MXPA00005186A - 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

Abstract

A process for preparing a coryne bacterium having an improved amino acid- or nucleic acid-productivity which comprises constructing mutants of the coryne bacterium by mutating the promoter sequence of an amino acid or nucleic acid biosynthesis gene on the chromosome of the coryne bacterium so as to bring it close to the consensus sequence or by genetic recombination, culturing the resultant mutants and then taking up therefrom a mutant producing the amino acid or the nucleic acid at a high yield. By using this process, the expression dose of a target gene can be adequately strengthened and controlled without resort to any plasmid and a mutant capable of producing an amino acid at a high yield can be constructed by genetic recombination or mutation.

Description

METHOD FOR BUILDING BACTERIAL SCAPS PRODUCERS OF AMINO ACIDS AND METHOD FOR PREPARING AMINO ACIDS BY FERMENTATION WITH PRODUCING BACTERIAL SCAPS BUILT AMINO ACIDS BACKGROUND OF THE INVENTION The present invention relates to a method for constructing a mutant strain capable of producing amino acids in a high yield, and a method for producing L-amino acids by fermentation with the mutant. Methods for constructing mutant strains useful for the production of amino acids by fermentation can be classified in two methods. One of them consists of introducing random mutations in the DNA with a chemical mutagen, and the other consists of genetic recombination. In the latter method, a strain that has an improved ability to produce a targeted substance can be developed by increasing a gene in a metabolic pathway related to the biosynthesis of a target substance, or by weakening an enzyme gene related to destruction. In connection with this, to increase a targeted gene, a plasmid capable of autonomously replicating independently of the chromosome in a cell has been used primarily. However, the method of increasing the target gene with a plasmid has problems. In particular, the degree of enrichment of the intended gene is variable depending on the number of copies of the plasmid itself. Therefore, for some types of genes intended, the copies often too many in number and as a result the expression becomes excessive, the growth is severely inhibited or the capacity to produce the intended substance is reduced. In such a case, although the degree of the increase of the targeted gene can be reduced by using a plasmid of a small number of the copies, the variety of the plasmid in many cases is limited and the intended control of the expression level of the intended gene is impossible. Another problem is that given the replication of the plasmid is often unstable, the plasmid is deleted. For example, the published unexamined Japanese patent application (hereinafter referred to as "JP KOKAI") No. 61-268185 describes a recombinant DNA comprising a DNA fragment containing a glutamate dehydrogenase (GDH) producing gene (GDH) ( glutamate dehydrogenase gene) derived from a glutamate-producing coryneform bacterium and a DNA fragment (plasmid) containing a gene necessary for autonomous replication in the cell. It also discloses that by reproducing the recombinant DNA in a cell, a GDH enrichment strain can be cultured 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 recombinant DNA described above is introduced into Corynebacterium to obtain a strain having the improved enzymatic activity, and L-glutamic acid is produced by fermentation with the strain. However, the production and yield of L-glutamic acid were still unsatisfactory. In this way, it was required to further improve the productivity of L-glutamic acid. It was reported that the requirement had been achieved by introducing a recombinant DNA containing two types of genes, that is, a glutamate dehydrogenase-producing gene derived from a coryneform glutamate-producing bacterium and a beta-dehydrogenase dehydrogenase (ICDH) gene, into a bacterium. glutamate producing corineforme. In addition, JP Kokai No. 6-502548 describes an expression system and secretion system of Corynebacterium comprising a strain of Corynebacterium and a secretory cassette comprising the first functional DNA sequence for expression in the strain, the second sequence of DNA encoding 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 selected protein element of PS1 and PS2 which guarantees the secretion of amino acids, polypeptides and / or proteins. Specifically, the secretion of polypeptides is described therein and in particular the mutagenesis of NTG was conducted with Corynebacterium and a mutant resistant to 4-fluoroglutamate (4FG) which is an analogue to glutamate is selected and subjected to transformation with PCGL141. It discloses that a strain having an increased expression of GDH can be obtained from the analog resistant bacteria. It is also disclosed therein that a mutation was observed in nucleotide sequence No. 251 to No. 266 of the GDH promoter.

BRIEF DESCRIPTION OF THE INVENTION The object of the present invention is to provide a method for constructing a mutant capable of appropriately increasing or controlling the expression of a gene destined without using a plasmid and also capable of producing amino acids in high yield, by gene recombination or mutation. Another object of the present invention is to provide a promoter for GDH capable of imparting a capacity to produce glutamic acid in high yield to a strain of Corynebacterium without severely increasing the amount of aspartic acid and alanine obtained as by-products. Another object of the present invention is to provide a GDH gene having a promoter sequence described above for GDH. A further object of the present invention is to provide a strain of Corynebacterium having the gene described above and capable of producing l-glutamic acid. A further object of the present invention is to provide a method for producing amino acids by fermentation wherein the amino acid producing microorganism thus constructed is used. Yet another object of the present invention is to provide a fermentation method for producing glutamic acid at low cost by increasing the yield of glutamic acid by using a coryneform bacterium producing glutamic acid. The present invention has been completed on the basis of a finding that the problems described above can be solved in an effective manner by varyingly modifying the promoter of amino acid biosynthesizing genes in a chromosome to control the amount of expression of the intended genes, particularly, the invention has been completed on the basis of a finding that the problem described above can be solved in an efficient manner by introducing a specific mutation into the -35 region or the -10 region which is a specific region of the promoter. That is, the present invention provides a method for producing coryneform bacteria having improved amino acid or nucleic acid productivity, comprising the steps of introducing mutations into a promoter sequence of amino acid biosynthesizing genes or nucleic acids on a chromosome of a coryne-forming bacterium. to make it close to a consensus sequence or to introduce a change in a promoter sequence of biosynthesizing genes of amino acids or nucleic acids in a chromosome of coryneform bacteria by recombining genes to make it close to a consensus sequence, to have mutants of the producer corineforme microorganism of amino acids or nucleic acids, cultivating the mutants and selecting a mutant capable of producing the amino acid or nucleic acid destined in large quantity.

The present invention also provides a promoter for a glutamate dehydrogenase (GDH) producing gene, having the sequence of (i) at least one DNA sequence selected from the group consisting of CGGTCA, TTGTCA, TTGACA and TTGCCA in the region - 35, (ii) TAT AAT sequence or the same TAT AAT sequence but in which the base of ATAAT is replaced by another base in region -10, or (iii) a combination of (i) and (ii), where the sequence does not inhibit the function of the promoter. The present invention also provides a glutamate dehydrogenase-producing gene having the promoter described above. The present invention also provides a coryneform microorganism producing L-glutamate having the gene described above. The present invention also provides a process for producing an amino acid by fermentation, comprising the steps of culturing a coryneform bacterium constructed by the method described above and having an improved amino acid producing ability in a medium to form and also to accumulate the amino acid intended in the middle, and collect the amino acid from the medium. The present invention also provides a process for producing L-glutamic acid by fermentation, comprising the steps of culturing a coryneform microorganism producing L-glutamic acid resistant to 4-fluoroglutamic acid in a liquid medium to form and also to accumulate L-acid. glutamic acid in the middle and collect L-glutamic acid from the medium.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a construction flow of GDH gene having a mutant promoter. Figure 2 shows a construction flow of CS gene having a mutant promoter. Figure 3 shows a shuttle vector construction flow carrying lacZ as a reporter gene.

DETAILED DESCRIPTION OF THE PREFERRED MODALITY The term "glutamic acid producing coryneform microorganism" as used herein also includes bacteria that are classified within 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 coryneproducer bacteria producing glutamic acid of the genus Brevibacterium or Corynebacterium which are shown below. The bacteria of the genus Corynebacterium and those of the genus Brevibacterium will be collectively known as "coryneform bacteria" as long as they do not refer to the productivity of glutamic acid.

Corynebacterium acetoacidophilum ATCC13870 Corynebacterium acetoglutamicum ATCC15860 Corynebacterium callunae ATCC15991 Corynebacterium glutamicum ATCC 13032 Brevibacterium divaricatum ATCC14020 Brevibacterium lactofermentum ATCC 13869 Corynebacterium lilium ATCC15990 Brevibacterium flavum ATCC14067 Corynebacterium melassecola ATCC 17965 Brevibacterium saccharolyticum ATCC14066 Brevibacterium immariophilum ATCC 14068 Brevibacterium reseum ATCC13825 Brevibacterium thiogenitalis ATCC19240 Microbacterium ammoniaphilum ATCC 15354 Corynebacterium thermoaminogenes AJ12310 (FERM 9246) The amino acids that are to be produced are not particularly limited as long as the genes related to the biosynthesis and promoters thereof have been elucidated. Examples of effective enzymes related to biosynthesis include GDH, citrate synthetase (CS), isocitrate synthetase (ICDH), pyruvate dehydrogenase (PDH) and aconitase (ACO) for fermentation of glutamic acid.

Enzymes for lysine fermentation including biosynthesis enzymes such as aspartate kinase (AK), dihydrodipicolinate synthetase, dihydrodipicolinate reductase, diaminopimelate dehydrogenase and diaminopimelate decarboxylase are also effective. The lysine eccrisis protein (lysE gene) related to lysine membrane eccrisis is also effective. Enzymes effective for arginine fermentation include N-acetylglutamate synthetase, N-acetylglutamate kinase, N-acetylglutamyl phosphate reductase, acetylomitine aminotransferase, N-acetylornitinase, omitino carbamyltransferase, argininosuccinate synthetase and argininosuccinane. Arginine is formed by a reaction catalyzed by these enzymes. These enzymes are effective. These enzymes are encoded by enzymes aerA, aegB, argC, argD, argE, argF, argG and argH, relatively. Enzymes effective for fermentation of serine include 3-phosphoglyceric acid dehydrogenase, phosphoserine transamylase, phosphoserine phosphatase and the like. Enzymes effective for phenylalanine fermentation include biosynthesising enzymes such as deoxyarabinohepturonic phosphate synthetase, 3-dehydrokinate synthetase, 3-dehydrocinic acid dehydroratase, shikimic dehydrogenase acid, shikimic kinase acid, 5-enol pyruvilshikimic acid-3-phosphate synthetase, synthetic corycinic acid enzymes , corisimato synthetase, corisimatto mutase, prefenate dehydroratase and the like. Metabolic sugar enzymes such as transcetorase transaldolase, synthetic phosphoenolpyruvic acid enzyme are also effective. Enzymes effective for tryptophan fermentation include enzymes belonging to the tryptophan operon, in addition to several enzymes effective in the aforementioned phenylalanine fermentation and several enzymes effective in the aforementioned serine fermentation. Preferred enzymes for proline fermentation include O-glutamyl kinase, O-glutamylcemialdehyde dehydrogenase, pyrrolino-5-carboxylate reductase, in addition to several enzymes effective in the aforementioned glutamic acid fermentation. Enzymes effective for glutamine fermentation include glutamine synthetase, in addition to several enzymes effective in the aforementioned glutamic acid fermentation. In the production of inosine, it is considered useful to increase the expression of 5-phosphoribosyl-1-diphosphate synthetase, 5-phosphoribosyl-1-diphosphate aminotransferase, phosphoribosylaminoimidazolecarboxamide formyltransferase and the like. In the production of guanosine, it is considered useful to increase the expression of 5'-isosinic acid dehydrogenase and 5-xanthicic aminase acid, in addition to 5-phosphoylibosyl-1-diphosphate synthetase, 5-phospholibosyl-1-diphosphate aminotransferase, phosphoribosilaminoimidazolecarboxamide formyltransferase and the like .

In the production of adenosine, it is considered useful to increase the expression of adenyrosuccinate synthetase, in addition to synthetic enzyme of 5-phosphoribosyl-1-diphosphoric acid, 5-phosphoribosyl-1-diphosphoric aminotransferase acid, phosphoribosylaminoimidazolecarboxamide formyltransferase and the like. In the production of nucleotides, it is considered useful to increase the expression of phosphoribosyl transferase, inosine kinase, guanosine kinase and adenosine kinase. In the present invention, a mutant of an amino acid-producing coryneform bacterium is obtained by introducing a mutation into a promoter sequence of desired amino acid biosynthesizing genes into a chromosome of an amino acid producing coryneform bacterium, such as the promoter sequence described above for GDH, to make it next to a consensus sequence with a chemical compound or introducing the mutation by genetic recombination to obtain a mutant of the amino acid producing coryneform microorganism. The term "consensus sequence" is a sequence that appears most frequently in several promoter sequences. Said 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. McCIure Nuc. Acid Res. 11: 2237-2255 (1983), and that of ß. subtilis is described in Charles et al. Mol. Gen. Genet 186: 339-346 (1982). The mutation can be caused either in a promoter sequence only such as that for GDH or two or more promoter sequences such as those for GDH, citrate synthetase (citrate synthesizing enzyme) (CS) and isocitrate synthetase (isocitrate synthesizing enzyme) ( ICDH). In the present invention, the mutant thus obtained is cultured to obtain the mutant capable of producing a large amount of the intended amino acid. It has already been elucidated that in the fermentation of glutamic acid, GDH derived from a glutamate-producing coryneform microorganism has its own promoter sequence in the region towards the 5 'end of it [Sahm et al., Molecular Microbiology (1992), 6, 317-326]. For example, the GDH promoter of the present invention, the GDH gene having the GDH promoter sequence and the L-glutamate producing Corynebacterium strain having this gene can be obtained, for example, by the following methods: know, the strain is subjected to a mutagenesis treatment such as irradiation with UV, X-rays or radiation, or treatment with a mutagen to obtain a strain resistant to 4-fluoroglutamic acid in an agar plate culture medium containing acid 4-fluoroglutamic. Namely, the mutagenized cells are disseminated on the agar plate culture medium containing 4-fluoroglutamic acid at such concentration that inhibits the growth of the parent and the mutant which grows in this way is separated. In addition, the promoter sequence of GDH genes can be replaced by variably modified sequences by site-directed mutagenesis, and the relationship between the respective sequence and GDH activity is examined to select those that have a high L-glutamate productivity. . It is particularly preferred in the present invention that the DNA sequence in the -35 region of the promoter for the 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 the -10 region of the promoter is TATAAT, or the bases of ATAAT in the TATTAT sequence in a -10 region is replaced by another base, while it does not inhibit the function of the promoter. The reason why the strain on which the bases of ATAAT in the TATAAT sequence in the -10 region is replaced by another base and the promoter function is not inhibited it can be selected as follows: because a remarkable increase in the specific activity of GDH was observed by simply replacing the first " C "of CATAAT by" T "in sequence -10 of wild type (refer to p6-4 in Table 1), it was considered that said replacement by another base is possible. The GDH gene promoter sequence is described, for example, in the previously described document Sahm et al., Molecular Microbiology (1992), 6, 631-326. In this it is described as Seq ID No. 1. The GDH gene sequence itself is also described in Sahm et al., Molecular Microbiology (1992), 6.6 317-326 as Seq ID No. 1.

Similarly, the mutation can be introduced into the promoter for citrate synthesizing enzyme (CS) or l-socitrate synthesizing enzyme (ICDH). In this manner, the promoters for GDH are those that have at least one DNA sequence in the region -10 selected from the group consisting of CGGTCA, TTGTCA, TTGACA and TTGCCA in the -35 region and / or a TATAAT sequence or the TATAAT sequence but in which the base of ATAAT is replaced by another base, in which it does not inhibit the function of the promoter. Genes are also provided to produce glutamate dehydrogenase, which have the promoter described above. The promoters for CS are those that have the sequence TTGACA in the -35 region and / or the TATAAT sequence in the region -10, which do not inhibit the promoter function. CS genes having the promoter described above are also provided. The promoters for ICDH are those that have the sequence TTGCCA or TTGACA in the first or second promoter of the -35 region and / or the TATAAT sequence in the first or second promoter of the -10 region that does not inhibit the function of the promoter. The icd genes having the promoter described above are also provided. The promoters for PDH are those that have the sequence TTGCCA in the -35 region and / or the TATAAT sequence in the -10 region, which does not inhibit the function of the promoter. PDH genes having the promoter described above are also provided.

The present invention also provides L-glutamate producing coryneform bacteria having the genes described above. The promoters for the algininosuccinate synthetase are those that have at least one DNA sequence selected from the group consisting of TTGCCA, TTGCTA and TTGTCA in the -35 region and / or the TATAAT sequence in the -10 region, or the ATAAT base. in the TATTAT sequence it is replaced by another base, which does not inhibit the promoter function. The argininosuccinate synthetase gene having the promoter described above is also provided. The present invention also provides arginine-producing coryneform bacteria having the genes described above. The 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 accumulate the intended amino acid, preferably L-glutamic acid, and collect the amino acid of the culture medium. The liquid culture medium used to cultivate 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. Carbon sources include carbohydrates such as glucose, fructose, sucrose, melases and starch hydrolyzates; alcohols such as ethanol and glycerol; and organic acids such as acetic acid. Sources of nitrogen include ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, ammonium acetate, ammonia, peptone, meat extract, yeast extract, and corn liquor. When the auxotrophic mutant is used, the required substances are added to the medium as the reagents or natural substances that contain them. Coryneform bacteria usually produce L-glutamic acid under a reduced biotin condition. Therefore, the amount of biotin in the medium is restricted or a substance inhibiting the biotin effect such as a surfactant or penicillin is added. The fermentation is preferably conducted by stirring the culture or by agitating the culture with aeration while in the pH of the culture liquid it is maintained on a scale of 5 to 9 for 2 to 7 days. The pH is preferably controlled with urea, calcium carbonate, gaseous ammonia, water with ammonia or the like. The culture temperature is preferably 24 to 37 ° C. The L-glutamic acid thus produced and accumulated in the culture liquid is collected by an ordinary method such as an ion exchange resin method or a recrystallization method. Specifically, the L-glutamic acid is separated by adsorption on an anion exchange resin or by crystallization by neutralization. In accordance with the present invention, the intended amino acid can be obtained in high yield by introducing a mutation is a promoter region of amino acid biosynthesizing genes of an amino acid producing coryneform bacterium to control the expression of the targeted genes. Furthermore, since any deletion of the intended gene does not occur in the bacterium according to the present invention, contrary to the cases where plasmid is used, the intended amino acid can be stably obtained in high yield. In this way, the industrial merit of the invention is greater. The present invention provides various promoters, particularly, promoters for GDH capable of imparting the power to produce amino acids, particularly glutamic acid, in high yield to strains of Corynebacterium without increasing the amount of aspartic acid and alanine obtained as by-products. In the present invention, a L-glutamate-producing coryneform bacterium is mutagenized, a strain in which the mutation introduced into a GDH gene promoter region and which is resistant to 4-fluoroglutamic acid is harvested, and the strain is grown for get glutamic acid in high yield. In this way, the present invention is industrially very advantageous. The following examples will illustrate the present invention.

EXAMPLE 1 Production of mutant GDH promoter A mutant GDH promoter was prepared by the site-directed mutagenesis method as follows: (1) Preparation of GDH genes having several mutant promoters The wild-type sequence in the -35 region and the -10 region of a GDH gene promoter of a coryneform bacterium is shown in sequence 1. The promoter sequence of wild type has already been reported [Molecular Microbilogy (1992), 6, 317-326]. The method for preparing a plasmid carrying the GDH gene having a mutant promoter is as follows: As shown in Figure 1, a chromosomal gene from a wild-type strain of an ATCC13869 coryneform bacterium prepared with Bacterial Genome DNA "(Advanced Genetic Technologies Corp.) was used as the template for PCR. Gene amplification was performed by PCR using GDH gene sequences towards the 5 'end and towards the 3' end. Both ends were slotted. The product thus obtained was inserted into the Smal site of the plasmid pHSG399 (a product of Takara Shuzo Co., Ltd). Then, an origin of replication taken from plasmid pSAK4 having the origin of replication capable of replicating in a coryneform bacterium was introduced into the Sal I site of the plasmid to obtain the plasmid pGDH. By this method, the GDH genes having each promoter sequence described above can be obtained by using an initiator having each of the Seq ID No. 1 to Seq ID No. 6 sequences shown in the Sequence Listing as the initiator to the 5 'end of the GDH gene, respectively. The fragment amplified by PCR was confirmed by sequencing that any mutation, other than the mutation introduced into the promoter sequence, did not occur in the amplified fragment. pSÁK4 is constructed in the following manner: the plasmid pHK4 previously obtained (J. P. KOKAI No. 5-7491) having an autonomous origin of replication derived from the plasmid pHM1519 [Agrie. Biol. Chem., 48, 2901-2903 (1984)], which is able to replicate autonomously in the Corynebacterium microorganism, is digested with restriction enzymes BamHI and Kpnl to obtain a DNA fragment having the origin of replication. Then, the fragment thus obtained is grooved at its ends with the DNA scoring equipment (Blunting kit from Takara Shuzo Co., Ltd.). After ligation with the Sali linker, the product thus obtained was inserted into the Sal I site of pHSG299 (a product of Takara Shuzo Co., Ltd.) to obtain the plasmid pSAK4. (2) Comparison of the degrees of expression of GDH that each promoter sequence has Each plasmid prepared as described above was introduced into the wild type strain of the coryneform bacterium ATCC13869 by the electroporation method (refer to KOKAI No. 2- 207791. To compare the degrees of expression of GDH for these strains, the specific activity of GDH was determined by the previously described method of Sahm et al. The results are shown in Table 1.

TABLE 1 ATCC 13869 / p6-2 to ATCC 13869p6-8 / corresponded to sequences from Seq ID No. 2 to Seq ID No. 6, respectively. These sequences were the same as sequence No. 1 (wild type) except that the underlined parts were changed as follows: Sequence No. 1 5-TTAATTCTTTGTGGTCATATCTGCGACACTGC CATAATTTGAACGT-3 'CGGTCA CATAAT 3. TGGTCA TATAAT 4. TTGACA TATAAT 5. TTGCCA TATAAT 6. TTGTCA TATATT These were the synthetic linear double-stranded DNA.

EXAMPLE 2 Preparation of mutant strains (1) Preparation of mutant strains resistant to 4-fluoroqlutamic acid AJ 13029 is a mutant producing glutamic acid strain and described 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 changed to 37 ° C. In this example, strain of Brevibacterium lactofermentum AJI3029 was used as the progenitor strain to prepare the mutant strains. In fact, any of the glutamic acid producing strains other than AJ13029 can be used as a progenitor strain to prepare mutant strains resistant to 4-fluoroglutamic acid. AJ13029 was grown on a CM2B agar medium (Table 2) at 31.5 ° C for 24 hours to obtain the bacterial cells. Cells were treated with 250 μg / 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 disseminated on agar plate culture medium (Table 3) containing 4-fluoroglutamic acid (4FG). The colonies were formed after incubating the plates at 31.5 ° C for 20 to 30 hours. In this experiment, an inclined medium containing 1 mg / ml of 4FG was prepared first, and then a layer of the same medium without 4FG was formed on it horizontally. Therefore, a concentration gradient of 4FG was obtained on the surface of agar medium. When the plate was inoculated with the obtained mutant cells as described, an adjoining line was formed at one edge of the growth limit of the strain. Bacterial strains that formed colonies were collected in an area containing 4FG of a concentration greater than that of the adjoining line. In this way, approximately 50 strains resistant to 4FG were obtained from approximately 10,000 mutagenized cells.

TABLE 2 CM2B agar medium Ingredient Concentration Polypeptone (Nippon Seiyaku Co.) 1.0% Yeast extract (Difco Co.) 1.0% NaCl 0.5% d-biotin 10 μg / l 1.5% agar (pH 7.2: adjusted with KOH) TABLE 3 agar medium Component Quantity in one liter of water Glucose 10 g MgS? 4 _ 7H2O 1 9 MnS04 _ 7H20 0.01 g MnSO _ 4-6H2O 0.01 g Thiamine hydrochloride 0.2 mg d-biotin 0.05 mg (NH4) 2SO4 5 g Na2HPO4 _ 12H2O 7.1 g TABLE 3 (continued) KH2P04 1.36 g Aqar 15 q (2) Confirmation of L-Qutamic Acid Production Capability of 4FG-Resistant Mutant Strains The glutamic acid production capacity of approximately 50 mutant strains obtained in (1) above and parental strain AJ13029 were confirmed as described below . AJ 13029 and mutant strains were each cultured with CM2B agar medium 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. Approximately 22 hours later, the fresh medium was added so that the final concentration was that of medium B shown in Table 4. The temperature was changed to 37 ° C and then the cultivation was continued for about 24 hours. After completion of the culture, this was examined with a biotic analyzer Biotic Analyzer (a product of Asahi Chemical Industry Co., Ltd.) to determine whether or not L-glutamic acid was produced. It was therefore found that when the 50 strains were grown, two strains that had a higher glutamic acid yield than that obtained from the progenitor strains and a high GDH activity were separated (strain A and strain B). The GDH activity of each of them was determined to find that the GDH activity of both had increased (Table 5). The activity of GDH was determined by the method of E. R. Bormann et al. [Molecular Microbiol., 6, 317-326 (1996)]. By sequencing GDH genes, it was identified that the mutation sites were found only in the GDH promoter region (Table 6). TABLE 4 Ingredient Medium A Medium B Glucose 3 g / dl 5 g / dl KH2P? 4 0.14 g / dl 0.14 g / dl MgSO4. 7H20 0.04 g / dl 0.04 g / dl FeS? 4. 7H 0 0.001 g / dl 0.001 g / dl MnS04. 4H O 0.001 g / dl 0.001 g / dl (NH4) 2 SO4 1.5 g / dl 205 g / dl Hydrolyzed soy protein solution 1.5 ml / dl 0.38 ml / dl Thiamine hydrochloride 0.2 mg / l 0.2 mg / l Biotin 0.3 mg / l 0.3 mg / l Anti foaming agent 0.05 ml / l 0.05 ml / l CaC? 3 5 gd / l 5 gd / l PH 7.0 (adjusted with KOH) TABLE 5 Glutamic acid formation and GDH activity of mutant strains Glu strain (g / dl) GDH specific activity Relative value AJ 13029 2.6 7.7 1.0 FGR1 2.9 23.1 3.0 FGR2 3.0 25.9 3.4 TABLE 6 DNA sequences in the GDH promoter region of mutant strains Sequence Promoter Sequence of GDH -35 -10 AJ 13029 TGGTCA TTCTGTGCGACACTGC CATAAT FGR1 TGGTCA TTCTGTGCGACACTGC TATAAT FGR2 TTGTCA T-CTGTGCGACACTGC TATAAT EXAMPLE 3 Introduction of mutation in the promoter region of CS gene of glutamate-producing coryneform bacterium In this example, a strain having an increased promoter for the genes encoding glutamate dehydrogenase (GDH) and citrate synthesizing enzyme (CS) was produced. (1) Cloning of the qltA gene The gltA gene sequence of a coryneform bacterium, which encodes citrate synthesizing enzyme, has already been elucidated [Microbial. 140, 1817-1828 (1994)]. Based on this sequence, primers shown in Seq ID No. 7 and Seq ID No. 8 were synthesized. On the other hand, the chromosomal DNA of Brevibacterium lactofermentum ATCC 13869 was prepared using a Bacterial Genome DNA Purification Kit (Advanced Genetic Technologies Corp.). Sterilized water was added to a mixture of 0.5 μg of chromosomal DNA, 10 pmol of each of the oligonucleotides, 8 μl of dNTP mixture (2.5 mM each), 5 μl of 10x Taq Buffer (Takara Shuzo Co., Ltd.) and 2 U from La Taq (Takara Shuzo Co., Ltd.) to obtain 50μl of PCR reaction mixture. The reaction mixture was subjected to PCR. PCR conditions were 30 cycles of denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 15 seconds and extension at 72 ° C for 3 seconds using Thermal Cycler TP240 (Takara Shuzo Co., Ltd.) to amplify approximately 3 Kbp of DNA fragments containing the gltA gene and the promoter thereof. The amplified fragments thus obtained were purified with SUPRECO2 (Takara Shuzo Co., Ltd.) and then scored at the ends. The slot was conducted with a slotting equipment from Takara Shuzo Co., Ltd. The fragment with slotted ends 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 see 2 (Takara Shuzo Co., Ltd.). After ligation was complete, the transformation was conducted with competent E. coli JM109 cells (Takara Shuzo Co., Ltd.). The cells were disseminated on a plate of L medium (containing 10 g / l of bactotriptone, 5 g / l of bacto-yeast extract, 5 g / l of NaCl and 15 g / l of agar; pH 7.2) containing 10 μg / ml of IPTG (isopropyl-β-D-thiogalactopyranoside), 40 μg / ml of X-Gal (5-bromo-4-cyoro-3-indolyl-β-D-galactoside) and μg / ml of chloramphenicol. After being grown overnight, white colonies were collected to obtain the transformed strains after a single colony isolation. From the transformed strains, plasmids were prepared by the alkaline 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 having the same restriction map as the map shown in Figure 2 was named "pHSG399CS". (2) Introduction of mutations in qltA promoter A Mutan-Super Express Km Expression Team (Takara) was used Shuzo Co., Ltd.) for the introduction of mutation in gltA promoter region. The method is specifically described below. PHSG399CS was completely digested with EcoRI and SalI to obtain an 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 SalI. After ligation was complete, the transformation was conducted with competent E. coli JM109 cells (Takara Shuzo Co., Ltd.). The cells were disseminated on L media plates containing 10 μg / ml IPTG, 40 μg / ml X-Gal and 25 μg / ml kanamycin. After being incubated overnight, white colonies were collected and transformants were obtained by isolation of individual colonies. From the transformants, plasmids were prepared and the plasmid containing the gltA gene was designated pKF19CS. PCR was conducted using pKF19CS as the template and 5'-phosphorylated synthetic DNA shown in the 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 E. coli MV1184 cells (Takara Shuzo Co., Ltd.) using the PCR product. The cells were disseminated on L media plates containing 25 μg / ml caramycin. After being incubated overnight, the colonies were harvested and transformants were obtained after isolation of individual colonies. From the transformants, plasmid DNA was prepared. The gltA promoter region sequence was determined by the method of Sanger [J. Mol. Biol., 143, 116 (1980)] using synthetic DNA having the sequence of Seq ID No. 12. Specifically, the sequence was determined by means of a Dye Termination Sequencing Kit (Dye Terminator Sequencing Kit (Applied Biosystems) and analyzed by means of a Genetic Analyzer Genetic Analyzer ABI310 (Applied Biosystems) The plasmids in which the gltA promoter region was replaced by the sequence shown in Table 7 were designated pKF19CS1, pKF19CS2 and pKF19CS4, respectively.

TABLE 7 region -35 region 10 pKF19CS ATGGCT TATAGC pKF19CS1 ATGGCT TATAAC pKF19CS2 ATGGCT TATAAT PKF19CS4 TTGACA TATAAT (3) Construction of mutant gltA plasmid pKF19CS, pKF19CS1, pKF19CS2 and pKF19CS4 constructed in step (2) was completely digested with SalI and EcoRI (Takara Shuzo Co., Ltd.).

On the other hand, plasmid pSFK6 (Japanese Patent Application No. 11-69896) having an origin of replication derived from plasmid pAM330 that can be replicated autonomously in a coryneform bacterium [Japanese Patent Publication for Opposition Purposes (hereinafter known as "JP KOKAI") No. 58-67699] was completely digested with EcoRI and Sali. The fragment obtained was ligated with approximately 2.5 kb of the fragment containing gltA. After completion of ligation, the transformation was conducted with competent E. coli JMI09 cells. The cells were disseminated on the plates of medium L containing 10 μl / ml of IPTG, 40 μg / ml of X-Gal and 25 μg (ml of kanamycin.) After being incubated overnight, the colonies were harvested and the transformants were harvested. obtained after isolation of individual colonies Plasmids were prepared from the transformants Plasmids containing gltA gene were designated pSFKC, pSFKCI, pSFKC2 and pSFKC4, respectively. (4) Determination of CS expression from mutant qltA plasmid in coryneform bacteria The plasmid constructed in step (3) above was introduced into Brevibacterium lactofermentum ATCC13869. Specifically, the treatment was conducted by the electric pulse method (J.P. KOKAI No. 2-07791). Transformants were selected at 31 ° C with CM2B media plate (comprising 10 g / 1 bactotriptone, 10 g / 1 bactolevadura extract, 5 g / 1 NaCl, 10 μg / l biotin and 15 g / 1 of agar; pH 7.0) containing 25 μg / ml kanamycin. After being incubated for two days, colonies were collected and the transformants containing pSFKC, pSFKCI, pSFKC2 and pSFKC4 were designated BLCS, BLCS1, BLSC2 and BLCS4, respectively, after isolation of individual colonies. A medium having a composition shown in Table 8 was inoculated with the transformant. The culture was continued at 31 ° C and finished before the glucose was completely consumed. The culture liquid was centrifuged to separate the cells. The cells were washed with 50 mM tris pH buffer (pH 7.5) containing 200m sodium glutamate and then suspended in the same buffer solution. After sonication with UD-201 (TOMY) followed by centrifugation (10,000g), the cells that remained undigested were removed to obtain a crude enzyme solution. The activity of citrate synthetase can be determined according to the Enzymol methods. 13, 3-11 (1969). Specifically, the crude enzyme solution was added to a reaction mixture containing 100 mM TisHCI. (pH8), 0.1 mM, of DTNB, 200 mM of sodium glutamate and 0.3 mM of acetii CoA, and the fundamental value was determined as the increase in absorbance at 412 nm at 30 ° C determined by Hitachi U-3210 spectrophotometer. In addition, oxaloacetic acid was added in such an amount that the final concentration thereof was 0.5 mM. The increase in absorbance at 412 nm was determined, from which the fundamental value was deduced to determine the activity of citrate synthetase. The concentration of protein in the crude enzyme solution was determined by Protein Assay protein test (BIO-RAD). Bovine serum albumin was used as the standard protein. The results are shown in Table 9. It was confirmed that the activity of the citrate synthetase of the gltA mutant promoters was increased as compared to the wild-type gltA promoter.

TABLE 8 Ingredient Concentration Glucose 50 g / l KH2P04 1 g / l MnS? 4 .7H2O 0.4 mg / l FeS0. 7H20 10 mg / l Hydrolyzed soy protein 20 ml / l Biotin 0.5 mg / l Thiamine hydrochloride 2 mg / l TABLE 9 Strain dABS / min / mg Relative activity Relative activity Wild type 4 6.8 1.0 BLCS00 38.8 5.7 1.0 BLCS01 57.1 8.4 1.21 BLCS02 92.5 13.6 1.9 BLCS04 239.5 35.2 4.8 (5) Introduction of mutant gltA gene into a plasmid sensitive to temperature To integrate mutant gltA promoter sequences into a chromosome, a method is known where a plasmid is used of which the replication in a coryneform bacterium is sensitive to temperature (J.P. KOKAI No. 5-7491). PSFKT2 (Japanese Patent Application No. 1 1-81693) was used as the plasmid vector, whose replication in a coryneform bacterium is sensitive to temperature. pKFCSI, pKFCS2 and pKFCS3 completely digested with Sali and BstPI and with the slotted ends were used as the promoter sequences of gltA mutants. They were ligated to pSFKT2 completely digested with Smal. After ligation was complete, the transformation was conducted with competent E. coli JM109 cells (Takara Shuzo Co., Ltd.). The cells were disseminated on L media plates containing 10 μg / ml IPTG, 40 μg / ml X-Gal and 25 μg / ml kacin. After being incubated overnight, white colonies were collected and transformants were obtained after isolation of individual colonies. Plasmids were prepared from the transformants. Temperature sensitive shuttle vectors containing gltA gene were designated pSFKTCI, pSFKTC2 and pSFKTC4, respectively. (6) Introduction of mutant qltA promoter into chromosome pSFKTCI, pSFKTC2 and pSFKTC4 were each introduced into strain FGR2 of Brevibacterium lactofermentum by the electric pulse method. Transformants were selected from CM2B media plates containing 25 μg / ml kacin at 25 ° C. After the introduction of each plasmid, each strain obtained was cultured in a liquid medium CM2B, disseminated on CM2B plates containing 25 μg / ml kacin, after dilution at a concentration of 10 ^ to 10§ cfu per plate and it was grown at 34 ° C. the strain having the temperature-sensitive plasmid became sensitive to kacin because the replication of the plasmid was inhibited at that temperature and therefore could not form colonies. On the other hand, the strain having plasmid DNA integrated into the chromosome could be selected because it formed the colonies. The colonies thus obtained were harvested and separated into respective colonies. Chromosomal DNA was extracted from the strain. PCR was conducted using the chromosomal DNA as the template and sequence primers shown in Seq ID No. 8 and Seq ID No. 13. Approximately 3 kb of amplified fragments were confirmed. Therefore, it was proved that in this strain, the gltA mutant gene derived from the temperature-sensitive plasmid was integrated near the gltA gene in the host chromosome by homologous recombination. Strains derived from pSFKTCI, 2 and 4 were designated BLCS11, BLCS12 and BLCS14, respectively. (7) Preparation of substituted qltA promoters First, kacin sensitive strains were obtained from strains BLCS11, BLCS12 and BLCS14 having the mutant gltA gene integrated therein by homologous recombination. Strains that had plasmid integrated in them were diluted and disseminated on CMM2B plates and then cultured at 34 ° C. After colony formation, plate replicates were made using CM2B plates containing 25 μg / ml kacin and cultured at 34 ° C. In this way, strains sensitized to kacin were obtained.

The chromosome was extracted from the kacin sensitive strain and PCR was conducted with primers having the sequence shown in Seq ID No. 7 and Seq ID No. 8 to prepare gltA gene fragments. The amplified fragments thus obtained were purified with SUPRECO2 (Takara Shuzo Co., Ltd.) and then subjected to the sequencing reaction using a Seq ID No. 13 primer to determine the sequence in the promoter region thereof. As a result, the strain having the same promoter sequence as pKF10CS1 of Table 7 was designated GB01, the strain having the same promoter sequence as that of pKF19CS2 was designated GB02 and the strain having the same promoter sequence as that of pKF19CS4. it was called GB03. It was indicated that in these strains, the wild-type gltA gene originally located on the chromosome was extracted together with the vector plasmid while the mutant gltA gene introduced by the plasma remained on the chromosome when the plasmid and the duplicated gltA gene were extracted from the chromosome. (8) Determination of citrate synthetase activity of mutant qltA promoter strains The citrate synthetase activities were determined by treating strains FGR2, GB01, GB02, GB03 and FGR2 / pSFKC obtained in step (7) in the same manner as those of Step 4). The results are shown in the Table . It was confirmed that the citrate synthetase activity of the substituted gltA promoter strain was greater than that of the progenitor strains thereof.

CLUADRCL10 Strain dABS / min / mq Relative activity FGR2 7.9 1.0 GB01 9.5 1.2 GB02 15.0 1.9 GB03 31.6 4.0 FGR2 / PSFKC 61.6 7.8 (9) Culture results of substituted qltA promoter strains Each of the strains obtained in step (7) described above was inoculated into a seed culture medium having a composition shown in Table 11. and the culture was shaken in a 39.5 ° C for 24 hours. 300 ml of a main culture medium having the composition shown in Table 11 s placed in a glass jar fermenters of 500 ml and then sterilized by heating and then inoculated with 40 ml of seeds grown as described above. The culture was started at a culture temperature of 31.5 ° C, while the agitation speed and the aeration rate were controlled at 800 to 1300 rpm and 1/2 to 1/1 wm, respectively. The pH of the culture liquid was maintained at 7.5 with gaseous ammonia. The temperature was changed to 37 ° C 8 hours after starting the culture. The culture was terminated when the glucose had been completely consumed in 20 to 40 hours and the amount of L-glutamic acid formed and accumulated in the culture liquid was determined. As a result, the greatest improvement in L-glutamic acid yield was confirmed when each of the GB02 and GB03 strains instead of GB01 and FGR2 / pSFKC was used as shown in Table 12. From these facts, it was found that good results were obtained introducing the mutation in the gttA promoter to increase the CS activity 2 to 4 times for the improvement in the glutamic acid yield produced by these strains.

TABLE 11 Concentration Ingredient Cultivation of seed Main crop Glucose 50 g / l 150 g / l KH2P04 i g / i 2 g / l MgSO4 .7H2O 0.4 g / l 1.5 g / l FeS? 4. 7H2O 10 mg / l 15 mg / l MnSO4 .4H2O 10 mg / l 15 mg / l Hydrolyzed soy protein 20 ml / l 50 ml / l Biotin 0.5 mg / l 2 mg / l Thiamine hydrochloride 2 mg / l 3 mg / l CUADRCL12 Strain L-glutamic acid (g / l) FGR2 8.9 GB01 9.1 GB02 9.4 GB03 9.4 - FGR2 / PSFKC 9.1 EXAMPLE 4 Introduction of mutation in the promoter region of the ICDH gene of glutamate-producing coryne-forming bacteria In this example, strains were produced that had promoters for genes encoding glutamate dehydrogenase, citrate synthetase and isocitrate dehydrogenase. (1) Cloning of the icd gene The DNA sequence of the icid gene of coryneform bacterium, which encodes citrate synthetase, is either elucidated [J. Bacteriol. 177, 774-782 (1995)].

Based on that sequence, primers shown in Seq ID were synthesized No. 14 and Seq ID No. 15. PCR was conducted using chromosomal DNA of Brevibacterium lactofermentum ATCC13869 as the template to amplify approximately 3 Kbp of DNA fragment encoding the cd gene and promoter thereof. The amplified fragment thus obtained was completely digested with EcoRI and mixed with that obtained by the complete digestion of pHSG399 (Takara Shuzo Co., Ltd.) with EcoRI to conduct the ligation. After completion of ligation, transformation was conducted using competent E. coli JM109 cells. The cells were disseminated on the L media plates containing 10 μg / ml IPTG, 40 μg / ml X-Gal and 40 μg / ml chloramphenicol. After being incubated overnight, white colonies were collected and transformants were obtained after isolation of individual colonies. The plasmid carrying the icd gene was named pHSG399icd. (2) Introduction of mutations in ¡cd promoter The precise localization of the ¡cd gene promoter has not yet been determined. The possibility of increasing the level of transcription of icd gene mRNA was investigated by artificially modifying the sequence towards the 5 'end of the gene encoding ICDH in the sequence similar to the promoter. Specifically, mutations were introduced in the region similar to -10 that existed in the DNA sequence approximately 190 bp towards the 5 'end (the first promoter) and approximately 70 bp (the second promoter) towards the 5' end of the first ATG of the ICDH protein.

A Mutan-Super Express Km Kit (Takara Shuzo Co., Ltd.) was used for the introduction of mutation in a region towards the 5 'end of the icd gene. The method is specifically described below. pHSG399icd was completely digested with PstI to obtain PstI fragment containing the promoter of the icd gene. The fragments were ligated with the fragment obtained by complete digestion of pKF18kM (Takara Shuzo Co., Ltd.) with PstI. After ligation was complete, the transformation was conducted with competent E. coli JM109 cells (Takara Shuzo Co., Ltd.). The cells were disseminated on medium L containing 10 μg / ml IPTG, 40 μg / ml X-Gal and 25 μg / ml kanamycin. After being incubated overnight, white colonies were collected and transformants were obtained after isolation of individual colonies. From the transformants, plasmids were prepared, and the plasmid containing the icd gene promoter was designated pKF18icd. PCR was conducted using pKF18icd as the template and d-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 initiator. These PCR products were used to transform competent E. coli JM109 cells. The cells were disseminated on plates of L medium containing 25 μg / ml kanamycin. After being incubated overnight, formed colonies were collected and transformants were obtained after isolation of individual colonies. From the transformants, the plasmid DNA was prepared and the sequence of the promoter region of cDd was determined using synthetic DNA shown in Seq ID No. 22 by the method of Sanger [J. Mol. Biol., 143, 161 (1980)]. Specifically, the DNA sequence was determined with the Dye Terminator Sequencing Kit (Applied Biosystems), and analyzed with the Genetic Analyzer Genetic Analyzer ABI310 (Applied Biosystems). Those obtained by replacing the promoter region of cd with a sequence shown in Table 7 were designated pKF18ICD1, pKF18ICD2, pKF18ICD3, pKF18ICD4, pKF18ICD5 and pKF18ICD6m, respectively. Among them, pKF18ICD2 was completely digested with PstI to obtain the PstI fragment containing the promoter of the icd gene. The fragment was ligated with the fragment obtained by digestion of complete PstI from pKF18kM (Takara Shuzo Co., Ltd.). After completion of ligation, transformation was conducted with competent E. coli JM 109 cells (Takara Shuzo Co., Ltd.). The cells were disseminated on L media plates containing 10 μg / ml IPTG, 40 μg / ml X-Gal and 25 μg / ml kanamycin. After being incubated overnight, white colonies were collected and the transformant strains were obtained after isolation of individual colonies. For the transformed strains, plasmids were prepared and the plasmid containing the icd gene promoter was designated pKF18ICDM2. PCR was conducted using pKF18ICDM2 as the template and 5-phosphorylated 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 PCR product. The cells were disseminated on the L media plates containing 25 μg / ml kanamycin. After being incubated overnight, colonies thus formed were collected and transformants were obtained after the isolation of individual colonies. For the transformants, DNA plasmids were prepared and the icd promoter region sequence was determined using the synthetic DNA shown in Seq ID No. 22. Those obtained by replacing the icd promoter region with the sequence shown in Table 13 were designated pKF18ICD25 and pKF18ICD26, respectively.

TABLE 13 Plasmid 1 er. promoter 2nd. promoter -35 -10 -35 -10 PKF181CD GCGACT GAAAGT TTTCCA CACCAT pKF18ICD01 GCGACT TATAAT TTTCCA CACCAT pKF18ICD02 TTGACA TATAAT TTTCCA CACCAT pKF18ICD03 TTGACT TAAAGT TTTCCA CACCAT pKF18ICD04 GCGACT GAAAGT I I CCA TATAAT PKF18ICD05 GCGACT GAAAGT TTGCCA TATAAT pKF18ICD06 GCGACT GAAAGT TTGACA TATAAT pKF18ICD25 TTGACA TATAAT TTGCCA TATAAT pKF18ICD26 TTGACA TATAAT TTGACA TATAAT (3) Plasmid construction for determination of promoter activity To easily determine the activity of the promoter, a possible method is the indirect determination of promoter activity using a reporter gene. The 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 reduced in a severe way, that the fundamental reaction does not occur and that it has a site of Restriction enzyme digestion for gene manipulation. Because β-galactosidase (LacZ) from E. coli is widely used as a reporter gene and bacteria of the genus Corynebacterium have no ability to assimilate lactose [J. Gen. Appl. Microbiol., 18, 399-416 (1972)], LacZ was determined to be the optimal reporter gene. Then, the pNEOL plasmid carrying LacZ as the reporter gene was constructed (see figure 3). The procedure for construction is described in detail below. PCR was conducted using a chromosomal DNA obtained from E. coli ME8459 (ME8459 was deposited at the National Institute of Genetics (Japan)) as the template with synthetic DNA shown in Seq ID No. 23 and Seq ID No. 24 as the initiator. The PCR product was completely digested with Saml and BamHI and then ligated with fragments obtained by digesting pKF3 (Takara Shuzo Co., Ltd.) with Hindlll and the ends were scored. After completion of ligation, transformation was conducted with competent E. coli JM 109 cells (Takara Shuzo Co., Ltd.). The cells were disseminated on the L media plates containing 25μg / ml kanamycin. After being incubated overnight, the colonies thus formed were harvested and separated into respective colonies to obtain the transformed strain. The plasmid obtained from the transformed strain was designated pKF3nptll. Then, the plasmid was digested with Sali. On the other hand, pSAK4 described in Example 1 (1) was completely digested with Smal and Sali and the ends were scored. These fragments were ligated together to construct a pNEO shuttle vector that can be replicated in a coryneform bacterium. The plasmid was able to impart resistance to chloramphenicol and resistance to kanamycin in the hosts. In addition, pNEO was completely digested with Smal and Sse8387l. The resulting fragments were ligated to those obtained by complete digestion of pMC1871 (Farmacia Biotech.) With PstI and Smal. In this way, the shuttle vector pNEOL that can be replicated in a coryneform bacterium and that has LacZ lacking 8 amino acids in N-terminus as the reporter gene was constructed (see figure 3). (4) Determination of the promoter activity of mutant icd The plasmids that have mutant icd promoter tenain constructed in step (2), described above, ie, pKF18ICD1, pKF18ICD2, pKF18ICD3, pKF18ICD4, pKF18ICD5, pKF18ICD6, pKF18ICD25, pKF18ICD26 and pKF18ICD, were completely digested with Sacll and Pstl and then the ends were scored. They were ligated with fragment obtained by digesting pNEOL with Smal. After the ligation was complete, the transformation was conducted with competent E. coli JM 109 cells. The cells were disseminated on plates of L medium containing IPTG, X-Gal and 40 μg / ml chloramphenicol. After being incubated overnight, blue colonies were collected and the transformed strains were obtained after isolation of individual colonies. From the transformed strains, plasmids were prepared. The plasmids that had a structure capable of producing a fused protein of ICDH and LacZ were designated pNEOICDI, pNEOICD2, pNEOICD3, pNEOICD4, pNEOICDd, pNEOICD6, pNEOICD25, pNEOICD26 and pNEOLICD, respectively. Each of these plasmids or pNEOL was introduced into Brevibacterium lactofermentum ATCC13869 by electrical pulse method. Transformants were selected using CM2B media plates (comprising 10 g / l of bactotriptone, 10 g / l of bacto-yeast extract, 5 g / l of NaCl, 10 μg / l of biotin and 15 g / l of agar and had a pH of 7.0) containing 25 μg / ml kanamycin and 40 μg / ml X-Gal at 31 ° C for two days. After completion of the introduction, the colonies thus formed were harvested and isolated as individual colonies. Transformants containing pNEOICDI, pNEOICD2, pNEOICD3, pNEOICD4, pNEOICDd, pNEOICD6, pNEOICD25, pNEOICD26 and pNEOLICD were designated BLAC1, BLAC2, BLAC3, VLAC4, BLAC5, BLAC6, BLAC25, BLAC26, BLAC and BNEOL, respectively. All transformants other than BNEO formed blue colonies. Crude enzyme solutions were prepared from the transformants in the same manner as step (4) in Example 3 except that "pH regulator Z" (comprising 10 mM KCl, 1 mM MgSO4, 270 μg. / 100 mM of 2-ME and NaPi and having a pH of 7.5) was used as a wash and suspension pH regulator. The activity of LacZ was determined as follows: pH regulator Z was mixed with the crude enzyme solution, ONPG in pH regulator Z having the final concentration of 0.8 mg / ml was added to the resulting mixture, and the increase in the absorbance at 420 nm at 30 ° C was determined with a Hitachi U-3210 spectrophotometer as the activity of LacZ. The concentration of protein in the crude enzyme solution was determined by the Protein Assay Protein Test (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 the icd promoter and expressing fused protein of ICDH-LacZ was higher than that expressing fused protein ICDH-LacZ of type wild.

TABLE 14 Strain dABS / min / mg Relative activity BNEOL Not detected 0.0 BNEOLI 42 1.0 BNEOLI-1 84 2.0 BNEOLI-2 168 4.0 BNEOLI-3 80 1.9 BNEOLI-4 126 3.0 BNEOLI-5 139 3.3 BNEOLI-6 84 2.0 BNEOLI-25 168 4.0 BNEOLI-26 170 4.0 (5) Introduction of mutant icd gene into the temperature-sensitive plasmid A vector of plasmid pSFKT2 (Japanese Patent Application No. 11-81693) whose replication in a coryneform bacterium was temperature sensitive was used. . pKF18lCD1, pKF18ICD2, pKF18ICD3, pKF18ICD4, pKF18ICD5, pKF18ICD6, pKF18ICD2d and pKF18ICD26 were completely digested with PstI and the obtained fragments were used as the promoter sequences of cD. The fragments thus obtained were ligated with pSFKT2 completely digested with PstI. After ligation was complete, the transformation was conducted with competent E. coli JM109 cells (Takara Shuzo Co., Ltd.). The cells were disseminated onto the L media plates containing 10 μg / ml IPTG, 40 μg / ml X-Gal and 25 μg / ml kanamycin. After overnight incubation, white colonies were collected and transformed strains were obtained after isolation of individual colonies. From the transformed strains, plasmids were prepared. The temperature sensitive shuttle vectors containing the cd promoter were designated pSFKTH, pSFKTI2, pSFKTI3, pSFKTI4, pSFKTId, pSFKTld, pSFKTI25 and pSFKTI26, respectively. (6) Integration of mutant cd promoter into chromosome The plasmids constructed in step (d) described above were each introduced into strain GB02 of Brevibacterium lactofermentum by the electric pulse method. Transformants were selected with CM2B medium plates (comprising 10 g / l of bactotriptone, 10 g / l of bacto-yeast extract, dg / l of NaCl, 10 μg / l of biotin and 15 g / l of agar and having a pH of 7.0) containing 25 μg / ml kanamycin at 25 ° C. After completion of the introduction, the obtained strains were cultured in liquid medium CM2B, disseminated on CM2B plates containing 2d μg / ml kanamycin after dilution at a concentration of 10 ^ to 10 ^ cfu per plate and cultured at 34 ° C. ° C. The strain having the temperature-sensitive plasmid became kanamycin sensitive because the replication of the plasmid was inhibited at that temperature and therefore could not form colonies. On the other hand, the strain that had plasmid DNA integrated into the chromosome could be selected because it could form colonies. The colonies thus obtained were harvested and separated into isolated colonies. Chromosomal DNA was extracted from the strain and PCR was conducted using chromosomal DNA as the template with primers shown in Seq ID No. 13 and Seq ID No. 1 d. Approximately 3 kb of amplified fragments were confirmed. It was proved that in this strain, the mutant cdd gene derived from the temperature-sensitive plasmid was integrated near the cd gene in the host chromosome by homologous recombination. (7) Preparation of strains having substituted cd promoter First, the kanamycin-sensitive strain was obtained from the strains having a cd gene integrated therein by the homologous recombination as described in step (6). ). Strains that had the plasmid integrated therein were diluted and disseminated on CM2B plates and then cultured at 34 ° C. After the formation of colonies, replicas of CM2B pineapples containing 2d μg / ml of kanamycin were made and incubated at 34 ° C. In this way, viable strains of kanamycin 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. 1 d to prepare icd gene fragments. The amplified fragments thus obtained were purified with SUPREC02 (Takara Shuzo Co., Ltd.) and then subjected to the sequencing reaction using an initiator shown in Seq ID No. 22 to determine the sequence of the promoter region thereof. As a result, strains that had ¡cd promoter sequences derived from pSFK ™, pSFKT! 2, pSFKTI3, pSFKTI4, pSFKTId, pSFKTI6, pSFKTI25 and? SFKTI26 were designated GC01, GC02, GC03, GC04, GC05, GC06, GC025 and GC026, respectively . In these strains, when the plasmid and the duplicated icd gene were cut from the chromosome, the wild type icd gene originally located on the chromosome was cut together with the vector plasmid, while the mutant cd gene introduced by the plasmid remained in the chromosome. (8) Determination of isocitrate dehydrogenase activity of mutant strains having mutant icd promoter A crude enzyme solution of ICDH was prepared using each of the 8 strains obtained in step (7) described above and strain GB02 in the same manner as that of step (7) in example 3. The activities of ICDH were determined as follows: the crude enzyme solution was added to a reaction solution containing 3d mM of TisHCI (pH 7.d ), 1.5 mM of MnS? 4, 0.1 mM of NADP and 1.3 mM of isocitric acid and the increase in absorbance at 340 nm at 30 ° C was determined to be a Hitachi U-3210 spectrophotometer as the activity of ICDH. The concentration of protein 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 cd promoter strains was greater than that of the progenitor strain.

TABLE 15 Strain dABS / min / mg Relative activity GB02 3.9 1.0 GC01 8.2 2.1 GC02 19.1 4.9 GC03 7.0 1.8 GC04 12.5 3.2 GC05 19.1 4.9 GC06 10.5 2.7 GC25 30.4 7.8 GC26 24.2 6.2 (9) Culture results of strains containing substituted cd promoter Each of the 8 strains obtained in step (7) described above was cultured in the same manner as those of step 9 of example 3. As a result, the improvement in the yield of L-glutamic acid was confirmed when any of the strains GC02, GC04, GC05, GC25 and GC26 were used as shown in Table 16. It was found that good results were obtained by introducing the mutation in the icd promoter to increase the ICDH activity at least 3-fold.

TABLE 16 Strain L-qyutamic acid (q / dl) GB02 9.2 GC01 9.0 GC02 9.5 GC03 9.1 GC04 9.4 GC05 9.6 GC06 9.2 GC25 9.9 GC26 9.8 EXAMPLE 5 Introduction of mutation in the promoter region of the pdh gene of coryneform glutamate producing bacteria (1) Cloning of pdhA gene from coryneform bacteria Initiators shown in Seq ID No. 25 and Seq ID No. 26 were synthesized by selecting regions that had high homology between subunits The pyruvate dehydrogenase (PDH) of Escherichia coli, Pseudomonas aeruginosa and Mycobacterium tuberculosis. PCR was conducted using chromosome from Brevibacterium lactofermentum ATCC 13869, prepared with a bacterial genomic DNA purification kit (Advanced Genetic Technologies Corp.), as the template under standard reaction conditions described on page 8 of PCR Technology (edited by H. Erlich and published by Stockton Press, 1989). The reaction solution was subjected to electrophoresis on an agarose gel to find that approximately 1.b kilobases of DNA fragment was simplified. The sequence of both ends of the DNA obtained was determined with synthetic DNA in Seq ID No. 25 t Seq ID No. 26. The sequence was determined by the method of Sanger [J. Mol. Biol., 143, 161 (1980)] with DNA Sequencing Equipment (Applied Biosystems Co.). The determined sequence was deduced for amino acids, and compared with E1 subunits of pyruvate dehydrogenase derived from each of Escherichia coli, Pseudomonas aeruginosa and Mycobacterium tuberculosis to find a high homology between them. Accordingly, it was determined that the DNA fragment amplified by PCR was a part of the pdhA gene encoding a subunit of pyruvate dehydrogenase from Brevibacterium lactofermentum ATCC13869. Cloning of the region towards the d 'end and towards the 3' end 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. An in vitro PCR Cloning Kit (Takara Shuzo Co., Ltd.) was used for cloning, using the sequences shown in Seq ID No. 27 and Seq ID No. 28 in the Sequence Listing as primers to clone the region towards the end d ', and the sequences shown in Seq ID No. 29 and Seq ID No. 30 as primers for cloning the region towards the 3 'end. After PCR using the kit, the DNA fragments of approximately 0.5, 2.5, 3.0, 1.5 and 1.8 kilobases were amplified for the region towards the d 'end of the fragments obtained by digestion with EcoRI, Hind III, Pst I, Sal I and Xba I, respectively; and DNA fragments of approximately 1.d, 3.d and 1.0 kilobases were amplified for the region towards the 3 'end from the fragments obtained by digestion BamHI, Hind III and Pst I, respectively. The sequences of these DNA fragments were determined in the same manner as described above. It was found that the amplified DNA fragments further contained an open reading frame of approximately 920 amino acids and also that a region assumed to be a promoter region was present in the region towards the d 'end. Because the amino acid sequence deduced from the DNA sequence of the open reading frame is highly homologous to the known subunit of pyruvate dehydrogenase such as that of E. coli, it was evident that the open reading frame was the pdhA gene It encodes the subunit The pyruvate dehydrogenase of Brevibacterium lactofermentum ATCC13869. The DNA sequence of the open reading frame was shown in Seq ID No. 31 in the Sequence Listing. In Seq ID No. 31 in the Sequence Listing, the amino acid sequence deduced from the DNA sequence is also shown. Since the N-terminal methionine residue of the protein is derived from ATG which is an initiation codon, it generally does not concern the essential function of the protein, and it is well known that the methionine residue is removed by the protein. Peptidase effect after translation. Therefore, in the protein described above, it is possible that the N-terminal meyionine residue has been removed. However, the GTG sequence is present in 6 bases towards the d 'end of ATG shown in Seq ID No. 31 in the Sequence Listing, and it is also possible that the amino acids are translated from this point. Pyruvate dehydrogenase from other microorganisms such as E. coli are composed of three subunits of E1, E2 and E3 and the genes that encode them constitute an operon in many cases. However, there was no open reading frame considered as an E2 and E3 subunit of pyruvate dehydrogenase in about 3 kilobases towards the 3 rd end of the pdhA gene. Instead, it was shown that a sequence that was supposed to be a terminator was present towards the 3 'end of the open reading frame. From these facts, it was assumed that the E2 and E3 subunits of pyruvate deshidogenase of Brevibacterium lactofermentum ATCC13869 were present in another region on the chromosome. (2) Construction of a plasmid to amplify pdhA It was evident that a strain obtained by introducing a gene encoding three subunits constituting E. coli PDH in Brevibacterium lactofermentum ATCC13869 gives an improved glutamic acid yield (JP No. 10-360619) . However, in PDH of Brevibacterium lactofermentum ATCC13869, only the pdhA gene encoding the El subunit has been cloned, and no examination had been done to know if the amplification of the gene is only effective in improving the yield of glutamic acid. Under these circumstances, the examination was made to know if the amplification of the pdhA gene alone is effective to improve the glutamic acid yield or not. The 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 using Brevibacterium lactofermentum ATCC13869 croosome 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 et al. published by Stockton Press, 1989) to amplify the pdhA gene. Among the primers thus synthesized, Seq ID No. 33 corresponded to the sequence of bases No. 1397 to No. 1416 in the 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 base sequences No. d3dd to No. 6374 in Seq ID No. 32 in the Sequence Listing that was represented from the d 'side. The PCR product thus obtained was purified by an ordinary method and reacted with restriction enzymes Sal I and EcoT22l. The fragment was ligated with pSFK (Patent Application No. 11-69896), digested with restriction enzymes Sal I and Pst I, with a ligation kit (Takara Shuzo Co., Ltd.). After transformation with competent cells (Takara Shuzo Co., Ltd.) of E. coli JM109, the cells were disseminated on L media plates (comprising 10 g / l of bactotriptone, dg / l of bacto-yeast extract, dg / l of NaCl and 1d g / l of agar having a pH of 7.2) containing 10 μg / ml of IPTG (isopropyl-β-D-thiogalactopyranoside), 40 μg / ml of X-Gal (5-bromo- 4-chloro-3-indolyl-β-D-galactoside) and 25 μg / ml kanamycin. After overnight incubation, white colonies were collected and transformed strains were obtained after isolation of individual colonies. 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. 10d, 1992). Restriction enzyme maps of DNA fragments inserted into the vectors were prepared and compared with the restriction enzyme map of the pdhA gene reported in sequence No. 32 of the Sequence Listing. A plasmid containing DNA fragments inserted therein that had the same restriction enzyme map as the pdhA gene was designated pSFKBPDHA. (3) Introduction of pASFKBPDHA in Brevibacterium lactofermentum ATCC13869 and GC2d and evaluation of the fermentation experiments Brevibacterium lactofermentum ATCC 13869 and GC2d were transformed with the plasmid pSFKBPDHA with the electric pulse method (JP KOKAI No. 2-207791) to obtain the strains transformed. The culture to produce L-glutamic acid was conducted with transformed strain ATCC13869 / pSFKBPDHA and GC2d / pSFKBPDHA obtained by introducing the plasmid pSFKBPDHA in Brevibacterium lactofermentum ATCC13869 and GC 2d as follows: the cells of ATCC13869 / pSFKBPDHA and GC2d / pSFKBPDHA obtained by culturing in Media plates CM2B containing 2d μg / ml kanamycin were inoculated into a medium (containing 1 liter of pure water containing 80 g of glucose, 1 g of KH2PO4, 0.4 g of MgSO4 7H2O, 30 g of (NH4) 2S04 , 0.01 g of FeS? 4 7H20, 0.01 g of MnSO4 7H2O, 1d ml of soy protein hydrolyzate, 200 μg of thiamine hydrochloride, 60 μg of biotin, 2d mg of kanamycin and dO g of CaC03; and that it had a pH adjusted to 8.0 with KOH). The culture was then stirred at 31 ° 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 (for GC2d / pSFK6 and GC2d / pSFKBDHA) or the medium eliminated biotin from the composition as described above (for ATCC13869 / pSFK &and ATCC13869 / pSFKBPDHA) in an amount of 5% and the shaking culture was conducted at 37 ° C until the sugar in the medium had been consumed. As a control, strains were obtained by transforming Brevibacterium lactofermentum ATCC13869 and GC2d with plasmid pSFK6 previously obtained able to replicate autonomously in coryneform bacteria by the electric pulse method (JP KOKAI No. 2-207791), it was cultivated in the same way as It was done previously. After the culture was completed, the amount of L-glutamic acid accumulated in the culture medium was determined with biotic analyzer Biotic Analyzer AS-210 (a product of Asahi Chemical Industry Co., Ltd). The results are shown in Table 17.

TABLE 17 Strain Yield of L-qytamic acid / g / dl) ATCC13869 / pSFK 3.6 ATCC13869 / pSFKBPDHA 3.8 GC2d / pSFK6 5.1 GC2d / pSFKBPDHA 5.3 From these results, it was evident that even the amplification of the pdhA gene is only effective enough to improve performance of Glu in Brevibacterium lactofermentum ATCC13869 and GC25. (4) Construction of plasmids for determination of mutated pdhA promoter activity To produce mutant of pyruvate dehydrogenase (PDH) promoter, the determination of the promoter region previously cloned from pdhA gel of Brevibacterium lactofermentum ATCC13869 was conducted and also the "determination of the difference in expression caused by the modification of the promoter region was driven by determining the β-galactosidase activity. The promoter region of the pdhA gene was assumed from the DNA sequence that had already been elucidated by cloning. As a result, it was assumed that base No. 2252 to No. 2257 and No. 2279 to No. 2284 in Seq ID No. 32 in the Sequence Listing was region -3d and region -10, respectively . Subsequently, the primers shown as Seq ID No. 3d and Seq ID No. 36 in the Sequence Listing were synthesized and the DNA fragments containing the pdhA gene promoter region were amplified by the PCR method using chromosomal DNA of Brevibacterium lactofermentum ATCC13869 as a template. Among the primers synthesized, Seq ID No. 3d corresponds to the sequence varying from base No. 2194 to base 2221 in Seq ID No. 32; but base No. 2198 had been replaced by C, and base No. 2200 and No. 2202 had been replaced by G, and the recognition sequence for Smal restriction enzyme had been inserted. Seq ID No. 36 corresponded to the sequence that varied from base No. 2373 to base No. 2398 in Seq ID No. 32; but base No. 2393 and No. 2394 had been replaced by G and the complementary strand of the DNA sequence having a restriction enzyme recognition sequence Smal inserted therein was represented from the end d '. PCR was conducted using Brevibacterium lactofermentum chromosome ATCC13869, prepared with Bacterial Genomic DNA Purification Kit 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 the pdhA gene. The PCR product thus obtained was purified by the ordinary method and reacted with restriction enzyme Sma I. The fragments were ligated with pNEOL lacking in the promoter region of lacZ gene that could be replicated in a coryneform bacterium and that had digested with restriction enzymes Sma I, (Example 4 (3) with a Ligation Kit Ligation Kit (Takara Shuzo Co., Ltd.) After transformation with competent cells (Takara Shuzo Co., Ltd.) coli JM109, the cells were disseminated on the plates of L medium (comprising 10 g / l of bactotriptone, dg / l of bactolevadura extract, dg / l of NaCl and 15 g / l of agar and having a pH of 7.2) containing 40 μg / ml of X-Gal (d-bromo-4-chloro-3-indolyl-β-D-galactoside) and 2d μg / ml of kanamycin After incubation overnight, blue colonies were collected and the transformed strains were obtained after isolation of individual colonies.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 the DNA fragments inserted into the vector by an ordinary method, the plasmid containing DNA fragment inserted therein was designated pNEOLBPDHAprol. In addition, primers indicated as Seq ID No. 37, Seq ID No. 38 and Seq ID No. 39 in the Sequence Listing were synthesized to construct plasmids in which a region that was supposed to be the promoter site was changed to the sequence of consensus. of promoters of coryneform bacteria. Using each of the primers and an initiator shown in Seq ID No. 36, DNA fragments in which the promoter region of the pdhA gene was changed to the consensus sequence were amplified by the PCR method using chromosomal DNA Brevibacterium lactofermentum ATCC13869 as a template. Among the primers synthesized, Seq ID No. 37 corresponded to the sequence that varied from base No. 2244 to base No. 2273 in Seq ID No. 32; Base No. 2255 had been replaced by C, and Base No. 2257 had been replaced by A; therefore, only the -35 region had been changed to the coryneform bacterium sequence. Seq ID No. 38 corresponds to the sequence that varied from base No. 2249 to base No. 2288 in sequence No. 32; Base No. 2279 and No. 2281 had been replaced by T; in this way only the -10 region had been changed to the consensus sequence of the coryneform bacterium. Sequence No. 39 corresponded to the sequence varying from base No. 2249 to base No. 2288 in Seq ID No. 32; Base No. 2255 had been replaced by C; base No. 2257 had been replaced by A and base No. 2279 and No. 2281 had been replaced by T; in this way, both the -35 region and the -10 region had been changed to the coryneform bacterium sequence. PCR was conducted using chromosome from Brevibacterium lactofermentum ATCC13869, prepared with a Bacterial Genomic DNA Purification Kit 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 pdhA gene promoter region with these primers so that the promoter region was changed to the consensus sequence. The PCR products thus obtained were purified by an ordinary method and reacted with Smal restriction enzyme. The fragments were ligated with pNEOL lacking the lacZ gene promoter region, which could be replicated in a coryneform bacterium and which had been digested with the Sma I restriction enzymes, with a Ligation Kit Ligation Kit (Takara Shuzo Co., Ltd.). After transformation with competent cells (Takara Shuzo Co., Ltd.) of E. coli JM109, the cells were disseminated on L media plates (comprising 10 g / l of bactotriptone, 5 g / l of bacto-yeast extract , dg / l of NaCl and 1d g / l of agar and having a pH of 7.2) containing 40 μg / ml of X-Gal (d-bromo-4-chloro-3-indolyl-β-D-galactoside) and 2d μg / ml kanamycin.

After being incubated overnight, blue colonies were collected and the transformed strains were obtained after isolation of individual colonies. 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. 10d, 1992). After sequencing DNA fragments inserted into the vector by an ordinary method, the plasmid containing DNA fragments wherein only the sequence in the -35 region had been changed to the consensus sequence, inserted therein was designated pNEOLBPDHApro3d; plasmids containing DNA fragments, wherein only the sequence in region -10 had been changed to the consensus sequence, was inserted therein and named pNEOLBPDHAprolO; and the plasmid containing DNA fragments, wherein the sequences in both the -3d region and the -10 region had been changed to the consensus sequence, inserted therein was designated pNEOLBPDHApro3d10. (d) The evaluation of the activity of the mutant pdhA promoter Brevibacterium lactofermentum ATCC 13869 was transformed with plasmids named pNEOLBPDHAprol, pNEOLBPDHAprolO and pNEOLBPDHApro3610 by the electric pulse method (J.P. KOKAI No. 2-207791) to obtain the transformant strains. The activity of the β-galactosidase of the transformants obtained was determined by the method described in example (4) 4. After changing the sequence in the promoter region to the cnosenso sequence, the activities of the β-galactosidase were as shown in Table 18, where the enzymatic activity of β-galactosidase having the promoter region of the pdhA gene was gave as 1.

TABLE 18 Strain β-galactosidase activity (relative value) ATCC13869 / pNEOLBPDHAprol 1 ATCC13869 / pNEOLBPDHAprolO 6 ATCC13869 / pNEOLBPDHApro3510 7.5 These results indicate that the putative promoter region was the promoter of the pdhA gene and that the expression of PdhA can be changed (increased) by changing the sequence in the region in the consensus sequence. This fact indicates that the expression can be changed, without using plasmid, by changing the promoter region of the pdhA gene. (6) Plasmid construction for the preparation of variant promoter strain Since it has been proven that the expression of pdhA can be changed by introducing mutations into the promoter, plasmids were constructed to prepare pdhA promoter-modified strains. Three constructs of the plasmid were constructed for the promoter-modified strains. Plasmids were where region -3d, region -10 and both regions were changed to the consensus sequence, respectively. Initiators shown in Seq ID No. 40 and Seq ID No. 41 were recently synthesized based on the DNA sequence that had already been cloned. Among the primers synthesized, Seq ID No. 40 was the complementary strand of the DNA sequence corresponding to the sequence varying from base No. 2491 to base No. 2621 in Seq ID No. 32, which was represented from the end 5 ', to which a sequence comprising three A followed by four T at the terminal 5' end. Seq ID No. 33 was the complementary strand of the DNA sequence corresponding to the sequence that varied from base No. 6020 to base No. 5039 of the pdhA gene in Seq ID No. 32, was represented from the 5 'end . PCR was conducted using Seq ID No. 33 and Seq ID No. 40 as the primers and chromosome of Brevibacterium lactofermentum ATCC13869 prepared with Bacterial DNA Genomic DNA Purification Kit 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). In addition, PCR was conducted using Seq ID No. 39 and Seq ID No. 41 and chromosome of Brevibacterium lactofermentum ATCC13869 as a template. The PCR products thus obtained were purified by an ordinary method. PCR was conducted using PCR products obtained using Seq ID No. 33 and No. 40, PCR products obtained by the use of Seq ID No. 39 and Seq ID No. 41 and Seq ID No. 33 and 41 as the initiators.

The PCR condition was as follows: the concentration of these four DNAs would be 10 μM in the reaction mixture and the taq (Takara Shuzo Co., Ltd.) was used without template. The 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 pSFKT2 plasmid sensitive to temperature with Sali, which can be replicated in a coryneform bacterium . Using Ligation Kit Ligation Kit (Takara Shuzo Co., Ltd.). After transformation with competent cells (Takara Shuzo Co., Ltd.) of E. coli JM109, the cells were disseminated on L media plates (comprising 10 g / l of bactotriptone, dg / l of bacto-yeast extract, dg / l of NaCl and 15 g / l of agar having a pH of 7.2) containing 10 μg / ml of IPTG (isopropyl-β-D-thiogalactopyranoside), 40 μg / ml of X-Gal (5-bromo- 4-chloro-3-indolyl-β-D-galactoside) and 25 μg / ml kanamycin. After being incubated overnight, white colonies were collected and transformants were obtained after isolation of individual colonies. 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.10d, 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 DNA fragments containing plasmid where only the sequences in the -3d region and in the promoter -10 region was changed to the consensus sequence of the coryneform bacterium, inserted therein was designated pSFKTPDHApro3d10.

A plasmid in which the -3d region of the pdhA gene promoter had been changed to the consensus sequence of coryneform bacteria, and also the plasmid wherein the -10 region of the pdhA gene promoter had been changed to the consensus sequence of coryneform bacteria was constructed in the same manner as that described above except that Seq ID No. 39 in the Sequence Listing was replaced by Seq ID No. 37 and 38, respectively. These plasmids were designated pSFKTPDHApro3d and pSFKTPDHAprol O, respectively. (7) Preparation of promoter-modified strains. Strains having a modified pdhA gene promoter were prepared by homologous recombination using the plasmid to prepare varied promoter strain constructed in step (6) described above. First, GC2d was transformed with plasmid pSFKTPDHApro3d10 to prepare a modified strain by promoter by the electric pulse method (see J.P. KOKAI No. 2-207791). The cells were disseminated on plates of CM2B medium (comprising 10 g / l of polypeptone, 10 g / l of bacto-yeast extract, dg / l of NaCl, 10 μg / ml of biotin and 15 g / l of agar and pH 7.2) and cultured at 25 ° C to obtain transformed strains. These transformants were grown in a CM2B liquid medium in a test tube overnight and then disseminated on CM2B medium plates containing 2d μg / ml kanamycin and cultured at 34 ° C to obtain a strain produced by a recombination that contained plasmid pSFKTPDHApro3d10 in its chromosome inserted by homologous recombination. After isolation of individual colonies, the strain was cultured in liquid medium CM2B in a test tube overnight. After adequate dilution, it was disseminated on plates with CM2B medium and cultured at 31 ° C. After the colonies began to appear, replicas were made on CM2B media plates containing 2dμg / ml kanamycin to obtain cnamicin-sensitive strains. Since two types of the strains, i.e., one strain having the sequence of the wild-type strain for the pdhA gene promoter region and the other strain having the mutation introduced therein, could occur, the region sequencing Therefore, a strain modified by promoter, where the mutation had been introduced into the promoter region of the pdhA gene, was obtained. In this strain, the -35 region and the -10 region of the pdhA gene promoter had been changed to the consensus sequence of coryneform bacteria. This strain was called GD3510. strains wherein the -35 or -10 region of the pdhA gene promoter had been changed to the consensus sequence of the coryneform bacteria was obtained in the same manner as that described above except that the plasmid described above pSFKTPDHApro3610 to produce the promoter-modified strain was replaced by the plasmid pSFKTPDHApro3d and pSFKTPDHAprolO to produce promoter-modified strains and was designated GD3d and GD10, respectively. (8) Evaluation of flask culture results of strains modified by pdhA gene promoter The flask culture to produce L-glutamic acid was conducted with three types of strains modified by pdhA gene promoter as described above. Each of the cells of the GD3610, GD3d, GD10 and GC2d promoter-modified strains obtained by plating CM2B medium was inoculated into a medium (comprising 1 liter of pure water containing 30 g of glucose, 1 g of KH2PÜ4, 0.4 g of MgS? 4 7H2O, 30 g of (NH4) 2S? 4, 0.01 g of FeS? 4 7H2O, 0.01 g of MnS? 4 7H2O, 15 ml of soy hydrolyzate, 200 μg of thiamine hydrochloride, 60 μg of biotin and 50 g of CaC 3, and which had a pH adjusted to 8.0 with KOH). Then, the culture was stirred 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 described above in an amount of d% and the agitation culture was conducted at 37 ° C until the sugar had been consumed. After completion of the culture, the amount of L-glutamic acid accumulated in the culture liquid was determined by means of a Biotic Analyzer 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) GC2d 1.9 GD35 2.0 GD10 2.0 GD3510 2.1 It is evident from the results that the promoter-modified strains obtained gave improved Glu yields.

EXAMPLE 6 Introduction of mutation in the gene promoter region of arginosuccinate synthetase (1) Determination of DNA sequence at the d 'end of the argG gene In order to amplify the argG gene of Brevibacterium flavum by PCR, the DNA sequences in the regions towards the d' end and towards the 3 'end of the ORF were determined. The determination of the DNA sequences was conducted by synthesizing an initiator based on the known DNA sequence (access AF030620 to the Gene Bank) of ORG of argG gene of Corynebacterium glutamicon and using an in vitro PCR cloning kit (Takara Shuzo Co., Ltd.) according to the instruction manual included in the equipment. As primers, an oligonucleotide (primers 1 and 2) having the DNA sequences set as Seq ID No. 42 and Seq ID No. 43 for the region towards the d 'end, and the oligonucleotide (primers 3 and 4) were specifically used. ) having the DNA sequences set as Seq ID No. 44 and Seq ID No. 4d for the region towards the 3 'end. The DNA sequences in the region towards the d 'end and towards the 3' end of argG were determined by completely digesting the chromosomal DNA of strain 2247 (ATCC14067), ie the wild-type strain of Brevibacterium flavum with an enzyme of EcoRI restriction, conducting first PCR with primer 2 or 3 (which had sequence No. 43 or 44), and conducting the second PCR with primer 1 or 2 (which had sequence No. 42 or 4d). (2) Promoting region prediction A promoter-shaped sequence at the 5-terminus of the ORF of the argG gene was searched for the sequences described above with commercially available software (GENETYX). The mutation was introduced into a region of the highest score (approximately 120 bp towards the 5 'end of the first ATG). Then, the activity of the promoter was measured. (3) Introduction of mutations in the promoter sequence and determination of the activity of the mutant promoters Mutation-introducing primers 9, 10, 11, 12 or 13 and 7 (which had the sequences No. 50, 51, 52, 53 , 54 or 48, respectively) for a region of the highest score was used and the first PCR was conducted with chromosomal DNA of a strain AJ 12092 as a template. The second PCR was conducted with the same chromosomal DNA as the template using the PCR product as the primer for the 3 'end and also using the primer 8 having the sequence No. 49 as the primer at the 5' end to obtain fragments of DNA that had the mutation introduced into the intended promoter region. To determine the activity of mutant promoters, these DNA fragments were inserted into the Saml site of the pNEOL promoter probe vector so they were in the same direction as the lacZ reporter gene to obtain the 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 strain AJ 12092 and primers 7 and 8 at the d end of the lacZ gene of pNEOL. -0, pNEOL-1, pNEOL-2, pNEOL-3, pNEOL-4 and pNEOL-7 were introduced into strain AJ 12092, respectively.The plasmids were introduced by electric pulse method (jP 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, dg of NaCl and 1 g of agar and having a pH of 7.2) containing 4 μg / ml of chloramphenicol, as strains resistant to chloramphenicol These strains were each disseminated on agar medium (containing Od g / dl glucose, 1 g / dl polypeptone, 1 g / dl of yeast extract, 0.5 g / dl of NaCl and 5 μg / l of chloramphenicol), and was cultivated at 31.5 ° C for 20 hours. 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 KH2PO4, 0.04 g / dl of MgS 4, 0.001 g / dl of FeS? 4, 0.01 g / dl of MnS? 4, 5 μg / dl of VB- | , 5 μg / dl of biotin and 4d mg / dl (in terms of N) of soy hydrolyzate]. Afterwards, it was cultured at 31 ° C for 18 hours, the activity of the β-galactosidase of the obtained cells was determined as described in example 4 (4). Since the activity of β-galactosidase was detected in AJ12092 / pNEOL-0 as shown in Table 20, it was found that the DNA fragment inserted at the d 'end of the lacZ structure gene functioned as a promoter. In addition, the activity of the β-galactosidase of each of the strains introduced in plasmid was greater than that of AJ12092 / pNEOL-0. It was found that the transcription activity was increased by introducing the mutation in the promoter-like sequence, as shown in Table 20.

TABLE_20 Relative activity (AJ12092 / pNEOL-0 = 1) AJI 12092 nd AJI12092 / pNEOL-0 1.0 AJI12092 / pNEOL-1 2.8 AJI12092 / pNEOL-2 2.7 AJI12092 / pNEOL-3 1.8 AJI12092 / pNEOL-4 0.8 AJI12092 / PNEOL-7 3.0 (4) Construction of a plasmid for mutation introduction Was conducted per using primers 14 and 15 (which had the Seq ID No. 55 and Seq ID No. 56 sequences) with chromosomal DNA of strain AJ12092 as the template. These DNA fragments thus obtained were inserted into a smal site at a cloning vector multiclonation site pHSG398 (a product of TaKaRa) to construct the pO plasmid. 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. The DNA fragments thus obtained were ligated to obtain p3 mutation-introducing plasmids (mutants derived from mutation-introducing primer 11) and p7 (mutation-inducing primer-derived mutant 13). (5) Introduction of mutation-introducing plasmids in Arq-producing bacteria Each of the plasmids thus obtained was introduced into Arg-producing bacteria of strain Brevibacterium lactofermentum AJ 12092 by electric pulse method (J. P. KOKAI No. 2-207791). Since these plasmids could not replicate autonomously in Brevibacterium, only the strains obtained by integrating these plasmids into the chromosome by homologous recombination could be selected as strains resistant to Cm. Strains in which the mutation-introducing plasmid was integrated into the chromosome were selected as chloramphenicol-resistant strains on CM2G medium plates (comprising 1 liter of pure water containing 10 g of polypeptone, 10 g of yeast extract, dg of glucose, dG of NaCl and 1 dg of agar, and having a pH of 7.2) containing 5μg / ml of chloramphenicol. Then, Cm-sensitive strains were selected in which the argG gene promoter region was replaced by the designated modified sequence. As a result, a strain substituted with sequence P3 (AJ12092-P3) and a strain substituted with sequence P7 (AJ12092-P7) were obtained. (8) Cloning of arqG gene Based on the DNA sequence as (1), oligonucleotides (primers 5 and 6) having the DNA sequence set in Seq ID No. 46 and Seq ID No. 47 were synthesized to conduct PCR using chromosomal DNA of Brevibacterium flavum as a template. The PCR reaction was conducted in 25 cycles, each cycle consisting of 94 ° C for 30 seconds, d5 ° C for one second and 72 ° C for 2 minutes and 30 seconds. The DNA fragment thus obtained was cloned into the Smal site in cloning vector multicloning site pSTV29 (Takara Shuzo Co., Ltd.) to obtain pSTVargG. In addition, pargG was prepared by inserting in the SalI site of pSTVargG a fragment containing the origin of replication obtained by treating pSAK4 fixed in Example 1 with Salí. (7) Introduction of pargG in Brev pargG was introduced in the strain Brevibacterium lactofermentum AJ12092. The plasmid was introduced by electric pulse method (J.P. KOKAI No. 2-207791). The transformant was selected as a strain resistant to chloramphenicol on plates of CM2G medium (comprising 1 liter of pure water containing 10 g of polypeptone, 10 g of yeast extract, 5 g of glucose, 5 g of NaCl and 1 g of agar and having a pH of 7.2) containing 4 μg / ml chloramphenicol. (8) ArgG activity of promoter-modified strains The ArgG activities of the two above-described types of strains modified by argG promoter and a strain obtained by amplifying argG with plasmid (AJ12092 / pargG) were determined. these strains were each disseminated on an agar medium (containing Od g / dl glucose, 1 g / dl polypeptone, 1 g / dl extract extract, 0.5 g / dl NaCl and d μg / l chloramphenicol) and were grown at 31.d ° C for 20 hours. One quart of the cells thus obtained was inoculated into a medium [containing 3 g / dl glucose], 1.5 g / dl of ammonium sulfate, 0.1 g / dl of KH P? 4, 0.04 g / dl of MgS? 4, 0.001 g / dl of FeS? 4, 0.01 g / dl of MnS? 4, 5 μg / dl of VB- | , d μg / dl of biotin and 4d mg / dl (in terms of N) of soy hydrolyzate]. After being cultured at 31 ° C for 18 hours, the ArgG activity of the cells obtained by the method described above was determined [Journal of General Microbiology (1990), 136, 1177-1183]. The ArgG activities of the two above-described types of strains modified by argG promoter and strain (AJ12092 / pargG) obtained by amplifying argG with plasmid as shown in Table 21. It is evident from Table 21 that by introducing the mutation in the promoter, the ArgG activity of AJ12092-P3 was increased to approximately two times greater than that of the parent strain, and the activity of AJ12092-P7 was increased to approximately three times that of the progenitor strain. The ArgG activity of AJ12092 / pargG was approximately 4.5 times greater than that of the parent strain.

CUADR0 21 Relative activity (AJ12092 = 1) AJÍ 12092 1.0 AJI12092-P3 2.1 AJI12092-P7 2.9 AJI12092 / parqG 4.4 (9) Production of Arg with promoter-modified strains The flask culture of each modified strain was conducted by promoter of argG. The progenitor strains were also cultured as controls AJ12092 and AJ12092 / pargG. These strains were inoculated each in a medium [which contained 0.1 g / dl of KH2P? 4, 0.04 g / dl of MgS? 4, 0.001 g / dl of FeS? 4, 0. 01 g / dl of MnS? 4, 5 μg / dl of VB- | , d μg / dl of biotin and 4d mg / dl (in terms of N) of soy hydrolyzate]; and then they spread on an agar medium (containing O.d g / dl of glucose, 1 g / dl of polypeptone, 1 g / dl of yeast extract, O.d g / dl of NaCl and dμg / l of chloramphenicol) and cultured at 31.d ° C during hours. One quart of the cells was cultured in a flask containing 4 g / dl of glucose and 6.d g / dl of ammonium sulfate at 31.d ° C until the glucose had been completely consumed. The absorbance (CD620) of the diluted culture liquid a concentration of 1/51 with 0.2 N HCl solution, the amount of arginine produced (concentration: g / dl) and culture time are shown in Table 22.

It can be seen from Table 22 that when the 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 also evident that the productivity of Arg thereof was greater than that of the strain amplified by plasmid.

TABLE 22 OD Arg Culture time Productivity (g / di) (h) (g / dl / h) AJI2092 0.502 1.25 48 0.026 AJI2092-P3 0.510 1.47 48 0.031 AJI2092-P7 0.514 1.43 48 0.030 AJI2092 / pargG 0.520 1.47 52 0.028 EXAMPLE 7 Introduction of mutation in promoter region of GDH gene of glutamate-producing coryne bacteria (1) Construction of mutant gdh plasmids Plasmids having the GDH promoter sequence of strain FGR1 and FGR2 described in Example 2 were constructed by site-directed mutagenesis. To obtain the GDH promoter sequence of the FGR1 strain, PCR was conducted using synthetic DNA shown in Seq ID No. 67 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 using synthetic DNA shown in Seq ID No. 68 and synthetic DNA shown in Seq ID No. 69 as the primers with chromosomal DNA of ATCC13869 as the template. In addition, PCR was conducted using synthetic DNAs shown in Seq ID No. 67 and Seq ID No. 68 as the primers with a mixture of these products.

PCR as the template. The PCR product thus obtained was inserted into the Smal site of pSFKT2 (Japanese Patent Application No. 11-69896) to construct pSFKTGU. To obtain the GDH promoter sequence of the FGR2 strain, PCR was conducted using synthetic DNA shown in Seq ID No. 67 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 using synthetic DNA shown in Seq ID No. 68 and synthetic DNA shown in Seq ID No. 61 as the primers and chromosomal DNA of ATCC13869 as the template.

In addition, PCR was conducted using synthetic DNA shown in Seq ID No. 67 and Seq ID No. 68 as the primers and a mixture of these PCR products as the template. The PCR product thus obtained was inserted into the SmaI site of pSFKT2 (Japanese Patent Application No. 1 1-69896) for construct pSFKTG07. The DNA sequences of the fragments inserted into the Smal sites of pSFKTGH and pSFKTG07 were determined to confirm that no mutation had been introduced in other regions than the promoter region in GDH. (2) Construction of strains modified by GDH promoter After, pSFKTGH and pSFKTG07 were introduced into the strain AJ 13029 by the electric pulse method and transformants were selected that grew on CM2B plates containing 25 μg / ml kanamycin at 25 ° C. Transformants were grown at 34 ° C to select strains that were resistant to kanamycin at 34 ° C. The fact that a strain is resistant to kanamycin at 34 ° C indicates that pSFKTG1 1 or pSFKTG07 was thus integrated on the chromosome of strain AJ 13029. The kanamycin-sensitive strains were obtained from the strains in which the plasmid was integrated into the chromosome. The GDH promoter sequences of these strains were determined. Strains having the same gdh promoter sequence as those of pSFKTGH and pSFKTG07 were designated GA01 and GA02, respectively. (3) L-glutamic acid productivity confirmation of gdh promoter-modified strains The glutamic acid productivities of strains GA01 and GA02 and progenitor strain AJ 13029 were confirmed in the same manner as for example 2 (2) given previously. As a result, a remarkable improvement in glutamic acid accumulation was recognized in GA01 and GA02 as shown in Table 23.

TABLE 23 Strain Glu (g / dl) Specific activity of GDH Relative value AJ 13029 2.6 7.7 GA01 3.0 22.3 2.9 Ga02 2.9 27.0 3.5 (4) Construction of self-cloning gdh plasmid First, the self-cloning vector pAJ220 was constructed. pAJ226 (J.P. KOKAI No. 61-152289) was treated with EcoRV and PstI to prepare a fragment containing a region that could replicate autonomously in a coryneform bacterium. The fragment was ligated with approximately 0.7 kb of DNA fragment obtained by treating pAJ224 (J.P. KOKAI No. Sho 61-152289) with EcoRV and PstI to obtain a plasmid pAJ220. This plasmid could be replicated autonomously in a coryneform bacterium, and could give the host resistance to trimethoprim. The PCR reaction was conducted using synthetic DNA shown in Seq ID No. 63 and Seq ID No. 64 as the primers and chromosomal DNA of ATCC13869 strain of wild-type coryneform bacteria as the template. The gdh gene fragment thus obtained was inserted into the Ball site of pAJ220 for construct pAJ220G. The promoter was present near the Ball site of pAJ220 and the expression of the inserted gene was increased depending on the direction of the gene inserted in the Ball site. PAJ220G and pGDH were introduced into strain ATCC13869 by the electric pulse method. The GDH activities of the strains thus constructed were determined by the method indicated in step (1) described above. As a result, the GDH activity of the strain into which pAJ220G was introduced was approximately 1.5 times greater than that of the strain into which dGDH had been introduced as shown in Table 24.

TABLE 24 Strain GDH-specific activity Relative value ATCC13869 7.7 1.0 ATCC13869 / pGDH 82.7 10.7 ATCC13869 / pAJ220G 120.1 15.6 (d) Investigations on the influence of gdh activity on yield and Asp obtained as byproduct pGDH and pAJ220G were introduced into AJ13029 by the method of electric pulse. Each of these strains and those obtained in step (2) described above were inoculated into a seed culture medium having a chewed composition in Table 2d and the shaking culture was conducted at 31 ° C for 24 hours. to obtain the sowing crop. 300 ml of primary culture medium having a composition shown in Table 25 was replaced in each of the 500 ml glass bottle fermenters and then sterilized by heating. 40 ml of the sow crops described above were inoculated into the medium. The culture was started at a temperature of 31.5 ° C while the agitation speed and the aeration rate were controlled at 800 to 1300 rpm and 1/2 to 1/1 wm, respectively. The pH of the culture liquid was maintained at 7.5 with gaseous ammonia. The temperature was changed to 37 ° C 8 hours after the start of the culture. The culture was terminated when the glucose had been completely consumed in 20 to 40 hours and the amount of L-glutamic acid produced and accumulated in the culture liquid was determined (Table 26). The activity of GDH to obtain the highest yield was approximately 3 times higher. When the activity of GDH rose more, the degree of improvement in performance was reduced. When the GDH activity was increased to approximately 16 times, the yield was further reduced. The amino acids produced as byproducts were analyzed with Hitachi Amino Acid Analyzer L-8500 Amino Acid Analyzer to find that the GDH activity was high, the amount of accumulated aspartic acid and alanine was increased. These results proved the following facts: to increase the yield of glutamic acid, it is necessary to adequately increase the activity of GDH so as not to cause a remarkable increase in the amount of aspartic acid and alanine. One of the effective methods for this consists in the introduction of several mutations in GDH promoter to control the GDH activity at approximately 3 times higher than that of the progenitor strain.

TABLE 25 Concentration Ingredient Cultivation of sowing Main cultivation Glucose 50 g / l 150 g / l KH2PO4 1 g / i 2 g / l MgS0. 7H20 0.4 g / l 1.5 g / l FeS04. 7H20 10 mg / l 15 mg / l MnS? 4. 4H2Ó 10 mg / l 15 mg / l Hydrolyzed soy protein 20 ml / l 50 ml / l Biotin 0.5 mg / l 2 mg / l Thiamine hydrochloride 2 mg / l 3 mg / l TABLE 26 Activity Glu Asp Relative Wing of Value Strain (g / di) (mg / dl) (mg / dl) Relative GDH AJ 13029 8.3 49 60 7.7 1.0 GA01 9.0 146 152 22.3 2.9 GA02 8.9 163 156 27.0 3.5 AJ13029 / pGDH 8.8 201 190 82.7 10.7 AJ13029 / pAJ220G 7.6 290 590 120.12 15.6 LIST OF SEQUENCES < 110 > Ajinomoto Co. Inc. < 120 > Method for building amino acid producing bacteria, and method for preparing amino acids by fermentation with bacteria producing amino acids constructed < 130 > OP 99052 < 1d0 > JP 10-271786 < 151 > 1998-9-26 < 1d0 > JP 10-271787 < 151 > 1998-9-25 < 160 > 6 < 210 > 1 < 211 > 46 < 212 > nucleic acid < 400 > 1 ttaattcttt gtggtcatat ctgcgacact gccataattt gaacgt < 210 > 2 < 211 > 46 < 212 > nucleic acid < 400 > 2 ttaattcttt gcggtcatat ctgcgacact gccataattt gaacgt < 210 > 3 < 211 > 46 < 212 > nucleic acid < 400 > 3 ttaattcttt gtggtcatat ctgcgacact gctataattt gaacgt < 210 > 4 < 211 > 46 < 212 > nucleic acid < 400 > 4 ttaattcttt gttgacatat ctgcgacact gctataattt gaaqgt < 210 > 5 < 211 > 46 < 212 > nucleic acid < 400 > 5 ttaattcttt gttgccatat ctgcgacact gctataattt gaacgt < 210 > 6 < 211 > 46 < 212 > nucleic acid < 400 > 6 ttaattcttt gttgtcatat ctgcgacact gctataattt gaacgt < 210 > 7 < 211 > 30 < 212 > nucleic acid < 220 > initiator A to clone gltA from Brevibacterium lactofermentum < 400 > 7 gtcgacaata gcctgaatct gttctggtcg < 210 > 8 < 211 > 30 < 212 > nucleic acid < 220 > initiator A to clone gltA from Brevibacterium lactofermentum < 400 > 8 aagcttatcg acgctcccct ccccaccgtt < 210 > 9 < 211 > 20 < 212 > nucleic acid < 220 > initiator 1 to introduce a mutation of gltA promoter < 400 > 9 atcggtataa cgtgttaacc < 210 > 10 < 211 > 20 < 212 > nucleic acid < 220 > initiator 2 to introduce a mutation of gltA promoter < 400 > 10 atcggtataa tgtgttaacc < 210 > 11 < 211 > 40 < 212 > nucleic acid < 220 > initiator 4 to introduce a mutation of gltA promoter < 400 > 11 gatttgacaa aaccgcattt atcggtataa tgtgttaacc < 210 > 12 < 211 > 28 < 212 > nucleic acid < 220 > gltA promoter sequence initiator < 400 > 12 agggatccgt ccagtctcag acagcatc < 210 > 13 < 211 > 17 < 212 > nucleic acid < 220 > universal initiator M13RV < 400 > 13 caggaaacag ctatgac < 210 > 14 < 211 > 20 < 212 > nucleic acid < 220 > initiator A to clone ICDH < 400 > 14 gaattcgctc ccggtgcagc < 210 > 15 < 211 > 20 < 212 > nucleic acid < 220 > initiator B to clone ICDH < 400 > 15 gatgcagaat tccttgtcgg < 210 > 16 < 211 > 28 < 212 > nucleic acid < 220 > initiator I to introduce an ICD promoter mutation < 400 > 16 tggattgctg gctataatgg tgtcgtga < 210 > 17 < 211 > 53 < 212 > nucleic acid < 220 > initiator 2 to introduce an ICD promoter mutation < 400 > 17 caacccacgt tcagttgaca actactggat tgctggctat aatggtgtcg tga < 210 > 18 < 211 > 53 < 212 > nucleic acid < 220 > initiator 3 to introduce an ICD promoter mutation < 400 > 18 caacccacgt tcagttgact actactggat tgctggctaa agtggtgtcg tga < 210 > 19 < 211 > 28 < 212 > nucleic acid < 220 > initiator 4 to introduce an ICD promoter mutation < 400 > 19 ggctgaaact gctataatag gcgccagc < 210 > 20 < 21 1 > 51 < 212 > nucleic acid < 220 > initiator 5 to introduce an ICD promoter mutation < 400 > 20 ggaaacacgg cgttgccatg cggggctgaa actgctataa taggcgccag c < 210 > 21 < 21 1 > 51 < 212 > nucleic acid < 220 > initiator 6 to introduce an ICD promoter mutation < 400 > 21 ggaaacacgg cgttgacatg cggggctgaa actgctataa taggcgccag c < 210 > 22 < 21 1 > 22 < 212 > nucleic acid < 220 > ICD promoter sequence starter < 400 > 22 gtgcgggtcc agatgatctt ag < 210 > 23 < 211 > 20 < 212 > nucleic acid < 220 > initiator A to amplify nptll < 400 > 23 gggatcccgg atgaatgtca < 400 > 24 < 211 > 23 < 212 > nucleic acid < 220 > initiator B to amplify nptll < 400 > 24 gcccggggtg ggcgaagaac tec < 210 > 25 < 211 > 23 < 212 > nucleic acid < 220 > initiator to amplify LA of pdhA gene Brevibacterium lactofermentum < 400 > 2d primer to amplify LA gene from pdhA from Brevibacterium lactofermentum aci gti tci atg ggi cti ggi ce < 210 > 26 < 211 > 23 < 212 > nucleic acid < 220 > < 400 > 26 ect tci ceg tti agi gti gti cg < 210 > 27 < 211 > 30 < 212 > nucleic acid < 220 > initiator for in vitro cloning LA gene of pdhA from Brevibacterium lactofermentum < 400 > 27 ttg cag tta acc acg aag gtc agg ttg tec < 210 > 28 < 211 > 30 < 212 > nucleic acid < 220 > initiator for in vitro cloning LA gene of pdhA from Brevibacterium lactofermentum < 400 > 28 tgg atg aga cea cgt gat tet ggc teg tec < 210 > 29 < 211 > 30 < 212 > nucleic acid < 220 > initiator to clone in vitro LA gene of pdhA from Brevibacterium lactofermentum < 400 > 29 here gat ect gca cga agg cat caca cga ggc < 210 > 30 < 211 > 30 < 212 > nucleic acid < 220 > initiator for in vitro cloning LA gene of pdhA from Brevibacterium lactofermentum < 400 > tea teg ctg cgg gta ect ect acg cea ecc < 210 > 31 < 211 > 2766 < 212 > nucleic acid < 213 > Brevibacterium lactofermentum ATCC13869 < 220 > LA gene of pdha from Brevibacterium lactofermentum ATCC13869 < 400 > 31 atg gcc gat ca gca aaa ctt ggt ggt aag ecc teg gat gac tet aac 4í Met Wing Asp Gln Wing Lys Leu Gly Gly Lys Pro Being Asp Asp Being Asn 1 5 10 15 tcc gcg atg ate cgc gat ggc gtg gca tet tat ttg aac gac tea gat 96 Phe Wing Met lie Arg Asp Gly Val Wing Being Tyr Leu Asn Asp Being Asp 20 25 30 ccg gag gag acc aac gag tgg atg gat tea etc gac gga tta etc cag 144 Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln 35 40 45 gag tet tet cea gaa cgt gct cgt tac etc atg ctt ct ttg ctt gag 192 Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu 50 55 60 cgt gca tet gca aag cgc gta tet ctt ecc cea atg acg tea acc gac 240 Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp 65 70 75 80 cgt gag cag ggt ggc att ccg tec tac ect cae cea cae ggt atg aag 624 Arg Glu Gln Gly Gly lie Pro Ser Tyr Pro His Pro His Gly Met Lys 195 200 205 gac ttc tgg gag ttc cea act gtg tec atg ggt ctt ggc cea atg gat 672 Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp 210 215 220 gee att tac cag gca cgt tcc aac cgc tac etc gaa aac cgt ggc ate 720 Wing lie Tyr Gln Wing Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly lie 225 230 235 240 aag gac acc tet gac cag falls gtc tgg gee ttc ctt ggc gac ggc gaa 768 Lys Asp Thr Ser Asp Gln His Val Trp Wing Phe Leu Gly Asp Gly Glu 245 250 255 atg gac gag cea gaa tea cgt ggt etc ate cag cag gct gca ctg aac 816 Met Asp Glu Pro Glu Ser Arg Gly Leu lie Gln Gln Ala Ala Leu Asn 260 265 270 aac ctg gac aac ctg acc ttc gtg gtt aac tgc aac ctg cag cgt etc 864 Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu 275 280 285 gac gga ect gtc cgc ggt aac acc aag ate ate cag gaa etc gag tec 912 Asp Gly Pro Val Arg Gly Asn Thr Lys lie lie Gln Glu Leu Glu Ser 290 295 300 ttc ttc cgt ggc gca ggc tgg tet gtg ate aag gtt gtt tgg ggt cgc 960 Phe Phe Arg Gly Wing Gly Trp Ser Val lie Lys Val Val Trp Gly Arg 305 310 315 320 gag tgg gat gaa ctt cg gag aag gac ggt ca ggt gca gtt gag gag 1008 Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu 325 330 335 ate atg aac aac acc tec gat ggt gac tac cag acc ttc aag gct aac 1056 lie Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Wing Asn 340 345 350 gac ggc gca tat gtt cgt gag drops ttc ttc gga cgt gac cea cgc acc 1104 Asp Gly Wing Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr 355 360 365 gca aag etc gtt gag aac atg acc gac gaa gaa ate tgg aag ctg cea 1152 Wing Lys Leu Val Glu Asn Met Thr Asp Glu Glu Lie Trp Lys Leu Pro 370 375 380 cgt ggc ggc falls gat tac cgc aag gtt tac gca gee tac aag cga gct 1200 Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Wing Tyr Lys Arg Wing 385 390 395 400 ctt gag acc aag gat cgc cea acc gtc ate ctt gct drops acc att aag 124 £ Leu Glu Thr Lys Asp Arg Pro Thr Val lie Leu Ala His Thr He Lys 405 410 415 ggc tac gga etc ggc falls aac ttc gaa ggc cgt aac gca acc falls cag 1296 Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Wing Thr His Gln 420 425 430 atg aag aag ctg acg ctt gat gat ctg aag ttg ttc cgc gac aag cag 1344 Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln 435 440 445 ggc ate cea ate acc gat gag cag ctg gag aag gat ect tac ctt ect 1392 Gly He Pro He Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro 450 455 460 ect tac tac cae ce ggt gaa gac gct ect gaa ate aag tac atg aag 1440 Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu He Lys Tyr Met Lys 465 470 475 480 gaa cgt cgc gca gcg etc ggt ggc tac ctg cea gag cgt cgt gag aac 14 Glu Arg Arg Wing Wing Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn 485 490 495 TAC GAT CCA ATT CAG GTT CCA CCA CTG GAT AAG CTT CGC TCT GTC CGT 1536 Tyr Asp Pro He Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg 500 505 510 aag ggc tec ggc aag cag cag ate gct acc act atg gcg act gtt cgt 1584 Lys Gly Ser Gly Lys Gln Gln He Wing Thr Thr Met Wing Thr Val Arg 515 520 525 acc ttc aag gaa ctg atg cgc gat aag ggc ttg gct gat cgc ctt gtc 1632 Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Wing Asp Arg Leu Val 530 535 540 cea ate att ect gat gag gca cgt acc ttc ggt ctt gac tet tgg ttc 1680 Pro He He Pro Asp Glu Wing Arg Thr Phe Gly Leu Asp Ser Trp Phe 545 550 555 560 cea acc ttg aag ate tac aac ccg falls ggt cag aac tac gtg ect gtt 1728 Pro Thr Leu Lys He Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val 565 570 575 gac falls gac ctg atg etc tec tac cgt gag gca ect gaa gga cag ate 1776 Asp His Asp Leu Met Leu Ser Tyr Arg Glu Pro Wing Glu Gly Gln He 580 585 590 ctg falls gaa ggc ate aac gag gct ggt tec gtg gca teg ttc ate gct 1824 Leu His Glu Gly He Asn Glu Wing Gly Ser Val Wing Being Phe He Wing 595 600 605 gcg ggt acc tec tac gee acc falls ggc aag gee atg att ccg ctg tac 1872 Wing Gly Thr Ser Tyr Wing Thr His Gly Lys Wing Met He Pro Leu Tyr 610 '615 620 ate ttc tac teg atg ttc gga ttc cag cgc acc ggt gac tech ate tgg 1920 He Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser He Trp 625 630 635 640 gca gca gee gat cag atg gca cgt ggc ttc etc ttg ggc gct acc gca 1968 Wing Wing Wing Asp Gln Met Wing Arg Gly Phe Leu Leu Gly Wing Thr Wing 645 650 655 ggt cgc acc action ctg acc ggt gag ggc etc cag falls atg gat gga falls 2016 Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His 660 665 670 tec ect gtc ttg gct tec acc aac gag ggt gtc gag acc tac gac cea 2064 Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro 675 680 685 tec ttt gcg tac gag ate gca falls ctg gtt falls cgt ggc ate gac cgc 2112 Being Phe Wing Tyr Glu He Wing His Leu Val His Arg Gly He Asp Arg 690 695 700 atg tac ggc cea ggc aag ggt gaa gat gtt ate tac tac ate acc ate 2160 Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Tyr Tyr He Thr He 705 710 715 720 tac aac gag cea acc cea cag cea gct gag cea gaa gga ctg gac gta 2208 Tyr Asn Glu Pro Thr Pro Gln Pro Wing Glu Pro Glu Gly Leu Asp Val 725 730 735 gaa ggc ctg falls aag ggc ate tac etc tac tec cgc ggt gaa ggc acc 2256 Glu Gly Leu His Lys Gly He Tyr Leu Tyr Ser Arg Gly Glu Gly Thr 740 745 750 ggc cat gag gca aac ate ttg gct tech ggt gtt ggt atg cag tgg gct 2304 Gly His Glu Ala Asn He Leu Ala Ser Gly Val Gly Met Gln Trp Ala 755 760 765 etc aag gct tec ate ctt gag gct gac tac gga gtt cgt gee aac 2352 Leu Lys Ala Ala Ser He Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn 770 775 780 att tac tec gct act tet tgg gtt aac ttg gct cgc gat ggc gct gct 2400 He Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala 785 790 795 800 cgt aac aag gca cag ctg cgc aac cea ggt gca gat gct ggc gag gca 2448 Arg Asn Lys Wing Gln Leu Arg Asn Pro Gly Wing Asp Wing Gly Glu Wing 805 810 815 ttc gta acc acc cag ctg aag cag acc tec ggc cea tac gtt gca gtg 2496 Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val 820 825 830 tet gac ttc tec act gat ctg cea aac cag ate cgt gaa tgg gtc cea 2544 Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln He Arg Glu Trp Val Pro 835 '840 845 ggc gac tac acc gtt etc ggt gca gat ggc ttc ggt ttc tet gat acc 2592 Gly Asp Tyr Thr Val Leu Gly Wing Asp Gly Phe Gly Phe Ser Asp Thr 850 855 860 cgc cea gct gct cgt cgc ttc ttc aac ate gac gct gag tec att gtt 2640 Arg Pro Wing Wing Arg Arg Phe Phe Asn He Asp Wing Glu Ser He Val 865 870 875 880 gtt gca gtg ctg aac tec ctg gca cgc gaa ggc aag ate gac gtc tec 2688 Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys He Asp Val Ser 885 890 895 gtt gct gct cag gct gct gag aag ttc aag ttg gat gat ect acg agt 2736 Val Wing Wing Gln Wing Wing Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser 900 905 910 gtt tec gta gat cea aac gct ect gag gaa 2766 Val Ser Val Asp Pro Asn Ala Pro Glu Glu < 210 > 32 < 211 > 8556 < 212 > nucleic acid < 213 > Brevibacterium lactofermentum ATCC13869 < 400 > 32 tcacgttacg gegatcaaca ccgcaaccac taegagaaga tctccaaacg agaccaagag 60 cgcttctaag cccgtctcat tttgcacctg ccattctgtg aggatatggc aggtgctttt 120 tcatgccact atcttggggt tctcggtatt agatettetg ataaaaaccc gatagttttc 180 ttgcgctaga cactaattac ggcaccgctt aagcatggtc gtgacacgta aaacctgact 240 taggccattt tgatgtggtg tagatcatat tgacgtcaat gaatgaagtg actaactccg 300 atcgtctaaa ccgaatccac gaccacgtaa aggcctgggc gaegagaaga agacgggcac 360 tcatcgacgc aactttacgg ctcatcgaca gcaatcgtcc cgtcacggtc aatgcagttg 420 tcaaagaaag cggagtggca cgtgcagcgg tttatcgacg ctggcccagg ctagtggatc 480 tagtagcgga agctttagat gccgggcgag ctccagttga aatagatacc ccaggggaca 540 tcaaagagac cttgattgat gggctgttta caaatcaggc gaaaaccact ggagtctcct 600 atcctcgtca gcgatttcgc aaacggctcg agttggtgat gtcagatcaa gaattacagc 660 tcgcctaatg gaattcacat gtgaagagac gtcgagaagc aaatattcgc gcgctgcaag 720 aaaaggccaa tcgcgcaaga atccgggcgg atctagacat cgaggcgtgc ctcgatgcaa 780 tccttggggt gttttattac caatcggtcg cgcgtggagt aaatttcacc gaccaaggta 840 caacgcaacg atgcaga gaa gccttggagg tgatctggca tggaatggaa ccttaaattc 900 aggttctgac gaggtgcgaa gcaagttgtc gcgcgccgca cctcagtatc cggatcaact 960 taatttcgaa gtgctgggtt ttctcgcgca tacccaatgc gtaccgatgt gcccatgagc 1020 gaaaaacagg ccacgataag tttcttaaaa cttatcgtgg cctgcttcta tatttgtgcg 1080 ccctgacggg ctcgaaccgc cgacctgctg ggtgtaaacc agctgctctt ccagctgagc 1140 taaaggcgcg cacgtgcttt tctagaacca ctcgaaagca ccttggtggc acgagtgaaa 1200 tactaacaca caatctccac tcgctgctca agacctaaaa attagcgatt ggccgtggaa 1260 cttgtttcca gttaaggctt cgctggacga ggcaagaacc ttgccaatta ccgagacgtt 1320 ccgccttggt ctgcacgaga cctgccagtt gtgctgattc agagataact ccaggagcca 1380 gggctccttc tttaccaatg ccaggagtca acacccagat acgaccattc tcagcgaggg 1440 atccacaagt agcggatgga ccgtcgacga gatcgccgtc caccagagca atcctcgcgc 1500 gcacgacatc gcacagctcg tcggtttctt catcgagtag ttcctcaccg attgcatctt 1560 cgatggactc gctgatcagc gtgtcggaat cttcatccca tccaatttct tgaacgatat 1620 gacccgattg aatgccgagt agttgagcat aatcctgggc accttgcttg actgcgcccg 1680 cactttaata gagcgtcggc TCCA tcctcg tgtgggcccc gatgtgtttt tcgattacat 1740 ggattcaaca tgaaaccgcg gggctattga tatatccgaa ttgcacatta ccgtccaacc 1800 ggtactttga accacctttc cctggaattt tttccttttc ctcccccttt acgctcaaga 1860 atcaatgaat tcaatcactg gccagcgatt aacttttcga gttttcagtc ttggatttcc 1920 acaattctct tcaaaataat tttttcatca ggtggctaga aaccctcacc aaaaggacat 1980 cagacctgta gttttatgcg attcgcgtca aaacatcaca aacgtgagag tctcacggga 2040 aactacccga taattctttg caaaactttg caaagggtaa tgaacatgca gctagtttcc 2100 tctttaaaaa gtagaaatgt atccacaaca attgccagga ttgatggata agcacaccga 2160 cctgaaatcc cagtgagcgc accactcccc ttacgtcaca gtctgtaaaa caaatcttcg 2220 tccttgttaa gtgttgcgta taacttatgc gttgacccat tcgtgcactt cggtgtgcca 2280 cgaccaagaa caattaggta tgggaccggg aaaccgggac gtataaacga aataaaacat 2340 ggtgtggaa tccaacagga atg gcc gat caca gca aaa ctt ggt ggt aag ecc 2392 Met Wing Asp Gln Wing Lys Leu Gly Gly Lys Pro 5 10 teg gat gac tet aac ttc gcg atg ate cgc gat ggc gtg gca tet tat 2440 Ser Asp Asp Ser Asn Phe Wing Met lie Arg Asp Gly Val Ala Ser Tyr 15 20 25 ttg aac gac tea gat ccg gag gag acc aac gag tgg atg gat tea etc. 2488 Leu Asn Asp Ser Asp Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu 30 35 40 gac gga tta etc cag gag tet tet cea gaa cgt gct cgt tac etc atg 2536 Asp Gly Leu Leu Gln Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met 45 50 55 ctt cgt ttg ctt gag cgt gca tet gca aag cgc gta tet ctt ecc cea 2584 Leu Arg Leu Leu Glu Arg Wing Being Wing Lys Arg Val Ser Leu Pro Pro 60 65 70 75 atg acg tea acc gac tac gtc aac acc att cea acc tet atg gaa ect 2632 Met Thr Ser Thr Asp Tyr Val Asn Thr He Pro Thr Ser Met Glu Pro 80 85 90 gaa ttc cea ggc gat gag gaa atg gag aag cgt tac cgt cgt tgg att 2680 Glu Phe Pro Gly Asp Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp lie 95 100 105 cgc tgg aac gca gcc ate atg gtt drops cgc gct cag cga cea ggc ate 2728 Arg Trp Asn Wing Wing He Met Val His Arg Wing Gln Arg Pro Gly He 110 115 120 ggc gtc ggc gga falls att tec act tac gca ggc gca gcc ect ctg tac 2776 Gly Val Gly Gly His He Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr 125 130 135 gaa gtt ggc ttc aac falls ttc tcc cgc ggc aag gat cae cea ggc ggc 2824 Glu Val Gly Phe Asn His Phe Phe Arg Gly Lys Asp His Pro Gly Gly 140 145 150 155 ggc gac cag ate ttc ttc cag ggc falls gca tea cea ggt atg tac gca 2872 Gly Asp Gln He Phe Phe Gln Gly His Wing Ser Pro Gly Met Tyr Wing 160 165 170 cgt gca ttc atg gag ggt cgc ctt tet gaa gac gat etc gat ggc ttc 2920 Arg Wing Phe Met Glu Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe 175 180 185 cgt cag gaa gtt tec cgt gag cag ggt gge att ccg tec tac ect cae 2968 Arg Gln Glu Val Ser Arg Glu Gln Gly Gly He Pro Ser Tyr Pro His 190 195 200 cea falls ggt atg aag gac ttc tgg gag ttc cea act gtg tec atg ggt 3016 Pro His Gly Met Lys Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly 205 210 215 ctt ggc cea atg gat gcc att tac cag gca cgt tcc aac cgc tac etc 3064 Leu Gly Pro Met Asp Wing He Tyr Gln Wing Arg Phe Asn Arg Tyr Leu 220 225 230 235 gaa aac cgt ggc ate aag gac acc tet gac cag falls gtc tgg gcc tcc 3112 Glu Asn Arg Gly He Lys Asp Thr Ser Asp Gln His Val Trp Wing Phe 240 245 250 ctt ggc gac ggc gaa atg gac gag cea gaa tea cgt ggt etc ate cag 3160 Leu Gly Asp Gly Glu Met Asp Glu Pro Glu Be Arg Gly Leu He Gln 255 260 265 cag gct gca ctg aac aac ctg gac aac ctg acc ttc gtg gtt aac tgc 3208 Gln Ala Ala Leu Asn Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys 270 275 280 aac ctg cag cgt etc gac gga ect gtc cgc ggt aac acc aag ate ate 3256 Asn Leu Gln Arg Leu Asp Gly Pro Val Arg Gly Asn Thr Lys He He 285 290 295 cag gaa etc gag tec ttc tcc cgt ggc gca ggc tgg tet gtg ate aag 3304 Gln Glu Leu Glu Ser Phe Phe Arg Gly Wing Gly Trp Ser Val He Lys 300 305 310 315 gtt gtt tgg ggt cgc gag tgg gat gaa ett ctg gag aag gac cag gat 3352 Val Val Trp Gly Arg Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp 320 325 330 ggt gca ctt gtt gag ate atg aac aac acc tec gat ggt gac tac cag 3400 Gly Ala Leu Val Glu He Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln 335 340 345 acc ttc aag gct aac gac ggc gca tat gtt cgt gag falls ttc ttc gga 3448 Thr Phe Lys Wing Asn Asp Gly Wing Tyr Val Arg Glu His Phe Phe Gly 350 355 360 cgt gac cea cgc acc gca aag etc gtt gag aac atg acc gac gaa gaa 3496 Arg Asp Pro Arg Thr Ala Lys Leu Val Glu Asn Met Thr Asp Glu 365 370 375 ate tgg aag ctg cea cgt ggc ggc falls gat tac cgc aag gtt tac gca 3544 He Trp Lys Leu Pro Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Wing 380 385 390 395 gcc tac aag cga gct ctt gag acc aag gat cgc cea acc gtc ate ctt 3592 Wing Tyr Lys Arg Wing Leu Glu Thr Lys Asp Arg Pro Thr Val He Leu 400 405 410 gct falls acc att aag ggc tac gga etc ggc falls aac ttc gaa ggc cgt 3640 Wing His Thr He Lys Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg 415 420 425 aac gca acc fall cag atg aag aag ctg acg ctt gat gat ctg aag ttg 3688 Asn Ala Thr His Gln Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu 430 435 440 tcc cgc gac aag cag ggc ate cea ate acc gat gag cag ctg gag aag 3736 Phe Arg Asp Lys Gln Gly He Pro He Thr Asp Glu Gln Leu Glu Lys 445 450 455 gat ect tac ctt ect ect tac tac falls cea ggt gaa gac gct ect gaa 3784 Asp Pro Tyr Leu Pro Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu 460 465 470 475 ate aag tac atg aag gaa cgc cgc gca gcg etc ggt ggc tac ctg cea 3832 He Lys Tyr Met Lys Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro 480 485 490 gag cgt cgt gag aac tac gat cea att cag gtt cea cea ctg gat aag 3880 Glu Arg Arg Glu Asn Tyr Asp Pro He Gln Val Pro Pro Leu Asp Lys 495 500 505 ctt cgc tet gtc cgt aag ggc tec ggc aag cag cag ate gct acc act 3928 Leu Arg Ser Val Arg Lys Gly Ser Gly Lys Gln Gln He Wing Thr Thr 510 515 520 atg gcg act gtt cgt acc ttc aag gaa ctg atg cgc gat aag ggc ttg 3976 Met Wing Thr Val Arg Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu 525 530 535 gct gat cgc ctt gtc cea ate att ect gat gag gca cgt acc ttc ggt 4024 Wing Asp Arg Leu Val Pro He He Pro Asp Glu Wing Arg Thr Phe Gly 540 545 550 555 ctt gac tet tgg ttc cea acc ttg aag ate tac aac ccg falls ggt cag 4072 Leu Asp Ser Trp Phe Pro Thr Leu Lys He Tyr Asn Pro His Gly Gln 560 565 570 aac tac gtg ect gtt gac falls gac ctg atg etc tec tac cgt gag gca 4120 Asn Tyr Val Pro Val Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala 575 580 585 ect gaa gga cag ate ctg falls gaa ggc ate aac gag gct ggt tec gtg 4168 Pro Glu Gly Gln He Leu His Glu Gly He Asn Glu Wing Gly Ser Val 590 595 600 gca teg ttc ate gct gcg ggt acc tec tac gcc acc falls ggc aag gcc 4216 Wing Being Phe He Wing Wing Gly Thr Ser Tyr Wing Thr His Gly Lys Wing 605 610 615 atg att ccg ctg tac ate ttc tac teg atg ttc gga ttc cag cgc acc 4264 Met He Pro Leu Tyr He Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr 620 625 630 635 ggt gac tec ate tgg gca gca gcc gat cag atg gca cgt ggc ttc etc 4312 Gly Asp Ser He Trp Wing Wing Wing Asp Gln Met Wing Arg Gly Phe Leu 640 645 650 ttg ggc gct action ggt ggt cgc acc acc action ggt gag ggc etc cag 4360 Leu Gly Wing Thr Wing Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln 655 660 665 falls atg gat gga falls tec ect gtc ttg gct tec acc aac gag ggt gtc 4408 His Met Asp Gly His Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val 670 675 680 gag acc tac gac cea tec ttt gcg tac gag ate gca falls etg gtt drops 4456 Glu Thr Tyr Asp Pro Ser Phe Wing Tyr Glu He Wing His Leu Val His 685 690 695 cgt ggc ate gac cgc atg tac ggc cea ggc aag ggt gaa gat gtt ate 4504 Arg Gly He Asp Arg Met Tyr Gly Pro Gly Lys Gly Glu Asp Val He 700 705 710 715 tac tac ate acc ate tac aac gag cea ac cea eag cea gct gag cea 4552 Tyr Tyr He Thr He Tyr Asn Glu Pro Thr Pro Gln Pro Wing Glu Pro 720 725 730 gaa gga ctg gac gta gag ggc ctg falls aag ggc ate tac etc tac tec 4600 Glu Gly Leu Asp Val Glu Gly Leu His Lys Gly He Tyr Leu Tyr Ser 735 740 745 cgc ggt gag ggc acc ggc cat gag gca aac ate ttg gct tech ggt gtt 4648 Arg Gly Glu Gly Thr Gly His Glu Wing Asn He Leu Wing Ser Gly Val 750 755 760 ggt atg cag tgg gct etc aag gct gca tec ate ctt gag gct gac tac 4696 Gly Met Gln Trp Wing Leu Lys Wing Wing Being He Leu Glu Wing Asp Tyr 765 770 775 gga gtt cgt gcc aac att tac tec gct act tet tgg gtt aac ttg gct 4744 Gly Val Arg Wing Asn He Tyr Ser Wing Thr Ser Trp Val Asn Leu Wing 780 785 790 795 cgc gat ggc gct gct cgt aac aag gca cag ctg cgc aac cea ggt gca 4792 Arg Asp Gly Ala Ala Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala 800 805 810 gat gct ggc gag gca ttc gta acc action cag ctg aag cag acc gcc tec 4840 Asp Wing Gly Glu Wing Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly 815 820 825 cea tac gtt gca gtg tet gac ttc tec act gat ctg cea aac cag ate 4888 Pro Tyr Val Ala Val Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln He 830 835 840 cgt gaa tgg gtc cea ggc gac tac acc gtt etc ggt gc gat gcc tcc 4936 Arg Glu Trp Val Pro Gly Asp Tyr Thr Val Leu Gly Wing Asp Gly Phe 845 850 855 ggt ttc tet gat ccc acc gcc gct cct cct gtc ttc aac ate gac 4984 Gly Phe Ser Asp Thr Arg Pro Wing Wing Arg Arg Phe Phe Asn He Asp 860 865 870 875 gct gag tec att gtt gtt gca gtg ctg aac tec ctg gca cgc gaa ggc 5032 Wing Glu Ser He Val Val Wing Val Leu Asn Ser Leu Wing Arg Glu Gly 880 885 890 aag ate gae gtc tec gtt gct gct cag gct gct gag aag ttc aag ttg 5080 Lys He Asp Val Ser Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu 895 900 905 gat gat ect acg agt gtt tec gta gat cea aac gct ect gag gaa taaat 5130 Asp Asp Pro Thr Ser Val Ser Val Asp Pro Asn Ala Pro Glu Glu 910 915 920 cacctcaagg gacagataaa tcccgccgcc agacgttagt ctggcggcgg gattcgtcgt 5190 aaagcaagct ctttttagcc gagaaaegee ttgtcagaca atgttgcgcc cttgatattg 5250 gcgaactcct gcagcaaatc gcgcacagtc aacttcgact tggtagcctg atctgcctgg 5310 tagacaatct ggccttcatg catcatgatc aggcgat gc ccaggcgaat tgcctgttcc 5370 atgttgtgcg tgaccataag cgtagtcaga gttccatctg ccacgatctt ttcggtcaag 5430 gtggtcacaa gctctgcacg ctgtggatca agcgctgcgg tgtgctcatc caacageatg 5490 attttaggtt gagtaaaacc agccatcagc agggacaatg cctgacgctg accgccagag 5550 agcaaaccaa ctttggcagt gagcctgttt tccagaccca gctcaaggcg ctcaagttcc 5610 tgcttgaatt gctcacggcg cttcgaggtc agtgcaaagc ccaatccacg gcgcttgccg 5670 cgcagcaacg cgatggccag attetettea atggtgagat tcggcgcggt gcctgccaaa 5730 ggatcctgaa aaacgcggcc gatgtagcgg gcacgcttgt gctctgacat cttgtttacc 5790 ttgttgccgt cgatggaaat ctcgccggaa tcaacaagea aacggccaga aacagcgttg 5850 agcagggtgg atttacccgc accgttagaa ccgatgacgg tgacaaaatc gccctcagcc 5910 atategagtt tgagctgctg caacgcgcgg cgctcattca cagtgccggg gaagaaggtt 5970 egttgatgga ttggaaattc taacatgtct taagcctcca ctgctactgg ttgcttaggc 6030 t tcggtgcct tggagaactt cgcacgccac etcggcagca gcatggcgac aaccaccaag 6090 ategeagaaa ttgeetteat atcgttgggg tcaaggccaa cgcgcagtgc tgcgaaaatg 6150 atcaggcggt acgcgatggc acegaegatg cagccaacca acagccaaca cacgcgacgc 6210 tgacegaaga tggcctggcc ccaaaataac cgatgcgaga ccgatcacga tgaggccaat 6270 atatctgcga acccatcgaa agccctggta ctgagcgatg agtgcaccgg caagaccaac 6330 agaaceattg gacagggaga tggtgaggat tttggtgaaa tcegttgaaa caccaaagga 6390 ctgcaccatc ggcccgttgt cgccggtgga tcgcagcgac agtcegatat cagtgttgag 6450 gaaccagatg acgatgagtc ccaaaaatcc cactgcaacg gcgaggatcg ccgggcctgc 6510 ccatgtgccg aggaggccgg cgtcgcgaag cggggtgaag aggttatcgg tgcgcaacaa 6570 gcgccaccca tggcacgttc tgatgcgcaa gttaaccgac tcatggtcaa cacaacgcaa 6630 aatacctgcg agcaaaccat cgatcttgcc cttggtgtgc agcaaaccgg tgatcatgee 6690 agcgataaag ecagtaaega aaccagcggc agtagecata agaggaggcc agecagacat 6750 aagagctgtc gcagctgttg ccgcgccagt ggtcaggctg ccgtcaacgg tgaggtcggg 6810 aaagttgagc acacggaacg tcaaatagac gcccaatgcg acaactccgt acaacaatcc 6870 gaactca aaa gcgccgatca tacgcgttcg gccttatcca aaatctcttg agggatctcc 6930 acgccctggc gctctgctgc atcttcgttg atcacgtagg tgaactcagt tgcagtctcc 6990 acaggcatgg ttgctgggtc ttcgccgtcc tgeagaatac geagagecat ctcgccagtc 7050 gctcggtgta tggcggccaa atcgataccc agggttgcca gtgcgccacc etcaacagtg 7110 ccggactcag caccgatcac agggatctgc ttctgctcag caacctgaac cagagaagaa 7170 caaccatgtt ataccggaaa acgtagatga gtcagttgga catcaacatc gcegagaget 7230 tcaacagcct gctgaatctc gttcacggta gtgacagtct gagtattaac ggacagcccc 7290 agtggctcag cagccttggt gacctcatcg acctgcacct gagagttgac ctcaccagac 7350 tgccgatgga gcgtagacga ctttgcgtca ggaaccaget gctgcaaaag ctccaactgc 7410 tgctcaateg gtgcgatatc agaagtaccg gtgacgtttc cgccaggtgc ttcattagaa 7470 tccaccagct ctgccgacac tgcatcggta actgcggtga acaggactgg gatatcagtg 7530 atattctgcg cagttgcctg tgctgctgga gttgcaacag ccaacacgag atccaaattg 7590 actgctgaga teagaagega aatagtcagt gcagtgccct gctcgccgtt agcgttttgc 7650 tcatcaaagg tgacgtcaac gcctgcctct tcaaaagctt ccttgaaacc agtggtcgct 7710 gcatcaagtg ca gggtgctg aacaagctgg ttgatgccaa ctcggtaaga gtcgccacct 7770 gcagcatcag tggaggtgga gctgtcactg gaatcgcttg ageaegaage caacgccaag • 7830 gcgccaacag taaagatgct tgcgagtacc ttcgaacggg aagaaaacat agcacatctc 7890 tattttcaaa cttaaagtgt aaggggcaga cagcgtcaac acatgtctcg gataaagaac 7950 catatgtgaa atgtctcatg atttaaacta cttgttctac cagtcatatg cgcaattccc 8010 ccgcaggaca cctggatatc tggacaaaat gggtggatag cgggtgcacc aattcaatct 8070 tttaaaggcc ctagacaccg cgatttcctt aatcgatcat taaagaggga tcctctcccc 8130 taacaaacct ccaaagacta gagtggggaa caccatgaac gtttcctcaa ataaacccag 8190 tgactctgac cgcgaatatc ttcaatcaga actcacccgg ctcgttggcc aggggcgact 8250 cgatctagat acttaccaag acgtggttga taccgtttgg tctactgatg atctaggcga 8310 gttgatgagg atccgtgccc gcttcctggg agggccgcag gtttcgcagc agcggcccca 8370 gcagccgcag caaccacatc agcggccgca acagcaaccg ccacagcatt atggacaacc 8430 cggctacggc caatcacctc aatatccacc gcagcagcct ccgcataatc agcccggcta 8490 ttaccccgat cccggccctg gccagcagca accaccgatg caccagccac caacgcgtcc 8550 aaatca < 210 > 33 < 211 > 20 < 212 > nucleic acid < 220 > initiator to construct LA gene amplification plasmid of pdhA from Brevibacterium lactofermentum < 400 > 33 aat gcc agg agt caá cae ce < 210 > 34 < 211 > 20 < 212 > nucleic acid < 220 > initiator to construct LA gene amplification plasmid of pdhA from Brevibacterium lactofermentum < 400 > 34 here tgg aac agg caá ttc ge < 210 > 35 < 211 > 28 < 212 > nucleic acid < 220 > initiator to introduce a LA gene promoter mutation of pdhA from Brevibacterium lactofermentum < 400 > 3d cgt ecc ggg ctg taa aac aaa tet teg g < 210 > 36 < 211 > 27 < 212 > nucleic acid < 220 > initiator to introduce an LA gene promoter mutation of pdhA from Brevibacterium lactofermentum < 400 > 36 ate ecc ggg ctt acc acc aag ttt tgc < 210 > 37 < 211 > 30 < 212 > nucleic acid < 220 > initiator to introduce a LA gene promoter mutation of pdhA from Brevibacterium lactofermentum < 400 > 37 ctt atg cgt tgc falls att cgt gca ctt cgg < 210 > 38 < 211 > 40 < 212 > nucleic acid < 220 > initiator to introduce a LA gene promoter mutation of pdhA from Brevibacterium lactofermentum < 400 > 38 gcg ttg acc cat teg tgc act teg gtg tgc tat aat tag g < 210 > 39 < 211 > 40 < 212 > nucleic acid < 220 > initiator to introduce a LA gene promoter mutation of pdhA from Brevibacterium lactofermentum < 400 > 39 gcg ttg cea cat teg tgc act teg gtg tgc tat aat tag g < 210 > 40 < 211 > 38 < 212 > nucleic acid < 220 > initiator to introduce a LA gene promoter mutation of pdhA from Brevibacterium lactofermentum < 400 > 40 ttt taa aac gtt ctg gag aag act ect gga gta ate cg < 210 > 41 < 211 > 20 < 212 > nucleic acid < 220 > initiator to introduce an LA gene promoter mutation of pdhA from Brevibacterium lactofermentum < 400 > 41 cga tet tgc ctt cgc gtg ce < 210 > 42 < 211 > 30 < 212 > nucleic acid < 400 > 42 agaccgccgg agtatgcaag aacgatgcgg < 210 > 43 < 211 > 30 < 212 > nucleic acid < 400 > 43 gacttcacca tcaatcatct tcttcaggta < 210 > 44 < 211 > 30 < 212 > nucleic acid < 400 > 44 accttcgacc agaccctggc taagggcttt < 210 > 4d < 211 > 30 < 212 > nucleic acid < 400 > 4d gctaacaagc gcgatcgcga agctggcaac < 210 > 46 < 211 > 25 < 212 > nucleic acid < 400 > 46 gcgatgacac cgtttttgtt ctcgc < 210 > 47 < 211 > 2d < 212 > nucleic acid < 400 > 47 ggcgacatcc ttgcccagat crawls < 210 > 48 < 211 > 2d < 212 > nucleic acid < 400 > 48 gacttcacca tcaatcatct tcttc < 210 > 49 < 211 > 24 < 212 > nucleic acid < 400 > 49 gccaggtaca actgtctgaa ttgc < 210 > d0 < 211 > 40 < 212 > nucleic acid < 220 > initiator to introduce a mutation < 400 > 50 gttaateget tgecaatgea ggcaggtaag gtataacccg < 210 > 51 < 211 > 40 < 212 > nucleic acid < 400 > 61 gttaateget tgetaatgea ggcaggtaag gtataacccg < 210 > d2 < 211 > 40 < 212 > nucleic acid < 400 > 62 gttaateget tgtcaatgca ggcaggtaag gtataacccg < 210 > d3 < 211 > 40 < 212 > nucleic acid < 400 > 63 gttaateget tgttaatgca ggcaggtaag gtataatccg < 210 > d4 < 211 > 40 < 212 > nucleic acid < 400 > 54 gttaateget tgtcaatgca ggcaggtaag gtataatccg < 210 > 5d < 211 > 30 < 212 > nucleic acid < 400 > dd gggttccagc ctcgtgcgga attegtggag < 210 > d6 < 211 > 2d < 212 > nucleic acid < 400 > 66 gcgttaccca gagctggatc etegg < 210 > d7 < 211 > 16 < 212 > nucleic acid < 400 > 67 cagttgtggc tgatcg < 210 > d8 < 211 > 17 < 212 > nucleic acid < 400 > 68 ctttcccaga ctctggc < 210 > d9 < 211 > 21 < 212 > nucleic acid < 400 > 69 gctataattt gacgtgagca t < 210 > 60 < 211 > 25 < 212 > nucleic acid < 400 > 60 gctcacgtca aattatagca gtgtc < 210 > 61 < 211 > d4 < 212 > nucleic acid < 400 > 61 ttgttgtcat tctgtgcgac actgctataa tttgaacgtg ageagttaac agee < 210 > 62 < 211 > 63 < 212 > nucleic acid < 400 > 62 gttaactgct cacgttcaaa ttatageaft gtegeacaga atgacaacaa agaattaaaa ttg < 210 > 63 < 211 > 2d < 212 > nucleic acid < 400 > 63 gctagcctcg ggagctctct aggag < 210 > 64 < 211 > 2d < 212 > nucleic acid < 400 > 64 gatctttccc agactetggc caege

Claims (17)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for producing coryneform bacteria having an improved amino acid or nucleic acid productivity, which consists of introducing a mutation in a promoter sequence of biosynthesizing genes of amino acids or nucleic acids in a chromosome of a coryneform bacterium to make it close to a sequence of consensus or introducing a change in a promoter sequence of biosynthesizing genes of amino acids or nucleic acids in a chromosome of a coryneform bacterium by recombining genes to make it close to a consensus sequence, to obtain a mutant of the coryneform microorganism producer of amino acids or nucleic acids , by culturing the mutant and selecting a mutant capable of producing the amino acid or nucleic acid destined in large quantity.

2. The method according to claim 1, further characterized in that the amino acid is selected from the group consisting of glutamic acid, lysine, arginine, serine, phenylalanine, proline and glutamine, and the nucleic acid is selected from the group consisting of Inosine, guanosine, adenosine and nucleotide.

3. The method according to claim 1, further characterized in that the amino acid is glutamic acid, and the promoter for the biosynthesizing gene is selected from the group consisting of a promoter for a glutamate dehydrogenase (GDH) gene, a promoter for a citrate synthetase (CS) gene, a promoter for an isocitrate synthetase gene (ICDH), a promoter for a pyruvate dehydrogenase (PDH) gene and a promoter for a aconitase producer (ACO).

4. The method according to claim 3, further characterized in that the promoter for the glutamate dehydrogenase (GDH) gene has a DNA sequence selected from the group consisting of (i) at least one DNA sequence selected from the group which consists of CGGTCA, TTGTCA, TTGACA and TTGCCA in the -3d region, (ii) the sequence of TATAAT in the same sequence of TATAAT but in which the base of ATAAT is replaced by another base in region -10, and ( iii) a combination of (i) and (ii), wherein the sequence does not inhibit the function of the promoter.

5. The method according to claim 4, further characterized in that the promoter for GDH has TGGTCA in the -35 region, and TATAAT in the -10 region; or TTGTCA in the -35 region and TATAAT in the -10 region.

6. The method according to claim 3, further characterized in that the promoter for CS has sequence (i) TTGACA in the -35 region, (ii) TATAAT sequence in the -10 region, or (iii) a sequence of the combination of (i) and (ii) and the sequence does not inhibit the function of the promoter.

7. A method according to claim 3, further characterized in that at least one of the first and second ICDH promoters has (i) TTGCCA sequence or TTGACA in the -3d region, (ii) TATAAT sequence in the -10 region. or (ii) a combination sequence of (i) and (i) and the sequence does not inhibit the function of the promoter.

8. The method according to claim 3, further characterized in that the PDH promoter has (i) TTGCCA sequence in the -3d region, (i) TATAAT sequence in the -10 region or (iii) a sequence of the combination of (i) and (ii) and the sequence does not inhibit the function of the promoter.

9. The method according to claim 1, further characterized in that the amino acid is arginine and the promoter for the biosynthesizing gene is an argininosuccinate synthetase promoter.

10. The method according to claim 9, further characterized in that the promoter for the argininosuccinate synthetase has a DNA sequence selected from the group consisting of (i) at least one DNA sequence selected from the group consisting of TTGCCA, TTGCTA and TTGTCA in region -36, (ii) TATAAT sequence or TATAAT sequence but in which the base of ATAAC is replaced by another base in region -10, and (iii) a combination of (i) and (ii) and the sequence does not inhibit the promoter function.

11. The method according to claim 10, further characterized in that the promoter for the argininosuccinate synthetase has a DNA sequence selected from the group consisting of (i) TTGTCA in the -35 region, (ii) sequence of TATAAT in the region -10, and (iii) a combination of (i) and (¡i).

12. - A glutamic acid synthesizing gene having a promoter according to any of claims 4 to 8.

13. An arginine synthetase gene having the promoter of claim 10.

14. A coryneform bacterium producing glutamic acid that has the glutamate synthetase gene of claim 12.

15. An arginine-producing coryneform bacterium having the arginine synthetase gene of claim 13.

16. A method for producing an amino acid or nucleic acid by fermentation, comprising the steps of culturing a coryneform bacterium constructed by the method of any of claims 1 to 11 and having an improved amino acid or nucleic acid productivity, or a coryneform bacterium of claims 14 or 15 in a culture medium to form and This way accumulate the amino acid or nucleic acid destined in the culture medium and collect it from the culture medium.

17. A method for producing L-glutamic acid by fermentation, comprising the steps of culturing a coryneform bacterium producing L-glutamic acid resistant to 4-fluoroglutamic acid in a liquid culture medium to produce and thus accumulate L-acid. glutamate in the culture medium and collect it from the culture medium.