AU757594B2 - 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

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant: AJINOMOTO CO., INC.

Invention Title: 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 C. 9

C

The following statement is a full description of this invention, including the best method of performing it known to me/us:

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 S. ~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 genewith 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.

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

The present invention also relates to a Corynebacterium strain having the above-described gene and capable of producing 1-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 -4efficiently solved by introducing a specific mutation into -35 region or region which is a specific region of the promoter.

According to a first aspect of the present invention, there is provided a glutamic acid synthesizing gene having a promoter which has a sequence selected from the group consisting of: CGGTCA, TTGTCA, TTGACA or TTGCCA in -35 region; (ii) TATAAT sequence or a derivative thereof in which one or more base of ATAAT is replaced with another base in -10 region; (iii) a combination of and and 10 (iv) TGGTCA in -35 region and TATAAT in -10 region, wherein the sequence does not inhibit the function of the promoter.

According to a second aspect of the present invention, there is provided an arginine synthesizing gene having the promoter which has a sequence selected from the group consisting of: TTGCCA, TTGCTA and TTGTCA in -35 region; (ii) TATAAT sequence or a derivative thereof in which one or ,more base of ATAAT is replaced with another base in -10 region, and (iii) a combination of and (ii), wherein the sequence does not inhibit the promoter function.

The invention also encompasses a coryneform bacterium having either one of their genes.

The present invention also provides a process for producing Lglutamic acid by the fermentation, which comprises the steps of culturing a coryneform L-glutamic H:\LeanneH\keep\speci\P42457.doc 6/11/02 acid-producing microorganism as described above in a liquid medium to form and also to accumulate L-glutamic acid in the medium, and collecting Lglutamic 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.

10 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 ATCC 14067 eT R4 Corynebacterium melassecola ATCC 17965 H: \LeanneH\keep\speci\P42457 .doc 6/11/02 Brevibacterium saccharolyticum ATCC14066 Brevibacterium immariophilum ATCC14068 Brevibacterium roseum ATCC13825 Brevibacteriurn thiogenitalis ATCC19240 Microbacterium ammoniaphilum ATCC 15354 Corynebacterium thermnoaminogenes AJ 12310(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 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 biosynthesis of glutamic acid.

5*'j5 Especially, "glutamic acid-synthesizing gene" includes glutamate dehydrogenase S (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, 20 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, 6 o* e* 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, y 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.

15 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 20 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 .15 186:339-346(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.

S. 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: 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 S 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 20 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 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 S DNA sequence selected from the group consisting of TTGCCA, TTGCTA, and S 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 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 37C.

*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 20 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 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: .20 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

S

Strain Promoter sequence Specific activity of GDH Relative -10 value ATCC13869 TGGTCA CATAAT 7.7 0.1 /pGDH TGGTCA CATAAT 82.7 /p6-2 CGGTCA CATAAT 33.1 0.4 /p6-4 TGGTCA TATAA.T 225.9 2.7 ATCC 1 3 86 9 /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:

S

10 0*S*

S

5*

S

.5.

Sequence No. 1 5'-TTAATTCTTTGTGGTCATATCTGCGACACTGC 2

CGGTCA

3.

TGGTCA

4.

TTGACA

5.

TTGCCA

6.

TTGTCA

CATAATTTGAACGT-3'

CATAAT

TATAAT

TATAAT

TATAAT

TATATT

*0.

*04 0@S 4r 15 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°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 AJ13029 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 31.50C for 24 hours to obtain the bacterial cells. The cells were treated with 250g/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 0 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 S 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 Table 2 CM2B agar medium

S.

00 6 0S@* OS@@.2 0* 0 0 0 Ingredient Polypeptone (Nippon Seiyaku Co.) Yeast extract (Difco Co.) NaCI d-Biotin Agar (fH 7.2: adiusted with KOH Concentration 10 pg/I Component Glucose MgSO4-7H 2 0 Table 3 Agar medium Amount in one liter of water 1g FeS0 4 -7H 2 0 0.01g MnS0 4 4-6H 2 0 0.01 g Thiamine hydrochloride 0.2 mg d-Biotin 0.05 mg

(NH

4 2SO 4 5 g Na 2

HPO

4 -12H 2 0 7.1g

KH

2 P0 4 1.36g Agar 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 0 C and then the culture was continued further for about 24 hours.

20 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).

Ingredient Glucose

KH

2

PO

4 MgSO 4 -7H 2 0 FeSO 4 -7H 2 0 MnSO 4 -4H 2 0 (N H 4 2 S0 4 Soybean protein hydrolyzate solution Thiamine hydrochloride Biotin Antifoaming agent CaCO, Table 4 Aledium A Mec 3 g/dl 0. 14 g/dl 0.04 g/dl 0.001 g/dl 0.001 g/dl 1.5 g/dl 1.5 mI/di 0.2 mg/I 0.3 mg/I 0. 05m I/I 5 gd/I adjusted with KOH) ium

B

5 g/dl 0. 14 g/dl 0.04 g/dl 0.001 g/dl 0.001 g/dl 2.5 g/dl 0.38 mI/dI 0. 2 mg/I 0.3 mg/I J. 5m I/I 5 gd/I *0 *0 0 0 ,20 0* b *0*0 0f, Table 5. Glutamic acid formation and GDH activity of mutant strains Strain AJ1 3029 FGR1 FGR2 G lu(a/dl) 2.6 2.9 GDH specific activity 7.7 23.1 Relative value 259 3.4 Table 6. DNA sequences in GDH promoter region of mutant strains Strain GDH promoter sequence i0 AJ1 3029 FGR1

TGGTCA

TGGTCA

TTCTGTGCGACACTGC

TTCTGTGCGACACTGC

CATAAT

TATAAT

FGR2 TTGTCA T T T Q A r A Tr-r TATAAT 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 pg of the chromosomal DNA, 10 pmol of each of the oligonucleotides, 8PI of S dNTP mixture (2.5 mM each), 5l1 of 10xLa Taq Buffer (Takara Shuzo Co., Ltd.) and 2 U of La Taq (Takara Shuzo Co., Ltd.) to obtain 501l 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 ,'20 extention at 72°C 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/1 of bactoyeast extract, 5 g/I of NaCI and 15 g/l of agar; pH 7.2) containing 10 pg/ml of IPTG (isopropyl-p-D-thiogalactopyranoside), 4 0p 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 gltA promoter: Mutan-Super Express Km (Takara Shuzo Co., Ltd.) was used for the introduction of mutation into gltA 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 pig/ml of IPTG, 40pg/ml of X-Gal and 25 4g/ml of 20 kanamycin. After overnight incubation, white colonies were takeFi 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 transformants, plasmid DNA was prepared. The sequence of gItA 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 gItA 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 pKF19CS4 TTGACA

TATAAT

o 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 '20 other hand, plasmid pSFK6 (Japanese Patent Application No.'1-69896) having a replication origin derived from plasmid pAM330 which can autonomously replicate in a S 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 gltA. After the completion of the ligation, transformation was conducted with competent cells of E. coli JM109. The cells was spread on the L-medium plates containing 10 ig/ml of IPTG, 40 g/ml of X-Gal and 25 pig/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 gltA gene were named pSFKC, pSFKC1, pSFKC2 and pSFKC4, respectively.

Determination of CS expression from mutant gItA 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, 10p.g/l of biotin and 15 g/1 of agar; pH containing 25 jg/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 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 mall/ Concentration 50 g/I 1 g/I 0.4 mg/I 10 mg/I 20 mi/I 0.5 mg/I 9 mn/I Thiamine hvdrochloride 2 ma/I 2 mn1l **20 2 Strain Wild type4

BLCSOO

BLCSO1 BLCS02 BR C.rn4 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 48

,'V

Introduction of mutant gItA 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 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 gItA gene were named pSFKTC1, pSFKTC2 and pSFKTC4, respectively.

Introduction of mutant gltA 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 25 pg/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 9 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 gltA 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 gltA 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 0 C. After the formation of colonies, the replicas of the plates were made using CM2B plates containing 25 g/ml of kanamycin and were cultured at 34 0 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 .20 by the plasmid was remained on the chromosome when the plasmid and duplicated gltA gene were excised from the chromosome.

Determination of activity of citrate synthase of mutant gItA 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/mg 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 gItA 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 i 15 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 370C 8 hours after the initiation of the culture. The culture was terminated when glucose had been completely consumed in 20 to 20 hours, and the quantity of L-glutamic acid formed and accumulated in the culture liquid was determined.

As a result, the larger improverment 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 25 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 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 Concentration Seed culture Main culture 50 g/1 150 g/l 1 g/l 2 g/l 0.4 g/l 1.5 g/l 10 mg/l 15 mg/l 10 mg/l 15 mg/I 20 ml/I 50 mi/I 0.5 mg/l 2 mg/l 2 man/ 3 mnll Thiamine~ hvrohlrde2 T. m/ Table12 1 r ft Strain L-glutamic acid (g/l) FGR2 8.9 GBO1 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 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 g g/ml of IPTG, 40 g g/ml of X-Gal and 40 p 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 *20 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 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 p 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 20 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 p g/ml of X-Gal and 25 p g/ml of kanamycin. After overnight 25 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 transformation of competent cells of E. coli JM109 was conducted with the PCR product. The cells were spread on the L-medium plates containing 25/ 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 pKF18ICDO1 GCGACT TATAAT TTTCCA CACCAT pKF18ICDO2 TTGACA TATAAT TTTCCA CACCAT pKF18ICDO3 TTGACT TAAAGT TTTCCA CACCAT pKF18ICDO4 GCGACT GAAAGT TTTCCA TATAAT pKF18ICD05 GCGACT GAAAGT TTGCCA TATAAT 20 pKF18ICDO6 GCGACT GAAAGT TTGACA TATAAT pKF18ICD25 TTGACA TATAAT TTGCCA TATAAT pKF18ICD26 TTGACA -TATAAT TTGACA TATAAT Plasmid construction for determination of promoter activity: 25 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 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 u 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 *.20 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 i. e. pKF181CD1, pKF181CD2, pKF181CD3, pKF181CD4, pKF181CD5, pKF181CD6, pKF181CD25, pKF181CD26 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 u 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, pNEOICD5, 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 15 (comprising 10 g/l of bactotryptone,10 g/l of bactoyeast extract, 5 g/l of NaCI, 10 g/l of biotin and 15 g/l of agar and having pH 7.0) containing 2 5 p g/ml of kanamycin and 40 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, 20 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 25 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 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

BNEOL

BNEOLI

BNEOLI-1 BNEOLI-2 BNEOLI-3 15 BNEOLI-4 BNEOLI-5 BNEOLI-6 BNEOT,T-2f dABS/min/mg Not detected 42 84 168 80 126 139 84 168 170 Relative activity 0.0 1.9 3.3 4.0 4* 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, 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 containing 10 i 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 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 p g/ml of kanamycin after the dilution to a concentration of 103 to 10 s cfu per plate and cultured at 34°C. The strain having *6 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 0

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, pSFKTI5, 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 goe the plasmid remained on the chromosome.

Determination of isocitrate-dehydrogenase activity of the mutant strains having mutant icd promoter: o..0.20 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 0** 25 absorbance at 340 nm at 30 0 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 GCO1 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 6.2

S.

20 s o* 0* 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.

Table16 Strain L-glutamic acid (g/dl) GB02 9.2 GC01 GC02 GC03 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 15 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 20 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 25 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 follows: 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 '20 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 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 15 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 20 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 25 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- 3 -D-thiogalactopyranoside), 40 p g/ml of X-Gal (5-bromo-4-chloro-3indolyl- 3 -D-galactoside) and 25 p 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 20 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 25 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 i 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 7HO, 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 protein hydrolyzate, 200 'U g of thiamine hydrochloride, 60 p 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 o r°r 20

C

C C 2 r oLI Strain Yield of L-glutamic acid (g/dl) ATCC13869/Psfk 3.6 ATCC13869/pSFKBPDHA 3.8 GC25/pSFK 6 5.1 GC25/pSFKBPDHA 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 3 -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 20 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 25 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 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/l of bactotryptone, 5 g/l of bactoyeast extract, 5 g/l of NaCI and g/l of agar and having pH 7.2) containing 4 0 g/ml of X-Gal (5-bromo-4-chloro-3indolyl-3 -D-galactoside) and 25p g/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 20 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.

25 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, 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/ of bactotryptone, 5 g/l of bactoyeast extract, g/ of NaCI and 15 g/l of agar and having pH 7.2) containing 40,u 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 20 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 sequence, was inserted therein was named pNEOLBPDHApro3510.

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.

0 -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, 3 -galactosidase activities were as shown in Table 18, wherein the enzymatic activity of 3 -galactosidase having the promoter region of pdhA gene was given as 1.

1 2 Table 18 Strain /3 -Galactosidase activity (relative value) ATCC13869/pNEOLBPDHAprol 1 ATCC13869/pNEOLBPDHAprolO 6 ATCC13869/pNEOLBPDHApro351 0 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.

S PCR was conducted by using PCR products obtained by using Seq ID No. 33 and S No.40, 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 20 concentration of these four DNA would be 10 p M in the reactionrcocktail 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 g/ml of IPTG (isopropyl- /3 -D-thiogalactopyranoside), 40 p g/ml of X-Gal (5-bromo-4-chloro-3indolyl- -D-galactoside) and 25 g 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 pSFKTPDHAprol0, respectively.

Preparation of promoter modified strains: Strains having modified pdhA gene promoter were prepared by the .20 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 25 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 i g /ml of kanamycin and cultured at 34 0 C to obtain a a strain caused by once-recombination which contains plasmid 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°C. After the colonies began to appear, the repllicas were made on CM2B medium plates containing 25 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 pSFKTPDHAprol0 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

PO

4 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 p g of thiamine hydrochloride, 60 p 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°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 0 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 9:0*0*:15 2 sot *600 *eat so 20 0

S..

*5 5 0 eS..

*6e 5

C)

S. S 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 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.

50, 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 p 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 g/l of chloramphenicol), and cultured at 31.5 0 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 S g/dl of MnSO 4 5 g/dl of VB,, 5 g /dl of biotin and 45 mg/dl (in terms of N) of 15 soybean hydrolyzate]. After the culture at 31.5°C for 18 hours, 3 -galactosidase activity of the obtained cells was determined as described in Example 4(4).

Since 3 -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

C

o Relative activity (AJ12092/pNEOL-0=1) AJ112092 nd AJI12092/pNEOL-0 AJ112092/pNEOL-1 2.8 AJI12092/pNEOL-2 2.7 AJI12092/pNEOL-3 1.8 AJI12092/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, pO 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 20 these plasmids into the chromosome by homologous recombination could be selected as Cm-resistant strains. Strains in which the mutation-introducing plasmid was S. 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 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.

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 940C 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.

S 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 o.

S 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 g/dl of VB,, 5 p 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 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 AJ112092-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 g g/dl of VB,, 5/j 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 0 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

J_

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/paraG 0.520 1.47 52 0,028

C

9 Example 7 Introduction of mutation into GDH gene promoter region of coryneform glutamate-producing bacterium: 20 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 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.

S S 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 0 C were selected. The transformants were cultured at 34°C to select strains which were resistant to kanamycin at 34 0 C. The fact that a strain is resistant to kanamycin at 34C 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 GA01 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 20 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.

Table 24 *4 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 31.5°C 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 31.5°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 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 MgSO 4 -7H 2 0 FeSO 4 -7H 2 0 MnSO 4 -4H 2 0 Soybean protein hydrolyzate Biotin 15E Thiamine hydroc~hloride Concentration Seed culture Main culture 50 g/l 150 g/l 1 g/l 2 g/l 0.4 g/l 1.5 g/l 10 mg/I 15 mg/I 10 mg/I 15 mg/I 20 mI/I 50 mI/I 0.5 mg/I 2 mg/I 2 ma/I 3 ma/I *0*egO @0*O see 4. S S

SI.S

S

S. S 4 5*

S.

S

Table 26 *fl.

Strain Glu Asp Ala Relative activity Relative (a/dl) (mg/dl) (mg/dl) of GDH value AJ 13029 8.3 49 60 7.7 GA01 9.0 145 152 22.3 2.9 GA02 8.9 153 155 27.0 AJ13029/pGDH 8.8 201 190 82.7 10.7 AJ13029/pAJ220G 7.5 290 590 120.12 15.6 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> JP 10-271786 <151> 1998-9-25 <150> JP 10-271787 <151> 1998-9-25 S <160> 6 <210> 1 <211> 46 <212> nucleic acid <400> 1 ttaattcttt gtggtcatat ctgcgacact gccataattt gaacgt e* <210> 2 <211> 46 2 12> nucleic acid <400> 2 ttaattcttt gcggtcatat ctgcgacact gccataattt gaacgt <210> 3 <211> 46 <212> nucleic acid <400> 3 ttaattcttt gtggtcatat ctgcgacact qctataattt gaacgt <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 25 <211> <212> nucleic acid S. <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 <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 S <212> nucleic acid <220> primer 4 for introducing a mutation of ICD promoter <400> 19 ggctgaaact gctataatag gcgccagc <210> *'25 <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

<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 S gggatcccgg atgaatgtca <400> 24 <211> 23 <212> nucleic acid <220> primer B for amplifying of nptII -*25 <400> 24 gcccggggtg ggcgaagaac tcc <210> <211> 23 <212> nucleic acid <220> primer for amiplifying of Brevibacterium lactofermentum pdhA gene LA <400> aci gti tci atg ggi cti ggi cc <210> 26

L

<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

tgg atg aga cca cgt gat tct ggc tcg tcc 5 <210> 29 <211> eoo: <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> <211> <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

C

f atg gcc gat caa gca aaa ctt ggt ggt aag Met Ala Asp Gln Ala Lys Leu Gly Gly Lys 1 5 10 ccc tcg gat gac tct aac 48 Pro Ser Asp Asp Ser Asn ttc gcg atg atc cgc gat Phe Ala Met Ile Arg Asp ggc gtg gca tct Gly Val Ala Ser 25 tat ttg aac Tyr Leu Asn gac tca gat 96 Asp Ser Asp r

I

I r25 ccg gag gag Pro Glu Glu acc aac gag Thr Asn Glu atg gat tca ctc gac gga tta Met Asp Ser Leu Asp Gly Leu ctc cag 144 Leu Gin gag tct Glu Ser 50 cgt gca Arg Ala tct cca gaa cgt Ser Pro Glu Arg tct gca aag cgc Ser Ala Lys Arg 70 get cgt tac ctc Ala Arg Tyr Leu atg ctt cgt ttg ctt gag 192 Met Leu Arg Leu Leu Glu gta tct ctt ccc Val Ser Leu Pro 75 cca atg acg tea acc gac 240 Pro Met Thr Ser Thr Asp tac gtc aac acc att cca acc tct atg gaa cct gaa ttc cca ggc gat 288 f Tyr Val Asn Thr Ile Pro Thr Ser Met Giu Pro Glu Phe Pro Gly Asp gag gaa atg gag aag Giu Giu Met Giu Lys 100 cgt tac cgt cgt Arg Tyr Arg Arg 105 tgg att cgc tgg aac gca Trp Ile Arg Trp Asn Ala 110 gcc 336 Al a atc atg gtt cac cgc gct Ile Met Val His Arg Ala 115 cag cga Gin Arg 120 cca ggc atc ggc gtc Pro Gly Ile Giy Val 125 ggc gga cac 384 Gly Gly His att tcc act tac gca ggc gca gcc cct Ile Ser Thr 130 cac ttc ttc His Phe Phe 145 Tyr Ala Gly Ala Ala Pro 135 ctg tac Leu Tyr 140 gaa gtt ggc ttc aac 432 Giu Vai Gly Phe Asn cgc ggc aag gat Arg Gly Lys Asp 150 cac cca ggc His Pro Giy 155 ggc ggc gac cag atc Gly Gly Asp Gin Ile 160 ttc 480 Phe gag 528 Glu ttc cag ggc cac gca tca cca ggt Phe Gin Gly His Ala Ser Pro Gly 165 atg tac gca Met Tyr Ala 170 cgt gca ttc atg Arg Ala Phe M et 175 25 ggt cgc ctt tct Gly Arg Leu Ser 180 cgt gag cag ggt Arg Glu Gin Giy 195 gac ttc tgg gag Asp Phe Trp Giu 210 gaa gac gat ctc gat Giu Asp Asp Leu Asp 185 ggc att ccg tcc tac Gly Ile Pro Ser Tyr 200 ggc ttc cgt cag Gly Phe Arg Gin 190 cct cac cca cac Pro His Pro His 205 gaa gtt tcc 576 Glu Val Ser ggt atg aag 624 Gly Met Lys cca atg gat 672 Pro Met Asp ttc cca act gtg tcc atg ggt ctt ggc Phe Pro Thr Val Ser Met Gly Leu Gly 215 220 gcc att tac cag gca cgt ttc aac cgc tac ctc gaa aac cgt ggc atc 720 Ile Tyr Gin Ala Arg 230 Phe Asn Arg Tyr Leu 235 Giu Asn Arg Gly Ile 240 aag gac acc tct gac Lys Asp Thr Ser Asp 245 atg gac gag cca gaa Met Asp Glu Pro Giu 260 aac ctg gac aac ctg Asn Leu Asp Asn Leu 275 cag cac gtc tgg gcc ttc Gin His Val Trp Ala Phe 250 ctt ggc gac Leu Gly Asp 255 ggc gaa 768 Giy Giu tca cgt ggt Ser Arg Gly 265 acc ttc gtg Thr Phe Val 280 ctc atc Leu Ile gtt aac Val Asn cag cag gct gca ctg aac Gin Gin Ala Ala Leu Asn 270 tgc aac ctg cag cgt ctc Cys Asn Leu Gin Arg Leu 285 816 864

I

,15 gac gga Asp Gly 290 ttc ttc Phe Phe 305 cct gtc cgc ggt aac Pro Vai Arg Gly Asn 295 cgt ggc gca ggc tgg Arg Gly Ala Gly Trp 310 gat gaa ctt ctg gag Asp Giu Leu Leu Giu 325 acc aag atc atc Thr Lys Ile Ile 300 tct gtg atc aag Ser Val Ile Lys 315 cag gaa ctc gag tcc 912 Gin Giu Leu Giu Ser gtt gtt tgg ggt cgc 960 Val Val Trp Gly Arg 320 gag tgg Giu Trp 25 aag gac Lys Asp 330 cag gat ggt gca ctt gtt Gin Asp Gly Ala Leu Val 335 gag 1008 Giu atc atg aac aac acc tcc gat ggt gac Ile Met Asn Asn Thr Ser Asp Gly Asp 340 345 tac cag acc ttc aag gct aac 1056 Tyr Gin Thr Phe Lys Ala Asn 350 gac ggc gca tat gtt cgt gag cac ttc ttc Asp Gly Ala -Tyr Val Arg Giu His Phe Phe 355 360 gca aag ctc gtt gag aac atg acc gac gaa Ala Lys Leu Val Giu Asn Met Thr Asp Giu gga Gi y cgt gac cca Arg Asp Pro cgc acc 1104 Arg Thr gaa atc tgg aag Giu Ile Trp Lys ctg cca 1152 Leu Pro 375 380 cgt ggc Arg Gly 385 ggo cac gat tac ogc aag gtt tac gca goc tac aag oga gct 1200 Gly His Asp Tyr Arg Lys Vai Tyr Ala Ala Tyr Lys Arg Ala 390 395 400 ott gag Leu Giu aco aag gat cgc cca Thr Lys Asp Arg Pro 405 aco gto atc Thr Val Ile 410 ott got cac acc att aag 1248 Leu Ala His Thr Ile Lys 415 ggo tao gga Gly Tyr Gly 4; oto ggc cac Leu Gly His aac ttc gaa Asn Phe Giu ggc cgt aao goa acc Gly Arg Asn Ala Thr 430 cac cag 1296 His Gin 25 atg aag aag ctg Met Lys Lys Leu 435 acg ott Thr Leu gat gat otg aag Asp Asp Leu Lys 440 ttg ttc ogc gao aag cag Leu Phe Arg Asp Lys Gin 1344 ggo ato Gly Ile 450 oot tao Pro Tyr 465 ooa ato aoo gat g Pro Ile Thr Asp G 455 rag oag otg gag aag gat ;iu Gin Leu Giu Lys Asp 460 oot tao ott Pro Tyr Leu oot 1392 Pro tao oao ooa ggt gaa Tyr His Pro Gly Giu 470 gao got oot gaa ato Asp Ala Pro Giu Ile 475 aag tao atg aag 1440 Lys Tyr Met Lys 480 00 0 00 000000 0 gaa ogt ogo goa gog oto ggt Giu Arg Arg Ala Ala Leu Gly 485 ggo tao Gly Tyr 490 otg ooa gag ogt ogt gag- aao 1488 Leu Pro Giu Arg Arg Glu Asn 495 TAG GAT CGA AT Tyr Asp Pro Il 500 'T GAG GTT GGA .e Gin Val Pro GGA GTG GAT Pro Leu Asp AAG GTT GG TGT Lys Leu Arg Ser GTG GGT 1536 Val Arg aag ggo too ggo aag oag oag Lys Gly Ser Gly Lys Gin Gin ato got aoo Ile Ala Thr act atg gog aot Thr Met Ala Thr gtt ogt 1584 Val Arg aco ttc Thr Phe 530 aag gaa otg atg cgc gat aag ggc ttg got gat cgc ctt gtc 1632 Lys Giu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val 535 540 oca atc Pro Ile 545 att oct gat gag gca cgt acc ttc ggt ctt Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu 550 555 gao tct tgg Asp Ser Trp 560 tac gtg cct Tyr Val Pro 575 tto 1680 Phe oca aco ttg aag ato tao aao Pro Thr Leu Lys Ile Tyr Asn 565 oog oao Pro His 570 ggt oag aao Gly Gin Asn gtt 1728 Val1 *025* gao oao gao otg Asp His Asp Leu 580 otg oao gaa ggo Leu His Giu Giy 595 gog ggt aco too Ala Gly Thr Ser 610 atg oto Met Leu ato aao Ile Asn too tao ogt Ser Tyr Arg 585 gag goa oct gaa gga oag Giu Aia Pro Giu Gly Gin 590 ato 1776 Ile gag got Giu Ala 600 ggt too gtg goa tog Gly Ser Vai Ala Ser 605 tto ato got 1824 Phe Ile Ala tac goc aco Tyr Ala Thr 615 cac ggo aag gc His Gly Lys Ala 620 tto oag ogo aco Phe Gin Arg Thr 635 atg att Met Ile ocg otg tao 1872 Pro Leu Tyr *0 0 0 00 000000 a ato tto tao tog atg tto gga Ile Phe Tyr Ser Met Phe Gly 625 -630 goa goa goc gat cag atg goa Ala Ala- Ala Asp Gin Met Ala 645 ggt gao Gly Asp t

S

00 ato er Ile 640 ct aco la Thr tgg 1920 Trp ogt ggo ttc oto ttg ggo g Arg Gly Phe Leu Leu Gly A 650 655 gca 1968 Al a ggt ogo aco aco otg aco ggt Gly Arg Thr Thr Leu Thr Gly gaa ggo oto cag cac atg gat gga cac 2016 Giu Gly Leu Gin His Met Asp Gly His tcc cct gtc ttg gct tcc acc aac Ser Pro Vai Leu Ala Ser Thr Asn 675 680 gag ggt gtc gag acc tac gac cca 2064 Glu Gly Val Glu Thr Tyr Asp Pro 685 tcc ttt gcg tac gag atc gca cac ctg gtt cac cgt ggc ato gac cgc 2112 Ser Phe Ala Tyr Giu Ile Ala His 690 695 Leu Val His 700 Arg Gly Ile Asp Arg atg tac ggc Met Tyr Gly 705 cca ggc aag ggt gaa Pro Gly Lys Gly Giu 710 gat gtt Asp Val 715 atc tac tac atc acc atc 2160 Ile Tyr Tyr Ile Thr Ile 720 tac aac gag cca acc Tyr Asn Glu Pro Thr 725 cca cag cca gct Pro Gin Pro Ala 730 ggc atc tac ctc Gly Ile Tyr Leu 745 gag cca gaa gga ctg gac gta 2208 Giu Pro Giu Gly Leu Asp Val 735 gaa ggc Giu Gly ctg cac aag Leu His Lys 740 tac tcc cgc ggt Tyr Ser Arg Gly 750 gaa ggc acc 2256 Giu Gly Thr cag tgg gct 2304 Gin Trp Ala ggc cat gag gca Giy His Giu Ala 755 aac atc ttg gct Asn Ile Leu Ala 760 tcc ggt gtt ggt atg Ser Gly Val Gly Met 765 ctc aag gct Leu Lys Ala 770 att tac tcc Ile Tyr Ser 785 gca tcc atc ctt gag gct gac tac gga gtt cgt gcc aac 2352 Ala Ser Ile Leu Giu Ala Asp Tyr Gly Vai Arg Ala Asn 775 780 gct act tct tgg gtt aac ttg gct cgc gat Aia Thr Ser Trp Val Asn Leu Ala Arg Asp 790 795 ggc gct gct 2400 Gly Ala Ala 800 cgt aac aag gca Arg Asn Lys Ala cag ctg cgc aac cca ggt gca gat gct ggc gag gca 2448 Gin Leu Arg Asn Pro Gly Ala Asp Ala Gly Giu Ala 805 810 ttc gta acc acc Phe Val Thr Thr 820 tct gac ttc tcc Ser Asp Phe Ser 835 cag ctg aag cag acc tcc ggc cca tac gtt gca gtg 2496 Gin Leu Lys Gin Thr Ser Gly Pro Tyr Val Ala Vai 825 830 act gat ctg cca aac Thr Asp Leu Pro Asn 840 cag ato cgt gaa Gin Ile Arg Giu 845 ggc ttc ggt ttc Gly Phe Gly Phe 860 tgg gtc cca 2544 Trp Val Pro ggc gac tac Gly Asp Tyr 850 acc gtt ctc Thr Val Leu 85E ggt gca gat Gly Aia Asp tct gat acc 2592 Ser Asp. Thr fee..: *eo4 09 .09.

04 9 009: 00 9.

0.99 foe* cgc cca gct Arg Pro Aia 865 gct cgt cgc ttc ttc aac Aia Arg Arg Phe Phe Asn 870 atc gac gct gag Ile Asp Ala Giu 875 tcc att gtt 2640 Ser Ile Val 880 gtt gca gtg ctg aac tcc ctg gca Val Ala Val Leu Asn Ser Leu Aia 885 gtt gct gct cag gct gct gag aag Val Ala Ala Gin Ala Aia Giu Lys 900 905 cgc Arq 890 gaa ggc aag atc gac gtc tcc 2688 Glu Giy Lys Ile Asp Vai Ser 895 ttc aag ttg gat gat Phe Lys Leu Asp Asp 910 cct acg agt 2736 Pro Thr Ser .9 9 *9 9 99 .999..

9 9 gtt tcc Val Ser gta gat cca aac gct cct gag gaa Val Asp Pro Asn Ala Pro Glu Giu 2766 <210> 32 <2-11> 8556 <212> nucleic acid <213> Brevibacterium lactofermentum ATCC13869 6

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<400> 32 tcacgttacg cgcttctaag .tcatgccact ttqcgctaga taggccattt ccgaatccac tcatcgacgc tcaaagaaag tagtagcgga tcaaagagac atcctcgtca tcgcctaatg tcgcgcaaga tccttggggt caacgcaacg aggttctgac taatttcgaa gaaaaacagg ccctgacggg taaaggcgcg tactaacaca gttaaggctt ccgccttggt gggctccttc agcggatgga gcacgacatc cgatggactc gacccqattg gagcgtcggc ggattcaaca ggtactttga atcaatqaat acaattctct cagacctgta gcgatcaacz cccgtctcat atcttggqgt cactaattac tgatgtggtg atcgtctaaa aactttacgg cggagtggca agctttagat cttgattgat gcgatttcgc gaattcacat aaaaggccaa gttttattac atgcagagaa gaggtgcgaa gtgctgggtt ccacgataag ctcgaaccgc cacgtgcttt caatctccac cttgtttcca ctgcacgaga tttaccaatg atccacaagt gcacagctcg gctgatcagc aatgccgagt cactttaata tgaaaccgcg accacctttc :caatcactg :caaaataat ;ttttatgcg kccgcaaccac :tttgcacctc itctcggtatt qqcaccgctt taqatcatat aggcctgggc ctcatcgaca cgtgcagcgg gccgggcqag gggctgttta aaacggctcg gtgaagagac atccgggcgg caatcggtcg gccttggagg gcaagttgtc ttctcgcgca tttcttaaaa cgacctgctg tctagaacca agacctaaaa cgctggacga cctgccagtt ccaggagtca ccgtcgacga tcggtttctt gtgtcggaat agttgagcat atcctcctcg gggctattga cctggaattt gccaqcgatt 4gtggctaga attcgcgtca tacqagaaga tctccaaacg ccattctgtg aggatatggc agatcttctg ataaaaaccc aagcatggtc gtgacacgta tgacgtcaat gaatgaagtg gaccacgtaa agacgggcac *gcaatcgtcc cgtcacggtc tttatcgacg ctggcccagg ctccagttga aatagatacc caaatcaggc gaaaaccact agttggtgat gtcagatcaa gtcgagaagc aaatattcgc atctagacat cqaggcgtgc cgcgtggagt aaatttcacc tgatdtggca tggaatggaa gcgcgccgca cctcagtatc tacccaatgc gtaccgatgt cttatcgtgg cctgcttcta ggtgtaaacc agctgctctt ccttggtggc ctcgaaagca tcgctgctca ggccgtggaa ggcaagaacc ttgccaatta gtgctgattc agagataact acacccagat acgaccattcI gatcgccgtc-atcctcgcgc catcgagtag ttcctcaccg cttcatccca tccaatttctt aatcctgggc accttgcttgz tgtgggcccc gatgtgttttt tatatccgaa ttgcacatta c tttccttttc ctcccccttt a aacttttcga gttttcagtc t tttttcatca aaccctcacc a aacgtgagag aaacatcaca t agaccaagag aggtgctttt gatagttttc aaacctgact actaactccg gacgagaaga aatgcagttg ctagtggatc ccaggggaca ggagtctcct gaattacagc gcgctgcaag ctcgatgcaa gaccaaggta ccttaaattc cggatcaact gcccatgagc tatttgtgcg ccagctgagc acgagtgaaa attagcgatt ccgagacgtt ccagg4agcca :cagcgaggg caccagagca ittgcatctt :gaacgatat ictgcgcccg :cgattacat :cg tcc aa cc Lcgctcaaga :tggatttcc aaaggacat :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 005 aactacccga gtagaaatgt cctgaaatcc gtgttgcgta caattaggta tccaacagga taattctttg caaaactttg caaagggtaa tgaacatgca gctagtttcc tctttaaaaa atccacaaca attgccagga agcacaccga ttgatggata cagtgagcgc accactcccc ttacgtcaca gtctgtaaaa caaatcttcg tccttgttaa taacttatgc gttgacccat tcgtgcactt cggtgtgcca cgaccaagaa tgggaccggg aaaccgggac gtataaacga aataaaacat ggtgtggaa atg gcc gat caa gca aaa ctt ggt ggt aag ccc Met Ala Asp Gin Ala Lys Leu Gly Giy Lys Pro 2100 2160 2220 2280 2340 2392 tcg gat gac Ser Asp Asp tct aac ttc gcg atg Ser Asn Phe Ala Met atc Ile 20 cgc gat ggc gtg gca tct tat Arg Asp Gly Val Ala Ser Tyr 2440 ttg aac Leu Asn gac gga Asp Gly gac Asp 30 tca gat Ser Asp ccg gag gag Pro Giu Giu 35 gag tct tct Giu Ser Ser 50 acc aac gag tgg atg Thr Asn Giu Trp Met cca gaa cgt gct cgt Pro Giu Arg Ala Arg gat tca ctc Asp Ser Leu tac ctc atg Tyr Leu Met 2488 2536 tta ctc cag Leu Leu Gin ctt Leu cgt ttg ctt gag Arg Leu Leu Giu gca tct gca aag Ala Ser Ala Lys cgc gta tct ctt ccc cca Arg Val Ser Leu Pro Pro 70 cca acc tct atg gaa cct Pro Thr Ser Met Giu Pro 2584 2632 atg acg Met Thr tca acc gac Ser Thr Asp cca ggc gat Pro Gly Asp tac gtc aac Tyr Vai Asn acc att Thr Ile 85 gaa ttc Giu Phe gag gaa atg Giu Giu Met gag aag cgt tac cgt cgt tgg att Giu Lys Arg Tyr Arg Arg Trp Ile 100 105 2680 cgc tgg aac gca gcc Arg Trp Asn Ala Ala 110 atc atg gtt cac cgc Ile Met Val His Arg 115 gct cag cga cca ggc atc Ala Gin Arg Pro Gly Ile 120 2728 ggc gtc ggc gga cac att tcc act tac gca ggc Gly Val Gly Gly His Ile Ser Thr Tyr Ala Gly 125 130 gca gcc cct ctg tac Ala Ala Pro Leu Tyr 135 gat cac cca qgc ggc Asp His Pro Gly Gly 155 2776 gaa gtt ggc ttc aac cac ttc ttc cgc Glu Val Gly Phe Asn His Phe Phe Arg 140 145 ggc aag Gly Lys 150 2824 ggc gac cag atc Gly Asp Gin Ile ttc ttc Phe Phe 160 gag ggt Giu Gly cag ggc cac gca Gin Gly His Ala 165 cgc ctt tct gaa Arg Leu Ser Giu 180 tca cca ggt Ser Pro Gly atg tac gca Met Tyr Ala 170 2872 cgt gca ttc atg Arg Ala Phe Met 175 gac gat ctc gat ggc ttc Asp Asp Leu Asp Gly Phe 185 2920 cgt cag gaa Arg Gin Giu 190 cca cac ggt Pro His Gly 205 gtt tcC cgt Val Ser Arg gag cag ggt Glu Gin Gly 195 ggc att ccg tcc tac cct cac Gly Ile Pro Ser Tyr Pro His 200 2968 atg aag gac Met Lys Asp tgg gag ttc cca act gtg tcc atg ggt Trp Giu Phe Pro Thr Vai Ser Met Gly 215 3016 S. S

SS

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ggc cca atg gat gcc att tac Gly Pro Met Asp Ala Ile Tyr 225 cag gca cgt Gin Ala Arg 230 ttc aac Phe Asn cgc tac ctc Arg Tyr Leu 235 3064 gaa aac cgt ggc atc aag gac acc tct gac cag cac gtc tgg gcc ttc 3112 Giu Asn Arg Gly Ile Lys Asp Thr 240 Ser Asp Gin His Vai Trp, 245 Ala Phe 250 atc cag Ile Gin ctt ggc gac ggc gaa atg gac gag cca gaa Leu Gly Asp Gly Giu Met Asp Giu Pro Glu 255 260 tca cgt ggt Ser Arg Gly ctc Leu 265 3160 cag gct gca ctg aac aac ctg gac aac ctg acc ttc gtg gtt aac tgc Gin Ala Ala Leu Asn Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys 270 275 280 3208 aac ctg cag Asn Leu Gin 285 cgt ctc gac Arg Leu Asp ccL gtc cgc ggt aac acc aag atc atc Pro Val Arg Gly Asn Thr Lys Ile Ile 295 3256 cag Gin 300 gaa ctc gag tcc ttc Giu Leu Giu Ser Phe 305 ttc cgt ggc gca Phe Arg Gly Aia tgg tct gtg atc aag Trp Ser Val Ile Lys 315 3304 gtt gtt tgg ggt cgc gag tgg gat gaa Vai Val Trp Giy Arg Giu Trp Asp Giu 320 ctg gag aag gac Leu Giu Lys Asp 000060 0 *0 .0 0* *0 cag gat Gin Asp 330 tac cag Tyr Gin 3352 3400 ggt gca ctt Gly Ala Leu gag atc atg aac aac acc tcc gat ggt Glu Ile Met Asn Asn Thr Ser Asp Gly 340 gac Asp 345 acc ttc aag Thr Phe Lys 350 cgt gac cca Arg Asp Pro 365 gct aac gac ggc gca tat Ala Asn Asp Gly Ala Tyr 355 gtt cgt Val Arg gag aac Giu Asn gag cac Giu His 360 ttc ttc gga Phe Phe Gly 3448 3496 cgc acc gca aag Arg Thr Ala Lys 370 ctc gtt Leu Val atg acc gac Met Thr Asp 375 gaa gaa Giu Giu atc tgg aag Ile Trp Lys 380 ctg cca cgt Leu Pro Arg 385 ggc ggc cac Gly Gly His gat tac Asp Tyr cgc aag Arg Lys gtt tac gca Val Tyr Ala 395 3544 gcc tac aag cga gct ctt Ala Tyr Lys Arg Ala Leu 400 gag acc aag gat cgc Giu Thr Lys Asp Arg 405 cca acc gtc atc ctt Pro Thr Val Ile Leu 410 3592 gct cac acc Ala His Thr att. aag ggc tac gga ctc ggc Ile Lys Gly Tyr Gly Leu Gly cac aac ttc qaa ggc cgt His Asn Phe Glu Gly Arg 425 ctt gat gat ctg aag ttg Leu Asp Asp Leu Lys Leu 440 3640 aac gca acc Asn Ala Thr 430 cac cag atg aag aag ctg His Gin Met Lys Lys Leu 435 3688 ttc cgc gac Phe Arg Asp 445 aag cag ggc atc Lys Gin Giy Ile 450 cca atc acc gat Pro Ile Thr Asp gag cag Giu Gin 455 ctg gag aag Leu Giu Lys 3736 ga t Asp 460 cct tac ctt cct cct tac tac Pro Tyr Leu Pro Pro Tyr Tyr 465 cac cca ggt His Pro Giy 470 a.

atc aag tac atg aag Ile Lys Tyr Met Lys 480 gaa cgt cgc gca gcg ctc Giu Arg Arg Ala Aia Leu 485 gaa gac gct. =t gaa Giu Asp Ala Pro Giu 475 ggt ggc tac ctg cca Giy Gly Tyr Leu Pro 490 cca cca ctg gat aag Pro Pro Leu Asp Lys 505 3784 3832 gag cgt cgt gag Giu Arg Arg Giu aac tac gat Asn Tyr Asp oca att.

Pro Ile 500 cag gtt Gin Val 3880 3928 ctt cgc tct gtc Leu Arg Ser Val 510 cgt aag ggc tcc ggc Arg Lys Gly Ser Gly 515 aag cag cag atc Lys Gin Gin Ile 520 gct acc act Ala Thr Thr atg gog act gtt cgt Met Ala Thr Val Arg 525 acc ttc Thr Phe 530 aag gaa ctg Lys Giu Leu atg cgc gat Met Arg Asp aag ggc ttg Lys Gly Leu 3976 gct Ala 540 gat cgc ctt gtc Asp Arg Leu Vai cca atc att cct gat Pro Ile Ile Pro Asp 545 gag gca cgt acc ttc ggt Giu Ala Arg Thr Phe Gly 4024 ott gac Leu Asp tct tgg tto cca acc ttg Ser Trp Phe Pro Thr Leu 560 aag atc tac aac cog cac ggt cag Lys Ile Tyr Asn Pro His Gly Gin 565 570 4072 aao tao Asn Tyr gtg cot gtt Val Pro Vai 575 gao cao gao Asp His Asp otg Leu 580 atg otc toc tao Met Leu Ser Tyr ogt gag gca Arg Giu Ala 585 ggt too gtg Gly Ser Val 4120 4168 cot gaa gga Pro Giu Gly 590 gca tog tto Aia Ser Phe 605 oag ato ctg cac gaa Gin Ile Leu His Glu 595 ggo ato aac gag got Gly Ile Asn Giu Ala 600 ato got Ile Ala gcg ggt Ala Gly 610 aco too tao gc Thr Ser Tyr Ala atg Met 620 att cog otg tao Ile Pro Leu Tyr ato Ile 625 tto tao tog atg ttc Phe Tyr Ser Met Phe 630 ac cac ggo aag gc Thr His Gly Lys Ala 615 gga tto cag cgo aco Gly Phe Gin Arg Thr 635 gca ogt ggc tto otc Ala Arg Gly Phe Leu 650 4216 4264 ggt gao too Gly Asp Ser ttg ggo got Leu Gly Ala cac atg gat His Met Asp 670 ato tgg gca Ile Trp Ala 640 gca ggt Ala Gly gca gco gat Ala Ala Asp cgo aco acc Arg Thr Thr 660 cct gto ttg Pro Val Leu 675 cag atg Gin Met 645 otg aco ggt gaa ggo otc cag Leu Thr Giy Glu Gly Leu Gin 665 got too aoo aac gag ggt gto Ala Ser Thr Asn Giu Gly Vai 680 4312 4360 4408 gga cac too Gly His Ser gag aco tao gac cca too Giu Thr Tyr Asp Pro Ser 685 ttt gog tao gag ato Phe Ala Tyr Giu Ile 690 gca cac otg gtt cao Ala His Leu Val His 695 4456

Z-

cgt ggc ate gac cgc atg tac ggc cca Arg Gly Ile Asp Arg Met Tyr Gly Pro 700 705 ggc aag ggt gaa gat gtt Gly Lys Gly Glu Asp Val 710 ate Ile 715 4504 tac tac ate acc ate tac aac gag Tyr Tyr Ile Thr Ile Tyr Asn Glu 720 cca acc Pro Thr 725 cca cag cca gct gag cca Pro Gin Pro Ala Glu Pro 730 4552 gaa Glu gga ctg Gly Leu gac gta Asp Val 735 gaa ggc ctg Glu Gly Leu cac His 740 aag ggc ate tac Lys Gly Ile Tyr ctc tac tec Leu Tyr Ser 745 4600 o

I

cgc ggt Arg Gly ggt atg Gly Met 765 gaa ggc acc ggc cat gag Glu Gly Thr Gly His Glu 750 755 cag tgg got ctc aag get Gin Trp Ala Leu Lys Ala 770 gca aac atc ttg gct tec ggt gtt Ala Asn Ile Leu Ala Ser Gly Val 760 4648 4696 gca tec atc Ala Ser Ile ctt Leu 775 gag get gac tac Glu Ala Asp Tyr oo* o gga Gly 780 gtt cgt gcc aac Val Arg Ala Asn att tac tec Ile Tyr Ser 785 gct act Ala Thr tgg gtt aac ttg Trp Val Asn Leu gct Ala 795 4744 cgc gat ggc gct get cgt aac aag gca Arg Asp Gly Ala Ala Arg-Asn Lys Ala 800 ctg cgc aac cca ggt gca Leu Arg Asn Pro Gly Ala 810 4792 gat get ggc gag Asp Ala Gly Glu 815 cca tac gtt gca Pro Tyr Val Ala 830 gca ttc gta acc acc Ala Phe Val Thr Thr 820 cag ctg aag cag Gin Leu Lys Gin acc tec ggc Thr Ser Gly 825 4840 gtg tct gac ttc tec act gat Val Ser Asp Phe Ser Thr Asp 835 ctg cca aac cag ate Leu Pro Asn Gin Ile 840 4888 cgt qaa tgg gtc cca ggc gac tac acc gtt ctc Arg Glu Trp Val Pro Gly Asp Tyr Thr Val Leu 845 850 ggt gca Gly Ala 855 gat ggc ttc Asp Gly Phe 4936 ggt Gly 860 ttc tct gat acc cgc Phe Ser Asp Thr Arg 865 gct gag tcc att gtt gtt Ala Giu Ser Ile Val Val 880 cca gct gct cgt cgc Pro Ala Ala Arg Arg 870 gca gtg ctg aac tcc Ala Val Leu Asn Ser 885 gct gct cag gct gct Ala Ala Gin Ala Ala 900 ttc ttc aac atc Phe Phe Asn Ile gac Asp 875 4984 ctg gca cgc gaa gqc Leu Ala Arg Giu Gly 890 aag atc gac gtc Lys Ile Asp Val 895 tcc gtt Ser Val gag aag Giu Lys ttc aag ttg Phe Lys Leu 905 gag gaa taaat Glu Giu 5032 5080 5130 gat gat cct acg agt gtt tcc Asp Asp Pro Thr Ser Vai Ser gta gat cca aac gct cct Val Asp Pro Asn Ala Pro 915 920 cacctcaagg aaagcaagct gcgaactcct 25 tagacaatct atgttgtgcg gtggtcacaa attttaggtt agcaaaccaa tgcttgaatt cgcagcaacg ggatcctgaa ttgttgccgt agcagggtgg atatcgagtt ttggaaattc 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 accgttagaa caacgcgcgg taacatgtct agacgttagt ttgtcagaca aacttcgact aggcgattgc gttccatctg agcgctgcgg agggacaatg tccagaccca agtgcaaagc atggtgagat gcacgcttgt tcaacaagca.

ccgatgacgg cgctcattca taagcctcca ctggcggcgg atgttgcgcc tggtagcctg ccaggcgaat ccacgatctt tgtgctcatc cctgacgctg gctcaaggcg ccaatccacg tcggcgcggt gctctgacat aacggccaga tgacaaaatc cagtgccggg ctgctactgg gattcgtcgt cttgatattg atctgcctgg tgc tgttcc 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 ttcggtgcct tggagaactt cgcacgccac ctcggcagca gcatggcgac aaccaccaag atcgcagaaa atcaggcggt tgaccgaaga acccatcgaa agaaccattg ctgcaccatc gaaccagatg ccatgtgccg tggcacgttc aatacctgcg agcgataaag aagagctgtc aaagttgagc gaactcaaaa acgccctggc acaggcatgg tggcggccaa ccggactcag ataccggaaa tcaacagcct agtggctcag gcgtagacga tgctcaatcg tccaccagct atattctgcg tcagaagcga tcatcaaagg gcatcaagtg gcagcatcag gcgccaacag cttaaagtgt catatgtqaa cctggatatc tttaaaqgcc taacaaacct ttgccttcat acgcgatggc tggcctggcc atatctgcga gacaggqaga ggcccgttgt acgatgagtc aggaggccgg gcgccaccca agcaaaccat ccagtaacga gcagctgttg acacggaacg gcgccqatca gctctgctgc ttgctgggtc gctcggtgta caccgatcac caaccatgtt gctgaatctc cagccttggt tgccgatgga gtgcgatatc ctgccgacac cagttgcctg act gct gaga tgacgtcaac cagggtgctg tggaggtgga taaagatgct tattttcaaa atgtctcatg ccgcaggaca ctagacaccg ccaaagacta atcgttgggg accgacgatg ccaaaataac agccctggta tggtgaggat cgccggtgga ccaaaaatcc cgtcgcgaag tgatgcgcaa cgatcttgcc aaccagcggc ccgcgccagt tcaaatagac tacgcgttcg atcttcgttg ttcgccgtcc atcgataccc agggatctgc gtcaqttgga gttcacggta gacctcatcg ctttgcgtca agaagtaccg tgcatcggta tgctgctgga aatagtcagt gcctgcctct aacaaqctgg gctgtcactg tqcgaqtacc aaggggcaga atttaaacta tqgacaaaat cgatttcctt gagtggggaa tcaaggccaa acagccaaca cgatgcgaga ctgagcgatg tttggtgaaa tcgcagcgac cactgcaacg cggggtgaag gttaaccgac cttggtgtgc agtagccata ggtcaggctg gcccaatgcg gccttatcca atcacqtagg tgcagaatac agggttgcca ttctgctcag acgtagatga gtgacagtct acctgcacct ggaaccagct gtgacgtttc actgcggtga gttgcaacag gcagtgccct tcaaaagctt ttgatgccaa gaatcgcttg ttcqaacggg cagcgtcaac cttgttctac gggtggatag aatcgatcat caccatgaac cgcgcagtgc cagccaacca ccgatcacga agtgcaccgg tccgttgaaa agtccgatat gcgaggatcg aggttatcgg cacaacgcaa agcaaaccgg agaggaggcc ccgtcaacgg acaactccgt aaatctcttg tgaactcagt gcagagccat gtgcgccacc caacctgaac catcaacatc gagtattaac gagagttgac gctgcaaaag cgccaggtgc acaggactgg ccaacacgag gctcgccgtt ccttgaaacc ctcggtaaga agcacgaagc aagaaaacat acatgtctcg cagtcatatg cgggtgcacc taaagaggqa qtttcctcaa tgcgaaaatg cacgcgacgc tgaggccaat caagaccaac caccaaagga cagtgttgag ccgggcctgc tgcgcaacaa tcatggtcaa tgatcatgcc agccagacat tgaggtcggg acaacaatcc agggatctcc tgcagtctcc ctcgccagtc ctcaacagtg cagagaagaa gccgagagct iggacagcccc ctcaccagac ctccaactgc ttcattagaa gatatcagtg 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 gcaqccgcag 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> 20 <212> nucleic acid <220> primer for construction of Brevibacterium lactofermentum pdhA gene LA amplification plasmid 4.

<400> 34 25 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 atc 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 S.<210> 38 S <211> **20 <212> nucleic acid <220> primer for introducing a mutation of Brevibacterium lactofermentum pdhA gene LApromoter <400> 38 gcg ttg acc cat tcg tgc act tcg gtg tgc tat aat tag g <210> 39 e <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 .e <211> <212> nucleic acid <400> 42 agaccgccgg agtatgcaag aacgatgcgg 25 <210> 43 <211> <212> nucleic acid <400> 43 gacttcacca tcaatcatct tcttcaggta <210> 44 <211> <212> nucleic acid <400> 44 accttcgacc agaccctgqc taagggcttt <210> <211> <212> nucleic acid <400> gctaacaagc gcgatcgcga aqctggcaac <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 qacttcacca tcaatcatct tcttc <210> 49 <211> 24 <212> nucleic acid <400> 49 gccaggtaca actgtctgaa 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 25 <211> <212> nucleic acid <400> 53 gttaatcgct tgttaatgca ggcaggtaag gtataatccg <210> 54 <211> <212> nucleic acid <400> 54 gttaatcgct tgtcaatgca ggcaggtaag gtataatccg <210> <211> <212> nucleic acid <400> gggttccagc ctcgtgcgga attcgtggag <210> 56 <211> <212> nucleic acid <400> 56 gcgttaccca gagctggatc ctcgg <210> 57 <211> 16 <212> nucleic acid 20 <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 gacqtgagca t <210> <211> <212> nucleic acid <400> qctcacqtca aattatagca gtgtc <210> 61 <211> 54 <212> nucleic acid <400> 61 ttgttgtcat tctgtgcgac actgctataa tttgaacgtg agcaqttaac agcc <210> 62 <211> 63 <212> nucleic acid <400> 62 :2Ogttaactgct cacqttcaaa ttatagcaft gtcgcacaga. atgacaacaa agaattaaaa ttg <210> 63 <211> <212> nucleic acid <400> 63 gctagcctcg ggagctctct aggag <210> 64 <211> <212> nucleic acid <400> 64 gatctttccc aqactctggc cacgc

Claims (5)

1. A glutamic acid synthesizing gene having a promoter which has a sequence selected from the group consisting of: CGGTCA, TTGTCA, TTGACA or TTGCCA in -35 region; (ii) TATAAT sequence or a derivative thereof in which one or more base of ATAAT is replaced with another base in -10 region; (iii) a combination of and and (iv) TGGTCA in -35 region and TATAAT in -10 region, wherein the 10 sequence does not inhibit the function of the promoter.

2. An arginine synthesizing gene having the promoter which has a sequence selected from the group consisting of: TTGCCA, TTGCTA and TTGTCA in -35 region; (ii) TATAAT sequence or a derivative thereof in which one or more base of ATAAT is replaced with another base in -10 region; and (iii) a combination of and (ii), wherein the sequence does not inhibit the promoter function.

3. A cornyeform glutamic acid-producing bacterium having the glutamate synthesizing gene of claim 1.

4. A cornyeform arginine-producing bacterium having the arginine synthesizing gene of claim 2. A method of producing L-glutamic acid by fermentation, which comprises the steps of culturing a coryneform L-glutamic acid-producing bacterium as claimed in claim 3 in a liquid culture medium to produce H:\LeanneH\keep\speci\P42457.doc 6/11/02 -91 and thereby to accumulate L-glutamic acid in the culture medium, and collecting it from the culture medium.

6. A method of producing an arginine, which comprises the steps of culturing the coryneform bacterium of claim 4 to accumulate the arginine in the culture medium, and collecting it from the culture medium. Dated this 6th day of November 2002 AJINOMOTO CO., INC. By their Patent Attorneys 10 GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia Trade Mark Attorneys of Australia o oooo ***ooo H:\LeaneH\keep\speci\P42457.doc 6/11/02