32; but the base No. 2198 had been replaced with C, and the base No.

2200 and No.2202 had been replaced with G, and recognition sequence for restriction enzyme Smal had been inserted. Seq ID No. 36 corresponded to the sequence ranging from base No. 2372 to base No. 2398 in Seq ID No. 32; but base No.2393 and No.2394 had been replaced with G, and the complementary strand of the DNA sequence having a recognition sequence of restriction enzyme Smal inserted therein was represented from the 5'-end. PCR was conducted by using chromosome of Brevibacterium lactofermentum ATCC13869, prepared with Bacterial Genomic DNA Purification kit (Advanced Genetic Technologies Corp.), as the template under standard reaction conditions described on page 8 of PCR Technology (edited by H.

Erlich and published by Stockton Press, 1989) to amplify the promoter region of pdhA gene. PCR product thus obtained was purified by an ordinary method and reacted with restriction enzyme Sma I. The fragments were ligated with pNEOL lacking in i, 41

SOF

promoter region of lacZ gene which could be replicate in a coryneform bacterium and which had been digested with restriction enzymes Sma I, (Example 4 with a Ligation Kit (Takara Shuzo Co., Ltd.). After the transformation with competent cells (Takara Shuzo Co., Ltd.) of E. coli JM109, the cells were spread on the L-medium plates(comprising 10,g/ of bactotryptone, 5 g/l of bactoyeast extract, 5 g/l of NaCI and g/l of agar and having pH 7.2) containing 40 p g/ml of X-Gal (5-bromo-4-chloro-3indolyl- f-D-galactoside) and 25 pg/ml of kanamycin. After overnight incubation, blue colonies were taken and the transformed strains were obtained after single colony isolation. From the transformants, plasmids were prepared by alkali method (Seibutsu Kogaku Jikken-sho edited by Nippon Seibutsu Kogaku-kai and published by Baifukan, p. 105, 1992). After sequencing DNA fragments inserted into the vector by an ordinary method, the plasmid containing the DNA fragment inserted therein was named pNEOLBPDHAprol.

Further, primers indicated as Seq ID No. 37, Seq ID No.38 and Seq ID NO.39 in the Sequence Listing were synthesized for constructing plasmids wherein a region supposed to be the promoter site was changed to the consensus sequence of promoters of coryneform bacteria. By using each of the primers and a primer shonw in Seq ID No. 36, DNA fragments wherein the promoter region of pdhA gene was changed to the consensus sequence were amplified by PCR method by using chromosomal DNA of Brevibacterium lactofermentum ATCC13869 as a template.

Among the synthesized primers, Seq ID No. 37 corresponded to the sequence ranging from base No. 2244 to base No. 2273 in Seq ID No. 32; base No. 2255 had been replaced with C, and base No. 2257 had been replaced with A; thus only region had been changed to the consensus sequence of the coryneform bacteria.

Seq ID No. 38 corresponded to the sequence ranging from base No. 2249 to base No.

2288 in sequence No. 32; base No. 2279 and No.2281 had been replaced with T; thus only -10 region had been changed to the consensus sequence of the coryneform bacteria. Sequence No. 39 corresponded to the sequence ranging from base No.

2249 to base No. 2288 in Seq ID No. 32; base No. 2255 had been replaced with C, 42

OFV\

base No. 2257 had been replaced with A, and base No. 2279 and No.2281 had been replaced with T; thus both -35 region and -10 region had been changed to the consensus sequence of the coryneform bacteria. PCR was conducted by using chromosome of Brevibacterium lactofermentum ATCC13869, prepared with a Bacterial Genomic DNA Purification Kit (Advanced Genetic Technologies Corp.), as the template under standard reaction conditions described on page 8 of PCR Technology (edited by H. Erlich and published by Stockton Press, 1989) to amplify the promoter region of pdhA gene with these primers so that the promoter region was changed to the consensus sequence. PCR products thus obtained were purified by an ordinary method and reacted with restriction enzyme Smal. The fragments were ligated with pNEOL lacking the promoter region of lacZ gene, which could replicate in a coryneform bacterium and which had been cleavaged with restriction enzymes Sma I, with a Ligation Kit (Takara Shuzo Co., Ltd.). After the transformation with competent cells (Takara Shuzo Co., Ltd.) of E. coli JM109, the cells were spread on the L-medium plates (comprising 10 g/l of bactotryptone, 5 g/ of bactoyeast extract, g/ of NaCI and 15 g/l of agar .and having pH 7.2) containing 40 g/ml of X-Gal bromo-4-chloro-3-indolyl- 3 -D-galactoside) and 25 p g/ml- of kanamycin. After overnight incubation, blue colonies were taken and the transformed strains were obatined after single colony isolation. From the transformed strains, plasmids were prepared by the alkali method (Seibutsu Kogaku Jikken-sho edited by Nippon Seibutsu Kogaku kai and published by Baifukan, p. 105, 1992). After sequencing DNA fragments inserted into the vector by an ordinary method, the plasmid containing DNA fragments, wherein only the sequence in -35 region had been changed to the consensus sequence, inserted therein was named pNEOLBPDHApro35; the plasmid containing DNA fragments, wherein only the sequence in -10 region had been changed to the consensus sequence, was inserted therein was named pNEOLBPDHAprolO; and the plasmid containing DNA fragments, wherein the sequences in both -35 region and -10 region had been changed to the consensus ,,equence, was inserted therein was named pNEOLBPDHApro3510.

_43 0 O F The elavuation of the mutated pdhA promoter activity: Brevibacterium lactofermentum ATCC13869 was transformed with plasmids named pNEOLBPDHAprol, pNEOLBPDHAprolO and pNEOLBPDHApro3510 by electrical pulse method P. KOKAI No. 2-207791) to obtain the transformed strains.

3 -galactosidase activity of the obtained transformants was determined by the method described in Example After changing the sequence in the promoter region to the consensus sequence, B -galactosidase activities were as shown in Table 18, wherein the enzymatic activity of' -galactosidase having the promoter region of pdhA gene was given as 1.

Table 18 Strain B -Galactosidase activity (relative value) ATCC13869/pNEOLBPDHAprol 1 ATCC13869/pNEOLBPDHAprolO 6 ATCC13869/pNEOLBPDHApro3510 These results indicate that the supposed promoter region was the promoter of pdhA gene and that the expression of PdhA can be changed (enhanced) by changing the sequence in this region into the consensus sequence. This fact indicates that the expression can be changed, without using plasmid, by changing the promoter region of pdhA gene.

Construction of plasmid for preparation of promoter varied strain: Since it had been proved that the expression of pdhA can be changed by introducing mutations into the promoter, plasmids for preparing a pdhA promoter modified strains were constructed. Three constructs of the plasmids for the promoter modified strains were constructed. They were plasmids wherein -35 region, region and both of them were changed to the consensus sequence, respectively.

Primers shown in Seq ID No. 40 and Seq ID No.41 were newly synthesized on the basis of the DNA sequence which had already been cloned. Among synthesized primers, Seq ID No. 40 was the complementary strand of the DNA sequence corresponding to a sequence ranging from base No. 2491 to base No. 2521 in Seq ID No. 32, which was represented from the 5'-end, and to which a sequence comprising three A's followed by four T's at the 5' terminal. Seq ID No. 33 was the complementary strand of the DNA sequence corresponding to the sequence ranging from base No. 5020 to base No. 5039 of pdhA gene in Seq ID No. 32, which was represented from the 5'-end. PCR was conducted by using Seq ID No. 33 and Seq ID No.40 as the primers and chromosome of Brevibacterium lactofermentum ATCC13869, prepared with Bacterial Genomic DNA Purification Kit (Advanced Genetic Technologies Corp.), as a template under standard reaction conditions described on page 8 of PCR Technology (edited by H. Erlich and published by Stockton Press. 1989). Further, PCR was conducted by using Seq ID No. 39 and Seq ID No. 41 and chromosome of Brevibacterium lactofermentum ATCC13869 as a template. The PCR products thus obtained were purified by an ordinary method.

PCR was conducted by using PCR products obtained by using Seq ID No. 33 and PCR products obtained by using Seq ID No. 39 and Seq ID No.41 and Seq ID No. 33 and 41 as the primers. The PCR condition was as follows: The concentration of these four DNA would be 10 u M in the reaction cocktail and La taq (Takara Shuzo Co., Ltd.) was used without template. PCR products were purified by an ordinary method, and reacted with restriction enzyme Sal I and Xho I. The fragments thus obtained were ligated with fragments obtained by digesting temperature-sensitive plasmid pSFKT2 with Sail, which can replicate in a coryneform bacterium, by using Ligation Kit (Takara Shuzo Co., Ltd.). After the transformation with competent cells (Takara Shuzo Co., Ltd.) of E. coli JM109, the cells was spread on the L-medium plates(comprising 10 g/l of bactotryptone, 5 g/l of bactoyeast extract, g/l of NaCI and 15 g/l of agar and having pH 7.2) containing 10 p1 g/ml of IPTG y^ (>is,propyl- 0 -D-thiogalactopyranoside), 40 p g/ml of X-Gal (5-bromo-4-chloro-3s b( indolyl- 1 -D-galactoside) and 25 p g/ml of kanamycin. After overnight incubation, white colonies were taken and transformants were obtained after single colon isolation. From the transformants, plasmids were prepared by the alkali method (Seibutsu Kogaku Jikken-sho edited by Nippon Seibutsu Kogaku-kai and published by Baifukan, p. 105, 1992). After sequencing DNA fragments inserted into the vector, the base sequence was compared with that of pdhA gene reported in sequence No.

32. The plasmid containing DNA fragments, wherein only the sequences in region and -10 region of the promoter were changed to the consensus sequence of the coryneform bacteria, inserted therein was named pSFKTPDHApro3510.

A plasmid wherein -35 region of the promoter of pdhA gene had been changed to the consensus sequence of coryneform bacteria, and also plasmid wherein region of the promoter of pdhA gene had been changed to the consensus sequence of coryneform bacteria were constructed in the same manner as that described above except that Seq ID No. 39 in the Sequence Listing was replaced with Seq ID No. 37 and 38, respectively. These plasmids were named pSFKTPDHApro35 and pSFKTPDHAprolO, respectively.

Preparation of promoter modified strains: Strains having modified pdhA gene promoter were prepared by the homologous recombination by using the plasmid for preparing promoter varied strain constructed in the above-described step First, GC25 was transformed with plasmid pSFKTPDHApro3510 for preparing promoter modified strain by electrical pulse method (refer to J. P. KOKAI No. 2- 207791). The cells were spread on CM2B mediu plates (comprising 10 g/l of polypeptone, 10 g/l of bactoyeast extract, 5 g/l of NaCI, 10 p g/ml of biotin and 15 g/l of agar, and having pH 7.2) and cultured at 25°C to obtain transformed strains. These transformants were cultured in CM2B liquid medium in a test tube overnight and then spread on CM2B medium plates containing 25 g /ml of kanamycin and cultured at 34°C to obtain a a strain caused by once-recombination which contains plasmid 46 1 pSFKTPDHpro3510 on its chromosome inserted by the homologous recombination.

After single colony isolation, this strain was cultured in CM2B liquid medium in a test tube overnight. After the suitable dilution, it was spread on CM2B medium plates and cultured at 31.5 0 C. After the colonies began to appear, the repllicas were made on CM2B medium plates containing 25p g/ml of kanamycin to obtain kanamycinsensitive strains. Since two kinds of the strains, i.e. a strain having the sequence of wild type strain for the promoter region of pdhA gene and another strain having the mutation introduced therein, could be occured, this region was sequenced. Thus, a promoter modified strain, wherein the mutation had been introduced into the promoter region of pdhA gene, was obtained. In this strain, -35 region and -10 region of promoter of pdhA gene had been changed to the consensus sequence of coryneform bacteria. This strain was named GD3510.

Strains wherein -35 region or -10 region of promoter of pdhA gene had been changed to the consensus sequence of coryneform bacteria were obtained in the same manner as that described above except that above described plasmid pSFKTPDHApro3510 for producing the promoter modified strain was replaced with plasmid pSFKTPDHApro35 and pSFKTPDHAprolO for producing promoter modified strains and they were named GD35 and GD10, respectively.

Evaluation of the results of flask culture of pdhA gene promoter modified strains: The flask culture for producing L-glutamic acid was conducted with three kinds of pdhA gene promoter modified strains obtained as described above. Each of the cells of the promoter modified strains GD3510, GD35, GD10 and GC25 obtained by the culture on CM2B medium plates was inoculated into a medium (comprising 1 liter of pure water containing 30 g of glucose, 1 g of KH 2 PO4, 0.4 g of MgSO 4 7H 2 0, 30 g of

(NH

4 2

SO

4 0.01 g of FeSO 4 7H 2 0, 0.01 g of MnSO 4 7H 2 0, 15 ml of soybean hydrolyzate, 200 g g of thiamine hydrochloride, 60 1 g of biotin and 50 g of CaCO 3; and having a pH adjusted to 8.0 with KOH). Then the culture was shaked at 31.5 0

C

until the sugar in the medium had been consumed. The obtained products were inoculated into the medium of the same composition as that described above in .an amount of and the shaking culture was conducted at 37°C until sugar in the medium had been consumed. After the completion of the culture, the amount of Lglutamic acid accumulated in the culture liquid was determined with Biotic Analyzer AS-210 (a product of Asahi Chemical Industry Co., Ltd.). The results are shown in Table 19.

Table 19 Strain L-glutamic acid (g/dl) 1.9 GD3510 2.1 It was apparent from the results that the obtained promoter modified strains provided improved Glu yields.

Example 6 Introduction of mutation into promoter region of arginosuccinate synthase gene: 1) Determination of DNA sequence in the upstream of argG gene: In order to amplify argG gene of Brevibacterium flavum by PCR, the DNA sequences in the upstream and downstream regions of the ORF were determined.

The determination of the DNA sequences was conducted by synthesizing a primer based on the known DNA sequence (Gen Bank accession AF030520) of ORF of argG gene of Corynebacterium glutamicom and using in vitro LA PCR cloning kit (Takara shuzo Co., Ltd.) in accordance with the instruction manual included in the kit. As primers, they were specifically used oligonucleotide (primers 1 and 2) having the DNA 48 sequences set out as Seq ID No. 42 and Seq ID No.43 for the upstream region, and oligonucleotide (primers 3 and 4) having the DNA sequences set out as Seq ID No.44 and Seq ID No.45 for the downstream region. The DNA sequences in the upstream and downstream region of argG were determined by completely digesting chromosome DNA of 2247 strain (ATCC14067), wild type strain of Brevibacterium flavum, with a restriction enzyme EcoRI, conducting first PCR with the primer 2 or 3 (having sequence No.43 or 44), and conducting second PCR with the primer 1 or 2 (having sequence No. 42 or 2) Prediction of promoter region: A promoter-like sequence in the upstream of ORF of argG gene was search for the above-described sequences with a commercially available software (GENETYX).

The mutation was introduced into a region of the highest score (about 120 bp upstream of the first ATG). Then, the promoter activity was measured.

3) Introduction of mutations into promoter sequence, and determination of activity of mutant promoters: Mutation-introducing primers 9, 10, 11, 12 or 13 and 7 (having sequence No.

51, 52, 53, 54 or 48,respectively) for a region of the highest score were used, and the first PCR was conducted with chromosomal DNA of AJ12092 strain as a template. The second PCR was conducted with the same chromosomal DNA as the template by using the PCR product as the primer for 3'-end and also using the primer 8 having sequence No. 49 as the primer on 5'-end to obtain DNA fragments having the mutation introduced in the intended promoter region. To determine the activity of the mutant promoters, these DNA fragments were inserted into Smal site of promoter probe vector pNEOL so that they were in the same direction with lacZ reporter gene to obtain plasmids pNEOL-1, pNEOL-2, pNEOL-3, pNEOL-4 and pNEOL-7. As a control for the activity, plasmid pNEOL-0 was constructed by inserting the DNA ,_fragment, obtained by PCR using chromosomal DNA of AJ12092 strain and primers 7 and 8, into the upstream of lacZ gene of pNEOL.

pNEOL-0, pNEOL-1, pNEOL-2, pNEOL-3, pNEOL-4 and pNEOL-7 were introduced into AJ12092 strain,respectivly. The plasmids were introduced by electrical pulse method P. KOKAI No. 2-207791). The transformants were selected on CM2G medium plates(comprising 1 liter of pure water containing 10 g of polypeptone, 10 g of yeast extract, 5 g of glucose, 5 g of NaCI and 15 g of agar, and having pH 7.2) containing 4 j g/ml of chloramphenicol, as chloramphenicol-resistant strains.

These strains were each spread 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 NaCI and 5 p g/l of chloramphenicol), and cultured at 31.5°C for 20 hours. One aze of the cells thus obtained was inoculated into a medium [containing 3 g/dl of glucose, 1.5 g/dl of ammonium sulfate, 0.1 g/dl of KH 2

PO

4 0.04 g/dl of MgSO 4 0.001 g/dl of FeSO 4 0.01 g/dl of MnSO 4 5 p g/dl of VB 1 5 g/dl of biotin and 45 mg/dl (in terms of N) of soybean hydrolyzate]. After the culture at 31.5 0 C for 18 hours, 3 -galactosidase activity of the obtained cells was determined as described in Example 4(4).

Since /-galactosidase activity was detected in AJ12092/pNEOL-0 as shown in Table 20, it was found that the DNA fragment inserted into the upstream of the gene of lacZ structure functioned as a promoter. In addition, 3 -galactosidase activity of each of the plasmid-introduced strains was higher than that of AJ12092/pNEOL-0. It was thus found that the transcription activity was increased by the introduction of the mutation into the promoter-like sequence, as shown in Table Table Relative activity (AJ12092/pNEOL-0=1) AJ112092 nd AJI12092/pNEOL-0 AJ112092/pNEOL-1 2.8 AJ112092/pNEOL-2 2.7 .fT^ AJI12092/pNEOL-3 1.8 AJ112092/pNEOL-4 0.8 AJ112092/pNEOL-7 4) Construction of a plasmid for introduction of mutation: PCR was conducted by using primers 14 and 15 (having the sequence of Seq ID No. 55 and Seq ID No.56) with chromosomal DNA of AJ12092 strain as the template. These DNA fragments thus obtained were inserted into a smal site in a multicloning site of cloning vector pHSG398 (a product of TaKaRa) to construct plasmid pO. Then, p0 was digested with restriction enzymes EcoRV and BspHI, and also pNEOL-3 and pNEOL-7 were digested with restriction enzymes EcoRV and BspHI. DNA fragments thus obtained were ligated to obtain mutation-introducing plasmids p3 (mutant derived from mutation-introducing primer 11) and p7 (mutant derived from mutation-introducing primer 13).

5) Introduction of mutation-introducing plasmids into Arg-producing bacterium: Each of the plasmids thus obtained was introduced into Arg-producing bacterium of the strain Brevibacterium lactofermentum AJ12092 by electrical pulse method P, KOKAI No. 2-207791). Since these plasmids could not autonomously replicate in Brevibacterium, only the strains obtained by integrating these plasmids into the chromosome by homologous recombination could be selected as Cm-resistant strains. Strains in which the mutation-introducing plasmid was integrated into the chromosome were selected as chloramphenicol-resistant strains on CM2G medium plates (comprising 1 liter of pure water containing 10 g of polypeptone, 10 g of yeast extract, 5 g of glucose, 5 g of NaCI and 15 g of agar, and having pH 7.2) containing 5 p g/ml of chloramphenicol. Then, Cm-sensitive strains were selected in which the promoter region of argG gene was replaced with the intended modified sequence.

As a result, a strain substituted with P3 sequence (AJ12092-P3) and a strain substituted with P7 sequence (AJ12092-P7) were obtained.

I.

Cloning of argG gene Based on the DNA sequence determined as in oligonucleotides (primers and 6) having the DNA sequence set out in Seq ID No. 46 and Seq ID No.47 were synthesized to conduct PCR using chromosomal DNA of Brevibacterium flavum as a template. The PCR reaction was conducted in 25 cycles, each cycle consisting of 94°C for 30 seconds, 55°C for one second and 72°C for 2 minutes and 30 seconds.

The thus-obtained DNA fragment was cloned to Smal site in multi-cloning site of cloning vector pSTV29 (Takara shuzo Co. Ltd.) toobtain pSTVargG. Furthermore, pargG was prepared by inserting into Sail site of pSTVargG a fragment containing the replication origin obtained by treating pSAK4 set out in Example 1 with Sail.

7) Introduction of pargG into Brev.: pargG was introduced into the strain Brevibacterium lactofernentum AJ12092.

Plasmid was introduced by electrical pulse method P, KOKAI No. 2-207791). The transformant was selected as chloramphenicol-resistant strain on CM2G medium plates(comprising 1 liter of pure water containing 10 g of polypeptone, 10 g of yeast extract, 5 g of glucose, 5 g of NaCI and 15 g of agar, and having pH 7.2) containing 4 p g/ml of chloramphenicol.

8) ArgG activity of promoter modified strains: ArgG activities of the above-described two kinds of argG promoter modified strains and a strain obtained by amplifying argG with plasmid (AJ12092/pargG) were determined. These strains were each spread on a 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 NaCI and 5 p g/l of chloramphenicol), and cultured at 31.5°C for 20 hours. One aze of the cells thus obtained were inoculated into a medium [containing 3 g/dl of glucose, 1.5 g/dl of ammonium sulfate, 0.1 g/dl of KH 2

PO

4 0.04 g/dl of MgSO 4 0.001 g/dl of FeSO 4 0.01 g/dl of MnSO 4 5 p g/dl of VB,, 51 g/dl of biotin and 45 mg/dl (in terms of N) of soybean hydrolyzate]. After the culture at 31.5°C for 18 hours, ArgG activity of the J^\S Si obtained cells was determined by the method described above [Journal of General Microbiology (1990), 136, 1177-1183]. ArgG activities of the above-described two kinds of ArgG promoter modified strains and the strain (AJ12092/pargG) obtained by amplifying argG with plasmid are shown in Table 21. It is apparent from Table 21 that by introducing the mutation into the promoter, ArgG activity of AJ12092-P3 was increased to about twice as high as that of the parent strain, and the activity of AJ12092-P7 was increased to about three times as high as that of the parent strain.

ArgG activity of AJ12092/pargG was about 4.5 times as high as that of the parent strain.

Table 21 Relative activity (AJ12092=1) AJ112092 AJI12092-P3 2.1 AJI12092-P7 2.9 AJI12092/pargG 4.4 9) Arg production by promoter modified strains: The flask culture of each of argG promoter modified strains was conducted. As controls, parent strain AJ12092 and AJ12092/pargG were also cultured. These strains were each inoculated into a medium [containing 0.1 g/dl of KH 2

PO

4 0.04 g/dl of MgSO 4 0.001 g/dl of FeSO 4 0.01 g/dl of MnSO 4 5 p g/dl of VB,, 5 g g/dl of biotin and 45 mg/dl (in terms of N) of soybean hydrolyzate]; and then spread 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 NaCI and 5 p g/l of chloramphenicol), and cultured at 31.5°C for 20 hours.

One aze of the cells were cultured in a flask containing 4 g/dl of glucose and 6.5 g/dl of ammonium sulfate at 31.5°C until glucose had been completely consumed. The absorbance (CD620) of the culture liquid diluted to a concentration of 1/51 with 0.2 N HCI solution, the quantity of arginine produced (concentration: g/dl) and culture time

I

were shown in Table 22.

It is apparent from Table 22 that when argG promoter modified strain was used, the yield of arg was increased to a level equal to that of argG amplified with plasmid. As for the promoter varied strains, both AJ12092-P3 and AJ12092-P7 had the culture time equal to that of the parent strain, while the culture time of the plasmid amplified strain was increased. It was thus apparent that Arg productivity thereof was higher than that of the plasmid amplified strain.

Table 22 OD Arg (g/dl) Culture time Productivity (g/dl/h) AJ12092 0.502 1.25 48 0.026 AJ12092-P3 0.510 1.47 48 0.031 AJ12092-P7 0.514 1.43 48 0.030 AJI2092/pargG 0.520 1.47 52 0.028 Example 7 Introduction of mutation into GDH gene promoter region of coryneform glutamate-producing bacterium: Construction of mutant gdh plasmids: Plasmids having GDH promoter sequence of FGR1 strain and FGR2 strain described in Example 2 were constructed by site directed mutagenesis. For obtaining GDH promoter sequence of FGR1 strain, PCR was conducted by using synthetic DNA shown in Seq ID No. 57 and synthetic DNA shown in No. 60 as the primers and chromosomal DNA of ATCC13869 as the template; and on the other hand, PCR was conducted by using synthetic DNA shown in Seq ID No. 58 and synthetic DNA shown in Seq ID No. 59 as the primers with chromosomal DNA of ATCC13869 as the template. Further, PCR was conducted by using synthetic DNAs shown in Seq ID Nos. 57 and Seq ID No.58 as the primers with a mixture of these 3 PCR products as the template. The PCR product thus obtained was inserted into Smal site of pSFKT2 (Japanese Patent Application No. 11-69896) to construct pSFKTG11. To obtain GDH promoter sequence of FGR2 strain, PCR was conducted by using synthetic DNA shown in Seq ID No. 57 and synthetic DNA shown in Seq ID No. 62 as the primers and chromosomal DNA of ATCC13869 as the template; and on the other hand, PCR was conducted by using synthetic DNA shown in Seq ID No. 58 and synthetic DNA shown in Seq ID No. 61 as the primers and chromosomal DNA of ATCC13869 as the template. Further, PCR was conducted by using synthetic DNA shown in Seq ID No. 57 and Seq ID No.58 as the primers and a mixture of these PCR products as the template. The PCR product thus obtained was inserted into Smal site of pSFKT2 (Japanese Patent Application No. 11-69896) to construct pSFKTG07. The DNA sequences of the fragments inserted into Smal sites of pSFKTG11 and pSFKTG07 were determined to confirm that no mutation was introduced into other refions than the promoter region in GDH.

Construction of gdh promoter modified strains: Then, pSFKTG11 and pSFKTG07 were introduced into AJ13029 strain by electrical pulse method, and transformants which grew on CM2B plates containing 25 p g/ml of kanamycin at 25°C were selected. The transformants were cultured at 34°C to select strains which were resistant to kanamycin at 34°C. The fact that a strain is resistant to kanamycin at 34°C indicates that pSFKTG11 or pSFKTG07 was thus integrated on the chromosome of AJ13029 strain. Kanamycin-sensitive strains were obtained from the strains in which the plasmid was integrated on the chromosome.

The GDH promoter sequences of these strains were determined. The strains having the same gdh promoter sequence as those of pSFKTG11 and pSFKTG07 were named GA01 and GA02, respectively.

Confirmation of L-glutamic acid-productivity of gdh promoter modified strains: The glutamic acid productivities of strains GAO1 and GA02 and the parent strain AJ13029 were confirmed in the same manner as that of Example 2 given above.

As a result, a remarkable improvement in the accumulation of glutamic acid was recognized in GA01 and GA02 as shown in Table 23.

Table 23 Strain Glu (g/dl) Specific activity of GDH Relative value AJ13029 2.6 7.7 GA01 3.0 22.3 2.9 GA02 2.9 27.0 Construction of self-cloning type gdh plasmid: First, self-cloning vector pAJ220 was constructed. pAJ226 P. KOKAI No.61- 152289) was treated with EcoRV and Pstl to prepare a fragment containing a region which could be autonomously replicated in a coryneform bacterium. The fragment was ligated with about 0.7 kb of the DNA fragment obtained by treating pAJ224 P.

KOKAI No. Sho 61-152289) with EcoRV and Pstl to obtain a plasmid pAJ220. This plasmid could autonomously replicate in a coryneform bacterium, and it could afford trimethoprim resistance to the host.

PCR reaction was conducted by using synthetic DNA shown in Seq ID No. 63 and Seq ID No.64 as the primers and chromosomal DNA of wild-type coryneform bacterium strain ATCC13869 as the template. The gdh gene fragment thus obtained was inserted in Ball site of pAJ220 to construct pAJ220G. The promoter was present near Ball site of pAJ220, and the expression of the inserted gene was increased depending on the direction of the gene inserted into Ball site. PAJ220G and pGDH were introduced into ATCC13869 strain by electrical pulse method.

GDH activities of the strains thus constructed were determined by the method stated in above-described step As a result, GDH activity of the strain into which pAJ220G had been introduced was about 1.5 times as high as that of the strain into which dGDH had been introduced as shown in Table 24.

56 -3 4

I

Table 24 Strain Specific activity of GDH Relative value ATCC13869 7.7 ATCC13869/pGDH 82.7 10.7 ATCC13869/pAJ220G 120.1 15.6 Investigations on influence of gdh activity on the yield and by-produced Asp: pGDH and pAJ220G were introduced into AJ13029 by electrical pulse method.

Each of these strains and those obtained in above-described step was inoculated into a seed culture medium having a composition shown in Table 25, and the shaking culture was conducted at 31.50C for 24 hours to obtain the seed culture. 300 ml of medium for main culture having a composition shown in Table 25 was placed in each of 500 ml glass jar farmenters and then sterilized by heating. 40 ml of the seed cultures as described above were inoculated into the medium. The culture was started at a temperature of 31.50C while the stirring rate and the aeration rate were controlled at 800 to 1300 rpm and 1/2 to 1/1 vvm, respectively. The pH of the culture liquid was kept at 7.5 with gaseous ammonia. The temperature was shifted to 370C 8 hours after the initiation of the culture. The culture was terminated when glucose had been completely consumed in 20 to 40 hours, and the quantity of L-glutamic acid produced and accumulated in the culture liquid were determined (Table 26). The GDH activity for obtaining the highest yield was about 3-times as high. When GDH activity was further elevated, the degree of the improvement in the yield was reduced.

When the GDH activity was elevated to about 16-times, the yield was rather reduced.

Amino acids produced as by-products were analyzed with Hitachi Amino Acid Analyzer L-8500 to find that as GDH activity was elevated, the amount of accumulated aspartic acid and alanine was increased. These results proved the following facts: For increasing the yield of glutamic acid, it is necessary to suitably increase GDH activity so as not to cause a remarkable increase in the amount of aspartic acid and alanine. One of the effective methods therefor comprises the introduction of various mutations into gdh promoter to control GDH activity to about 3-times as high as that of the parent strain.

Table Ingredient Glucose

KH

2

PO

4 MgSO4 7H 2 0 FeSO4 7H 2 0 MnSO4 4H 2 0 Soybean protein hydrolyzate Biotin Thiamine hydrochloride Concentration Seed culture Main culture 50 g/ 150 g/l 1 g/l 2 g/l 0.4 g/l 1.5 g/l 10 mg/l 15 mg/I 10 mg/l 15 mg/l 20 mill 50 ml/I 0.5 mg/l 2 mg/I 2 ma/l 3 mall Table 26 Strain AJ 13029 GA01 GA02 AJ13029/pGDH AJ13029/pAJ220G Glu (g/dl) 8.3 9.0 8.9 8.8 7.5 Asp (mg/dl) 49 145 153 201 290 Ala (mg/dl) 60 152 155 190 590 Relative activity of GDH 7.7 22.3 27.0 82.7 120 12 Relative value 2.9 10.7 156 590 12019 156

S.

0

S

SSr

S

S..

5 0 4. 4

S

S. S 0* 4

S

0 In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprising" is used in the sense of "including", i.e. the features specified may be associated with further features in various embodiments of the invention.

It is to be understood that a reference herein to a prior art document does not constitute an admission that the document forms part of the common general knowledge in the art in Australia or in any other country.

Sequence Listing <110> Ajinomoto Co. Inc.

<120> Method of constructing amino acid producing bacteria, and method of pre paring amino acids by fermentation with the constructed amino acid producing bacteria <130> OP 99052 <150> <151> <150> <151> JP 10-271786 1998-9-25 JP 10-271787 1998-9-25 <160> 6 <210> <211> <212> 1 46 nucleic acid <400> 1 ttaattcttt gtggtcatat ctgcgacact gccataattt gaacgt <210> 2 <211> 46 <212> nucleic acid <400> 2 ttaattcttt gcggtcatat ctgcgacact gccataattt gaacgt S<210> 3 <211> 46 <212> nucleic acid <400> 3 ttaattcttt gtqgtcatat ctgcgacact gctataattt gaacqt <210> 4 <211> 46 <212> nucleic acid <400> 4 ttaattcttt gttgacatat ctgcgacact gctataattt gaacgt <210> <211> 46 <212> nucleic acid <400> ttaattcttt gttgccatat ctgcgacact gctataattt gaacgt <210> 6 <211> 46 <212> nucleic acid <400> 6 ttaattcttt gttgtcatat ctgcgacact gctataattt gaacgt <210> 7 <211> <212> nucleic acid <220> primer A for cloning of gltA from Brevibacterium lactofermentum <400> 7 gtcgacaata gcctgaatct gttctggtcg <210> 8 <211> <212> nucleic acid <220> primer B for cloning of gltA from Brevibacterium lactofermentum <400> 8 aagcttatcg acgctcccct ccccaccgtt <210> 9 <211> <212> nucleic acid <220> primer 1 for introducing a mutation of gltA promoter <400> 9 atcggtataa cgtgttaacc <210> <211> <212> nucleic acid <220> primer 2 for introducing a mutation of gltA promoter <400> atcggtataa tgtgttaacc <210> 11 <211> <212> nucleic acid <220> primer 4 for introducing a mutation of gltA promoter <400> 11 gatttgacaa aaccgcattt atcggtataa tgtgttaacc <210> 12 <211> 28 <212> nucleic acid <220> gltApromoter sequenceprimer S4Q <400> 12 agggatccgt ccagtctcag acagcatc <210> <211> <212> <220> 13 17 nucleic acid universal primer M13RV <400> 13 caggaaacag ctatgac <210> <211> <212> <220> 14 nucleic acid primer A for cloning of ICDH <400> 14 gaattcgctc ccggtgcagc <210> <211> <212> <220> nucleic acid primer B for cloning of ICDH <400> gatgcagaat tccttgtcgg <210> <211> <212> <220> 16 28 nucleic acid primer 1 for introducing a mutation of ICD promoter <400> 16 tggattgctg gctataatgg tgtcgtga <210> 17 <211> 53 <212> nucleic acid <220> primer 2 for introducing a mutation of ICD promoter <400> 17 caacccacgt tcagttgaca actactggat tgctggctat aatggtgtcg tga <210> 18 <211> 53 <212> nucleic acid <220> primer 3 for introducing a mutation of ICD promoter <400> 18 caacccacgt tcagttgact actactggat tgctggctaa agtggtgtcg tga <210> 19 <211> 28 <212> nucleic acid <220> primer 4 for introducing a mutation of ICD promoter <400> 19 ggctgaaact gctataatag gcgccagc <210> <211> 51 <212> nucleic acid <220> primer 5 for introducing a mutation of ICD promoter <400> ggaaacacgg cgttgccatg cggggctgaa actgctataa taggcgccag c <210> 21 <211> 51 <212> nucleic acid <220> primer 6 for introducing a mutation of ICD promoter

L-A

LI')

<400> 21 ggaaacacgg cgttgacatg cggggctgaa actgctataa taggcgccag c <210> 22 <211> 22 <212> nucleic acid <220> ICD promoter sequence primer <400> 22 gtgcgggtcc agatgatctt ag <210> 23 <211> <212> nucleic acid <220> primer A for amplifying of nptII <400> 23 gggatcccgg atgaatgtca <400> 24 <211> 23 <212> nucleic acid <220> primer B for amplifying of nptII <400> 24 gcccggggtg ggcgaagaac tcc <210> <211> <212> <220> 23 nucleic acid primer for amiplifying of Brevibacterium lactofermentum pdhA gene LA <400> aci gti tci atg ggi cti ggi cc 1<21 0> 26 2 -77 <211> 23 <212> nucleic acid <220> primer for amiplifying of Brevibacterium lactofermentum pdhA gene LA <400> 26 cct tci ccg tti agi gti gti cg <210> 27 <211> <212> nucleic acid <220> primer for in vitro cloning of Brevibacterium lactofermentum pdhA gene

LA

<400> 27 ttg cag tta acc acg aag gtc agg ttg tcc <210> 28 <211> <212> nucleic acid <220> primer for in vitro cloning of Brevibacterium lactofermentum pdhA gene

LA

<400> 28 tgg atg aga cca cgt gat tct ggc tcg tcc <210> 29 <211> <212> nucleic acid <220> primer for in vitro cloning of Brevibacterium lactofermentum pdhA gene

LA

<400> 29 aca gat cct gca cga agg cat caa cga ggc <210> -2 l-3 0 <212> nucleic acid <220> primer for in vitro cloning of Brevibacterium lactofermentum pdhA gene

LA

<400> tea tcg ctg cgg gta cct cct acg cca ccc <210> 31 <211> 2766 <212> nucleic acid <213> Brevibacterium lactofermentum ATCC13869 <220> Brevibacterium lactofermentum ATCC13869 pdhA gene LA <400> 31 atg gcc gat caa gca aaa ctt ggt ggt aag ccc tcg gat gac tct aac 48 Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn 1 5 10 ttc gcg atg atc cgc gat Phe Ala Met Ile Arg Asp ccg gag gag acc aac gag Pro Glu Glu Thr Asn Glu ggc gtg gca Gly Val Ala 25 tct tat ttg aac gac tca gat 96 Ser Tyr Leu Asn Asp Ser Asp atg gat Met Asp tca ctc gac gga Ser Leu Asp Gly tta ctc cag 144 Leu Leu Gln gag tct tct cca gaa cgt Glu Ser Ser Pro Glu Arg 50 55 get cgt tac ctc Ala Arg Tyr Leu atg ctt Met Leu cgt ttg ctt Arg Leu Leu gag 192 Glu cgt gca tct gca aag cgc gta tct ctt ccc Arg Ala Ser Ala Lys Arg Val Ser Leu Pro 70 75 cca atg acg Pro Met Thr tca acc Ser Thr gac 240 Asp acc att cca acc tct atg gaa cct gaa ttc cca ggc gat 288 Tyr Val Asn Thr Ile Pro Thr Ser Met Giu Pro Giu Phe Pro Gly Asp 90 gag gaa Giu Glu atg gag aag Met Giu Lys 100 cgt tac cgt cgt tgg att cgc tgg Arg Tyr Arg Arg Trp Ile Arg Trp 105 110 aac gca gcc 336 Asn Ala Ala atc atg gtt cac cgc Ile Met Val His Arg 115 gct cag cga Ala Gin Arg 120 cca ggc atc ggc gtc Pro Gly Ile Gly Val 125 ggc gga cac 384 Gly Gly His att tcc Ile Ser 130 cac ttc His Phe 145 ttc cag Phe Gin act tac gca Thr Tyr Ala ggc gca Gly Ala 135 gcc cct ctg tac Ala Pro Leu Tyr 140 cac cca ggc ggc His Pro Gly Gly 155 gaa gtt Glu Val ggc ttc aac 432 Gly Phe Asn ttc cgc ggc aag gat Phe Arg Gly Lys Asp 150 ggc gac Gly Asp cag atc Gin Ile 160 ttc 480 Phe gag 528 Glu ggc cac gca Gly His Ala 165 tca cca Ser Pro ggt atg Gly Met 170 ctc gat Leu Asp 185 tac gca cgt gca ttc atg Tyr Ala Arg Ala Phe Met 175 ggt cgc ctt tct Gly Arg Leu Ser 180 gaa gac gat Glu Asp Asp ggc ttc cgt cag Gly Phe Arg Gin gaa gtt Glu Val tcc 576 Ser cgt gag cag Arg Giu Gin 195 gac ttc tgg Asp Phe Trp 210 ggt ggc att ccg tcc Gly Gly Ile Pro Ser 200 gag ttc cca act gtg Glu Phe Pro Thr Val 215 tac cct cac cca cac ggt atg Tyr Pro His Pro His Gly Met 205 aag 624 Lys tcc atg ggt ctt ggc cca atg gat 672 Ser Met Gly Leu Gly Pro Met Asp 220 att tac cag gca cgt ttc aac cgc tac ctc gaa aac cgt ggc atc 720 68 di Ala 225 Ile Tyr Gin Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Giy Ile 230 235 240 aag Lys gac acc tct gac Asp Thr Ser Asp 245 cag cac gtc Gin His Val atg gac gag Met Asp Glu 2' aac ctg gac Asn Leu Asp 275 gac gga cct Asp Giy Pro 290 :a gaa tca cgt ggt :o Giu Ser Arg Gly 265 c otg acc ttc gtg n Leu Thr Phe Val 280 tgg gc ttc ctt ggc gac ggc gaa 768 Trp Ala Phe Leu Gly Asp Gly Glu 250 255 ctc atc cag cag gct gca otg aac 816 Leu Ile Gin Gin Ala Ala Leu Asn 270 gtt aac tgo aac ctg cag ogt ctc 864 Val Asn Cys Asn Leu Gin Arg Leu 285 gtc cgc ggt aac Val Arg Gly Asn 295 aco aag atc ato Thr Lys Ile Ile 300 cag gaa Gin Glu ctc gag Leu Glu tcc 912 Ser ttc ttc cgt ggc Phe Phe Arg Gly 305 gca ggo Ala Gly 310 tgg tct gtg atc aag Trp Ser Val Ile Lys 315 gtt gtt Val Val tgg ggt Trp Gly 320 cgo 960 Arg gag tgg gat gaa ott Glu Trp Asp Giu Leu 325 ato atg aac aao acc Ile Met Asn Asn Thr 340 ctg gag aag gac cag gat ggt gca ctt gtt gag 1008 Leu Giu Lys Asp Gin Asp Gly Ala Leu Val Glu 330 335 tcc gat ggt gao tac cag acc tto aag got aac 1056 Ser Asp Gly Asp Tyr Gin Thr Phe Lys Ala Asn 345 350 gao ggc gca tat Asp Gly Ala Tyr 355 gca aag otc gtt Ala Lys Leu Vai s7 C gtt cgt gag cac ttc ttc Vai Arg Giu His Phe Phe 360 gag aac atg aco gac gaa Glu Asn Met Thr Asp Glu gga cgt gac cca cgc acc 1104 Gly Arg Asp Pro Arg Thr 365 gaa atc tgg aag ctg oca 1152 Glu Ile Trp Lys Leu Pro cgt Arg 385 ggc ggc cac gat tac cgc Gly Gly His Asp Tyr Arg 390 380 aag gtt tac gca gcc tac aag cga gct 1200 Lys Val Tyr Ala Ala Tyr Lys Arg Ala 395 400 ctt gag acc aag gat cgc cca Leu Giu Thr Lys Asp Arg Pro 405 acc gtc atc ctt gct cac acc att Thr Val Ile Leu Ala His Thr Ile 410 415 aag 1248 Lys ggc tac gga ctc ggc cac Gly Tyr Gly Leu Gly His 420 aac ttc gaa Asn Phe Glu 425 ggc cgt aac gca acc Gly Arg Asn Ala Thr 430 cac cag 1296 His Gin aag cag 1344 Lys Gin atg aag aag Met Lys Lys 435 ctg acg ctt gat gat Leu Thr Leu Asp Asp 440 atc acc gat gag cag Ile Thr Asp Giu Gin 455 ctg aag ttg ttc Leu Lys Leu Phe 445 cgc gac Arg Asp ggc Gly atc cca Ile Pro 450 ctg gag aag Leu Giu Lys 460 gat cct tac Asp Pro Tyr cct 1392 Pro cct Pro 465 tac tac cac cca ggt Tyr Tyr His Pro Gly 470 gaa gac gct Giu Asp Ala gaa atc aag tac atg Giu Ile Lys Tyr Met 480 aag 1440 Lys gaa cgt cgc gca gcg ctc ggt Giu Arg Arg Ala Ala Leu Gly 485 ggc tac Gly Tyr 490 ctg cca gag cgt cgt gag aac 1488 Leu Pro Giu Arg Arg Giu Asn 495 GAT AAG CTT CGC TCT GTC CGT 1536 Asp Lys Leu Arg Ser Val Arg ,510 TAC GAT CCA ATT CAG Tyr Asp Pro Ile Gin 500 GTT CCA CCA CTG Val Pro Pro Leu 505 aag ggc tcc ggc aag cag cag atc gct Gly Ser Gly Lys Gin Gin Ile Ala acc act Thr Thr atg gcg act gtt cgt 1584 Met Ala Thr Val Arg acc ttc aag gaa Thr Phe Lys Giu 530 ctg atg cgc Leu Met Arg 535 gat aag Asp Lys ggc ttg gct gat cgc ctt gtc 1632 Gly Leu Ala Asp Arg Leu Val 540 cca atc att cct gat gag Pro Ile Ile Pro Asp Glu 545 550 cca acc ttg aag atc tac Pro Thr Leu Lys Ile Tyr .565 gca cgt acc Ala Arg Thr ttc ggt ctt.

Phe Gly Leu gac tct tgg ttc 1680 Asp Ser Trp, Phe 560 tac gtg cct gtt 1728 Tyr Val Pro Val 575 aac ceg cac Asn Pro His 570 ggt cag aac Gly Gin Asn gac cac gac Asp His Asp ctg atg ctc tcc tac cgt Leu Met Leu Ser Tyr Arg 585 gag gca cct. gaa Glu Ala Pro Glu 590 gga cag atc 1776 Gly Gin Ile ctg cac gaa Leu His Giu 595 gcg ggt acc Ala Gly Thr 610 ggc atc aac gag gct Giy Ile Asn Giu Ala 600 ggt tcc gtg gca. tcg ttc atc gct 1824 Gly Ser Vai Ala Ser Phe Ile Ala 605 tcc tac gcc acc Ser Tyr Ala Thr 615 cac ggc aag gcc His Gly Lys Ala 620 atg att ccg ctg tac 1872 Met Ile Pro Leu Tyr atc ttc tac tcg atg ttc gga ttc cag cgc acc ggt gac tcc atc tgg 1920 Ile Phe 625 gca gca Ala Ala Tyr Ser Met P1 630 he Gly Phe Gin Thr Gly Asp Ser Ile Trp 640 gcc gat cag atg Ala Asp Gin Met 645 gca cgt ggc ttc Ala Arg Giy Phe 650 ctc ttg ggc gct acc gca 1968 Leu Leu Gly Ala Thr Ala 655 ggt cgc acc acc ctg acc gy Arg Thr Thr Leu Thr ggt gaa ggc Gly Glu Giy ctc-cag cac Leu Gin His atg gat gga cac 2016 Met Asp Gly His 670 tcc cct gtc ttg gct Ser Pro Val Leu Ala 675 tcc acc aac gag ggt gtc gag acc tac gac cca 2064 Ser Thr Asn Giu Gly Vai Giu Thr Tyr Asp Pro 680 685 tcc ttt Ser Phe 690 gcg tac gag atc gca cac ctg gtt cac cgt Ala Tyr Giu Ile Ala His Leu Val His Arg 695 700 ggc atc gac Gly Ile Asp cgc 2112 Arg atg tac ggc Met Tyr Gly 705 cca ggc aag Pro Gly Lys 710 ggt gaa gat gtt Gly Glu Asp Vai 715 atc tac tac atc acc Ile Tyr Tyr Ile Thr 720 atc 2160 Ile tac aac gag cca acc Tyr Asn Glu Pro Thr 725 cca cag cca gct Pro Gin Pro Ala.

730 gag cca gaa gga ctg Giu Pro Giu Gly Leu 735 gac gta 2208 Asp Val gaa ggc ctg cac Giu Gly Leu His 740 aag ggc atc tac ctc Lys Gly Ile Tyr Leu 745 tac tcc cgc ggt Tyr Ser Arg Gly 750 gaa ggc Giu Gly acc 2256 Thr ggc cat gag gca aac atc ttg gct Gly His Giu Ala Asn Ile Leu Ala 755 760 tcc ggt qtt ggt atg Ser Gly Val Gly Met 765 cag tgg gct 2304 Gin Trp Ala ctc aag gct Leu Lys Ala 770 gca tcc atc ctt gag gct gac tac gga gtt cgt gcc aac 2352 Ala Ser Ile Leu Glu 775 Ala Asp Tyr 780 aac ttg gct Asn Leu Ala 795 Gly Val Arg Ala Asn att tac tcc gct Ile Tyr Ser Ala 785 act tct tgg gtt Thr Ser Trp Val 790 cgc gat Arg Asp ggc gct Gly Ala 800 gct 2400 Al a cgt aac aag gca cag ctg cgc aac cca ggt qca gat gct ggc gag gca 2448 RA4Arg Asn Lys Ala Gin Leu Arg Asn Pro Gly Ala Asp Ala Gly Giu Ala ttc gta acc acc cag ctg aag cag acc Phe Val Thr Thr Gin Leu Lys Gin Thr 820 825 tct gac ttc tcc act gat ctg cca aac Ser Asp Phe Ser Thr Asp Leu Pro Asn 835 840 tcc ggc cca tac Ser Gly Pro Tyr 830 cag atc cgt gaa Gin Ile Arg Glu 845 gtt gca gtg 2496 Vai Ala Val tgg gtc cca 2544 Trp Val Pro ggc gac Gly Asp 850 tac acc gtt ctc C Tyr Thr Val Leu C 855 ;gt gca gat ggc ttc ;iy Ala Asp Gly Phe 860 ggt ttc tct gat acc 2592 Gly Phe Ser Asp Thr cgc cca Arg Pro 865 gct gct cgt cgc Ala Ala Arg Arg 870 ttc ttc aac Phe Phe Asn atc gac gct Ile Asp Ala 875 gag. tcc att gtt 2640 Giu Ser Ile Val 880 gtt gca gtg ctg aac tcc Val Ala Vai Leu Asn Ser 885 gtt gct gct cag gct gct Val Ala Ala Gin Ala Ala ctg gca cgc Leu Ala Arg 890 gag aag ttc Giu Lys Phe 905 gaa ggc aag atc gac gtc tcc 2688 Giu Gly Lys Ile Asp Val Ser 895 aag ttg gat gat cct acg Lys Leu Asp Asp Pro Thr 910 agt 2736 Ser gtt tcc gta gat cca aac gct cct gag gaa Val Ser Val Asp Pro Asn Ala Pro Giu Giu 2766 <210> 32 <211> 8556 <212> nucleic acid <213> Brevibacterium lactofermentum ATCC13869 <400> 32 tcacgttacg gcgatcaaca ccgcaaccac tacgagaaga tctccaaacg agaccaagag cgcttctaag cccgtctcat tcatgccact atcttggggt ttgcgctaga cactaattac taggccattt tgatgtggtg ccqaatccac atcgtctaaa tcatcgacgc aactttacgg tcaaagaaag cggagtggca tagtagcgga agctttagat tcaaagagac cttgattgat atcctcgtca gcgatttcgc tcgcctaatg gaattcacat tcgcgcaaga aaaaggccaa tccttggggt gttttattac caacgcaacg atgcagagaa aggttctgac gagqtgcgaa taatttcgaa gtgctgggtt qaaaaacagg ccacqataag ccctgacggg ctcgaaccgc taaaggcgcg cacgtgcttt tactaacaca caatctccac gttaaggctt cttgtttcca ccgccttggt ctgcacgaga gggctccttc tttaccaatg agcggatgga atccacaagt gcacgacatc qcacagctcg cgatggactc gctgatcagc gacccgattg aatgccgagt gagcgtcggc cactttaata ggattcaaca tgaaaccgcg ggtactttga accacctttc atcaatgaat tcaatcactg acaattctct tcaaaataat r gctt gttttatgcg RK2 14 tttgcacctg tctcggtatt ggcaccgctt tagatcatat agqcctgggc ctcatcqaca cgtgcagcgg gccgggcgag gggctgttta aaacggctcg gtgaagagac atccgggcgg caatcggtcg gccttggagg gcaagttgtc ttctcgcgca tttcttaaaa cgacctgctg tctagaacca agacctaaaa cgctggacga cctgccagtt ccaggagtca ccgtcgacga tcggtttctt gtgtcggaat agttgagcat atcctcctcg gggctattga cctggaattt gccagcgatt qgtggctaga attcqcgtca ccattctgtg agatcttctg aagcatggtc tgacgtcaat gacca cgtaa gcaatcgtcc tttatcqacg ctccagttga caaatcaggc agttggtgat gtcgagaagc atctagacat cgcgtggagt tgatctggca gcgcgccgca tacccaatgc cttatcgtgg ggtgtaaacc ccttggtggc tcgctgctca ggcaagaacc gtqctgattc acacccagat gatcgccgtc catcgagtag cttcatccca aatcctgqgc tgtgggcccc tatatccgaa tttccttttc aacttttcga tttttcatca aacgtgagag aggatatggc ataaaaaccc gtgacacgta gaatgaagtg agacgggcac cgtcacggtc ctggcccagg aatagatacc gaaaaccact gtcagatcaa aaatattcgc cgaggcgtgc aaatttcacc tggaatgqaa cctcagtatc gtaccgatgt cctgcttcta agctgctctt ctcgaaagca ggccgtggaa ttgccaatta agagataact acgaccattc atcctcgcgc ttcctcaccg tccaatttct accttgcttg gatgtttttI ttgcacatta ctcccccttt gttttcagtc aaccctcaccz aaacatcacat aggtgctttt gatagttttc aaacctgact actaactccg gacgagaaga aatgcagttg ctagtggatc ccaggggaca ggagtctcct gaattacagc gcgctgcaag ctcgatgcaa gaccaaggta ccttaaattc cggatcaact gcccatgagc tatttgtgcg tcagctgagc acgagtgaaa attagcgatt ccgagacgtt ccaggagcca tcagcgagqg caccagagca St tg cat ct t tgaacgatat actgcgcccg tcgattacat ccgtccaacc icgctcaaga -tggatttcc iaaaggacat :ctcacggga 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 aactacccga gtaqaaatgt cctgaaatcc gtgttgcgta caattaggta tccaacagga taattctttg caaaactttg tctttaaaaa atccacaaca cagtgagcgc accactcccc tccttgttaa taacttatgc cgaccaagaa tgggaccggg ggtgtggaa atg gcc gat Met Ala Asp caaagggtaa tgaacatgca gctagtttcc.

attgccagga agcacaccga ttgatggata ttacgtcaca gtctgtaaaa caaatcttcg gttgacccat tcgtgcactt cggtgtgcca aaaccgggac gtataaacga aataaaacat caa gca aaa ctt ggt ggt aag ccc Gin Ala Lys Leu Gly Gly Lys Pro 2100 2160 2220 2280 2340 2392 tcg gat gac tct aac ttc gcg atg Ser Asp Asp Ser Asn Phe Ala Met atc cgc gat ggc gtg Ile Arg Asp Gly Val gca tct tat Ala Ser Tyr 2440 ttg aac gac tca Leu Asn Asp Ser gat ccg gag gag acc aac Asp Pro Glu Giu Thr Asn 35 gag tgg atg gat tca ctc Glu Trp Met Asp Ser Leu 2488 gac gga Asp Gly tta ctc cag gag tct Leu Leu Gin Glu Ser 50 tct cca gaa cgt Ser Pro Giu Arg gct cgt tac ctc atg Ala Arg Tyr Leu Met gta tct ctt ccc cca Val Ser Leu Pro Pro 2536 2584 cgt ttg ctt gag Arg Leu Leu Glu gca tct gca aag Ala Ser Ala Lys cgc Arg atg acg tca acc gac Met Thr Ser Thr Asp tac gtc aac acc att Tyr Val Asn Thr Ile 85 cca acc tct atg gaa cct Pro Thr Ser Met Giu Pro 2632 gaa ttc cca ggc Giu Phe Pro Giy gat gag gaa atg gag aag cgt tac cgt cgt tgg att Asp Giu Giu Met Giu Lys Arg Tyr Arg Arg Trp Ile 100 105 2680 cgc tgg aac Arg Trp Asn ,IAZ-,110 gca gcc atc atg gtt cac cgc Aia Ala Ile Met Val His Arg 115 gct cag cga cca ggc atc Ala Gin Arg Pro Gly Ile 120 2728 ggc gto ggc gga cac att tcc act Gly Vai Gly Gly His Ile Ser Thr 125 130 tac gca ggc gca gcc cot ctg tac Tyr Ala Gly Ala Ala Pro Leu Tyr 135 2776 gaa Glu 140 gtt ggc ttc aac cac Val Gly Phe Asn His 145 ttc ttc cgc ggc aag Phe Phe Arg Gly Lys 150 cag ggc cac gca tca Gin Gly His Ala Ser 165 gat cac cca Asp His Pro ggo ggc Gly Gly 155 2824 2872 ggc gao cag atc ttc ttc Gly Asp Gin Ile Phe Phe 160 cca ggt atg tac gca Pro Gly Met Tyr Ala 170 cgt gca ttc atg gag ggt cgc ctt tct Arg Ala Phe Met Giu Gly Arg Leu Ser 175 180 gaa gac gat otc gat ggo ttc Glu Asp Asp Leu Asp Giy Phe 185 2920 ogt cag gaa Arg Gin Glu 190 gtt too cgt gag cag Val Ser Arg Glu Gin 195 ggt ggo att cog too tac cct cac Gly Gly Ile Pro Ser Tyr Pro His 200 2968 oca cac Pro His 205 ggt atg aag gao tto Gly Met Lys Asp Phe 210 cca atg gat gc att Pro Met Asp Ala Ile 225 tgg gag ttc cca act Trp Giu Phe Pro Thr 215 tao cag gca ogt tto Tyr Gin Ala Arg Phe 230 ott Leu 220 ggo Gly gtg too atg ggt Val Ser Met Gly aac cgo tac otc Asn Arg Tyr Leu 235 gto tgg gc tto Val Trp Ala Phe 250 3016 3064 gaa aac cgt Glu Asn Arg ggc ato Gly Ile 240 aag gao aco Lys Asp Thr tct gao Ser Asp 245 cag cac Gin His 3112 ott ggc gao Leu Gly Asp gaa atg gao gag oca Glu Met Asp Giu Pro 260 gaa toa ogt ggt Glu Ser Arg Gly otc ato cag Leu Ile Gin 265 3160 cag gct Gin Ala gca ctg aac aac ctg gac aac ctg acc ttc gtg gtt aac tgc Ala Leu Asn Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys 270 275 280 3208 aac ctg Asn Leu 285 cag cgt ctc gac Gin Arg Leu Asp gga cct Gly Pro 290 gtc cgc ggt Val Arg Gly cag Gin 300 gaa ctc gag tcc ttc Giu Leu Giu Ser Phe 305 ttc cgt ggc gca ggc Phe Arg Gly Ala Gly 310 tgg gat gaa ctt. ctg Trp Asp Giu Leu Leu 325 aac acc Asn Thr 295 tgg tct Trp Ser gag aag Giu Lys aag atc atc Lys Ile Ile gtg atc aag Val Ile Lys 315 gac cag gat Asp Gin Asp 330 3256 3304 3352 gtt gtt tgg ggt cgc gag Vai Vai Trp Giy Arg Giu 320 ggt gca ctt gtt Gly Ala Leu Vai 335 gag atc Giu Ile atg aac Met Asn aac As n 340 acc tcc gat ggt Thr Ser Asp Gly ga c Asp 345 tac cag Tyr Gin 3400 acc ttc aag Thr Phe Lys 350 gct aac gac ggc gca Ala Asn Asp Gly Ala tat gtt cgt Tyr Val Arg gag cac Giu His 360 ttc ttc gga Phe Phe Gly 3448 cgt gac Arg Asp 365 cca cgc acc gca aag Pro Arg Thr Ala Lys 370 ctc gtt gag aac atg acc gac gaa gaa Leu Val Glu Asn Met Thr Asp Giu Giu 375 3496 atc Ile 380 tgg aag ctg cca cgt Trp Lys Leu Pro Arg 385 ggc ggc cac gat Gly Giy His Asp cgc aag Arg Lys gtt tac gca Vai Tyr Ala 395 gtc atc ctt Vai Ile Leu 410 3544 gcc tac aag cga gct ctt -Ala Tyr Lys Arg Ala Leu 4400 gag acc aag Giu Thr Lys cgc cca acc Arg Pro Thr 3592 get cac acc Ala His Thr aag ggc tac gga ctc Lys Gly Tyr Gly Leu 420 ggc cac aac ttc gaa ggc cgt Gly His Asn Phe Glu Gly Arg 425 acg ctt gat gat ctg aag ttg Thr Leu Asp Asp Leu Lys Leu 440 3640 aac gca acc cac Asn Ala Thr His 430 cag atg aag Gin Met Lys aag ctg Lys Leu 435 3688 ttc cgc gac Phe Arg Asp 445 aag cag ggc atc Lys Gin Gly Ile 450 cca atc acc gat Pro Ile Thr Asp gat Asp 460 cct tac ctt cct cct tac tac Pro Tyr Leu Pro Pro Tyr Tyr 465 cac cca ggt His Pro Gly 470 gag cag ctg gag aag Glu Gin Leu Glu Lys 455 gaa gac gct cct gaa Glu Asp Ala Pro Glu 475 ggt ggc tac ctg cca Gly Gly Tyr Leu Pro 490 cca cca ctg gat aag Pro Pro Leu Asp Lys 505 3736 3784 atc aag tac atg aag Ile Lys Tyr Met Lys 480 gag cgt cgt gag aac Glu Arg Arg Glu Asn 495 gaa cgt Glu Arg tac gat Tyr Asp cgc gca Arg Ala cca att Pro Ile 500 gcg ctc Ala Leu 485 cag gtt Gin Val 3832 3880 ctt cgc tct gtc Leu Arg Ser Val 510 cgt aag ggc tcc ggc Arg Lys Gly Ser Gly 515 aag cag cag atc get acc act Lys Gin Gin Ile Ala Thr Thr 520 3928 atg gcg Met Ala 525 act gtt Thr Val cgt acc ttc Arg Thr Phe 530 aag gaa Lys Glu ctg atg cgc Leu Met Arg 535 gat aag ggc ttg Asp Lys Gly Leu 3976 gct gat cgc ctt gtc cca Ala Asp Arg Leu Val Pro 40 545 ate att cct gat Ile Ile Pro Asp gca cgt Ala Arg acc ttc ggt Thr Phe Gly 555 4024 ctt gac tct tgg tte cca Leu Asp Ser Trp Phe Pro 560 acc ttg aag atc tac aac ccg Thr Leu Lys Ile Tyr Asn Pro 565 cac ggt eag His Gly Gin 570 egt gag gca Arg Giu Ala 585 4072 aac tac gtg Asn Tyr Vai ect gtt Pro Val 575 gac cac gac ctg Asp His Asp Leu 580 atg etc tcc tac Met Leu Ser Tyr 4120 cct gaa gga Pro Giu Gly 590 gca teg ttc Ala Ser Phe 605 eag atc ctg cac gaa ggc Gin Ile Leu His Glu Gly 595 atc aac gag gct ggt tcc gtg Ile Asn Giu Ala Gly Ser Val 600 tae gee ace eac ggc aag gcc Tyr Ala Thr His Gly Lys Ala 615 4168 4216 atc gct gcg ggt Ile Ala Ala Gly 610 acc tcc Thr Ser atg Met 620 att ccg ctg tac Ile Pro Leu Tyr atc ttc Ile Phe 625 tac tcg Tyr Ser atg ttc Met Phe 630 cag atg Gin Met 645 gga tte cag cgc ace Gly Phe Gin Arg Thr 635 4264 ggt gac tce ate tgg gca Gly Asp Ser Ile Trp Ala 640 ttg ggc gct acc gca ggt Leu Gly Ala Thr Ala Gly 655 gca gcc gat Ala Ala Asp cgc acc acc Arg Thr Thr 660 cct gte ttg Pro Vai Leu 675 gca cgt ggc Ala Arg Gly ttc ctc Phe Leu 650 4312 ctg acc ggt gaa ggc ctc cag Leu Thr Giy Giu Gly Leu Gin 665 get tc acc aac gag ggt gte Ala Ser Thr Asn Giu Gly Val 680 4360 cae atg His Met gag ace Giu Thr L-RA/ 685 gat Asp 670 gga cac tee Gly His Ser 4408 4456 tac gac cca te ttt Tyr Asp Pro Ser Phe 690 gcg tae gag ate gca Ala Tyr Giu Ile Ala 695 cac ctg gtt cac His Leu Vai His ggc atc gac cgc atg tac ggc cca Gly Ile Asp Arg Met Tyr Gly Pro 705 ggc aag ggt Gly Lys Giy 710 gaa gat gtt atc Giu Asp Val Ile 715 4504 tac tac atc acc Tyr Tyr Ile Thr atc tac Ile Tyr 720 aac gag Asn Glu cca acc Pro Thr 7 25cca cag cca gct gag cca Pro Gin Pro Aia Giu Pro 730 4552 gaa gga ctg gac Giu Gly Leu Asp 735 gta gaa ggc ctg cac Val Glu Gly Leu His 740 aag ggc atc tac ctc tac tcc Lys Gly Ile Tyr Leu Tyr Ser 745 4600 cgc ggt gaa ggc acc ggc cat gag Arg Gly Giu Gly Thr Gly His Giu 750 755 gca aac atc ttg gct tcc ggt gtt Ala Asn Ile Leu Ala Ser Gly Val 760 4648 ggt atg cag Gly Met Gin 765 tgg gct ctc aag Trp Ala Leu Lys 770 gct gca tcc atc ctt gag gct gac tac Aia Ala Ser Ile Leu Glu Ala Asp Tyr 775 4696 gga Gi y 780 gtt cgt gcc aac Val Arg Ala Asn att taG tcc gct act Ile Tyr Ser Ala Thr 785 cgt aac aag gca cag Arg Asn Lys Ala Gin 805 t ct Ser 790 tgg gtt aac Trp Vai Asn ttg gct Leu Ala 795 4744 cgc gat ggc gct Arg Asp Giy Ala gct Al a 800 ctg cgc aac cca ggt gca Leu Arg Asn Pro Gly Aia 810 4792 gat gct ggc gag gca ttc gta Asp Aia Giy Giu Ala Phe Val 815 acc acc cag ctg aag cag acc Thr Thr Gin Leu Lys Gin Thr 820 825 tcc ggc Ser Gly 4840 cca tac Pro Tyr gtt gca gtg tct gac Val Ala Val Ser Asp 830 tcc act Ser Thr gat ctg cca Asp Leu Pro 840 aac cag atc Asn Gin Ile 4888 cgt gaa tgg Arg Giu Trp 845 ggt ttc tct Gly Phe Ser 860 gtc cca ggc gac tac Val Pro Gly Asp Tyr 850 gat acc cgc cca gct Asp Thr Arg Pro Ala 865 acc gtt ctc Thr Val Leu ggt gca Gly Ala gat ggc ttc Asp Gly Phe 4936 gct cgt cgc Ala Arg Arg 870 ttc ttc aac atc gac Phe Phe Asn Ile Asp 875 ctg gca cgc gaa ggc Leu Ala Arg Giu Gly 890 4984 gct gag tc& att gtt gtt gca Ala Glu Ser Ile Val Val Ala 880 gtg ctg aac tcc Val Leu Asn Ser 885 5032 aag atc gac Lys Ile Asp gat gat cct Asp Asp Pro gtc Val1 895 tcc gtt gct gct Ser Val Ala Ala cag gct gct Gin Ala Ala 900 gag aag Glu Lys ttc aag ttg Phe Lys Leu 905 gag gaa taaat Giu Giu 5080 5130 acg agt gtt tcc gta gat cca aac gct cct Thr Ser Val Ser Val Asp Pro Asn Ala Pro cacctcaagg aaagcaagct gcgaactcct tagacaatct atgttgtgcg gtggtcacaa attttaggtt agcaaaccaa tgcttgaatt cgcagcaacg ggatcctgaa ttgttgccgt agcagggtgg ttuggaaattc gacagataaa ctttttagcc gcagcaaatc ggccttCatg tgaccataag gctctgcacg gagtaaaacc ctttggcagt gctcacggcg cgatggccag aaacgcggcc cgatggaaat atttacccgc tgagctgctg cgttgatgga tcccgccgcc gagaaacgcc gcgcacagtc catcatgatc cgtagtcaga ctgtggatca agccatcagc gagcctgttt cttcgaggtc attctcttca gatgtagcgg ctcgccggaa accqttagaa caacgcgcgq taacatgtct agacgttagt ttgtcagaca aacttcgact aggcgattgc gttccatctg agcgctgcgg agggacaatg tccagaccca agtgcaaagc atggtgagat gcacqcttgt tcaacaagca ccgatqacgg cgctcattca taagcctcca ctggcggcgg atgttgcgcc tggtagcctg ccaggcgaat ccacgatctt tgtgctcatc cctgacgctg gctcaaggcg ccaatccacg tcggcgcggt gctctgacat aacggccaga tgacaaaatc cagtgccggg ctgctactgg gattcgtcgt cttgatattg atctgcctgg tgcctgttcc ttcggtcaag caacagcatg accgccagag ctcaagttcc gcgcttgccg gcctgccaaa cttgtttacc aacagcgttg gccctcagcc gaagaaggtt ttgcttaggc 5190 5250 5310 5370 5430 5490 5550 5610 5670 5730 5790 5850 5910 5970 6030 a I.

ttcggtgcct tqgagaactt atcgcagaaa ttgccttcat atcaggcggt acgcgatgqc tgaccgaaga tggcctggcc acccatcgaa atatctgcga agaaccattg gacagggaga ctgcaccatc ggcccgttgt gaaccagatg acgatgagtc ccatqtgccg aggaggccgg tggcacgttc gcgccaccca aatacctgcg agcaaaccat agcgataaag ccagtaacga aagagctgtc gcagctgttg aaagttqagc acacggaacg gaactcaaaa gcgccgatca acgccctggc gctctgctgc acaggcatgg ttqctgggtc tggcggccaa gctcqgtgta ccggactcag caccgatcac ataccggaaa caaccatgtt tcaacagcct gctgaatctc agtggctcaq cagccttggt gcgtagacga tgccgatgga tgctcaatcg gtgcqatatc tccaccagct ctgccqacac atattctgcg cagttgcctg tcagaaqcga actgctgaga tcatcaaagg tgacgtcaac gcatcaagtg caqggtgctg gcagcatcag tgqaggtgga gcgccaacag taaagatgct cttaaagtgt tattttcaaa catatqtgaa atgtctcatg cctggatatc ccgcaggaca -tttaaaggcc ctagacaccg aaacct ccaaagacta cgcacgccac atcgttgggg accgacgatg ccaaaataac agccctggta tggtgaggat cgccqgtgga ccaaaaatcc cgtcgcgaag tgatgcgcaa cgatcttgcc aaccagcggc ccgcgccagt tcaaatagac tacgcgttcq atcttcgttg ttcgccgtcc atcqataccc agggatctgc gtcagttgga gttcacggta gacctcatcg ctttgcgtca agaagtaccg tgcatcggta tgctgctgga aatagtcagt gcctgcctct aacaaqctgg gctgtcactg tgcgagtacc aaggggcaga atttaaacta tqgacaaaat cgatttcctt gagtggggaa ctcggcagca tcaaggccaa acagccaaca cgatgcgaga ctgagcgatg tttggtgaaa tcgcagcgac cactgcaacg cggggtgaag gttaaccgac cttggtgtgc agtagccata ggtcaggctg gcccaatgcg gccttatcca atcacgtagg tgcagaatac agggttgcca ttctgctcag acgtagatga gtgacagtct acctgcacct ggaaccagct gtgacgtttc actgcggtga gttgcaacag gcagtgccct tcaaaagctt ttgatgccaa gaatcgcttg ttcgaacggg cagcgtcaac cttgttctac gggtgqatag aatcqatcat caccatgaac gcatggcgac cgcgcagtgc cagccaacca ccgatcacga agtgcaccgg tccgttgaaa agtccgatat gcgaggatcg aqqttatcgg cacaacgcaa agcaaaccgg agaqgaggcc ccgtcaacgg acaactccgt aaatctcttg tgaactcagt gcagagccat gtgcgccacc caacctgaac catcaacatc gagtattaac gagagttgac gctgcaaaag cgccaggtgc acaggactgg ccaacacgag gctcgccgtt ccttgaaacc ctcggtaaga agcacgaagc aagaaaacat acatgtctcq cagtcatatg cgggtgcacc taaagaggga gtttcctcaa aaccaccaag tgcqaaaatg cacgcgacgc tgaggccaat caaqaccaac caccaaagga cagtgttgag ccgqgcctgc tgcgcaacaa tcatggtcaa tgatcatgcc agccagacat tgaggtcggg acaacaatcc agggatctcc tgcagtctcc ctcgccagtc ctcaacagtg cagagaagaa gccgaqagct ~ggacagcccc ctcaccagac ctccaactgc ttcattagaa qatatcagtg atccaaattg agcgttttgc agtggtcgct gtcgccacct caacgccaag agcacatctc gataaagaac cgcaattccc aattcaatct tcctctcccc ataaacccag 6090 6150 6210 6270 6330 6390 6450 6510 6570 6630 6690 6750 6810 6870 6930 6990 7050 7110 7170 7230 7290 7350 7410 7470 7530 7590 7650 7710 7770 7830 7890 7950 8010 8070 8130 8190 tgactctgac cgcgaatatc ttcaatcaga actcacccgg ctcgttggcc aggggcgact 8250 cgatctagat acttaccaag acgtggttga taccgtttgg tctactgatg atctaggcga 8310 gttgatgagg atccgtgccc gcttcctggg agggccgcag gtttcgcagc agcggcccca 8370 gcagccgcag caaccacatc agcggccgca acagcaaccg ccacagcatt atggacaacc 8430 cggctacggc caatcacctc aatatccacc gcagcagcct ccgcataatc agcccggcta 8490 ttaccccgat cccggccctg gccagcagca accaccgatg caccagccac caacgcgtcc 8550 aaatca <210> 33 <211> <212> nucleic acid <220> primer for construction of Brevibacterium lactofermentum pdhA gene LA amplification plasmid <400> 33 aat gcc agg agt caa cac cc <210> 34 <211> <212> nucleic acid <220> primer for construction of Brevibacterium lactofermentum pdhA gene LA amplification plasmid <400> 34 aca tgg aac agg caa ttc gc <210> <211> 28 <212> nucleic acid <220> primer for introducing a mutation of Brevibacterium lactofermentum pdhA gene LA promoter <400> cgt ccc ggg ctg taa aac aaa tct tcg g 210> 36 <211> 27 <212> nucleic acid <220> primer for introducing a mutation of Brevibacterium lactofermentum pdhA gene LA promoter <400> 36 ate ccc ggg ctt acc acc aag ttt tgc <210> 37 <211> <212> nucleic acid <220> primer for introducing a mutation of Brevibacterium lactofermentum pdhA gene LA promoter <400> 37 ctt atg cgt tgc cac att cgt gca ctt cgg <210> 38 <211> <212> nucleic acid <220> primer for introducing a mutation of Brevibacterium lactofermentum pdhA gene LApromoter <400> 38 gcg ttg ace cat tcg tgc act tcg gtg tgc tat aat tag g <210> 39 <211> <212> nucleic acid <220> primer for introducing a mutation of Brevibacterium lactofermentum pdhA gene LA promoter <400> 39 gcg ttg cca cat tcg tgc act tcg gtg tgc tat aat tag g 210> <211> 38 <212> nucleic acid <220> primer for introducing a mutation of Brevibacterium lactofermentum pdhA gene LA promoter <400> ttt taa aac gtt ctg gag aag act cct gga gta atc cg <210> 41 <211> <212> nucleic acid <220> primer for introducing a mutation of Brevibacterium lactofermentum pdhA gene LA promoter <400> 41 cga tct tgc ctt cgc gtg cc <210> 42 <211> <212> nucleic acid <400> 42 agaccgccgg agtatgcaag aacgatgcgg <210> 43 <211> <212> nucleic acid <400> 43 gacttcacca tcaatcatct tcttcaggta <210> 44 <211> <212> nucleic acid 4 o0 0 44 accttcgacc agaccctggc taagggcttt <210> <211> <212> nucleic acid <400> gctaacaagc gcqatcgcga agctggcaac <210> 46 <211> <212> nucleic acid <400> 46 gcgatgacac cgtttttgtt ctcgc <210> 47 <211> <212> nucleic acid <400> 47 ggcgacatcc ttgcccagat gatca <210> 48 <211> <212> nucleic acid <400> 48 gacttcacca tcaatcatct tcttc <210> 49 <211> 24 <212> nucleic acid <400> 49 gccaggtaca actqtctgaa ttgc <210> <211> <212> nucleic acid <220> primer for introducing a mutation <400> gttaatcgct tgccaatgca ggcaggtaag gtataacccg <210> 51 <211> <212> nucleic acid <400> 51 gttaatcgct tgctaatgca ggcaggtaag gtataacccg <210> 52 <211> <212> nucleic acid <400> 52 gttaatcgct tgtcaatgca ggcaggtaag gtataacccg <210> 53 <211> <212> nucleic acid <400> 53 gttaatcgct tgttaatgca ggcaggtaag gtataatccg <210> 54 <211> <212> nucleic acid <400> 54 .gttaatcgct tgtcaatgca ggcaggtaag gtataatccg '87 I 87

TO

<210> <211> <212> nucleic acid <400> gggttccaqc ctcgtgcgga attcgtggaq <210> 56 <211> <212> nucleic acid <400> 56 gcgttaccca gagctggatc ctcgg <210> 57 <211> 16 <212> nucleic acid <400> 57 cagttgtggc tgatcg <210> 58 <211> 17 <212> nucleic acid <400> 58 ctttcccaga ctctggc <210> 59 <211> 21 <212> nucleic acid <400> 59 gctataattt gacgtgagca t <210> <211> <212> nucleic acid <400> gctcacgtca aattatagca gtgtc <210> 61 <211> 54 <212> nucleic acid <400> 61 ttgttgtcat tctgtgcgac actgctataa tttgaacgtg agcagttaac agcc <210> 62 <211> 63 <212> nucleic acid <400> 62 gttaactgct cacgttcaaa ttatagcaft gtcgcacaqa atgacaacaa aqaattaaaa ttg <210> 63 <211> <212>. nucleic acid <400> 63 gctagcctcg ggagctctct aggag <210> 64 <211> <212> nucleic acid <400> 64 gatctttccc agactctqgc cacgc

Claims (4)

1. A method of producing coryneform bacteria having an improved amino acid- or nucleic acid-productivity, which comprises the steps of introducing a mutation in a promoter sequence of amino acid- or nucleic acid-biosynthesizing genes on a chromosome of a coryneform bacterium to make it close to a consensus sequence or introducing a change in a promoter sequence of amino acid- or nucleic acid- biosynthesizing genes on a chromosome of a coryneform bacterium by gene recombination to make it close to a consensus sequence, to obtain a mutant of the coryneform amino acid- or nucleic acid-producing microorganism, culturing the mutant and selecting a mutant capable of producing the intended amino acid or nucleic acid in a large amount.

2. The method of claim 1, wherein the amino acid is selected from the group consisting of glutamic acid, lysine, arginine, serine, phenylalanine, proline and glutamine, and nucleic acid is selected from the group consisting of inosine, guanosine, adenosine and nucleotide.

3. The method of claim 1, wherein the amino acid is glutamic acid, and the promoter for the biosynthesizing gene is selected.from the group consisting of a promoter for glutamate dehydrogenase (GDH) gene, a promoter for citrate synthase (CS)

20. gene, .a promoter for isocitrate synthase (CDH) gene, a promoter for pyruvate S° dehydrogenase (PDH) gene and a promoter for aconitase (ACO)-producing gene. 4. The method of claim 3, wherein the promoter for glutamate dehydrogenase (GDH) gene has a DNA sequence selected from the group consisting of at least one DNA sequence selected from the group consisting of CGGTCA, TTGTCA, TTGACA and TTGCCA in -35 region (ii) TATAAT sequence or the same TATAAT sequence but in which the base of ATAAT is replaced with another base in -10 region, and (iii) a combination of and wherein the sequence does not inhibit the function of the promoter. The method of claim 4, wherein the promoter for GDH has TGGTCA in -35 region, 7 Si and TATAAT in -10 region; or TTGTCA in -35 region, and TATAAT in -10 region. 6. The method of claim 3, wherein the promoter for CS has TTGACA sequence in region, (ii) TATAAT sequence in -10 region, or (iii) a sequence of the combination of and and the sequence does not inhibit the function of the promoter. 7. The method of claim 3, wherein at least one of first and second promoters for ICDH has TTGCCA or TTGACA sequence in -35 region, (ii) TATAAT sequence in region, or (iii) a sequence of the combination of and and the sequence does not inhibit the function of the promoter. 8. The method of claim 3, wherein the promoter for PDH has TTGCCA sequence 10 in -35 region, (ii) TATAAT sequence in -10 region, or (iii) a sequence of the combination of and and the sequence does not inhibit the function of the promoter. 9. The method of claim 1, wherein the amino acid is arginine, and the promoter for the biosynthesizing gene is a promoter for argininosuccinate synthase. 10. The method of claim 9, wherein the promoter for the argininosuccinate synthase has a DNA sequence selected from the group consisting of at least one DNA tl sequence selected from the group consisting of TTGCCA, TTGCTA and TTGTCA in region, (ii) TATAAT sequence or TATAAT sequence but in which the base of ATAAC is replaced with another base in -10 region, and (iii) a combination of and (ii) and the sequence does not inhibit the promoter function. 11. The method of claim 10, wherein the promoter for the argininosuccinate synthase has a DNA sequence selected from the group consisting of TTGTCA in region, (ii) TATAAT sequence in -10 region, and (iii) a combination of and (ii). 12. A method of producing an amino acid or nucleic acid by fermentation, which comprises the steps of culturing a coryneform bacterium constructed by the method of any one claims 1 to 11 and having an improved amino acid- or nucleic acid-productivity, in a culture medium to form and thereby to accumulate the intended amino acid or nucleic acid in the culture medium, and collecting it from the culture medium. l S 9 1 OPF\CP( Dated this 7th day of June 2001 AJINOMOTO CO.. INC. By their Patent Attorneys GRIFFITH HACK C. C ~~uSr< z 92